Factors Affecting the Recovery of Orchids in a Post-Mining Landscape
FACTORS AFFECTING THE RECOVERY
OF ORCHIDS
IN A POST-MINING LANDSCAPE
Margaret Thora Collins BSc. Honours (Science), MSc. (Science)
This Thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia School of Earth and Geographical Sciences Discipline of Soil Science 2007 Photograph on title page: Prasophyllum hians Rchb.f. in flower. Abstract
Currently, Alcoa World Alumina Australia (Alcoa) mines and undertakes procedures to rehabilitate approximately 550 ha of jarrah forest each year at two open-cut bauxite mines in South-West Western Australia. Alcoa aims to establish a self-sustaining jarrah forest ecosystem that maintains the functions of the landscape prior to mining, including biodiversity, on areas that have been mined for bauxite. Indigenous terrestrial orchids form a significant proportion of the indigenous geophytic plant species that either fail to colonise rehabilitated areas or do so very slowly. Terrestrial orchids are considered to be particularly sensitive to competition from weeds and disturbance, which combined with the obligate nature of the orchid-mycorrhizal fungus association suggests that orchids would colonise rehabilitation areas only when both microhabitat sites and soil microflora have established. Occurrence of certain orchids may therefore be expected to be useful as indicators of ecosystem health, the success of vegetation establishment and the recovery of edaphic conditions suitable for orchid mycorrhizal fungi.
Vegetation surveys were undertaken to compare orchid species richness and population size of a chrono-sequence of rehabilitation areas with adjacent unmined forest. Total orchid and clonal orchid population sizes of rehabilitation areas had returned to densities not significantly different to those of the adjacent unmined forest within five years of rehabilitation establishment. The total number of orchid species and clonal species richness had also returned to levels that were not significantly different to those of adjacent unmined forest within the same time frame. However, orchid populations in rehabilitation areas often contained large numbers of species identified as disturbance opportunists, which were absent from 25+ year old rehabilitation areas and undisturbed jarrah forest sites. Six orchid taxa were identified as recalcitrant species; Cryptostylis ovata R.Br., Cyrtostylis heugelii Endl. In Lehm., Eriochilus dilatatus Lindl., Prasophyllum parvifolia Lindl., Pyrochis nigricans (R.Br.) D.L.Jones and M.A.Clem. and Thelymitra crinita Lindl.. These taxa were either very rare (compared to unmined forest populations), or absent from rehabilitated areas.
The role of orchid mycorrhizal fungi (OMF) in the recruitment of orchids in the rehabilitated landscape was investigated using a seed baiting technique in a subset of the rehabilitation survey areas. These consisted of single transects that were 1, 10 and 26
i years old (established in 2001, 1992 and 1976) and their adjacent unmined Jarrahforest. Fungal baits consisted of buried six chambered nylon mesh packets containing seed of the six jarrah forest orchid taxa; Caladenia flava R.Br. subsp. flava (C. flava), Disa bracteata Sw., Microtis media R.Br. subsp. media, Pterostylis recurva Benth., Pyrorchis nigricans and Thelymitra crinita. Germination of seed and development of protocorms to stage 4 (initiation of shoot primordia) was regarded as evidence of the presence of a particular orchid’s mycorrhizal fungus. Detection of orchid mycorrhizal fungi was infrequent, especially in the youngest rehabilitation site examined, where only mycorrhizal fungi associated with P. recurva were detected. Mycorrhizal fungi of the other orchid taxa were widespread but sparsely distributed in older rehabilitation and forest areas. Detection of mycorrhizal fungi varied between taxa and baiting sites for the two survey years (2002 and 2004).
Caladenia flava and T. crinita mycorrhizal fungi were the most frequently detected. The presence of C. flava mycorrhizal fungi was correlated with high levels of leafy litter cover, and high maximum depth, and soil moisture at the vegetation type scale (50 x 5 m belt transects), as well as tree and litter cover at the microhabitat scale (1 m2 quadrats). The presence of T. crinita mycorrhizal fungi was positively correlated with soil moisture in rehabilitation areas and low shrub cover in forest. The frequency of detection of orchid mycorrhizal fungi in both rehabilitated sites (15 - 25% of baits) and unmined forest (15 - 50% of baits) tended to increase with rehabilitation age as vegetation recovered. The failure of some orchid taxa to reinvade rehabilitation areas is unlikely to be entirely due to absence of the appropriate mycorrhizal fungi. However, the infrequent detection of orchid mycorrhizal fungi suggests that they occur in isolated patches of soil, so the majority of dispersed orchid seeds are likely to perish especially in recently disturbed habitats.
The ecological specificity of mycorrhizal associations of the three orchid taxa: Caladenia flava, Thelymitra crinita and Disa bracteata were determined. The disturbance opportunist D. bracteata appears to be promiscuous in its mycorrhizal associations, although the promiscuity is limited to fungi within the Tulasnallales. The two common sympatric orchids C. flava and T. crinita used fungi from different taxonomic groups as mycorrhizal fungi which suggests that they are not competing directly for the same nutrient resources. Several orchid plants contained more than one ii mycorrhizal fungus, and in two individual orchids the second endophyte was a fungus more commonly associated with an ericoid mycorrhiza or ectomycorrhiza. Phylogenetic analysis using ITS rDNA region sequences of orchid mycorrhizal fungi (OMF) isolated in this study were generally in agreement with previous studies: C. flava was associated with fungi from the Sebacinales; and T. crinita and D. bracteata were associated with fungi from the Tulasnellales.
Evidence from this study indicates that with increasing age, the vegetation of rehabilitation areas develops structural and soil surface cover characteristics similar to that of unmined forest. Younger rehabilitation areas (between 1 and 15 years old) are very different to unmined forest in both vegetation structure and plant species assemblages, while the oldest rehabilitation areas examined (26 and 27 years old) were not significantly different to adjacent areas of unmined forest. It is evident that vegetation of the post-mining landscape is more homogeneous than that of unmined forest as there were fewer plant species and species assemblages present. Plant species assemblages of 1 to 15 year old rehabilitation areas were characterised by the presence of disturbance opportunist plant species. However, both the cover and species richness of these disturbance opportunist species had returned to unmined forest levels in rehabilitation areas over the chrono-sequence examined. The post-mining landscape appears to be developing a ‘new’ jarrah forest ecosystem that is structurally similar to unmined jarrah forest, but appears, floristically, less species rich and with fewer plant species assemblages. This lower species richness is in part due to the lower cover and species richness of tufted, rhizomatous and herbaceous species.
Orchid taxa present in each vegetation assemblage were generally not exclusive to these assemblages, with the following broad exclusions: D. bracteata was found only in species assemblages associated with rehabilitation areas; and Eriochilus sp. and T. crinita were found only in species assemblages associated with unmined forest. No single orchid species appears to be an indicator of ecosystem recovery. However, the presence of populations of C. flava, P. sp. crinkled leaf (G.J.Keighery 13426) or P. recurva in combination with the absence of the disturbance opportunist orchid taxa D. bracteata and M. media appears to be a measure of the maturity of the rehabilitation vegetation.
iii Orchid species richness and clonal orchid population size were correlated with changes in vegetation structure, but apart from the absence of orchids in 1 year old rehabilitation areas, these orchid population characteristics did not show any direct relationship with rehabilitation age or vegetation maturity. Only two orchid taxa appeared to have potential as indicators of vegetation characteristics: T. crinita as an indicator of undisturbed jarrah forest; and D. bracteata as an indicator of disturbed ecosystems.
The results of this study suggest that most jarrah forest orchid taxa will readily colonise the post bauxite mining landscape, but that the unassisted colonisation by recalcitrant orchid taxa may be a prolonged process. It is recommended that field-based transplantation and/or seeding trials be undertaken with these recalcitrant taxa to determine if these procedures will enhance recruitment. The results of this work have applications not only in the management of post-mining landscapes but also in vegetation monitoring and conservation work in Western Australia and elsewhere.
iv Table of Contents
Abstract i Table of Contents v Acknowledgments viii Candidates Declaration ix Definitions and terminology used in this document x Orchid Nomenclature xiii Fungal Nomenclature xiii Plant Nomenclature xiii Papers arising from this thesis xiv
INTRODUCTION 1
CHAPTER 1 LITERATURE REVIEW 5 Introduction 5 Jarrah forest 5 Bauxite Mining 7 Orchidaceae 9 Recovery of Orchids in Disturbed Habitats 12 Mycorrhizal Associations 14 Identification of Orchid Mycorrhizal Fungi 40 Thesis Outline and Research Approach 43
CHAPTER 2 RECRUITMENT OF TERRESTRIAL ORCHIDS IN THE POST- 45 MINING LANDSCAPE Introduction 45 Materials and Methods 47 Results 49 Discussion 55 Conclusions 60
CHAPTER 3 COLONISATION OF BAUXITE MINE REHABILITATION SITES OF 63 SOUTH-WEST WESTERN AUSTRALIA BY ORCHID MYCORRHIZAL FUNGI Introduction 63 Materials and Methods 66 Results 72 Discussion 81 Conclusions 86
CHAPTER 4 DIVERSITY OF ORCHID MYCORRHIZAL ENDOPHYTES 87 Introduction 87 Materials and Methods 90 Results 99 Discussion 108 Conclusions 114
v CHAPTER 5 THE RELATIONSHIP BETWEEN ORCHID DISTRIBUTION, 115 VEGETATION STRUCTURE AND PLANT SPECIES ASSEMBLAGES OF BAUXITE MINE REHABILITATION AREAS IN SOUTH-WEST WESTERN AUSTRALIA Introduction 115 Materials and Methods 119 Results 125 Discussion 156 Conclusions 165
CHAPTER 6 GENERAL DISCUSSION 167 Introduction 167 Orchid colonisation of the post-mining landscape 168 Detection of orchid mycorrhizal fungi 171 OMF identification 172 Vegetation establishment 173 Limitations of study 175 Future studies 176 Management recommendations 178
REFERENCES 181
APPENDICES 201
APPENDIX 1: ORCHIDS OF THE JARRAH FOREST 201 TABLE A1.1 A complete list of the indigenous orchid taxa of the Northern 201 jarrah forest. TABLE A1.2 Descriptions of taxa used as study species. 203 TABLE A1.3 Descriptions of orchid taxa identified within survey transects 206 at the study site during the course of the project. TABLE A1.4 Descriptions of orchid taxa observed in forest at the study site 209 during the course of the project but not found within survey transects. TABLE A1.5 Source and weights of orchid seed collected for use in 211 experimental work and in confirmation of mycorrhizal capacity of putative mycorrhizal fungi over the period of the study. TABLE A1.6 Specimens of Disa bracteata Sw. (syn. Monadenia bracteata, 212 M. micrantha, M. australis) in the Western Australian Herbarium, Department of Conservation and Land Management, South Perth, W.A. (Date accessed May 2005).
APPENDIX 2: VEGETATION OF THE JARRAHDALE MINE SITE 214 TABLE A2.1 A cumulative list of Western Australian indigenous plant taxa 214 identified in forest and rehabilitation area transects during vegetation surveys at the Jarrahdale bauxite-mine site in 2002. TABLE A2.2 A cumulative list of alien plant taxa identified during 218 vegetation surveys of forest and rehabilitation area transects at the Jarrahdale bauxite-mine site in 2002. vi FIGURE A2.1 Output matrix from TWINSPAN analysis of transect species 219 presence/absence data. FIGURE A2.2 Prevalence of rare species (number/transect) in rehabilitation 220 areas and unmined forest. FIGURE A2.3 Changes in mean percentage cover for ‘Lomandra and sedge- 220 like’ species over a chrono-sequence of rehabilitation areas compared to the mean value for unmined forest.
APPENDIX 3 FORMULAE FOR MEDIA 223 SEED GERMINATION MEDIUM 223 Oatmeal Agar 223 ISOLATION AND GROWTH MEDIA 223 Soil Solution Equivalent Agar (SSE) 223 Fungal Isolation medium (FIM) 224 Potato Dextrose Agar (PDA) 224 Nutrient Broth 224 SSE Broth 224 Modified Melin Norkans (MMN) 225 10% V8/50% SSE Broth 225
APPENDIX 4 PUBLICATIONS 227
Collins, M. T. (2005) How do you determine when orchid seed germination 227 has been successful? The Orchadian, 15 (2) 60-71
Collins, M., Koch, J., Brundrett, M. and Sivasithamparam, K. (2005) 239 Recovery of terrestrial orchids in the post-mining landscape. Selbyana, 26, 255-264
Collins, M., Brundrett, M., Koch, J. and Sivasithamparam, K. (2007) 249 Colonisation of jarrah forest bauxite-mine rehabilitation areas by orchid mycorrhizal fungi. Australian Journal of Botany, 55 653-664
vii Acknowledgments
This PhD project was jointly funded through ARC linkage project LP0221070 by Alcoa Worldwide Alumina – Australia, Botanic Gardens and Parks Authority and The University of Western Australia. I gratefully acknowledge the generous support of Alcoa Worldwide Alumina Australia in provision of both monetary and technical assistance to the project. I also thank the Australian Federation of University Women (WA) Inc. for provision of travel funding from the Mary and Elsie Stevens Bursary. This funding enabled me to attend the second International Orchid Conservation Congress in Sarasota, Florida, and visit the Jodrell Laboratories, Royal Botanic Gardens, Kew, UK in 2004.
I thank my supervisors Professor Krishnapillai Sivasithamparam (UWA), Dr. Mark Brundrett (UWA) and Dr. John Koch (Alcoa) for technical and academic advice.
In particular I thank my friends (and former colleagues) Krystina Haq, Benedict Killigrew and Sato Juniper for their editorial feedback on this thesis. I also thank my colleagues Titiek Yulanti, Nura Abdul Karim, Nicolyn Short, Ryan Hooper, Linda Maccarone, Harsh Garg and Aravinda Mutukumarana for their friendship and good humour during the time we shared an office. This was vital to maintaining my sanity.
Thanks also to Dr Andrew Batty of Kings Park and Botanic Garden and Andrew Brown of the Department of Environment and Conservation for advice and discussions on orchid biology and taxonomy and everyone who provided valuable field work assistance. I also thank Kristian Pollock from the Department of Environment and Conservation for providing information on planned control burns in the Jarrahdale area.
I also apologise to Lewis Carroll and John Tenniel for the repeated misuse of their work in this thesis.
viii Candidate’s Declaration
This thesis contains published work and/or work prepared for publication, some of which has been co-authored. Except where duly acknowledged, all the work presented in this thesis was performed by the PhD candidate.
Professor Krishnapillai Sivasithamparam; Dr Mark C Brundrett and Dr John M Koch as supervisors played an active role in overseeing the project and reviewing manuscripts. Dr Brundrett advised on the use of seed baits and the ex-situ baiting technique.
Margaret Thora Collins Professor K. Sivasithamparam PhD Candidate Coordinating Supervisor
ix Definitions and terminology used in this document
Alien: a conservation status applied by Florabase (http://florabase.calm.wa.gov.au/) to exotic plant species growing in Western Australia.
Anamorph: asexual phase of fungal life cycle.
Apogeotropic: growing away from the ground. It is used in this thesis to describe subterranean roots growing upwards.
Axenic growth: growth of an organism in the absence of all other ‘contaminating’ organisms. (Note: in this thesis the term is used to describe orchids growing in the absence of microbial symbionts.)
Climax species: a plant species found in ‘mature’ or ‘old growth’ vegetation that has reached a state of dynamic equilibrium with its environment. (Note: in this thesis the term is used to describe orchid taxa found only in unmined forest or rehabilitation areas over 25 years old and not in its original and accurate sense.)
Clonal orchid: an orchid taxon that primarily reproduces vegetatively, that is by tuber multiplication.
Collar: a swollen underground region of stem found in some terrestrial orchid genera that is heavily infected with mycorrhizal fungus.
Disturbance opportunist: an orchid taxon that exhibits an increase in recruitment in response to habitat disturbance.
Dropper: the underground stem in terrestrial orchids. Tubers form at the end of droppers.
x Endophyte: an organism living inside a plant. The word is derived from the Greek: endon meaning within; and phyton meaning plant. In this thesis the term is used in its broadest sense to describe fungi that can be detected within the tissues of apparently healthy plants.
Fire weed: a plant species with a relatively short life span that germinates abundantly only after its habitat has been burnt in a hot summer fire. Holomorph: the complete fungus, encompassing both phases of life cycle.
K-strategist traits: large size, slow development, long life span, and low fecundity.
Mallet: a Eucalyptus growth form. The term is applied to tall, high-branched, single stemmed Eucalyptus species of SW Western Australia. This category includes the following taxa; E. astringens, E clivicola, E. falcata, E. gardneri, E. ornate, E. spathulata and E. spathulata subsp. spathulata.
Mycorrhiza: an association between a fungus and a plant root where at least one of the partners in the association benefits nutritionally from the association. The word is derived from the Greek: mykes meaning fungus; and rhiza meaning root.
Myrmecochore: plant taxa whose seeds or fruits are dispersed by ants. Seeds or fruit of these taxa have nutrient rich, ant-attracting, tissues or structures, called elaiosomes.
‘New’ jarrah forest: is used in this document to describe the jarrah forest ecosystem that develops on rehabilitated sites as a replacement for the natural forest that was present prior to mining.
Orchid mycorrhizal fungus: an orchid fungal endophyte capable of germinating seed of the species from which it was obtained and supporting the growth of the protocorm up to the development of a leaf primordium.
OMF: an acronym for orchid mycorrhizal fungi or fungus. OMF refers to that suite of fungal endophytes capable of forming mycorrhizal associations with an orchid taxon and therefore may refer to more than one fungal taxon.
xi Peloton: an intra-cellular branched coil of hyphae formed by mycorrhizal endophytes within cortical cells of the root, underground stem or root-tuber of terrestrial orchids.
Pioneer species: a plant species that colonises previously un-colonized land. (Note: in this thesis the term is used to describe orchid taxa found to have colonised 5 year old rehabilitation areas and not in its original and accurate sense.)
Protocorm: that developmental stage between germination and seedling that is peculiar to the Orchidaceae.
Recalcitrant: a plant species that is difficult to re-establish in the post-mining landscape. (The dominant form of reproduction for these species is usually vegetative as they produce few seeds of low viability and have poor rates of germination success.) r-strategist traits: small size, rapid development, short life-span, high fecundity and ability to disperse offspring widely.
Secondary coloniser: a plant species that will not colonize previously un-colonized land until the habitat has been modified by the establishment of early colonizing ‘pioneer species’. (Note: in this thesis the term is used to describe orchid taxa found to have colonised ten and fifteen year old rehabilitation areas and not in its original and accurate sense.)
Teleomorph: sexual phase of fungal life cycle.
Weed: an r-strategist plant species that is not normally found in mature vegetation. Weeds may be an indigenous taxa; for example fire-weeds (see definition above) or invasive alien taxa.
xii Orchid Nomenclature Orchid nomenclature is consistent with the taxonomy of Florabase (http://florabase.calm.wa.gov.au/, accessed 26th July 2007). For most orchid taxa the author is given in the text following the species epithet of the scientific name when the taxon is first mentioned. However, for taxa occurring only in tables the author has been omitted to maintain clarity of the table. Author names for these taxa are available on Florabase. The following abbreviations have been used in the text: Caladenia flava for Caladenia flava subsp. flava; Caladenia reptans for Caladenia reptans Lindl. subsp. reptans ; Eriochilus dilatatus for Eriochilus dilatatus Lindl. subsp. multiflorus; Microtis media for Microtis media subsp. media; and Pterostylis sp. crinkled leaf for Pterostylis sp. crinkled leaf (G.J.Keighery 13426). (N.B. G.J.Keighery 13426 is the collector’s specimen number.)
Fungal Nomenclature Fungal nomenclature is as presented in cited references. For species occurring in the text the author is given following the species epithet of the scientific name when the taxon is first mentioned. However, for taxa occurring only in tables the author has been omitted to maintain clarity of the table. Author names for orchid mycorrhizal fungi can be found in Roberts (1999).
Plant Nomenclature Non-orchid plant nomenclature is consistent with the taxonomy of Florabase (http://florabase.calm.wa.gov.au/, accessed 26th July 2007). For species occurring in the text the author is given following the species epithet of the scientific name when the taxon is first mentioned. However, for taxa occurring only in tables the author has been omitted to maintain clarity of the table. Author names for these taxa are available on Florabase (http://florabase.calm.wa.gov.au/).
xiii Papers arising from this study
The following papers arising from the whole or parts of chapters of this thesis have been accepted for publication:
CHAPTER 1 Collins, M. T. (2005) How do you determine when orchid seed germination has been successful? The Orchadian, 15 (2) 60-71
CHAPTER 2 Collins, M., Koch, J., Brundrett, M. and Sivasithamparam, K. (2005) Recovery of terrestrial orchids in the post-mining landscape. Selbyana, 26, 255-264
CHAPTER 3 Collins, M., Koch, J., Brundrett, M. and Sivasithamparam, K. (2007) Colonisation of jarrah forest bauxite-mine rehabilitation areas by orchid mycorrhizal fungi. Australian Journal of Botany, 55 (6) 653-664
xiv Dulce est desipere in loco
Horace - BC
xv The Porpoise
“They were obliged to have him with them,” the Mock Turtle said. “No wise fish would go anywhere without a porpoise.”
“Wouldn’t it, really?” said Margaret, in a tone of great surprise
“Of course not,” said the Mock Turtle. “Why, if a fish came to me, and told me he was going on a journey, I should say ‘With what porpoise?’ ”
“Don’t you mean ‘purpose’?” said Margaret.
“I mean what I say,” the Mock Turtle replied, in an offended tone. And the Gryphon added “Come, let’s hear some of your adventures.”
(Apologies to Lewis Carroll and John Tenniel)
xvi INTRODUCTION
The terrestrial orchids of the South West botanical province of Western Australia are a highly visible part of the spring flora and are capable of inducing intense emotive responses in many people. This high public profile means that for many people their presence is an important visual indicator of ecosystem health. The South West botanical province is known for its high floristic biodiversity and endemism including approximately 400 terrestrial orchid taxa (Myers et al. 2000; Paczkowska and Chapman 2000; Hopper and Gioia 2004). The Jarrah (Eucalyptus marginata Donn ex Smith) forest is an ecosystem unique to this botanical province and contains over 900 taxa of flowering plants, including at least 76 terrestrial orchid taxa (A. Brown pers. comm. 2005; Florabase, http://florabase.calm.wa.gov.au/, accessed July 2007). This ecosystem is threatened by fragmentation, the pathogen causing ‘Jarrah dieback’ (Phytophthora cinnamomi Ronds.), land clearing for housing, agriculture and mining, and recreational activities (Havel 1989; Mills 1989; Mc Dougall et al. 2002). Other threats that are of increasing importance include weed invasion, increasing fire frequency, rising soil salinity, changing water tables, and grazing by feral animals.
Areas of the northern Jarrah forest with lateritic soils are mined for bauxite. Bauxite mining is an open cut mining process that causes severe disturbance of local flora, fauna and soil biota, as well as the soil structure. Alcoa World Alumina Ltd. has been mining for bauxite and rehabilitating mine sites in the Northern Jarrah forest for over 35 years. Their rehabilitation processes aim to re-establish a functional and self-sustaining Jarrah forest ecosystem in the post-mining landscape that substantially fulfils the functions of the pre-mining landscape (Elliott et al. 1996). The majority of propagules for vegetation re-establishment in these areas are provided by respread topsoil and sown seed. However, some recalcitrant taxa, particularly those in the Families Cyperaceae, Restionaceae and Dasypogonaceae, are propagated in vitro and replanted by hand (Willyams 2005).
There is little or no information on the recovery of indigenous orchids in the post- bauxite mining landscape. Orchid seed does not survive long in the soil seed bank and it is not included in seed mixes so that neither topsoil nor sown seed is a potential source of propagules (Batty et al. 2000). The recovery of orchids in mine site rehabilitation areas has been expected to occur through the natural dispersal of seed from plants in the 1 surrounding unmined forest (Koch et al. 1994). However, long term rehabilitation monitoring data indicates that certain orchid taxa appear to be severely affected by the mining and rehabilitation process (Grant and Koch 2003). Germination of orchid seed is dependent upon the presence of the appropriate mycorrhizal fungi and the survival of seedlings and mature plants on establishment of microhabitats that provide suitable environmental and edaphic conditions for survival of orchids and their fungal endophytes. Orchid colonisation of the post-mining landscape therefore reflects the recovery of both vegetation and soil microflora, and their presence or absence may have potential as completion indicators for the success of vegetation establishment and ecosystem recovery.
OBJECTIVES
This study is the first to attempt to understand some of the processes involved in the establishment of native terrestrial orchids in the post-mining landscape. A vegetation survey of a temporal sequence of mine-site rehabilitation areas and suitable forest reference areas was undertaken to provide basic information on the species richness and abundance of Jarrah forest orchids in both rehabilitation areas and unmined forest, and a time frame for the recovery process. Vegetation structure and species richness characteristics were examined to determine if these characteristics were associated with the presence of individual orchid species in both rehabilitation areas and unmined forest, and if rehabilitation areas are returning to a Jarrah forest-like state. The soils of rehabilitation areas and unmined forest were examined to determine the diversity and abundance of orchid mycorrhiza fungi of the following six orchid taxa: Caladenia flava R.Br. subsp. flava; Disa bracteata Sw.; Microtis media R.Br. subsp. media; Pterostylis recurva Benth.; Pyrorchis nigricans (R.Br.) D.L.Jones and M.A.Clem.; and Thelymitra crinita Lindl.. Isolation of mycorrhizal fungi four orchid taxa: C. flava subsp. flava; Cryptostylis ovata R.Br.; D. bracteata; and T. crinita were attempted and the resulting fungal collection examined to determine if the specificity of the orchid-mycorrhizal endophyte relationship was associated with either ease of colonisation, or abundance of these taxa in both mined and unmined sites.
The overall objectives of this study were to: i) compare the temporal and spatial colonisation patterns for native terrestrial orchids in bauxite mined areas of Jarrah forest and the adjacent unmined
2 forest; ii) determine the temporal and spatial colonisation patterns of orchid mycorrhizal fungi in rehabilitation areas and unmined Jarrah forest; iii) evaluate the role of specificity of orchid mycorrhizal relationships in determining relative abundance of orchid taxa during vegetation establishment after mining and in undisturbed forest; iv) determine temporal changes in the vegetation structural and species richness characteristics associated with a chrono-sequence of rehabilitation areas, and compare these characteristics with adjacent forest reference sites; v) compare vegetation structural characteristics and species assemblages associated with native terrestrial orchids in rehabilitation areas to adjacent forest reference sites; and vi) evaluate the value of native terrestrial orchids as indicators for the completion of the rehabilitation process.
JUSTIFICATION OF RESEARCH
Currently, there is limited information about the recovery of terrestrial orchids in the post-bauxite mining landscape and the diversity of fungi that associate with WA terrestrial orchids. Such information is essential for the identification of suitable orchid habitats and to gain an understanding of the factors determining the success of seedling establishment. This project will provide key information on factors limiting the establishment of terrestrial orchids in the post-mining landscape and on the mycorrhizal relationships of terrestrial orchids in Jarrah forest ecosystems. This will help to guide management strategies in the establishment of sustainable forest ecosystems on bauxite mine-sites, and enable a more comprehensive range of ecologically significant taxa to be able to be grown. The maintenance of all aspects of biodiversity of the Jarrah forest ecosystem in rehabilitated mine-sites will provide the mining industry with a higher environmental profile, while effective rehabilitation is required for access to new mineral resources both nationally and internationally. The results of this work will also indicate the value of orchid species as indicators of the recovery of the rehabilitated soil ecosystem. Thus, this project will help to establish criteria for measuring the success of the rehabilitation processes in previously bauxite-mined Jarrah forest and other disturbed ecosystems.
3 The key questions that will be answered are: • Do orchid mycorrhizal fungal populations in rehabilitated mine-sites and natural forests differ? • Does the occurrence of the appropriate fungi limit orchid establishment in rehabilitated mine-sites? • Does mycorrhizal fungal diversity increase with time since disturbance? • Is orchid species richness correlated with orchid mycorrhizal fungal diversity? • Do sympatric orchid species utilise the same fungi in mycorrhizal associations? • Do orchids that occur early in the rehabilitation of disturbed habitats share mycorrhizal fungi with those that arrive later? • Is there a relationship between orchid mycorrhizal fungus diversity and orchid abundance? • Do multiple orchid mycorrhizal fungi occur in one plant? • Are individual terrestrial orchid taxa associated with particular vegetation structural and species richness characteristics, and are these associations similar in rehabilitation areas and adjacent forest reference sites? Answers to these basic questions are required to understand why orchids become established in some sites and fail to re-establish in others.
4 CHAPTER 1
LITERATURE REVIEW
INTRODUCTION
This literature review provides a brief description of the study ecosytem; the Jarrah (Eucalyptus marginata D.Donn ex Sm) forest of Western Australia, and an introduction to Alcoa World Alumina Australia’s (Alcoa) bauxite mining and rehabilitation processes in the northern Jarrah forest. Existing literature on terrestrial orchids in the post-mining landscape is then examined. Species richness within the family Orchidaceae is described, in particular in relationship to the terrestrial orchid flora of the south-west botanical province of Western Australia. Two aspects of orchid biology important to successful recruitment of new plants are discussed in detail. They are: fungus-mediated germination of orchid seed, including criteria for identification of successful germination; and the breadth of specificity of the plant-fungal endophyte relationship in Australian terrestrial orchid mycorrhizal associations. Techniques used for the identification of orchid mycorrhizal fungi and orchid taxonomy are also discussed.
JARRAH FOREST
Two major forest ecosystems occur within South West Botanical province of Western Australia; Jarrah forest and Karri (Eucalyptus diversicolor F. Muell.) forest. Karri forest is restricted to the wetter areas of the lower south west of the state, while Jarrah forest is more extensive (Fig. 1.1). The boundaries of the latter are delimited by the Darling scarp in the west, the 750 mm isohyet in the east (limit of mallet (a term covering several Eucalyptus spp.)-wandoo (Eucalyptus wandoo Blakely) woodlands in north and of Marri (Corymbia calophylla (Lindl.) K.D.Hill and L.A.S.Johnson syn. E. calophylla- wandoo woodlands in south) and the climatic boundary between dry and moderate Mediterranean climates in the south (Fig. 1.1) (Erickson et al. 1983; Beard 1990).
Jarrah forest is classified as medium open forest on the basis of height and canopy density (10-30 m height and 30-70% canopy cover) (Muir 1977). It is dominated by two tree species; Jarrah and Marri. The climate is dry-Mediterranean with mean annual rainfall of 1220 mm falling mainly in the winter months of June, July and August. It is
5 FIGURE 1.1 South West of Western Australia showing extent of Alcoa mining lease in relation to Jarrah and Karri forest. Map provided by Alcoa World Alumina Australia. 6 divided into northern and southern sub-regions based on understorey flora. The boundary between these sub-regions is a poorly defined line where ironstone gravels become less prevalent and the understorey flora becomes more like that of the Karri forest (Beard 1990).
Soils of the northern Jarrah forest are shallow, highly leached, low nutrient, overlying a lateritic duricrust, and contain pisolitic and ironstone nodules that can exceed 75% of the soil mass (Dell et al. 1989). Mycorrhiza and other nutrient scavenging mechanisms (e.g. proteoid roots) are extremely important for Jarrah forest species and in nutrient cycling within this ecosystem (Brundrett and Abbott 1991).
BAUXITE MINING
Alcoa has mined bauxite in the northern Jarrah forest since 1963 at three open cut mines; Jarrahdale, Willowdale and Huntly (Fig. 1.1). The mine at Jarrahdale operated from 1963 to 1998, and was decommissioned in 2001 after the rehabilitation process was completed (Grant and Koch 2003; Grant and Koch 2007). Bauxite mining is confined to the laterite mantled uplands that correspond to Havel’s Jarrah forest site- vegetation types P, S and T (Havel 1975a; b). Each of these vegetation types has a Jarrah and Marri over-storey but differs in the structure and species composition of the understorey (Table 1.1). Bauxite mining does not directly disturb wetlands, watercourses or granite outcrops (Koch, J, 2002 pers. comm.).
Mining and rehabilitation procedures The area to be mined is cleared of vegetation and the soil covering the bauxite is removed as two layers. Firstly, the topsoil (top 10-15 cm) is removed and directly redistributed in another area undergoing rehabilitation, a process referred to as ‘direct return’. The overburden (40 cm deep) is then removed and stockpiled next to the pit, then the duricrust and the bauxite layer (approximately 4 m thick) removed (Grant and Koch 2003). After the bauxite has been removed the pit walls are battered down and the area landscaped. Overburden and topsoil are then respread in correct order, the area ripped to reduce erosion, seeded and fertilised. The seed mix is composed of the dominant tree species, Jarrah and Marri, and over sixty under-storey species (Grant and Koch 2003). Currently over 550 ha of Jarrah forest is rehabilitated each year at the two active mine sites at Huntly and Willowdale (J. Koch pers. comm. 2004).
7 Originally, the aim of rehabilitation was to establish vegetation cover as quickly as possible to limit erosion and often used fast growing exotic species or non-indigenous Australian species. Alcoa adopted a process of continuous improvement in its environmental management and rehabilitation standards over the period of its mining lease (Grant and Koch 2007), and the current aim of rehabilitation is to re-establish a functional and self-sustaining Jarrah forest ecosystem in areas that have been mined for bauxite (Elliott et al. 1996). However, many indigenous herbaceous and annual plant species either fail to re-establish or do so very slowly within these rehabilitation areas (Koch and Ward 1994; Koch et al. 1996; Grant and Koch 1997). Indigenous terrestrial orchids form a significant proportion of these difficult to re-establish species (Nichols et al. 1991).
TABLE 1.1 List of indicator species for site-vegetation types of the northern Jarrah forest that are mined for bauxite. Site vegetation types were designated: P, S and T by Havel (1975a; 1975b). + = always present, +/- = sometimes present, - = absent.
Site-Vegetation Type Plant Species P S T
Tree stratum Corymbia calophylla + + + Eucalyptus marginata + + + Second storey Banksia grandis + + +/- Casuarina fraseriana + +/- - Eucalyptus patens - - +/- Persoonia longifolia + - + Under storey Acacia strigosa + + +/- Acacia urophylla - +/- + Adenanthos barbigera + + +/- Bossiaea aquifolium - +/- + Chorizema ilicifolium - - +/- Clematis pubescens - - + Davesia pectinata +/- +/- - Grevillea wilsonii + - - Hakea lissocarpha - - +/- Hakea ruscifolia +/- - - Hovea chorizemifolia + + - Lasiopetalum floribundum +/- +/- + Lechenaultia biloba + +/- - Lepidosperma angustatum + +/- - Leucopogon capitellatus - + + Leucopogon propinquis - + + Leucopogon verticillatus - +/- + Macrozamia riedlei - + + Patersonia rudis + + +/- Phyllanthus calycinus +/- + + Pteridium esculentum - - + Styphelia tenuiflora + + +/- Trimalium ledifolium +/- - -
8 THE ORCHIDACEAE
The Orchidaceae is the largest and most diverse of angiosperm families. Recent studies based on DNA sequence data estimate in excess of 24,900 orchid species (Chase et al. 2003). This represents almost 10% of the world’s flowering plants, and indicate that Orchidaceae is an extremely successful family that has adapted to a wide range of habitats. Orchids are thought to have evolved in the tropics but there are few accepted fossil records and the earliest that are most likely to be orchidaceous are from the Eocene (Schmid and Schmid 1977). It has even been proposed that in Orchidaceae, the terrestrial life form may have evolved from the epiphytic form, though this is a controversial view (Robinson and Burns-Balogh 1982; Ackerman 1983; Bennett 1983; Benzing and Atwood 1984; Chase et al. 2003). Certainly at least some terrestrial species (notably in the genera Epidendrum and Oncidium) have literally ‘come down from the trees’ as they retain an epiphytic morphology (Benzing and Atwood 1984). Orchids are very diverse vegetatively as well as floristically and can be epiphytic, lithophytic or terrestrial. They may, for example, be large vines many metres long (e.g. Vanilla), tiny epiphytes only 3-4 mm tall (e.g. Bulbophyllum), leafless, achlorophyllous, tuberous terrestrials (e.g. Gastrodia) or completely subterranean (e.g. Rhizanthella). The enormous floral diversity in Orchidaceae is believed to be a reflection of rapid pollinator-orientated evolution (Darwin 1904; Benzing 1981; Dressler 1981). Phylogenetic groups within Orchidaceae can be difficult to delineate and this is believed to be evidence that the group is actively evolving (Dressler 1993).
Range Orchids occupy a wide geographical range, from the far north of Europe and North America to Macquarie Island (part of Australia’s Antarctic territory). The range of habitats they occupy is diverse, including Alpine meadows, tropical rain forests and the arid rangelands of Western Australia. The greatest diversity of genera and species occurs in tropical regions where stable warm humid conditions favour epiphytic life forms; 60.7% of orchid genera contain epiphytes and 73% of all orchid species are epiphytic (Atwood 1986). Terrestrial orchids have perennating tubers or rhizomes that enable them to survive climatic extremes and predominate in the more climatically variable temperate regions.
9 Orchids in Western Australia Australia has a relatively low level of orchid diversity compared to many other countries, with orchids making up approximately 4% of the flora (Jones 1988). There is, however, a high degree of endemism with over 70% of taxa not found anywhere else in the world. Australia is an extremely arid continent (second only to Antarctica) with about 80% of the land area falling into arid or semi-arid climatic zones. The result of this aridity is an orchid flora that is predominantly terrestrial (over 75% of Australian orchids are terrestrial) (Jones 1988). Epiphytic and lithophytic species occur only in the tropical and sub-tropical areas of the north and east of the continent.
Western Australia’s orchid flora contains 416 taxa (http://florabase.calm.wa.gov.au/ accessed July 2007). There are only two indigenous Western Australian epiphytic species, Cymbidium canaliculatum R. Br. and Dendrobium affine (Decne) Steud., both confined to the sub-tropical north, the remaining taxa are all terrestrial. The South West Botanical province (Fig. 1.2) with 400 taxa (96.6% of the total orchid flora) (http://florabase.calm.wa.gov.au/ accessed July 2007) has one of the most diverse terrestrial orchid floras in the world and is comparable with that of one of the worlds botanical hot spots, the Cape flora of South Africa (Linder et al. 2005). All but one of the orchids of South West Botanical province are terrestrial, tuberous and summer deciduous. The single exception is Cryptostylis ovata, which is terrestrial and tuberous but is also evergreen.
Habitats of terrestrial orchids in WA Orchids of the South West Botanical province occur in a wide variety of habitats. Some rare species are restricted to small areas where they are confined to a specific habitat while others are more adaptable and are widespread (Hoffman and Brown 1998). For example Caladenia winfieldii Hopper and A.P. Brown is known from only two populations in winter-wet depressions adjacent to seasonal creeks near Manjimup, while C. flava occurs throughout the province in a broad range of habitats including winter- wet swamps and Banksia woodland on the coastal plain, Jarrah and Karri forest of the south west, and granite outcrops of inland areas. In the arid inland areas of the Goldfields orchids are confined to moist areas around salt lakes or run-off areas around granite outcrops. The greatest concentration of orchid taxa occurs in higher rainfall areas of the southwest (http://florabase.calm.wa.gov.au/ accessed July 2007) (Hoffman and Brown 1998).
10 Fire is an important environmental factor in Australia and much of the flora has adapted morphologically and physiologically to recurrent fire (Kemp 1981; Singh et al. 1981). In the Western Australian terrestrial orchid flora it plays an important role in stimulating flowering, which can be profuse following a summer bushfire (Hoffman and Brown 1998). Certain taxa, for example Pyrorchis nigricans and many of the Prasophyllum species, have become dependent on fire to stimulate flowering and will fail to do so unless their habitat has been burnt during the preceding summer (Hoffman and Brown 1998). Mass flowering following fire means that seed production and dispersal will be maximized at times when competition for light and nutrients from other vegetation has been greatly reduced thereby enhancing recruitment in the following wet season.
FIGURE 1.2 The limits of the Botanical provinces of Western Australia. Terrestrial orchids are found predominantly in the South West Province and South West Interzone. The orchid flora of these regions contains over 96% of all Western Australian orchid taxa (http://florabase.calm.wa.gov.au/ accessed July 2007).
11 RECOVERY OF ORCHIDS IN DISTURBED HABITATS
Most Western Australian studies on biological aspects of post-mining landscapes have been directed towards the rehabilitation needs of the mining industry, that is, to rapidly establish vegetation to limit erosion, provide ecological habitats for wildlife and re- establish biological and functional diversity (Nichols et al. 1991; Koch et al. 1994; Ward 2000; Grant and Koch 2007). Bauxite mining necessarily causes a severe localised impact on vegetation and a lesser secondary impact through fragmentation of surrounding unmined areas. The selective removal of P, S and T site-vegetation types (Table1.1) from the Jarrah forest may affect populations of plant and animal taxa limited to, or found primarily within these site-vegetation types through both loss of habitat and fragmentation of populations. There have been few Western Australian studies examining the effect of fragmentation on plant population viability and regeneration success, except in the severely fragmented remnant vegetation of the Western Australian wheat-belt (Hobbs and Yates 2003). There is very little published information on the establishment of terrestrial orchids in these disturbed habitats and, to date, the only publication specifically examining this issue in Western Australia is a retrospective examination of vegetation monitoring data (Grant and Koch 2003).
The main sources of plant propagules for revegetation of rehabilitation areas are respread topsoil, the seeding mixture and natural seed dispersal (Grant and Koch 2007). Previous work on orchid soil seed-banks in Western Australian has revealed a short–lived seed bank with viable seed not persisting through the wet season following dispersal (Batty et al. 2000). If this result is typical for Western Australian terrestrial orchids, the soil seed bank present in respread topsoil cannot be expected to be significant in the recruitment of new individuals. Orchid seed is not included in the seed mixes used for rehabilitation, but it is minute and is readily wind dispersed (Gandawijaja and Arditti 1983; Rasmussen 1995). Most orchid seed is believed to fall within ten metres of the parent plant (Murren and Ellison 1998; Chung et al. 2004), however there are instances of orchid seed dispersing over tens or hundreds of kilometres; for example the Javan mainland was found to be the predominant source of orchid seed for colonisation of Krakatau, 40 km away (Partomihardjo 2003). This ease of dispersal suggests orchid seed will disperse to rehabilitation areas provided that orchid populations in adjacent areas of unmined Jarrah forest flower and produce seed.
12 In 1991, Orchidaceae was identified as one of seven poorly represented plant families in rehabilitation areas as only eight of 18 orchid species recorded in pre-mining surveys had re-established (Nichols et al. 1991). The youngest rehabilitation areas found to contain orchids were 27 months old, with the number of orchid species present in rehabilitation areas increasing with time since establishment (Nichols et al. 1991). Koch and Ward (1994) examined very early bauxite mine rehabilitation areas (nine months after establishment) and found that for those sites where pre and post mining vegetation data were available, 48 plant species did not re-occur after mining. Of these, 14 species were tuberous geophytes including 10 orchid species.
Orchids belong to a group of plant species (Type 1N) that invade rehabilitation areas with time (Koch et al. 1994). Natural invasion is considered the most appropriate and realistic means of establishment due to the need for the presence of a mycorrhizal symbiont. Koch et al. (1994) postulated that this can only be re-established with litter and soil development, and was the probable reason why orchids only established in older rehabilitation areas.
Few other publications mention terrestrial orchids in the post-mining landscape. Grant and Loneragan (1999) examined the effect of fire on vegetation in 11-13 year old rehabilitation areas and found an increase in orchid numbers. Grant and Koch (1997), in a study of the effect of heat and smoke treatments on soil seed banks, revealed a positive effect of smoke on germination in the introduced orchid species, D. bracteata. A study on topsoil return techniques found a single un-named orchid species to be one of the most abundant species in four year old rehabilitation areas after direct whole return of topsoil (Nichols and Michaelsen 1986).
Grant and Koch (2003) examined data from Alcoa’s long-term vegetation monitoring of unmined Jarrah forest and rehabilitation areas, of between 1 and 31 years old, and found a total of 23 orchid species. Three species, Cryptostylis ovata R.Br., Lyperanthus serratus Lindl., and Prasophyllum elatum R.Br. occurred only in unmined forest while Diuris carinata Lindl., occurred only in rehabilitated areas (all species are listed in Table 2.1, Chapter 2). This study found that the overall density of orchids in unmined forest (13,755 plants ha-1) was ten times that of rehabilitation areas (1381 plants ha-1) and, with the exception of Caladenia flava R.Br., the most abundant species were different for unmined forest and rehabilitation areas.
13 The major difference between vegetation establishment following mining and regeneration after natural disturbance in dry sclerophyll vegetation, such as the Jarrah forest, is a reduction in the abundance of re-sprouting species in the rehabilitation area (Grant and Loneragan 1999). Mining removes all existing vegetation so that vegetative resprouting is largely eliminated as a source of plants (Bellairs and Bell 1993). Clonal or rhizomatous geophytes (including some orchids) can be present in large numbers or cover large areas in unmined forest but are particularly vulnerable to mining as reproduction in these plants may be predominantly vegetative. Recalcitrant common Jarrah forest geophytes, for example taxa in Dasypogonaceae; Restionaceae and Cypercaeae, are now propagated in vitro and planted in rehabilitation areas to ensure establishment (Willyams 2005; Grant and Koch 2007).
MYCORRHIZAL ASSOCIATIONS
Definition and Function The term mycorrhiza means literally fungus root (being derived from the Greek words mykes meaning fungus and rhiza meaning root). It describes a fungal association with the underground parts of terrestrial plants, most commonly with the roots. Plant-fungus symbiotic relationships are recognised as forming a continuum of interactions between mutualism and parasitism (Bronstein 1994; Johnson et al. 1997). These interactions are summarised in the interaction matrix in Fig. 1.3 (Johnson et al. 1997). Mycorrhizal relationships are generally regarded as symbiotic in the sense that they are mutualistic, with both partners deriving benefits from the relationship (Fig 1.3) (Johnson et al. 1997). The mutualism occurs as a bi-directional transfer of nutrients, the fungus extracts nutrients and minerals from the soil which are transported to the plant which provides carbon (as photosynthate) to the fungus (Smith and Read 1997). The mycorrhizal relationship enhances a plant’s ability to extract nutrients from the soil because the external mycelial network provides a massive extension to both the surface area available for nutrient exchange and the volume of soil from which nutrients can be extracted (Read 1992). Mycorrhizal associations are therefore particularly valuable to plants in low nutrient soils such as those of southwest Western Australia (Brundrett and Abbott 1991).
Seven types of mycorrhiza have been identified (Table 1.2). The two most common types are arbuscular and ecto-, which have broad host ranges, while the others are
14 generally more specific and occur within particular plant families. The orchid mycorrhizal association is an example of one of these more specific relationships.
Orchid Mycorrhiza Orchid mycorrhizal relationships have generally been regarded as atypical mycorrhiza as nutrient transfer is thought to be predominantly unidirectional; from fungus to plant (Alexander and Hadley 1985; Smith and Read 1997). However, recent work on nutrient transfer between Goodyera repens (L.) R.Br. and its mycorrhizal fungus Ceratobasidium cornigerum Bourdot) D.P.Rodgers has shown that photo-assimilated carbon (C) can be transported to extraradical mycelium in vitro under nutrient limited conditions (Cameron et al. 2006). In this study, photo-assimilated carbon was the only C source available to the fungus and it is not known if this potential for mutualism is fulfilled in nature where the fungus has access to multiple carbon sources. Certainly previous nutrient transport studies have failed to detect any evidence of mutualism (Hadley and Purves 1974, Alexander and Hadley 1985).
All orchids are myco-heterotrophic, where the orchid is literally parasitic on its mycorrhizal endophyte/s, for at least the germination and early growth phase of its life (Rasmussen and Whigham 1993; Leake 1994; Rasmussen 1995; Roberts 1999; Leake 2005). Terrestrial orchids are thought to rely heavily on their mycorrhiza throughout their life (Harley and Smith 1983; Gebauer and Meyer 2003; Julou et al. 2005). This is especially true for Australian taxa, many of which have few or no roots and relatively small leaves. In these orchids, the term mycorrhiza is applied to a fungal association that functions as a root system (i.e. an organ absorbing nutrients and water from the soil) rather than in conjunction with or as an extension of a root system. This association begins and functions from early in the orchid’s life cycle (prior to any evidence of root formation) to provide the nutrients required for germination and early growth that are believed to be absent from the seed because of the highly reduced (or lack of) endosperm (Peterson et al. 1998). These associations best fulfil criteria for relationships in the shaded area of Fig 1.3 where the plant benefits nutritionally from the relationship but under most circumstances the fungus does not. These relationships have been described as epiparasitic or exploitative (Brundrett 2004).
15 16
TABLE 1.2 Summary of different types of mycorrhizal associations (adapted from Harley and Smith (1983); Brundrett et al.(1996))
Structure Arbuscular Ecto Ectendo* Arbutoid* Monotropoid* Ericoid Orchid
Septate hyphae - (+) +/- +/- + + + +
Hyphae in cells + - + + + + +
Hyphal coils +/- - + + - + +
Arbuscules + ------
Mantle - + (-) + (-) + + - -
Hartig net - + + + + - -
Vesicles +/------
Host plant Vascular plants Gymnosperms Gymnosperms Ericales Monotropaceae Ericales Orchidaceae and and Angiosperms Angiosperms
Chlorophyll + + + + (-) - + + (-)
Fungal Ascomycetes Basidiomycetes Basidiomycetes Basidiomycetes Basidiomycetes Ascomycetes Basidiomycetes associate and mostly, some mostly, some mostly, some mostly, some and Glomeromycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes Basidiomycetes and and and and Zygomycetes, Zygomycetes, Zygomycetes, Zygomycetes, Note: - = absent, + = present, (-) = occasionally absent, (+) = occasionally present, +/- = present or absent * Brundrett believes these to be categories of Ectomycorrhiza (Brundrett 2004) Gradient of Gradient of plant responses fungal responses + 0 –
+ Mutualism Commensalism Parasitism (+,+) (0,+) (–,+)
0 Commensalism Neutralism Amensalism (+,0) (0,0) (–,0)
_ Parasitism Amensalism Competition (+,–) (0,-) (–,–)
FIGURE 1.3. Interaction matrix of plant/fungus interactions in mycorrhizal associations. (modified from Johnson et al.1997) The left hand column of plant responses represents the continuum of possible interactions in orchid mycorrhizae. It should be noted that though the potential for mutualism (diagonal stripping) has been demonstrated in vitro it has not been shown to occur in nature (Cameron et al. 2006). Response gradient : + = positive; 0 = neutral; – = negative.
The Role of Orchid Mycorrhizal Fungus in Seed Germination Terrestrial orchid seed is minute and consists of a thin testa and a simple embryo, containing little if any endosperm (Wirth and Withner 1959; Arditti and Ernst 1984; Peterson et al. 1998). To develop into a viable plant in nature an orchid seed needs to germinate and form an effective fungal association (mycorrhiza) capable of providing the necessary nutrients for growth beyond imbibition, cracking of the testa and early protocorm development. Stimulation of orchid seed germination by a fungus has commonly been regarded as an indication of the capacity of that fungus to establish an orchid mycorrhizal association (Arditti and Ernst 1984; Tsutsui and Tomita 1986; Masuhara and Katsuya 1989; Currah 1991; Masuhara and Katsuya 1994; Rasmussen 1995).
In most plant species the physical manifestations of germination - imbibition, swelling of the embryo and splitting of the testa - also indicate metabolism of stored nutrients in the endosperm that support the growth of the embryo (Jackson and Jacobs 1985; Lambers et al. 1998). It is generally accepted that the predominant metabolic activity in storage tissues during germination is the production of sugars from lipids (Ching 1972). In orchid seed the embryo functions as its own nutrient reservoir, however, until an appropriate fungal association is formed the metabolism of nutrients that is the
17 physiological manifestation of germination does not occur (Harrison 1977; Harrison and Arditti 1978; Peterson et al. 1998). Uetake et al. (1992) demonstrated, in Spiranthes sinensis (Pers.) Ames, that fungal invasion of the embryo was associated with the disappearance of endogenous lipid and hypothesised that fungal invasion may alter embryo metabolic activity providing a stimulus to growth. Richardson et al. (1992) also found fungal invasion stimulated protocorm development and concomitant utilisation of lipid and protein reserves in Platanthera hyperborea (L.) Lindl. In vitro studies have shown that orchid seed will not germinate asymbiotically unless provided with a carbon source in the form of simple sugars (Burgeff 1959; Harrison and Arditti 1978; Arditti and Ernst 1984). In nature this carbon source is provided by the fungus in a mycorrhizal association (Smith and Read 1997; Peterson et al. 1998).
Defining successful germination Much of the literature on orchid seed germination and orchid mycorrhizas fails to indicate what the author considers successful germination, rather, regarding this as self- evident (Warcup 1971; 1973; Arditti et al. 1982; Arditti and Ernst 1984; Rasmussen 1995; Kulikov and Philippov 2003). Orchid seed passes though a developmental stage that is unique to orchids between imbibition and rupture of the testa and the development of a seedling. This stage is called the protocorm. The developmental stages of the protocorm have been variously defined by different authors, as has the protocorm growth stage representing successful germination (these are listed in Table 1.3). The lack of standard criteria defining successful germination in orchids makes it difficult to compare studies on seed germination and/or the mycorrhizal capacity of orchid endophytes.
Hadley (1970) observed that development of protocorms to a stage frequently regarded as indicative of successful germination, namely rupture of testa and/or development of trichomes (see references cited in Table 1.3), could be followed by cessation of growth, parasitism by the fungus and subsequent necrosis. Warcup (1983) has reported similar observations. Both authors regarded only substantial growth stimulus, resulting in the production of a leaf, as an indication of successful germination and formation of a functional mycorrhiza. Thus, while rupture of testa and/or development of trichomes indicates germination (see Table 1.3) it does not necessarily represent a successful germination capable of producing a healthy plant (Tsutsui and Tomita 1988; Masuhara and Katsuya 1989; Rasmussen and Rasmussen 1991; Rasmussen et al. 1991; Hayakawa
18 et al. 1999; Batty et al. 2000; McKendrick et al. 2002; Hollick et al. 2005). This requires establishment of an effective mycorrhizal association capable of supporting growth of the protocorm and nutrition of the seedling. In this study the minimum requirement for successful germination is regarded as development of the shoot primordium (Stage 4, Table 1.3)
The post-germination mycorrhizal relationship Mature terrestrial orchids are putatively autotrophic but are thought to retain some dependency on their mycorrhiza for at least partial nutrition (Taylor and Bruns 1999; Gebauer and Meyer 2003). In most types of mycorrhizal relationships plants gain a nutritional advantage through infection with the fungus, but can survive independently provided they have adequate nutrition. Both epiphytic and terrestrial orchids can be grown axenically in vitro, and may even flower (Arditti 1977; Harrison and Arditti 1978; Arditti et al. 1982; Collins and Dixon 1992). However, axenic growth has not been observed in nature and attempts to transfer axenic terrestrial orchid plantlets to soil generally fail unless the plantlets have already formed tubers (Batty et al. 2006).
The degree of dependence on the mycorrhiza and the specificity of the plant-fungus relationship is thought to be variable with some species regarded as particularly vulnerable due to their dependence on a highly specific fungus (Andersen and Rasmussen 1996; Zettler et al. 2003; Brundrett 2004). This dependence is presumably linked to the underground morphology of each orchid taxon. Those with roots can probably extract some nutrients from the soil. However, taxa with few or no roots must be very limited in their capacity to do so and therefore very dependent on their mycorrhizal relationship. Ramsay et al. (1986) examined the underground parts of 144 Western Australian terrestrial orchid taxa and identified five patterns of endophyte infection, each associated with a particular group of orchid genera and a particular set of underground morphologies. Some examples of these morphologies include: Thelymitra and Diuris, which have infected root systems; Caladenia, Elythranthera and Cyanicula, which have no roots but have a stem collar infection; and Gastrodia and Rhizanthella which have no roots and are also achlorophyllous, but have stem tuber infections. Orchids in these latter two genera are myco-heterotrophic and have obligate symbiotic associations with their mycorrhizal fungi (Leake 1994). Certainly, a critical dependence on a specific Sebacina-like fungus has been shown in the achlorophyllous European
19 20 TABLE 1.3. Comparison of criteria used in the literature by different authors to define successful germination and describe protocorm development stages of autotrophic terrestrial orchids. *These definitions are compared with development stages described in the current study in the column labelled Stage.
Definition of Successful Germination Reference Development Stages Described in Reference Description Stage* Stages defined in this study enlargement of protocorm and initiation of 4 0 unimbibed seed (Collins 2005) leaf primordium 1 imbibed seed, embryo swollen 2 ruptured testa, occasional trichome 3 enlarged protocorm, numerous trichomes 4 further enlargement of protocorm and initiation of leaf primordium 5 leaf elongation 6 seedling with green leaf and initiation of dropper 7 tuber and root formation Hadley 1970 marked growth stimulus 4-5 0 no infection s compatible infection S marked growth stimulus Warcup 1973 not defined not defined 0 no germination 1 cracked testa 2 2-3 x original volume 3 protocorms longer than seed coat 4 protocorm larger 5 shoot differentiation initiated 6 green leaf Clements 1981 green leaf 5 0 no germination 1 cracked testa 2 2-3 x original volume 3 protocorms longer than seed coat 4 protocorm larger 5 shoot differentiation initiated 6 green leaf TABLE 1.3 continued.
Definition of Successful Germination Reference Development Stages Described in Reference Description Stage* Warcup 1983 shoot formation 4-5 0 no symbiosis P parasitism (+) partial development of protocorm + protocorms with shoot formation Clements et al. 1986 not defined not defined 0 ungerminated seed 1 germinated seed with rupture of testa 2 production of rhizoids 3 production of leaf primordium 4 production of first green leaf 5 production of root initial Ramsey et al. 1986 first signs of greening and leaf emergence 4-5 1 seed coat split, embryo slightly swollen 2 embryo enlarged, few trichomes 3 embryo protocorms-like, many trichomes,5-10 x seed volume 4 highly enlarged protocorms, numerous trichomes 5 first signs of greening and leaf emergence Clements 1988 green leaf 5 stages 0-6 as defined by Clements 1981 Tsutsui and Tomita 1988 broken testa 2 none defined Masuhara and Katsuya 1989 torn testa 2 none defined Muir 1989 rupture of testa 2 stages 0-5 as defined by Clements et al. 1986 Rubluo et al. 1989 protocorms formation 3 none defined Wilkinson et al. 1989 first signs of greening and leaf emergence 4-5 stages 1-5 as defined by Ramsey et al. 1986 Rasmussen and Rasmussen 1991 testa ruptured and/or rhizoids developed 2-3 none defined Rasmussen 1992 not defined not defined none defined Richardson et al. 1992 rupture of testa, enlarged embryo and 2-3 none defined
21 growth of epidermal hairs 22 TABLE 1.3 continued.
Definition of Successful Germination Reference Development Stages Described in Reference Description Stage* Masuhara et al. 1993 protocorm development 3 - no germination + protocorms ++ shoot Masuhara and Katsuya 1994 rupture of seed coat 2 none defined Perkins and McGee 1995 swollen seed, cracked testa 2 + swollen seed, cracked testa ++ protocorm with apical meristem and trichomes Perkins et al. 1995 swollen seed, cracked testa 2 - protocorms killed by fungus + swollen seed, cracked testa ++ protocorm with apical meristem and trichomes +++ green leaf Zettler and Hofer 1998 rhizoid production 1-2 0 no germination 1 rhizoid production 2 rupture of testa 3 appearance of promeristem 4 appearance of first true leaf 5 elongation of leaf and root formation Hayakawa et al. 1999 protocorm hair formation 2-3 - no germination + protocorm hair formation ++ protocorm formation +++ shoot formation Batty et al. 2000 enlargement of protocorm and production of 3 stages 1-5 as defined by Ramsey et al. 1986 trichomes TABLE 1.3 continued.
Definition of Successful Germination Reference Development Stages Described in Reference Description Stage* Batty et al. 2001a enlargement of protocorm and production of 3 0 unimbibed seed first trichomes 1 imbibed seed with cracked testa 2 first trichomes 3 enlargement of protocorm and initiation of leaf primordium 4 protocorm enlargement with first green leaf 5 seedling with green leaf and initiation of dropper Batty et al. 2001b enlargement of protocorm and production of 3 stages 1-5 as defined by Ramsey et al. 1986 trichomes Kulikov and Filipov 2001 not defined not defined 1 seed 2 rupture of testa and rhizoid formation 3 initiation of leaf primordium 4 formation of primary shoot 5 formation of assimilating leaves, roots and storage organs McKendrick et al. 2002 torpedo shaped protocorm, no rhizoids 2-3 0 ungerminated seed 1 changes before rupture of testa; 1a embryo becomes translucent 1b fungal penetration of embryo 1c expansion of embryo with formation of pelotons 2 changes after rupture of testa; torpedo shaped protocorm, no rhizoids 3 changes after branching of protocorm; rootlets develop, differentiated shoot bud
23 develops Bonnardeaux 2003 trichome production 3 stages 1-5 (Ramsey et al. 1986) 24 TABLE 1.3 continued.
Definition of Successful Germination Reference Development Stages Described in Reference Description Stage* Brundrett et al. 2003 not defined not defined 1 split seed 2 protocorms 3 leaf primordium 4 leaves 5 droppers Otero et al. 2004 swelling of embryo 1-2 0 no germination 1 swelling of embryo 2 development of radical hairs 3 development of leaf projection 4 development of first leaf 5 development of second leaf 6 development of roots Huynh et al. .2004 green leaf 5 stages 1 - 6 (Warcup 1973) stages 1 - 5 (Ramsay et al. 1986) Esitken et al. 2005 production of rhizoids 1-2 stages 0 - 5 (Zettler and Hofer 1998) Hollick et al. 2005 trichome development 3 none defined Abdul Karim 2005 production of leaf primordium 4 stages 0-6 (Clements et al.1986 and Ramsay et al. 1986) terrestrial orchid Neottia nidus-avis (L.) L.C.M. Rich. (McKendrick et al. 2002; Selosse et al. 2002).
Fungi associated with orchid mycorrhizas The endophytic fungi associated with orchid mycorrhizae are predominantly Basidiomycetes, generally regarded as belonging to the form-genus Rhizoctonia (Currah 1991; Sneh et al. 1991; Currah and Zelmer 1992; Andersen and Rasmussen 1996). Rhizoctonia contains a taxonomically diverse assemblage of anamorphic fungi belonging to several different orders of basidiomycetes (Sneh et al. 1991; Stalpers and Andersen 1996). Many are important plant pathogens, for example Rhizoctonia solani Kühn, the anamorph of Ceratobasidium cereale D.I.Murray and Burpee and Thanatephorus cucumeris (Frank) Donk, has been identified as the causal agent of root rots and foliar diseases of many crop plants (Sen et al. 1999). However, most Rhizoctonia strains that form orchid mycorrhizae are not known to be plant pathogens, although pathogenic strains can germinate seed and support the growth of orchid seedlings in vitro (Warcup 1981; Hadley and Pegg 1989; Masuhara et al. 1993; Currah et al. 1997; Sen et al. 1999).
Specificity Specificity exists in the relationship between an orchid taxa and its mycorrhizal endophytes if there is a limitation on the phylogenetic breadth of that association (Taylor and Bruns 1999). A continuum of degrees of specificity exists, ranging from highly specific (one orchid species - one fungal taxa), through narrowly specific (one orchid species -few fungal taxa) to broad (one orchid species - many fungal taxa). Warcup (1981; 1988) found specificity of orchid mycorrhizal associations occurred at different fungal taxonomic levels, from species to subtribe, however, narrow specificity in orchid mycorrhiza is now believed to be a common phenomenon (Dearnaley 2007). Specificity is divided into two categories; ecological specificity which refers to the specificity of the plant-fungus interaction under natural conditions and potential specificity which is that which occurs under laboratory conditions (Harley and Smith 1983; Masuhara and Katsuya 1994). Potential specificity is generally broader than ecological specificity and may account for the perception by some researchers that there is generally low specificity in orchid-fungal mycorrhizal associations (Curtis 1939; Harvais and Hadley 1967; Hadley 1970; Masuhara et al. 1993; Rasmussen 1995). In addition, the criteria used by the researcher to define germination, and the techniques
25 used to delimit fungal and orchid taxonomy, will affect the observed specificity of orchid-fungal endophyte relationship.
In the past, non-photosynthetic orchids were believed to be associated with highly specific mycorrhizal associations due to their life-long myco-heterotrophism, while photosynthetic taxa were thought to have broad specificity (Rasmussen 2002), however the situation appears to be more complex (Dearnaley 2007). Taylor and Bruns (1999) examined the effect of geography, habitat and phenotype on specificity in a study of two congeneric, achlorophyllous orchids, Corallorhiza maculata Raf. and C. mertensiana Bong. Different factors were found to influence the breadth of specificity for each species. Corallorhiza maculata showed strong influences from habitat and phenotype, while C. mertensiana showed an influence due to geography. Interestingly, when growing sympatrically, these species were each associated with several Russula spp. but had none in common, indicating an underlying specificity based on genotype that was independent of habitat. Fungal endophytes from different genera within the Russulaceae have also been found to be associated with individual plants of the achlorophyllous orchid Dipodium hamiltonianum F.M.Bailey collected from different populations (Dearnaley and Le Brocque 2006). McCormick et al. (2004) examined four orchid species of different growth habits and found that neither photosynthetic capacity, nor evergreen habit, was necessarily linked to a greater diversity of mycorrhizal associates. Thus, it appears that there is some degree of flexibility, or perhaps opportunism, even in those associations that have previously been regarded as the most specific of orchid mycorrhizal relationships.
Ecological specificity of Australian terrestrial orchid mycorrhizal associations has been examined by a number of researchers since Warcup first published in this area in 1967 (Table 1.4). Unfortunately, it is difficult to compare the results from different studies: some authors have failed to identify orchid taxa to species level; many have assumed that fungal endophytes are mycorrhizal without undertaking confirmatory tests; and no attempt has been made to identify a number of the fungal associates (Table 1.4). Also, the taxonomy of the Rhizoctonia complex is currently uncertain, as there appears to be no clear definition of genus, species or strain (Brundrett 2007). Australian Orchidaceae have been undergoing a major taxonomic revision and many new genera and species have been described (Hopper and Brown 2000; 2001; Kores et al. 2001; Hopper and Brown 2004). Examples include: the reinstatement of the genus Leptocerus; the
26 erection of the genus Cyanicula, for the small, blue flowered orchids previously included in Caladenia; and the erection of the monotypic genus Praecoxanthus (Hopper and Brown 2000). Changes to or uncertainty in taxonomy of orchid and their fungal associates may alter the apparent breadth of specificity of the listed associations (Table 1.4) at various taxonomic levels.
Endophytes of Australian terrestrial orchids that have been identified and confirmed as mycorrhizal fungi, generally belong to the following families; Ceratobasidiaceae, Sebacinaceae or Tulasnellaceae (Tables 1.4 and 1.5), although achlorophyllous orchids are associated with a broader range of fungi, including taxa from the Russulaceae and Tricholomataceae (Dearnaley 2007). Some very general affinities are evident amongst these associations. Sebacina vermifera Oberw. (syn. Serendipita vermifera), for example, is very commonly associated with orchid species in the Caladenia alliance, which includes the genera Caladenia, Cyanicula, Elythranthera, Eriochilus, Glossodia, Leptocerus and Praecoxanthus (Sneh et al. 1991). However, it has also been found associated with Acianthus and Microtis, so that this is not an exclusive association. Ceratobasidium cornigerum is very commonly associated with the orchid genera Pterostylis and Prasophyllum, but again this is not exclusive. Tulasnella species appear to form mycorrhizae with a number of orchid genera; Acianthus, Arthrochilus, Calochilus, Caleana, Chiloglottis, Corybas, Cryptostylis, Drakea, Diuris, Microtis, Orthoceras, Pyrorchis and Thelymitra, and, in a single case, with a Caladenia species (C. reticulata Fitzg.) (see Table 1.4). However, there appear to be some cases of absolute specificity; orchids of the genus Diuris and of the species Orthoceras strictum R.Br., Acianthus exsertus R.Br. and A. fornicatus R.Br. appear to form mycorrhizae exclusively with the fungal species Tulasnella calospora (Boud.) Juel, although this fungus is quite promiscuous in its associations. Absolute specificity also exists between the underground orchid Rhizanthella gardneri R.S.Rogers and its mycorrhizal endophyte Thanatephorus gardneri Warcup (Table 1.4) (Warcup 1991; Mursidawati 2003). Roberts (1999) regarded this identification as doubtful, however inclusion of this mycorrhizal fungus in Thanatephorus has been recently been confirmed by molecular techniques (Table 1.5) (Mursidawati 2003). Thus, it appears that the ecological specificity of the orchid mycorrhizal association is highly variable between and within orchid genera. Any exclusivity that does exist appears to be determined by the individual orchid species, while the associated fungal taxa are quite promiscuous in their associations.
27 TABLE 1.4 Fungal associates of temperate Australian terrestrial orchids with references. Mycorrhizal status is confirmed if isolate has been shown to support germination and growth of protocorm to stage 4 (see Fig 1.3 for description). Specificity as follows: high = one fungal taxon/orchid taxon; narrow = 2-3 fungal taxa/orchid taxon; broad ≥ 4 fungal taxa/orchid taxon. (*Both fungal and orchid nomenclature in this table is as given in the cited reference. Table 1.5 give teleomorph and anamorph names, and proposed changes described by Roberts (1999).)
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Acianthus reniformis Ceratobasidium obscurum Not indicated Narrow Perfect states described from Warcup and Talbot 1967 Sebacina vermifera fruiting cultures.
Diuris spp. (2 species) Tulasnella calospora Not indicated High A. exertus
C. reticulata Tulasnella calospora Not indicated Narrow S. vermifera
T. luteocilium T. asymmetrica Not indicated High
T. grandiflora Tulasnella sp. Not indicated High
Caladenia spp. (4 species) S. vermifera Not indicated High Glossodia major Microtis unifolia
T. antennifera Thanatephorus sterigmaticus Not indicated Narrow T. calospora
Pterostylis spp. (4 species) C. cornigerum Not indicated High Prasophyllum spp. (2 species) Caladenia spp. (18 species) S. vermifera Confirmed High Perfect states described from Warcup 1971 Elythranthera spp (2 species) fruiting cultures Glossodia major TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity C. flava Hyphodontia sp. Confirmed Narrow As above Warcup 1971 contd. S. vermifera
Caladenia reticulata S. vermifera Confirmed Narrow T. calospora
C. deformis S. vermifera Confirmed Narrow C. menziesii Unidentified isolates C. dilatata
Eriochilus cucullatus T. calospora Confirmed Narrow S. vermifera Unidentified isolate.
Diuris spp. (5 species) T. calospora Confirmed High (Rhizoctonia repens)
Orthocerus strictum T. calospora Confirmed High Thelymitra spp. (4 species) Tulasnella asymmetrica Not indicated High Perfect states described from Warcup and Talbot 1971 fruiting cultures. Corybas dilatatus T. allantospora Not indicated High
Acianthus caudatus T. cruciata Not indicated High T. fusco-lutea
T. aristata T. asymmetrica Not indicated Narrow T. violea TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids (** indicates secondary infection)
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity T. pauciflora T. asymmetrica Not indicated Narrow As above Warcup and Talbot 1971 T. cruciata contd.
P. mutica Ceratobasidium sp Not indicated High Pterostylis mutica Ceratobasidium Not indicated High Perfect states described from Warcup and Talbot 1980 angustisporum fruiting cultures
Calanthe triplicata C. globisporum Not indicated High
Prasophyllum macrostachyum Thanatephorus anomala Not indicated High var ringens (Ypsilonidium anomalum sp. nov.) Acianthus exertus T. calospora Confirmed High Perfect states described from Warcup 1981 A. fornicatus fruiting cultures. Diuris spp. (6 species) Orthocerus sp.
A. caudatus S. vermifera Confirmed Narrow T. cruciata
A. reniformis S. vermifera Confirmed High/Narrow (**C. cornigerum)
Arthrochilus spp. (2 species) Tulasnella sp. Confirmed High Caleana sp. TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids (** indicates secondary infection)
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Caladenia spp. (22species) S. vermifera Confirmed Narrow/Broad As above. Warcup 1981 contd. C. cornigerum (**T. calospora, unidentified orchid mycorrhizal fungus)
Calochilus spp. (2 species) Tulasnella sp. Confirmed Narrow Unidentified orchid mycorrhizal fungus
Chiloglottis spp. (4 species) T. allantospora Confirmed Narrow Tulasnella sp.
Corybus spp (4 species) T. calospora Confirmed Broad T. allantospora Tulasnella sp. Unidentified orchid mycorrhizal fungus
Cryptostylus spp. (3 species) T. asymmetrica Confirmed Narrow Tulasnella sp.
Drakeae spp. (2 species) T. violea Confirmed Narrow Tulasnella sp.
Glossodia spp. (2 species) S. vermifera Confirmed High Elythranthera spp. (2 species) Eriochilus spp. (2 species) TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids (** indicates secondary infection)
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Leporella sp. S. vermifera Confirmed Narrow As above. Warcup 1981 contd. (**unidentified orchid mycorrhizal fungus)
Lyperanthus nigricans T. calospora Confirmed Broad (syn. Pyrorchis nigricans) Thanatephorus sp. Rhizoctonia spp. (4 isolates)
Lyperanthus suaveolans Unidentified orchid Confirmed High mycorrhizal fungus
Microtis spp. (2 species) S. vermifera Confirmed Narrow T. calospora
Prasophyllum spp. (10 C. cornigerum Confirmed Broad species) Thanatephorus cucumeris Pterostylis spp. (16 species) Ceratobasidium sp. Unidentified orchid mycorrhizal fungus
Thelymitra spp. (15 species) T. calospora Confirmed Broad T. asymmetrica T. cruciata T. violea Tulasnella sp Unidentified orchid myorrhizal fungus TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Rhizanthella gardneri Rhizoctonia sp. Confirmed High Perfect states described from Warcup 1985 (classification unresolved) fruiting cultures. Acianthus spp (2 species) Group 1: putatively Confirmed Narrow Mycorrhizal isolates grouped Ramsey et al. 1986 Diuris spp (6 species) C. cornigerum but not formally identified. Gastrodia sesamoides T. calospora Grouping based on cultural Prosophyllum spp (12 taxa) characteristics and nuclei Pterostylis spp (15 taxa) number.
Drakea spp (5 species) Group 2: putatively Confirmed High/Narrow Paracaleana spp (2 species) Tulasnella spp Spiculea ciliata
Caladenia spp (55 taxa) Group 3: putatively Confirmed High Corybas spp (2 species) S. vermifera Cryptostylis ovata Elythranthera spp (2 species) Eriochilus spp (2 species) Lyperanthus serratus Microtis spp (4 species) Monadenia bracteata (syn. Disa bracteata) T. villosa
Leporella fimbriata Group 4: no putative Confirmed Indeterminate Lyperanthus nigricans assignment (syn. Pyrorchis nigricans) Thelymitra spp (3 species) TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Thelymitra spp (2 species) Group 5: no putative Confirmed Indeterminate As above Ramsey et al. 1986 contd. assignment
Thelymitra spp (2 species) Group 4 and 5: no putative Confirmed Indeterminate assignment
Pterostylis rogersii Group 6: putatively Confirmed High/Narrow Thanatephorus spp
Calochilus robertsonii Group 8: no putative Confirmed Indeterminate assignment Pterostylis aff. rufa Group 1: putatively Confirmed High As for Ramsay et al. 1986 Ramsey et al. 1987 P. aff. scabra C. cornigerum Group 1 isolates separated P. barbata into 8 anastomosis groups. P. cycnocephala P. dilatata P. mutica P. nana P. vittata P. vittata var. subdifformis P. vittata var. vittata
P. allantoidea Group 6: putatively Confirmed High/Narrow P. angusta Thanatephorus spp P. pusilla P. sargentii TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity P. aff plumosa Group 1 and 6 Confirmed Narrow As above Ramsey et al. 1987 P. recurva P. rogersii P. scabra var. robusta Rhizanthella gardneri Thanatephorus gardneri sp. Confirmed High Perfect states described from Warcup 1991 nov. fruiting cultures. Pterostylis acuminata Rhizoctonia solani AG6 Confirmed High Culture characteristics and Perkins and McGee 1995 anastomosis tests. Microtis parviflora Epulorhiza repens Confirmed Narrow Culture characteristics and Perkins et al. 1995 Epulorhiza sp. (syn. R. hyphal morphology. globularis) Pterostylis acuminata Rhizoctonia solani AG6 and Not indicated High/Narrow Culture characteristics, hyphal Carling et al. 1999 AG12 morphology and anastomosis testing Pterostylis acuminata Rhizoctonia solani AG6 and Confirmed High/Narrow Sequencing of ITS region of Pope and Carter 2001 AG12 rRNA followed by alignment with published sequences in GenBank. Rhizanthella gardneri Thanatephorus sp. Confirmed High Culture characteristics and Mursidawati 2003 hyphal morphology. Sequencing of ITS region of rRNA followed by alignment with published sequences in GenBank. TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Calochilus sp. (I) Unidentified fungus Confirmed High Culture characteristics, hyphal Abdul Karim 2005 morphology, pectic Calochilus sp. (II) Epulorhiza sp. (1 isolate) Confirmed Narrow zymogram analysis and Fusarium solani sequencing of ITS region of rRNA followed by alignment Nervilia holochila Fusarium solani Not indicated High with published sequences in GenBank. Liparis habenaria Fusarium solani Not indicated Narrow Unidentified fungi (2 isolates)
Eulophia bicallosa Fusarium solani Not indicated High
Geodorum terrestre Fusarium solani Confirmed Broad Unidentified fungus
Unidentified fungi (3 isolates) Not indicated
Habenaria triplonema Epulorhiza sp. Confirmed Narrow/ Fusarium solani Broad Unidentified fungi (2 isolates)
Habenaria sp. (I) Fusarium solani Confirmed Narrow
Epulorhiza sp. Not indicated TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Acianthus exsertus Phaeosphaeria phragmiticola Not indicated High Sequencing of ITS region of Bougoure et al. 2005 rRNA followed by alignment A. pusillus Phaeosphaeria phragmiticola Not indicated High with published sequences in GenBank. Caladenia carnea Sebacina vermifera sensu Not indicated High Oberwinkler
Microtis parviflora Steccherinum ochraceum Not indicated High
Pterostylis longifolia Rhizoctonia sp. Not indicated High
P. nutans Rhizoctonia sp. Not indicated High
P. obtusa Rhizoctonia sp. Not indicated High Erythrorchis cassythoides Sebacina vermifera Not indicated Broad Sequencing of ITS region of Dearnaley 2006 Russula mustelina rRNA followed by alignment Metarhizum album with published sequences in Coltricia perennis GenBank. Gymnopus luxurians Ericoid mycorrhizal sp. Dipodium hamiltonianum Gymnomyces fallax Not indicated Broad Sequencing of ITS region of Dearnaley and Le Brocque Russula lepidicolor rRNA followed by alignment 2006 Penicillium dalae with published sequences in GenBank. Disa bracteata Epulorhiza sp. Am8 Confirmed Broad Sequencing of ITS region of Bonnardeaux et al. 2007 (syn. Monadenia bracteata) Tulasnella sp. 224 rRNA followed by alignment Tulasnella sp. JTO307 with published sequences in Tulasnella pruinosa GenBank. TABLE 1.4 contd. Fungal associates of Australian terrestrial orchids
Orchid Taxa Fungal Associate/s* Mycorrhizal Putative Identification Method Reference Status Specificity Disa bracteata Ericoid mycorrhizal sp. Confirmed Broad Sequencing of ITS region of Bonnardeaux et al. 2007 (syn. Monadenia bracteata) rRNA followed by alignment contd. Nectria mauritiicola Not indicated with published sequences in Leptodontidium orchidicola GenBank.
Pyrochis nigricans Rhizoctonia sp. Eab-S4 Not indicated Broad (syn. Lyperanthus nigricans) Thanatephorus cucumeris Tulasnella danica Ericoid mycorrhizal sp. Sd9
Prasophyllum giganteum Tulasnella calospora Not indicated High
Diuris magnifica (syn. Diuris Tulasnella calospora Confirmed High corymbosa)
Caladenia falcata Sebacina vermifera Confirmed High
Microtis media Sebacina vermifera Confirmed High
Pterostylis sanguinea Vouchered mycorrhizae Confirmed High (Ceratobasidium) Pterostylis recurva Vouchered mycorrhizae (Ceratobasidium) Confirmed High TABLE 1.5 Teleomorph and anamorph names of fungi that form mycorrhiza with Australian terrestrial orchids. The list is based on data obtained from literature cited in Table 1.4 and Roberts (1999). Recent synonyms and putative identities of doubtful identifications have been included. Note: * the inclusion of T. gardneri in Thanatephorus has recently been confirmed by molecular techniques (Mursidawati 2003, J. Bougoure pers. comm. 2006). ** Serendipita is not in common usage and these fungi are usually referred to as Sebacina.
Teleomorph Anamorph Genus Species Genus Species Synonyms/Putative identifications Ceratobasidium Ceratorhiza C. bicorne unnamed Ypsilonium anomalum Thanatephorus anomala C. cornigerum unnamed C. globisporum unnamed C. obscurum unnamed Thanatephorus obscurus probably conspecific with C. cornigerum (Roberts 1999) C. pseudocornigerum unnamed C. angustisporum
Thanatephorus Moniliopsis T. cucumeris M. solani Rhizoctonia solani T. gardneri unnamed possibly Coniophora sp. (Roberts 1999)* T. sterigmaticus unnamed
Serendipita** Unnamed S. vermifera unnamed Sebacina vermifera
Tulasnella Epulorhiza T. cruciata unnamed T. deliquescens E. repens T. calospora Rhizoctonia repens T. pinicola unnamed T. asymmetrica T. rubropallens unnamed T. allantospora T. violea unnamed IDENTIFICATION OF ORCHID MYCORRHIZAL FUNGI
Classical methods Classification of Rhizoctonia spp. is based on cytomorphology of hyphae and the morphology of colonies in cultures, with supporting evidence provided by the morphology of teleomorphs and affinities for hyphal anastomosis (Sneh et al. 1991; Carling 1996). Rhizoctonia associated with orchid mycorrhizae characteristically exhibit short inflated segments resembling spores (called monilioid hyphae) and loose aggregates of hyphae regarded as poorly developed sclerotia or resting bodies in culture (Hadley 1982). Those orchid mycorrhizal fungi which have been induced to form sexual stages have been assigned to the teleomorph genera Ceratobasidium, Sebacina (syn. Serendipita), Thanatephorus and Tulasnella (Warcup and Talbot 1967; 1971). However, it is usually very difficult to induce the perfect state in orchid mycorrhizal fungi and alternatives such as DNA base sequence homology have become popular as identification tools.
Molecular identification techniques The polymerase chain reaction (PCR) made possible the amplification of targeted nucleotide sequences from very small amounts of template DNA (Mullis and Faloona 1987). Relatively impure DNA extracts, such as cell lysates or mixtures of plant and fungal DNA can be used for PCR enabling the amplification of DNA from obligate parasites, symbionts and rare taxa (Gardes and Bruns 1993). PCR techniques have been widely adapted for use in the biological and medical sciences (McPherson et al. 1993). RFLP (Random Fragment Length Polymorphism), sequencing or oligonucleotide probing can be used to analyse PCR products, enabling the construction of molecular phylogenies and identification of unknown organisms by comparison with existing databases.
The nuclear ribosomal DNA (rDNA) ITS region has become a convenient target region for the molecular identification or allocation of fungi at the species or subspecies level (Bruns et al. 1991). The internal transcribed spacer (ITS) consists of two variable, non- coding regions of DNA lying within the rDNA repeats, separating the highly conserved genes coding for the rRNA small subunit (18S), the rRNA 5.8S subunit and the rRNA large subunit (28S) (Fig 1.4). Gardes and Bruns (1993) identified the following four
40 reasons for the convenience of this region as target for the molecular identification of fungi. They are: “i) the entire region is between 600 – 800 bp in length and is readily amplified with ‘universal primers’ complementary to conserved flanking sequences within the rRNA genes; ii) the multicopy nature of the rDNA repeat makes amplification easy from small, dilute or degraded samples; iii) the ITS region is often highly variable between morphologically distinct fungi while intraspecific variation is low; and iv) ITS species –specific probes can be produced quickly without the need to produce a chromosomal library” (Gardes and Bruns 1993).
ITS 1
ITS ITS 18S 5.8S 28S . ITS 4
FIGURE 1.4 Diagram of internal transcribed spacer (ITS) region in relation to rDNA genes encoding the rRNA small subunit (18S), rRNA 5.8S subunit and rRNA large subunit (28S) (not to scale). Labelled arrows show binding sites for ITS 1 and 1TS 4 primers and direction of primer extension for amplification of ITS region (after Gardes and Bruns 1993).
Molecular techniques have been widely used to discriminate between fungal endophytes of orchid species (Taylor and Bruns 1997; Sen et al. 1999; Kristiansen et al. 2001; Pope and Carter 2001; Otero et al. 2002; Shan et al. 2002; McCormick et al. 2004; Shefferson et al. 2005; Dearnaley and Le Brocque 2006). In Western Australia, PCR amplification of rDNA ITS regions have been used to successfully discriminate between fungal endophytes of Diuris spp. (Quay, L. 2001 pers. comm.), D. bracteata and P. nigricans, R. gardneri and R. slateri (Rupp.) M.A.Clements and P.J.Cribb (Mursidawati 2003; Bonnardeaux et al. 2007), several Caladenia species and hybrids (Hollick 2004) and have also been used to examine ectomycorrhizal fungi associated with Eucalyptus ecosystems (Glen et al. 2001b; a).
Each of the various techniques used to identify these fungi: descriptions of fruiting bodies; cultural characteristics; anastomosis grouping; pectic zymogram analysis; AFLP
41 and RFLP grouping; rDNA ITS region, rLSU or rSSU sequencing; may provide support for existing taxonomy or yield different taxonomic groupings (Sharon et al. 2006). Ideally identification would use a consensus of several different techniques to confirm taxonomy, however in most studies this is generally limited by logistical constraints.
Recent molecular studies of orchid mycorrhizal fungi have found diversity within the mycorrhizal Rhizoctonia that suggests the taxonomy of these fungi is poorly resolved. Fungi previously regarded as species may encompass broader taxonomic groups: for example; Weiss et al. (2004) suggested that the fungus Sebacina vermifera sensu Warcup and Talbot was a broad complex of species, and states that ‘Sebacina appears to be polyphyletic’ and ‘current species concepts within Sebacinaceae are questionable’. Weiss believed that the taxonomic distinction and diversity within the Sebacinoid fungi was sufficient to establish a new order; the Sebacinales (Weiss et al. 2004). Recent phylogenetic analyses suggest that mycorrhizal Rhizoctonia form three polyphyletic clades: Ceratobasidiales; Sebacinaceae (i.e. the recently established Sebacinales); and Tulasnellales (Taylor et al. 2003; Binder et al. 2005), however there are many taxonomic issues still to be resolved (Suarez et al. 2006).
42 THESIS OUTLINE AND RESEARCH APPROACH
The following thesis chapters describe the results of studies examining the broad objectives of this project (see Introduction). Time constraints required the use of different age rehabilitation sites as ‘proxies’ for repeated long-term surveys monitoring changes in: orchid colonization; vegetation structure; and species composition within individual sites. The study site was Alcoa’s decommissioned mine site at Jarrahdale approximately 55 km south east of Perth, the capital city of Western Australia (see Fig. 1.1). The approximate positions of survey sites are shown on the map of the Jarrahdale mine site in Figure 1.6.
First, an orchid survey was undertaken to establish orchid species richness and population sizes across a replicated chrono-sequence of bauxite mine rehabilitation areas within the Jarrahdale mine site, and to compare these to adjacent unmined forest (Chapter 2). The presence and frequency of occurrence of orchid mycorrhizal fungi in mine-site rehabilitation areas and adjacent unmined forest were then examined using a seed baiting technique (Chapter 3). Mycorrhizal endophytes of three Jarrah forest orchid taxa: Caladenia flava subsp. flava, Disa bracteata and Thelymitra crinita were isolated and identified. The identities of these fungi and the breadth of mycorrhizal specificity for each orchid taxa were compared with one another and with previous studies of the mycorrhizal fungi of Australian terrestrial orchids (Chapter 4).
In Chapter 5 temporal changes in vegetation of the post-mining landscape were examined to determine age related changes in: vegetation structural characteristics; soil surface cover; species richness; and species cover characteristics, as a ‘new’ Jarrah forest ecosystem develops. Orchid species richness and population size of rehabilitation sites were examined in relationship to these vegetation characteristics and compared with unmined forest. Major functional and growth habit groups of Jarrah forest plant taxa were also examined to determine if they were related to orchid colonisation of the post-mining landscape.
Finally, Chapter 6 contains a general discussion examining the overall results in relation to the objectives of the project, comments on successes and failures, and makes suggestions for future studies and recommendations to the bauxite mining industry.
43 FIGURE 1.6 Site of Alcoa’s Jarrahdale mine site showing distribution of paired transects across the study site. Sites are coded with numbers indicating the rehabilitation area age in years followed by the replicate number. For example; the second replicate ten year old rehabilitation transect and adjacent unmined forest transect are at the position marked 10-2. Two additional transects were placed through populations of Cryptostylis ovata and these are labeled Cov-1 and Cov-2. N.B. This map covers the area of mined and rehabilitated Jarrah forest next to the Jarradale label in Fig. 1.1
44 CHAPTER 2
RECRUITMENT OF TERRESTRIAL ORCHIDS IN THE POST-MINING
LANDSCAPE
INTRODUCTION
Alcoa’s minesite rehabilitation currently aims to re-establish, as near as is practicable, the pre-mining vegetation structure, species richness and forest function (Elliott et al. 1996). Many indigenous geophytic plant species, however, either fail to re-establish, or do so very slowly within bauxite mine rehabilitation areas (Koch and Ward 1994; Koch et al. 1996; Grant and Koch 2003). Indigenous terrestrial orchids make up approximately 8.3% of the flora of the Jarrah forest of Western Australia but represent 20% of geophytic species that are difficult to re-establish. The recruitment of orchids in mine site rehabilitation areas has been expected to occur through the natural dispersal of seed from plants in the surrounding unmined forest (Koch et al. 1994). Therefore, it is dependent on the presence of flowering stimuli, flowering, pollination, seed production and seed dispersal. Western Australian terrestrial orchids produce large numbers of seed per capsule but fruit set is often low (Dafni and Calder 1987; Elliott and Ladd 2002; M. Brundrett pers. comm. 2006). Wind tunnel and laboratory experiments have shown that the distance orchid seed is dispersed from the parent plant is dependent on both wind speed and turbulence, but most seeds are dispersed less than 8m (Murren and Ellison 1998; Chung et al. 2004). However, orchid seed dispersal distances of up to several hundred kilometres have been reported (Gandawijaja and Arditti 1983; Rasmussen 1995; Partomihardjo 2003), therefore dispersal was not considered to be a limiting factor in recruitment.
Recruitment of orchids in undisturbed bushland is dependent on both availability of viable seed and an adequate inoculum potential of the appropriate orchid mycorrhizal fungi in the soil of suitable microhabitats at the beginning of the wet season (May). Batty et al. (2001a) observed low recruitment rates of terrestrial orchids from natural seed dispersal in Western Australian bushland. This was suspected to be the result of the patchy distribution of the mycorrhizal fungi in the soil and the scarcity of suitable micro-habitats in the landscape (Batty et al. 2001a; McKendrick et al. 2002). Thus,
45 orchid recruitment in the highly disturbed post-mining landscape will be dependent on not only the production and dispersal of viable seed from remnant bushland but also the recovery of the inoculum potential of the appropriate mycorrhizal fungi in the soil and the re-establishment of suitable microhabitats through regrowth of vegetation.
In this study the re-establishment of orchid population size and species richness was examined by surveying a temporal sequence of bauxite mine rehabilitation areas and the adjacent unmined forest within the Jarrahdale mine site. The aims of the study were: a) to investigate the chronology of terrestrial orchid species return to the post- mining landscape; b) to determine if orchid population size and species richness in rehabilitation areas approached that of unmined forest within the temporal sequence examined; and c) to determine if rehabilitation areas and adjacent unmined jarrah forest had species in common, that is, if the adjacent forest was a likely source of seed.
46 MATERIALS AND METHODS
Study Site Alcoa’s Jarrahdale bauxite mine is located along the Darling Scarp near the town of Jarrahdale, 55 km SE of Perth in Western Australia. The climate is dry Mediterranean with a mean annual rainfall of 1220 mm falling over mainly the winter-wet season. Mining commenced at Jarrahdale in 1963 and the region was actively mined until 1998, rehabilitation was completed in 2001 and the site decommissioned in 2002. In the post- mining rehabilitation process the pit is landscaped, overburden and topsoil are respread in the correct order, and the area is ripped to 1.5 m depth then seeded and fertilized. The current seeding mixture contains seed of the two dominant tree species, Eucalyptus marginata D.Donn ex Sm (jarrah), Corymbia calophylla (Lindl.) K.D. Hill and L.A.S. Johnson syn. Eucalyptus calophylla (marri), and in excess of 60 understorey species (Nichols et al. 1991).
Orchid Survey Single belt transects 5 m by 50 m, each consisting of ten contiguous 5 m x 5 m quadrats, were in established in each of four replicates of 1, 5, 10 and 15 year old rehabilitation areas (established in 2001, 1997, 1992 and 1987, respectively) and in adjacent unmined forest at Jarrahdale in autumn 2002 (see Chapter 1; Fig. 1.6). A further two transects were established in 27 year old rehabilitation areas, with an over-storey of marri and a single transect in a 26 year old rehabilitation area, each was paired with transects in adjacent unmined forest. These three transects are included in the category of 25+ year old rehabilitation area. Two additional transects were also established through forest populations of Cryptostylis ovata. These transects were used to examine vegetation associations of this species as it was rare in both forest and rehabilitation areas. In total 40 transects were established, each in a separate forest or rehabilitation area. Each transect was scored for the number of orchid species and number of plants of each species present in each quadrat over the 2002 growing season.
Species Identification and Nomenclature Orchid species identification and nomenclature was consistent with taxonomy of orchids in Florabase (http://florabase.calm.wa.gov.au/ ; accessed 26th July 2007). Voucher specimens were collected from the orchid species: Caladenia flava, Cyanicula sericea; Cyrtostylis huegelii; Disa bracteata; Diuris brumalis; Microtis media;
47 Prasophyllum hians; Pterostylis barbata; P. vittata; P. recurva; P. sp. crinkled leaf; Thelymitra crinita and T. macrophylla; for submission to the Western Australian Herbarium. Appendix 1, Table A1.1 contains a complete list of indigenous orchid taxa of the northern Jarrah forest. Tables A1.2 to A1.4 in Appendix 1 contain brief descriptions of all orchids observed within the Jarrahdale site over the term of the study.
Data Analysis Orchid population density and diversity data were log transformed before analysis by ANOVA using GenStat Version 7, Lawes Agricultural Trust, Rothamsted Experimental Station.
48 RESULTS
Orchid Species Succession Orchids were found to have re-established in all rehabilitation areas except for 1 year old rehabilitation areas (established 2001). Twenty one orchid species were found in the combined forest and rehabilitation areas. These were separated into three groups based on apparent chronology of invasion: ‘Pioneer species’ first appear in 5 year old rehabilitation areas; ‘Secondary colonisers’ in 10 to 15 year old sites; and ‘Tertiary colonisers’ or ‘Climax species’ in 25 years or older. Species that only occurred in unmined forest are included in this last group.
TABLE 2.1 A cumulative list of orchid species observed in forest and rehabilitation areas at the Jarrahdale study site. Numbers indicate the number of transects where an orchid species was observed; the number of transects in brackets indicate that a species was present at a site but found only outside the survey transect, dashes indicate the species was not observed. Total number of transects for unmined forest = 21; for rehabilitation areas 1, 5, 10 and 15 years old = 4; for rehabilitation areas 25+ year old = 3. N.B. Taxonomy is consistent with Florabase (http://Florabase.calm.gov.au/, accessed 26th July 2007)
Age of Rehabilitation Area Orchid Species Forest 1 yrs 5 yrs 10 yrs 15 yrs 25+ yrs Pioneer Species Caladenia flava 15 - 3 - 3 3 Disa bracteata* (2)✝ - 1 4 3 - Microtis media - - 1 3 2 1 Pterostylis sp. crinkled leaf 5 - 2 1 2 - Pterostylis recurva 7 - 3 2 1 1 Pterostylis sanguinea - - 1 - 2 -
Secondary Colonisers Caladenia longiclavata (1) - - (1) - 1 Diuris brumalis 1 - - 1 1 3 Pterostylis barbata 3 - - 1 1 - Thelymitra macrophylla 2 - - 1 - 2 Thelymitra benthamiana - - - 1 - 1 Caladenia macrostylis - - - - 1 - Cyanicula sericea 8 - - - 3 - Pterostylis vittata 4 - - - (1) 1 Thelymitra crinita 19 - - - (1) -
Climax Species Eriochilus dilatatus 8 - - - - (1) Caladenia reptans (1) - - - - - Cryptostylis ovata 2 - - - - - Cyrtostylis huegelii 3 - - - - - Prasophyllum parvifolia 1(1) - - - - - Pyrochis nigricans 4 - - - - -
Number of species 18 0 6 9 11 9 * Indicates an exotic species ✝ Note: D. bracteata was found only in locally disturbed areas in unmined forest.
49 The six orchid species first found in 5 year old rehabilitation areas are listed under ‘Pioneer species’ in Table 2.1. The nine orchid species which first colonise in ten and fifteen year old sites are listed as ‘Secondary colonisers’ and the six species found only in the oldest rehabilitation areas (25+ years) and forest are listed under ‘Climax species’. Several species were sporadically absent from the chrono-sequence of rehabilitation areas after their first appearance.
Comparison of species richness and population density Orchid species richness (measured as the mean number of orchid species present) was significantly different in 1 year old rehabilitation areas (at P ≤ 0.05) to all other rehabilitation and forest areas (Fig. 2.1A). The mean numbers of orchid species in forest adjacent to the 25+ year old transects was significantly lower than in forest adjacent to five year old rehabilitation area but not to any of the other forested areas nor to their adjacent rehabilitation areas.
The data for clonal orchids was examined separately. Clonal orchids, in this study, are those that primarily reproduce vegetatively, that is, by multiplication of tubers. High variance in the species richness of clonal orchids in forest transects adjacent to 1 year old rehabilitation areas meant that these forest sites were not significantly different to the 1 year old rehabilitation areas (Fig. 2.1B.) (at P ≥ 0.05). The 15 and 25+ year old rehabilitation areas had a higher mean number of species present but were only significantly different to the 1 year old rehabilitation area and its adjacent forest (Fig. 2.1B.).
Orchid density was found to be highest in the oldest rehabilitation area transects (Fig. 2.2A). However, only 1 year old and 5 year old rehabilitation areas were significantly different to this site (at P ≤ 0.05). The orchid density of 1 year old rehabilitation areas was significantly less than all other age rehabilitation sites. Orchid density in unmined forest was highly variable and patchy. Thus, despite the large differences in the mean density of orchids, none of the forested areas was significantly different to one another, nor to their adjacent rehabilitation areas.
The mean density of clonal orchids in the 25+ year old rehabilitation area was significantly higher (at P ≤ 0.05) than all other rehabilitation areas and forest areas except for those adjacent to 15 and 25+ year old rehabilitation areas. These forest areas
50 were not, however, significantly different to any of the other rehabilitation area or forested areas (Fig. 2.2B). It should be noted that the apparently high mean values for 25+ year old rehabilitation areas (Fig. 2.2A) are due to one transect with an extremely high count for the clonal orchid C. flava. Statistical analysis was carried out on log transformed counts of orchids/transect and this value was not found to be significantly different to those for 10 and 15 year old rehabilitation areas.
Orchid species identified as disturbance opportunists - D. bracteata; M. media; and C. macrostylis - are taxa that exhibit an increase in recruitment in response to habitat disturbance. The mean number of disturbance opportunists (Fig. 2.2C) in rehabilitation areas that were 1, 5 and 25+ year old were significantly lower than 10 and 15 year old rehabilitation areas (at P ≤ 0.05). No disturbance opportunists were found in any of the forest area transects.
51 10.0 A. Rehabilitation
Adjacent Forest 8.0
b 6.0 b b
4.0 b
2.0
a 0.0
B. b b
Number Species/transect of 3.0
b 2.0
b
1.0
a 0.0 1 5 10 15 >25
Years since rehabilitation establishment
FIGURE 2.1. Species richness (s) measured as the mean number of orchid species found in a temporal sequence of transects in rehabilitation areas and adjacent unmined forest (for 1, 5, 10 and 15 year old rehabilitation areas n = 4, for 25+ year old n = 3). A. All orchid species. B. Clonal orchid species. Vertical bars represent ± 1s.e. Data are untransformed means. (N.B. Data were log10(1+n) transformed for analysis by ANOVA.) Columns identified by the same letter are not significantly different (P > 0.05). Transect area 0.025 ha (5 m x 50 m). For each orchid category the dashed line is the mean value for all forest transects and the broad coloured bar represents ± 1 s.e.
52
Rehabilitation c 20000 A. Adjacent Forest
15000
10000
5000 bc
b bc a 0 B. 20000 b
15000
10000 Number of plants/hectare 5000
a a a a 0 C. b
3000
2000
b
1000
a a a 0 1 5 10 15 25+ Years since rehabilitation establishment
FIGURE 2.2. Orchid population size of a temporal sequence of transects in rehabilitation areas and adjacent unmined forest (for 1, 5, 10 and 15 year old rehabilitation areas n = 4, for 25+ year old n = 3). A. All orchids. B. Clonal orchids. C. Disturbance opportunists. Vertical bars represent ± 1 s.e. Data are untransformed means. (N.B. Data were log10(1+n) transformed for analysis by ANOVA. Mean densities per transect have been converted to mean number of plants ha-1.) Columns identified by the same letter are not significantly different (P > 0.05). (N.B. no disturbance opportunists were found in any forest transects.). For each orchid category the dashed line is the mean value for all forest transects and the broad coloured bar represents ± 1 s.e. 53 Comparison of Orchid Populations in Rehabilitation Areas with Adjacent Forest Orchid species found in rehabilitation areas generally did not also occur in adjacent forest (Table 2.2). In fact, seven of the 16 orchid species found in the rehabilitation sites were not found in any of the adjacent unmined forest sites. Unfortunately, data for most species were sparse and it is not possible to say definitively whether the adjacent forest was or was not the primary source of orchid seed for population re-establishment. Only one species, C. flava, appears to be more likely to occur in rehabilitation areas if there is a population in the adjacent forest. This species was found in forest next to seven of the nine rehabilitation sites where it had re-established (Table 2.2). However, C. flava is one of the most common orchids in both rehabilitation areas and forest so it is not clear if short-range seed dispersal is important as other factors probably favour the presence of this species. Thus, it appears that for most orchid species short-range seed dispersal is less important than other environmental or edaphic factors in successful invasion of rehabilitation areas.
TABLE 2.2 Observations of co-occurrence of orchid species in rehabilitation areas and adjacent forest. Data are for 19 paired rehabilitation area/forest sites and includes orchids observed in the survey sites but outside survey transects. Numbers in brackets indicate the number of transects where a species was observed at a particular site but only outside the survey transect. N.B. Taxonomy is consistent with Florabase (http://Florabase.calm.gov.au/, accessed 26th July 2007)
Number of Transects with Orchid Populations Orchid Species Rehabilitation Forest Co-occurring Clonal Species Caladenia flava 9 13 7 Caladenia reptans 0 (2) 0 Cyrtostylis huegelii 0 2 0 Diuris brumalis 6 2 1 Microtis media 7 0 0 Pterostylis sp. crinkled leaf 5 3 0 Pterostylis sanguinea 3 0 0 Pterostylis vittata 1 4 0 Pyrochis nigricans 0 4 0 Other Species Caladenia longiclavata (1) (2) (1) Caladenia macrostylis 1 0 0 Cyanicula sericea 3 7 1 Disa bracteata 8 (1)* 1 Eriochilus dilatatus (1) 7 0 Prasophyllum parvifolia 0 1 (1) 0 Pterostylis barbata 2 3 1 Pterostylis recurva 8 5 4 Thelymitra macrophylla 4 5 2 Thelymitra benthamiana 2 0 0 Thelymitra crinita (1) 18 0 * Note: a single D. bracteata plant was found in a locally disturbed area within unmined forest.
54 DISCUSSION
The establishment of flora in post-bauxite mining rehabilitation areas is thought to follow the “initial floristic composition” model (Koch and Ward 1994; Grant and Loneragan 2001), that is, the flora composition immediately following disturbance determines future shifts in dominance. In the post mining landscape, this initial vegetation composition is determined by the composition of both the seeding mixture and the soil seed bank in respread topsoil. Alcoa’s earliest rehabilitation procedures consisted of planting exotic tree species (e.g. Pinus pinaster Aiton) with few understorey species. This was later modified to seeding with non-indigenous eucalypt species (e.g. Eucalyptus resinifera Sm.) and understorey species dominated by legumes. Since 1988 rehabilitation areas have been seeded with indigenous tree species, a broader range of understorey species and planted with tissue cultured recalcitrant taxa (Grant and Koch 2003; Willyams 2005; Grant and Koch 2007). Both the seeding mix and the soil seed-bank in respread topsoil are important sources of propagules for revegetation for many plant species. However, orchid seed has never been included in seed mixtures used for rehabilitation and the orchid soil seed bank in South West Western Australia is known to be short lived (Batty et al. 2000). It is therefore unlikely that respread topsoil is a significant source of viable orchid propagules. Successful germination of orchid seed requires the presence of fungi capable of forming mycorrhizal associations and thus requires the recovery of soil microflora. Orchids were therefore not expected to reappear rapidly in the post-mining landscape.
In terrestrial orchids seed production per pod is high (e.g. Diuris spp are known to produce approximately 5,000 seeds/pod (M.T. Collins unpubl. data) and C. arenicola Hopper and A.P.Br. approximately 30,000 seeds/pod) but natural recruitment is low (Batty 2001). Recruitment in rehabilitation areas will thus be dependent on: seed set in surrounding forest; adequate seed dispersal; and the recovery of soil microflora with the progressive build up of organic matter. This study found that within five years of rehabilitation area establishment the total number of orchids and orchid species recruited was not significantly different from adjacent unmined forest. Due to the dependence of terrestrial orchids on their mycorrhizal associations for germination of seed, nutrition of protocorms, seedlings and adult plants their presence may be regarded as an indicator of the recovery of their mycorrhizal fungi in the edaphic environment (Rasmussen 1995). Orchid mycorrhizal partners are Rhizoctonia-like fungi that are
55 primarily found in the coarse fraction of soil organic matter i.e. partially decomposed litter (Brundrett et al. 2003). Invasion by these fungi may therefore be expected to be coincident with litter accumulation.
The establishment of orchid taxa with differing growth habits will influence the future species richness and size of orchid populations within the rehabilitation areas. Once established, clonal orchids can increase population size rapidly through vegetative reproduction. In this study these species were absent from 1 year old rehabilitation areas (2001) but present in areas of five years or older. Grant and Koch (2003) found the three clonal species; C. huegelii, C. flava and P. sp. crinkled leaf, had the highest densities of any indigenous orchids in forest areas (Table 2.3). Two of these species, C. flava and P. sp. crinkled leaf, re-established in rehabilitation areas with densities reaching approximately 10% that of unmined forest in >10 year old rehabilitation area. However, occurrence of other orchid species (disturbance opportunists excluded) was generally at much lower percentages of unmined forest population densities.
The current study found similar trends in forest species, as five of the six most populous forest species were clonal orchids. However, in >10 year old rehabilitation areas, both clonal and non-clonal species that had successfully re-established often reached population densities that were higher than the surrounding unmined forest (Table 2.3). In the cases of C. flava and D. bracteata this was probably due to the rehabilitation area survey including one extremely populous transect for each species. Placement of forest transects in this study was biased towards visibly undisturbed areas and this may also have affected the population counts.
There does appear to be a ‘succession’ of orchid species colonising the rehabilitation areas. Six pioneer species, including two known disturbance opportunists, D. bracteata and M. media, re-established within five years. The oldest rehabilitation areas (25+ years) examined had fewer species than 15 year old areas and only four of these species were common to both. In part, this is due to the absence of one of the disturbance opportunist species (D. bracteata) in the oldest sites examined, but probably also to changes in habitat with vegetation growth (Chapter 5). The total number of orchid species present in cumulative species lists for rehabilitation areas and unmined forest were similar with 16 and 18 species, respectively (Table 2.1). However, three species; C. macrostylis Fitzg., M. media and T. benthamiana Rch..f., present in rehabilitation
56 -1 TABLE 2.3. Mean density of orchids (plants ha ) found in a retrospective study of data from permanent vegetation monitoring plots by Alcoa (adapted from Grant and Koch 2003) compared with those found in survey transects in the current study. Species lists have been separated on growth habit and listed in decreasing forest density found by Grant and Koch (2003). Species names in bold were identified as disturbance opportunists based on the Grant and Koch paper. Note: Species observed in survey sites but not found inside transects have not been included.
Orchid Species Mean Density (plants ha-1) Grant and Koch (2003) Current Study Scientific Name Common Name Forest All Rehab >10yr old Forest All Rehab >10yr old Clonal Species Cyrtostylis huegelii Mosquito orchid 3515.8 0.2 0.0 2177.1 0.0 0.0 Caladenia flava Cowslip orchid 2870.5 138.5 271.0 613.3 1772.6* 4771.4* Pterostylis sp. crinkled leaf Slender snail orchid 2694.8 133.3 250.0 123.8 128.4 45.7 Leporella fimbria Hare orchid 607.0 0.4 0.0 0.0 0.0 0.0 Pterostylis vittata Banded greenhood 520.3 60.5 119.3 20.9 8.4 22.9 Pyrochis nigricans Red beaks 237.6 21.3 42.0 360.0 0.0 0.0 Caladenia reptans Little pink fairy orchid 129.5 1.4 2.3 0.0 0.0 0.0 Cryptostylis ovata Slipper orchid 46.2 0.0 0.0 316.2** 0.0 0.0 Microtis media Common mignonette orchid 3.4 889.5 1742.7 0.0 6.1 120.0 Diuris brumalis Winter donkey orchid 0.0 0.0 0.0 19.0 181.1 445.7 Pterostylis sanguinea Dark banded greenhood 0.0 0.0 0.0 0.0 12.6 19.1 Other Species Thelymitra crinita Blue lady orchid 1183.6 1.5 3.1 817.1 2.1 0.0 Eriochilus dilatatus Common bunny orchid 709.6 2.7 5.0 53.3 0.0 0.0 Thelymitra macrophylla Scented sun orchid 399.8 0.4 0.8 15.2 181.1 485.7 Pterostylis recurva Jug orchid 171.2 17.6 34.8 66.7 61.1 28.6 Disa bracteata1 South African orchid 41.7 210.4 336.6 0.0 595.8 1057.1 Cyanicula sericea Silky blue orchid 22.5 0.2 0.4 51.4 12.6 34.3 Prasophyllum elatum Tall leek orchid 20.3 0.0 0.0 0.0 0.0 0.0 Pterostylis barbata Dwarf bird orchid 16.9 0.8 0.0 13.3 4.2 5.7 Prasophyllum brownii Christmas leek orchid 15.8 0.2 0.0 0.0 0.0 0.0 Caladenia macrostylis Leaping spider orchid 13.5 17.6 28.7 0.0 4.2 11.4 Lyperanthus serratus Rattle beaks 11.3 0.0 0.0 0.0 0.0 0.0 Elythranthera brunonis Purple enamel orchid 2.3 0.4 0.8 0.0 0.0 0.0 Diuris carinata2 Tall bee orchid 0.0 1.2 2.3 0.0 0.0 0.0 Caladenia longiclavata Clubbed spider orchid 0.0 0.0 0.0 0.0 2.1 5.71 Prasophyllum parvifolia Autumn leek orchid 0.0 0.0 0.0 1.8 0.0 0.0 Thelymitra benthamiana Leopard orchid 0.0 0.0 0.0 0.0 65.3 171.4 1 indicates an exotic weed species. 2 indicates an indigenous species not normally found in jarrah forest. * Value high due to one very high transect population. ** Value high due to biased sampling. areas were not found in the forest and five species: C. reptans Lindl., C. ovata, C. huegelii, P. parvifolia Lindl. and P. nigricans, present in forest were not found in the rehabilitation areas. Thus, several further decades may need to elapse for orchid species richness to be re-established and for the populations of these species to return to pre- mining densities.
Orchid species data from transect surveys were grouped into the following species categories: clonal species; disturbance opportunists; exotic species; and other species, for comparison with data from Grant and Koch (2003) (Table 2.4). The results of each study were generally similar for each species category with the exception that the current study did not find the exotic species D. bracteata in forest transects. This may be an artefact of the smaller sampling area of the present study or to this species responding to forest disturbance due to causes other than mining in the earlier study. D. bracteata was observed only twice in the unmined forest in the current study, once growing in an area of bare ground in a Phytophthora affected area and once in soil on top of a fallen log. Its absence from older rehabilitation areas and rarity in unmined forest indicates a preference for recent disturbance.
Three species were identified as disturbance opportunists from Grant and Koch’s study of long term vegetation survey work; Microtis media, Disa bracteata and Caladenia macrostylis (Table 2.3). In the current study, these disturbance opportunist species were found to be most abundant in 10 and 15 year old rehabilitation areas, with population densities dropping dramatically in the oldest rehabilitation areas. Bonnardeaux (2007) found a high diversity of fungal endophytes forming mycorrhiza with D. bracteata (syn. Monadenia bracteata). This naturalized South African species is one of the most common of the disturbance opportunists in the rehabilitation areas. It may be that the ability to form mycorrhiza with a wide diversity of fungi is advantageous in the early recruitment of orchid flora. Other edaphic qualities or vegetation structural elements present only in young rehabilitation areas may also provide a conducive environment to these species, for example: low soil organic matter; dense shrubs; intense shade; or lack of competition. The ecology of D. bracteata and other disturbance opportunists needs to be examined further to determine whether these species have the potential to be used as indicator species of the progress of vegetation establishment.
58 TABLE 2.4. Comparison of the number of orchid species of differing growth strategies found in an Alcoa retrospective study (Grant and Koch, 2003) with data from transects surveyed in this study. Data for the Alcoa retrospective study were acquired from 75 unmined and 334 rehabilitation areas over 11 years, monitoring plots measured 20 m x 20 m. Data for the current study were acquired from 21 unmined and 19 rehabilitation area belt transects over a single growing season. Transects measured 5 m x 50 m.
Number of orchid species Alcoa Survey Current Study Category Forest All Rehab >10yr old Forest All Rehab >10yr old Clonal 8 7 5 7 5 5 Disturbance opportunists 2 2 2 0 2 2 Exotic species* 1 1 1 0 1 1 Other species 10 9 7 7 5 5 Total 21 19 15 14 13 13
Total Area Surveyed (ha) 4.44 25.88 13.08 0.525 0.475 0.175 * Note: the exotic species D. bracteata is also a disturbance opportunist.
59 CONCLUSIONS
There does appear to be a ‘succession’ of orchid species colonising rehabilitation areas. However, some orchid species have failed to establish even in the oldest rehabilitation sites examined. The sporadic absence of an orchid species from the rehabilitation area chrono-sequence after their first appearance may be a reflection of the naturally sparse occurrence of these orchid species or be an artefact of limited sample size. Orchids were absent from 1 year old rehabilitation areas, but population size and species richness in all other age rehabilitation areas were not significantly different to adjacent unmined forest sites. This rapid colonisation was, in part, due to invasion by disturbance opportunist species. Most of the orchid species found in the rehabilitation areas returned within five to fifteen years suggesting that seed dispersal is not limiting orchid recruitment. However, for all orchid species, except C. flava, there was no evidence of co-occurrence of that species, that is, its presence in both a rehabilitation area and the adjacent forest. This suggests that the primary source of seed for most orchid species may be at some distance, and that unidentified environmental and/or edaphic factors, other than seed dispersal, are limiting successful invasion of rehabilitation areas.
Five orchid species were not observed in rehabilitation areas during this study, however, three of these species were found in rehabilitation areas by Grant and Koch’s survey of data from Alcoa’s long term monitoring plots (Table 2.3). A more extensive survey that included rehabilitation areas over 27 years old may have detected these species. However, four orchid species, C. ovata, P. parvifolia, P. elatum R.Br. and L. serratus Lindl., were not detected in rehabilitation areas in either study. These orchid species may be naturally rare or factors other than those associated with the establishment of vegetation may be limiting recruitment such as the absence of specific pollinators or flowering stimuli. For example, C. ovata populations are sparse in the Jarrahdale area and close to the northern limit of its habitat. Pollination in this species occurs only as a result of pseudo-copulation with the male ichneumon wasp, Lissopimpla semipunctata Kirby (Hoffman and Brown 1998). Successful seed production in this orchid therefore requires habitat capable of supporting viable pollinator populations within flight distance of the extant orchid populations. The orchid P. elatum flowers only in response to a summer bushfire, and therefore without fire at the appropriate time seed will not be produced. Absence of summer fire not only stops colonisation of rehabilitation areas by this species but also hinders identification and the accuracy of plant surveys. Future
60 work could be directed towards these areas of study, that is, examination of the survival and/or recovery of pollinator populations in the post-mining landscape for those orchid species where highly specific relationships exist and, the effect of fire on seed production and recruitment of orchids.
61 62 CHAPTER 3
COLONISATION OF BAUXITE MINE REHABILITATION SITES OF
SOUTH-WEST WESTERN AUSTRALIA BY ORCHID MYCORRHIZAL
FUNGI.
INTRODUCTION
Recruitment of indigenous terrestrial orchids in the highly disturbed post-bauxite mining landscape in Western Australia is dependent on (i) the dispersal of seed from adjacent undisturbed bushland, (ii) an adequate inoculum potential of the appropriate orchid mycorrhizal fungi and (iii) the re-establishment of suitable microhabitats through regrowth of vegetation. Orchid seed is known to be dispersed widely by wind, however, most seed falls within a few metres of the parent plant (Murren and Ellison 1998; Arditti and Ghani 2000; Chung et al. 2004). For most jarrah forest orchid taxa seed dispersal is not likely to prevent recovery, but recruitment rates may be very low in the first few years as seed would not be present unless topsoil was spread prior to seed release in surrounding unmined areas of forest. The structural characteristics of the rehabilitation area vegetation may also be unsuitable for some orchid taxa during the first decade due to the intense competition from shrubs, lack of tree canopy and altered soil conditions.
Orchid mycorrhizal fungi are required for the germination of seed, and provide almost complete nutrition of protocorms and seedlings at least until the development of photosynthetic capacity (Rasmussen 1995). The extent of dependence on the orchid mycorrhizal fungi (OMF) and the specificity of this relationship are thought to vary between taxa. Hereafter the acronym OMF (orchid mycorrhizal fungus or fungi) is used to refer to that suite of fungi capable of forming mycorrhizal associations with a particular orchid taxon. Terrestrial orchids are regarded as particularly vulnerable to environmental disturbance due to their dependence on highly specific fungal associations (Table 1.4) (Masuhara and Katsuya 1994; Masuhara et al. 1994; Perkins et al. 1995; Andersen and Rasmussen 1996; Carling et al. 1999; Pope and Carter 2001; Mursidawati 2003). Soil disturbance, associated with stripping and respreading of
63 topsoil during mining, is known to reduce the inoculum potential of arbuscular mycorrhiza and ericoid mycorrhizal fungi (Jasper et al. 1989a; b; Hutton et al. 1997) and is expected to have a similar effect on OMF.
Low orchid recruitment rates from natural seed dispersal have been observed in undisturbed bushland in Western Australia and are suspected, in part, to be the result of patchy distribution of the mycorrhizal fungi in the soil and the scarcity of suitable micro-habitats across the landscape (Batty et al. 2001a). The presence or absence of particular OMF and their frequency of occurrence are thus important limiting factors in the successful recruitment of orchid taxa. The dependency of orchids on a fungal association for seed germination provides a simple means for detection of OMF in the field. In 1993, Rasmussen and Whigham first used seed packets to observe orchid seed germinating in situ. Their technique has been modified and widely used for the detection of fungi capable of germinating orchid seed in natural and disturbed ecosystems (van der Kinderen 1995; Batty et al. 2001a; McKendrick et al. 2002; Brundrett et al. 2003).
A preliminary survey has shown that orchids invade mine site rehabilitation areas within five years of establishment with orchid numbers and species diversity similar to that of adjacent unmined forest (Collins et al. 2005). However, orchid numbers were boosted by high populations of disturbance opportunists, some orchid species were found only in either forest or rehabilitation areas, and populations of individual species were generally very low compared with natural forest populations (Grant and Koch 2003; Collins et al. 2005). This study was undertaken to determine whether the absence of the appropriate orchid mycorrhizal fungi was affecting the invasion of rehabilitation areas by orchids.
The seed packet baiting technique was used to survey mine-site rehabilitation areas soils of three ages: 1, 10 and 26 years after establishment; and adjacent unmined forest for the presence of orchid mycorrhizal fungi. Vegetation structural characteristics, soil surface cover and litter quality characteristics were also examined at all survey sites. The aims were to determine the following: (a) the frequency of occurrence of orchid mycorrhizal fungi in rehabilitation areas and adjacent unmined forest,
64 (b) the diversity of orchid mycorrhizal fungi indirectly through simultaneously baiting with the seed of six species orchid taxa in multi-chambered baits, (c) whether re-establishment of orchid mycorrhizal fungi was associated with the recovery of particular vegetation structure and soil surface cover characteristics, and (d) whether the chronological sequence of the return of orchid mycorrhizal fungi drives the chronology of orchid taxa to rehabilitation areas.
65 MATERIALS AND METHODS
Species selection Orchid species were selected for inclusion in orchid mycorrhizal fungi (OMF) baits based on density of orchids in permanent vegetation monitoring plots by Alcoa World Alumina Australia (Grant and Koch 2003) and by visual assessment in the field in 2001/2002. The six species selected were: Caladenia flava subsp. flava (hereafter referred to as C. flava), Disa bracteata, Microtis media subsp. media (hereafter referred to as M. media), Pterostylis recurva, Pyrorchis nigricans, Thelymitra crinita (Table 3.1). Taxonomy of orchids is consistent with Florabase (http://florabase.calm.wa.gov.au/)
Orchid seed Orchid seed was collected from naturally and hand pollinated wild plants in Spring/Summer of 2001, 2002 and 2003 just prior to dehiscence. Seed was cleaned by passing through a fine metal sieve (0.075 mm), dried over silica gel for 24 h at room temperature and stored in 1.5 mL Nunc tubes at 4oC. Local provenance orchid seed collected from the Jarrahdale mine-site was used whenever possible, and when this was unavailable, seed of known provenance and age was obtained from the seed collection at Kings Park and Botanic Gardens, Perth, Western Australia. Pyrorchis nigricans seed obtained from this source was collected in jarrah forest approximately 50km north of the study site. (A complete list of provenance and weight of orchid seed collected during this study is contained in Appendix 1, Table A1.5.)
TABLE 3.1. Growth habits and occurrence of orchid species selected for inclusion in orchid mycorrhizal fungus detection baits.
Orchid taxa Clonal1 Disturbance Occurs in opportunist forest rehabilitation
Caladenia flava subsp. flava + - + + Disa bracteata* - + - + Microtis media subsp. media + + - + Pterostylis recurva - - + + Pyrorchis nigricans + - + - Thelymitra crinita - - + -
1 Clonal refers to species that reproduce vegetatively and the term disturbance opportunist to species that were more common in disturbed areas than undisturbed forest. * D. bracteata is a South African species that has naturalised in Western Australia.
66 Orchid Mycorrhizal Fungi detection baits (OMF baits) Multiple-species OMF detection baits were prepared with 6 x 15 cm strips of 90 µm nylon mesh utilising the method described by Brundrett et al. (2003). The mesh was folded length-wise and heat-sealed at 2.4 cm intervals to create six pockets and an area for attaching the coloured location tag. Approximately 0.25 g of a mixture of seed and sterile white sand containing about 125 seeds from the appropriate orchid species was added to each pocket and the top edge of the baits heat-sealed (Fig. 3.1).
FIGURE 3.1. Orchid seed bait containing seed of six orchid species used to detect orchid mycorrhizal fungi in the field. Baits were prepared from 90 µm nylon mesh using the method of Brundrett et al. (2003). Each of the six pockets contain approximately 0.25 g of orchid seed mixed with white silica sand and contain about 125 seeds. A coloured location tag is attached to one end for ease of identification at harvest. Bar = 5 cm.
Baiting sites Single transects in each of three different age rehabilitation areas (1, 10 and 26 years old) and adjacent forest within the Jarrahdale bauxite mine area were selected for orchid mycorrhiza detection with OMF detection baits. Each transect was 50 m long. The closest point of each rehabilitation transect to unmined forest was approximately 20 m. Transects in unmined forest were a similar distance from the adjacent rehabilitation area. A spade was used to make vertical cuts in the soil at 90o to the direction of the transect and with minimal soil disturbance. In areas with litter, baits were placed vertically in the cuts at a point corresponding to the interface between the litter layer and the soil ‘A’ horizon. In areas with little or no litter, baits were placed at 4-5 cm depth. Twenty baits were placed at 2.5-m intervals along each transect in late autumn of
67 2002. The position of each bait was marked with a numbered plant label and a coloured location tag attached to the bait for ease of identification at collection. In early winter 2004 fresh baits were placed as close as practicable to the position of the 2002 baits. Baits were recovered after 20 weeks, placed between damp paper towels in a sealed container for transport to the laboratory and stored at 4oC prior to scoring. Baits were stored for up to 3 weeks in 2002 and 1 week in 2004 because of the time required for scoring.
Scoring of germination stages The baits were cut open one pocket at a time. Each pocket was opened and the contents washed off the nylon mesh into a sterile Petri dish with deionised water. The contents were then examined under a dissection microscope at 20x magnification. In 2002, baits were scored for the number of seeds recovered and germination stage of each recovered seed in each bait pocket. Descriptions of germination stages are as defined by Collins (2005) (Table 3.2). In 2004, baits were scored for recovered seed numbers and number of successful germinations. In this study successful germination is defined as attainment of stage 4, initiation of shoot primordium (Table 3.2). The number of orchid species with seed germinating in each multi-species bait is used as an indirect measure of OMF diversity.
TABLE 3.2. Descriptions of orchid seed germination stages used in this study Stages are as defined by Collins (2005) and are based on those of Clements et al (1986), Ramsey et al. (1986), Zettler and Hofer (1998) and Batty et al (2001b)
Stage Description
0 unimbibed seed 1 imbibed seed, embryo swollen 2 ruptured testa, occasional trichome 3 enlarged protocorm, numerous trichomes 4 initiation of leaf primordium (shoot beginning to develop) 5 leaf elongation 6 initiation of dropper 7 tuber and root formation
Vegetation associations In 2002 vegetation cover, litter cover, type and depth were determined in 1 m2 quadrats surrounding each bait made from PVC pipe (20 mm). Quadrats were placed at baiting positions along the transects, centred on the bait and parallel to the transect. 68 Vegetation in each quadrat was scored for percentage cover provided by each of the following structural categories: trees > 30 m; trees 10-30 m; trees < 10 m; shrubs > 2 m; shrubs 1-2 m; shrubs < 1 m and herbs/sedges/grasses. Vegetation structure categories are based on those of Keighery (1994), Specht (1970) and Muir (1977). The category “herbs, sedges and grasses” includes all low tufted plants and ground covers. The structural category trees < 10 m includes saplings as these provide much of the vegetation cover in younger rehabilitation areas.
In addition, data were collected for soil surface cover in the following categories: leafy litter (leaves and fine twigs); litter - min. depth; litter - max. depth; woody litter (large branches > 2.5 cm diameter and logs) and bare ground. In both forest and rehabilitation sites three samples of topsoil and decomposing litter to 4-5 cm depth (50-800 g dry wt.) were taken at random intervals approximately 20 cm distance from each bait for moisture content determination.
In rehabilitation areas the position of the bait on the rip line was scored (bottom, side or top). The rip lines are a series of parallel furrows and mounds, approximately 1.5 m apart and 25 cm in amplitude, created by deep-ripping during the rehabilitation process. The bottom of rip lines consisted of the lower 10 cm of the furrows. The top of the rip line was the upper 10 cm of mounds. The sides consisted of the region between the top and bottom.
Vegetation cover, litter cover, type and depth were determined in belt transects in 2002. Each bait transect followed one side of a belt transect. Belt transects measured 5 x 50 m. Each was scored for vegetation and litter cover in a series of ten contiguous quadrats (5 m x 5 m). Vegetation strata and litter were categorised as given previously for bait quadrats.
Litter analysis Leafy litter was collected from each of three 1 m2 bait quadrats in each of five transects shortly after the 2004 bait recovery. The site containing the sixth transect had been burnt by a natural bush-fire shortly after bait recovery so litter was not available. Litter was separated into two fractions, an upper layer of loose superficial material and a deeper layer of composted litter. Litter was dried at 70oC and then weighed. Soil and stones were separated from organic matter, and the organic material weighed. Twigs
69 and leafy material were then passed through a garden mulcher (Viking® GE220) to break up larger material and mix samples before sub-sampling and grinding. All chemical analysis of ground litter was carried out by CSBP, Bibra Lake, Western Australia. Nitrogen was analysed using a Leco FP-428 combustion analyser (Sweeney and Rexroad 1987). The elements; P (total), K, S, Na, Ca, Mg, Cu, Zn, Mn, Fe, Mo and
B were analysed by ICP-AES (McQuaker et al. 1979), and, NO3 and Cl were analysed colormetrically in a Lachat Flow Injection Analyser (Zall et al. 1956)., Total organic carbon analysis was carried out in a Leco CHN-1000 elemental analyser (Black 1965).
Climate information Climate information for the Bureau of Meteorology weather station at Karnet 16.25 km south of the Jarrahdale study site was provided by the Brian Kowald, Climate & Consultative Services, Bureau of Meteorology, PO Box 1370, West Perth, 6872 (Fig. 3.2).
Data analysis Unless otherwise indicated, percentage data were arcsine square root transformed before analysis. Percentage cover for vegetation and litter obtained from belt transect data were compared with data collected in the same year (2002) on the number of orchid species OMF detected and percentage of recovered seed that had germinated successfully for each test species, using regression analysis to construct a matrix of correlation coefficients. The number of adult orchid plants present in each transect (for species included in the baits) was included in the analysis. Regression analysis of the percentage of germinating seeds reaching stage 2 (and higher) and the percentage reaching stage 4 (and higher) was carried out to determine whether the data supported the use of stage 4 as an indicator of successful germination. Litter load and chemical composition of the loose and composted fractions, was compared with data on: detection of OMF of C. flava and T. crinita; frequency of detection of OMF; and OMF diversity (the number of orchid species OMF detected); at the vegetation type scale (transect) in the second survey year (2004) by constructing a matrix of correlation coefficients. Data used in comparison of position on rip lines were log10 transformed prior to analysis using ANOVA. Data were analysed by GenStat Version 7 (Lawes Agricultural Trust, Rothamsted Experimental Station).
70 50 300 A Annual Means 250 40
200 30 150 20 100
10 50
0 0 B 2002 250 40 C
° 200 30 150 20 100 Rainfall mm Temperature
10 50
0 0 C 2004 250 40
200 30 150 20 100
10 50
0 0 J F M A M J J A S O N D
Month
o FIGURE 3.2 Monthly rainfall (mm) and temperature maximum and minimum ( C) for Karnet: A. annual means, B. 2002 and C. 2004. Arrows indicate times of burial and recovery of baits. Karnet is 16.25 km south of the Jarrahdale minesite and is the Bureau of Meteorology weather station closest to the study site.
71 RESULTS
Detection of Orchid Mycorrhizal Fungi In the first year of baiting (2002) the frequency of a fungal bait detecting OMF of one or more orchid species was generally lower in the rehabilitation areas (15-25% of baits) than in unmined forest (15.8-42.1% of baits) (Table 3). In the second survey year (2004) OMF baits were established approximately 6 weeks later than in 2002, and a lower overall frequency of OMF detection observed, namely, 5-20% and 1-25% of baits in rehabilitation and forest sites, respectively. The low frequency of detection of OMF of most of the study species precluded statistical comparisons between orchids (Table 3.3) and detection of OMF of a particular orchid was rarely repeated at the same position for both sampling years (Figure 3.3). The percentage of seeds in seed baits reaching each stage 2 (and higher) was not correlated (R2 = 0.031) with the percentage reaching stage 4 (and higher) in 2002.
TABLE 3.3 Frequency of detection the OMF of six orchid species in three different age rehabilitation sites compared with adjacent unmined forest for two survey years. The term % baits detecting OMF refers to the percentage of baits detecting one or more orchid mycorrhizal fungus. Each transect was baited 20 times, a single bait was missing from forest sites adjacent to both 1976 and 1992 rehabilitation areas in 2002.
Survey Year 2002 2004 Orchid species Year rehab. Rehabilitation Adjacent Rehabilitation Adjacent Established area Forest area Forest C. flava 2001 0 4 0 5 D. bracteata 0 1 0 1 M. media 0 1 0 0 P. recurva 3 0 1 0 P. nigricans 0 1 0 1 T. crinita 0 1 0 0 % Baits detecting OMF 15 30 5 25 C. flava 1992 2 2 1 1 D. bracteata 0 0 1 0 M. media 0 1 1 0 P. recurva 0 2 0 0 P. nigricans 1 2 1 0 T. crinita 0 3 0 1 % Baits detecting OMF 15 42.1 15 10 C. flava 1976 4 1 2 1 D. bracteata 0 0 0 0 M. media 0 0 1 0 P. recurva 0 0 0 2 P. nigricans 0 0 0 0 T. crinita 1 2 0 0 % Baits detecting OMF 25 15.8 20 15
72 Site Sampling Year 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2001 Rehab 2002 2004
Adjacent 2002 Forest 2004
1992 Rehab 2002 2004
Adjacent 2002 Forest 2004
1976 Rehab 2002 2004
Adjacent 2002 Forest 2004
Number of species germinating: 1 = 2 = 3 =
FIGURE 3.3 Diagrammatic representation of transects showing positions where OMF were detected in 2002 and 2004, and number of species for which OMF were detected. Positive baits rarely occurred in the same position in both baiting years and few baits detected more than one OMF. Twenty baits were placed at 2.5 intervals over 50 m transects starting 1.25 m from one end.
In 2002, the OMF of all study species were detected at least once in one forest site and, for all study species except the two disturbance opportunists, D. bracteata and M. media, at least once in one rehabilitation site (Table 3.3). OMF of the following orchid species were detected in the forest adjacent to the 1 year old rehabilitation area: D. bracteata; M. media; C. flava; T. crinita; and P. nigricans. OMF of five species: M. media; C. flava; T. crinita; P. recurva; and P. nigricans, were detected in the forest adjacent to the 10 year old rehabilitation area, and OMF of two species: C. flava; and T. crinita, were detected in the forest site adjacent to the 26 year old rehabilitation area. OMF of only one species, P. recurva, was detected in the 1 year old rehabilitation area, two species, C. flava and P. nigricans, in the 10 year old rehabilitation area, and two species, C. flava and T. crinita, in the 26 year old rehabilitation area (Table 3.3).
Two years later (2004) OMF for all study species except M. media were detected at least once in one forest site and those for all study species except T. crinita at least once in one rehabilitation site (Table 3.3). OMF of C. flava and P. nigricans were detected in the forest site adjacent to the 2001 rehabilitation area, two species C. flava and T. crinita, in the forest site adjacent to the 1992 rehabilitation area, and two species C.
73 flava and P. recurva, in forest adjacent to the 1976 rehabilitation area. OMF of only one species, P. recurva, was detected in the 2001 rehabilitation area, four species, D. bracteata, M. media, C. flava and P. nigricans, in the 1992 rehabilitation area, and two species, M. media and C. flava, in the 1976 rehabilitation area.
Vegetation and litter There were significant differences in vegetation structure (P ≤ 0.05) across the three different age rehabilitation areas for most vegetation and litter characteristics (Table 3.4). Significant differences (P ≤ 0.05) were also found between the three forest sites in relation to the following characteristics: trees > 30 m, trees 10-30 m, trees < 10 m, shrubs >2 m, shrubs 1-2 m, shrubs < 1 m, herbs, sedges and grasses, leafy litter cover and minimum litter depth (Table 3.4). Temporal changes in vegetation and litter structure for the three rehabilitation sites are presented in Fig. 3.3 A & B and clearly display the structural changes that occur with increasing site age. (A more detailed examination of the recovery of post-mining vegetation is contained in Chapter 5) The most obvious vegetation changes are the reduction in cover of the category “herbs, sedges and grasses” (in 1 year old rehabilitation areas this is predominantly from ground cover by short-lived Kennedia spp.) and increasing height and cover from tree species. Soil surface cover from leafy litter (leaves and fine twigs) increased with rehabilitation area age as vegetation cover increase, while woody litter (branches > 2.5 cm diameter) reached a maximum in 10 year old rehabilitation areas after most short-lived leguminous species had died.
The orchid mycorrhizal fungi detected most frequently in 2002 were those of C. flava and T. crinita. Correlation between transect vegetation structure and soil surface cover characteristics, and numbers of orchid plants present (for species used in OMF detection baits) with detection of C. flava and T. crinita OMF, frequency of a positive detection bait and the number of species OMF detected are shown in Table 3.5. Detection of C. flava OMF was positively correlated with leafy litter cover, maximum litter depth and soil moisture content. There was no correlation between the presence of adult plants of either C. flava or T. crinita with detection of their respective OMF. Significant positive correlations were found between both the frequency of a positive bait and OMF diversity, and minimum litter depth. Frequency of a positive bait and OMF diversity were both correlated with the presence of adult plants of T. crinita (P ≤ 0.01).
74 TABLE 3.4 Comparison of vegetation structure and soil surface cover characteristics for the three rehabilitation and three adjacent unmined forest areas used as baiting sites in this study. Values are mean percentage cover for 10 contiguous (5 m)2 quadrats along each 5 m x 50 m belt transect. For each row cover values followed by same letter are not significantly different (P > 0.05). Data were arcsine square root transformed prior to analysis using ANOVA. Note: total litter exceeded 100% in some cases because woody litter (branches > 2.5 cm diameter and logs) can overlay leafy litter (leaves and fine twigs).
Year rehabilitation area established Adjacent unmined forest
Cover Characteristic 2001 1992 1976 2001* 1992 1976 Vegetation Strata Trees >30 m 0.00 a 0.00 a 0.00 a 23.00 b 0.00 a 0.00 a Trees 10-30 m 0.00 a 17.50 bd 47.50 c 18.00 bd 5.70 ad 26.00 b Trees <10 m 2.75 ac 26.10 b 4.35 ac 1.75 c 3.80 ac 11.00 a Shrubs >2 m 0.00 a 3.15 b 0.00 a 0.00 a 0.50 ab 3.05 b Shrubs 1-2 m 5.60 ab 7.60 a 3.00 b 7.50 a 7.00 a 2.30 b Shrubs <1 m 9.60 a 3.70 b 0.90 c 25.90 d 20.30 d 13.50 a Herbs, sedges & grasses 44.70 a 1.35 b 1.55 b 11.70 c 20.00 d 13.70 cd Litter Characteristic Leafy litter 0.50 a 56.00 b 96.80 cd 98.00 d 90.10 c 90.20 c Minimum litter depth (cm) 0.00 a 0.30 b 0.90 bc 1.80 d 0.50 bc 0.50 bc Maximum litter depth (cm) 0.30 a 5.70 bc 6.60 b 4.20 cd 4.00 d 4.00 d Woody litter (>2.5 cm diameter) 0.95 a 7.50 b 5.90 b 5.55 b 11.00 b 3.00 ab Bare ground 58.50 a 44.00 b 3.20 c 1.90 c 9.90 c 9.80 c * Year refers to the year adjacent rehabilitation area was established 100 A Vegetation Structure Trees 10 - 30m Trees < 10m Shrubs > 2m 80 Shrubs 1 - 2m Shrubs < 1m Herbs/sedges/grasses
60
40
20
0
% Cover B Soil Surface Cover Leafy Litter Woody Litter 120 Bare ground
100
80
60
40
20
0 1 10 26
Years since rehabilitation area established
FIGURE 3.4 Temporal changes in cover characteristics of baited rehabilitation areas for (a) vegetation structural classes and (b) litter cover classes. All values are percentage cover. Leafy litter consists of leaves and fine twigs and woody litter of branches >2.5 cm diameter and logs. N.B. Total litter can be >100% because woody litter (branches) often overlies leafy litter forming ‘standing’ litter.
76 TABLE 3.5 Correlation of transect vegetation structure and soil surface cover characteristics, and numbers of orchid plants present (for species used in OMF detection baits) with detection of C. flava and T. crinita OMF, frequency of a positive detection bait and the number of species OMF detected. Values are Pearson correlation coefficients (r). Data from all unmined forest and rehabilitation area transects were combined for the analysis. Strongest correlations (P ≤ 0.05) are listed in bold. (✝ = P ≤ 0.10; * = P ≤ 0.05; ** P ≤ 0.01). Number of belt transects n = 6, d.f. = 4, r = 0.729, P ≤ 0.10; r = 0.811, P ≤ 0.05; r = 0.917, P ≤ 0.01. Data were arcsine square root transformed prior to analysis.
Cover Characteristic C. flava T. crinita Frequency of Number of OMF OMF positive baits species OMF detected Vegetation Cover Trees >30m 0.396 0.210 0.295 0.542 Trees 10-30m 0.803 ✝ 0.280 - 0.003 0.055 Trees <10m 0.005 - 0.401 - 0.509 - 0.352 Shrubs >2m 0.053 - 0.273 - 0.159 - 0.030 Shrubs 1-2m 0.021 - 0.233 0.383 0.521 Shrubs <1m - 0.050 0.508 0.515 0.658 Herbs, sedges and grasses - 0.791 ✝ - 0.016 0.024 - 0.081 Soil Surface Cover Leafy litter cover 0.892 * 0.741 ✝ 0.542 0.634 Min. depth 0.473 0.799 ✝ 0.930 ** 0.854 * Max. depth 0.881 * 0.350 0.201 0.308 Woody litter (>2.5cm diam.) 0.724 0.459 0.672 0.684 Bare ground - 0.807 ✝ - 0.783 ✝ - 0.601 - 0.634 Soil moisture content (% H2O) 0.818 * 0.725 ✝ 0.683 0.689
Number of plants present Disa bracteata 0.056 - 0.577 - 0.391 - 0.230 Microtis media 0.482 - 0.096 - 0.159 - 0.304 Caladenia flava 0.648 0.178 0.135 0.022 Thelymitra crinita 0.349 0.616 0.962 ** 0.937 ** Pterostylis recurva 0.688 0.119 0.372 0.458 Pyrorchis nigricans 0.458 0.079 0.413 0.613 Total all orchid species 0.806 ✝ 0.370 0.487 0.374
The correlation analysis for detection of C. flava and T. crinita OMF was repeated using cover data for vegetation and litter characteristics obtained at the microhabitat scale (1 m2 bait quadrats) with forest sites and rehabilitation area data analysed separately (Table 3.6). In rehabilitation areas, significant positive correlations were found between the frequency of detection of C. flava mycorrhizal fungi and tree cover at 10-30 m, leafy litter cover, minimum litter depth, maximum litter depth, soil moisture content and OMF diversity. Detection of T. crinita mycorrhizal fungi was positively correlated with OMF diversity and soil moisture content. In unmined forest, there was a significant negative correlation between the frequency of detection of C. flava OMF and maximum litter depth, and a positive correlation with the total number of OMF detected. Detection
77 of T. crinita mycorrhizal fungi was positively correlated with shrubs < 1 m and the total number of species OMF detected.
Position on rip line No significant differences were found for the soil surface cover categories: leafy litter cover; maximum litter depth; woody litter cover; bare ground; and moisture content, between top and bottom of rip lines. Rip lines are a series of parallel mounds and furrows created by deep ripping of soil during rehabilitation and are approximately 1.5 metres apart with an approximate amplitude of 25 cm. However, litter minimum depth was significantly higher in the bottom of rip lines (P < 0.05). There were also a significantly higher number of species OMF detected by baits placed in the bottom compared with the top of rip lines (P < 0.05) (Table 3.7).
Chemical composition of litter Significant differences (P < 0.05) in litter chemical composition with age of rehabilitation area occurred for K, S, Cu and Fe in loose litter, for N, P, K, S, Na, Ca, Zn, Mn, B, and C: N in composted litter and for total litter weight (t ha-1) for both loose and composted litter. Detection of C. flava OMF was positively correlated with sodium content of composted fraction and with litter weight of both litter fractions. No highly significant correlations (P < 0.05) were found for T. crinita OMF. No one chemical characteristic was correlated with the frequency of detection of OMF or OMF diversity. The number of positive baits was negatively correlated with sulphur and zinc in the loose litter fraction, and negatively correlated with zinc and positively correlated with sodium in the composted litter fraction. OMF diversity was negatively correlated with phosphorus and sodium content, and positively correlated with manganese content of the composted litter fraction.
78 TABLE 3.6 Correlations (r) between bait quadrat vegetation structure and soil surface cover characteristics with frequency of detection of OMF of C. flava and T. crinita, and soil moisture. Forest and rehabilitation data were analysed separately. Strongest correlations (P ≤ 0.05) are listed in bold (✝ = P ≤ 0.10, * = P ≤ 0.05, ** = P ≤ 0.01). For rehabilitation areas, n = 60, d.f. = 58, r = 0.257, P ≤ 0.05; r = 0.329, P ≤ 0.01; for forest, n = 58, d.f. = 56, r = 0.260, P ≤ 0.05; r = 0.338, P ≤ 0.01.
Cover Characteristic Caladenia flava OMF Thelymitra crinita OMF Soil Moisture (%H2O) Rehabilitation Forest Rehabilitation Forest Rehabilitation Forest Vegetation Cover Trees >30 m n.a. A 0.107 n.a. - 0.139 n.a. 0.587 ** Trees 10-30 m 0.345 ** - 0.172 0.198 0.158 0.572 ** - 0.107 Trees <10 m - 0.009 - 0.027 - 0.063 - 0.013 - 0.130 - 0.141 Shrubs >2 m - 0.039 - 0.043 - 0.017 - 0.041 - 0.012 0.043 Shrubs 1-2 m - 0.052 - 0.132 - 0.065 0.001 - 0.195 - 0.178 Shrubs <1 m - 0.008 - 0.170 - 0.092 0.284 * - 0.238 ✝ 0.239 ✝ Herbs, sedges and grasses - 0.147 - 0.252 ✝ - 0.069 0.039 - 0.295 * - 0.242 ✝ Soil Surface Cover Leafy litter cover 0.266 * - 0.163 0.115 0.061 0.596 ** 0.071 Min. depth 0.387 ** - 0.209 0.246 ✝ 0.115 0.474 ** - 0.076 Max. depth 0.323 * - 0.273 * 0.160 0.125 0.646 ** - 0.042 Woody litter (>2.5cm diam.) 0.148 - 0.019 0.209 - 0.031 0.522 ** 0.308 * Bare ground - 0.260 * 0.065 - 0.165 - 0.075 - 0.657 ** - 0.356 ** Soil moisture content (% H2O) 0.241 * 0.066 0.333 ** 0.121 1.000 ** 1.000 **
Number of species OMF detected 0.626 ** 0.466 ** 0.275 * 0.464 ** 0.221 ✝ 0.248 ✝ A n.a. = not applicable TABLE 3.7 Orchid mycorrhiza detection in relation to position on rip-line and soil surface cover characteristics. Values are means for 1 m2 quadrats surrounding OMF detection baits. Data from all age rehabilitation areas was combined for analysis. Rip lines are approximately 1.5 metres apart and with an amplitude of approximately 25 cm. Top refers to top 10 cm of mounds and bottom to lower 10 cm of troughs. For both positions, n = 16. (n.s. = P > 0.05)
Position on Rip-line Soil Surface Cover Characteristic Bottom Top P Mean SE Mean SE Leafy litter (% cover) 57.09 ± 9.73 40.25 ± 10.54 n.s Min. depth (cm) 1.50 ± 0.38 0.44 ± 0.16 < 0.05 Max. depth (cm) 5.38 ± 1.03 3.66 ± 0.89 n.s. Woody litter (% cover) 2.94 ± 1.04 1.75 ± 0.51 n.s. Bare ground (%) 31.6 ± 8.70 46.44 ± 9.46 n.s Soil moisture content (% H2O) 17.9 ± 3.71 10.29 ± 1.67 n.s Number of species OMF detected 0.38 ± 0.13 0.06 ± 0.06 < 0.05
TABLE 3.8 Correlations (r) between transect litter load and chemical composition for loose and composted litter fractions with detection of OMF of C. flava and T. crinita, frequency of a positive detection bait and the number of species OMF detected in 2004. Data from all transects has been combined. Only elements with correlations significant at P ≤ 0.10 for either loose litter or composted litter are shown. Strongest correlations (P ≤ 0.05) are listed in bold (n = 5, d.f. = 3, ✝ = P ≤ 0.10, * = P ≤ 0.05, ** = P ≤ 0.01).
Element C. flava T. crinita Frequency of Number of OMF OMF OMF positive baits detected Loose Litter: Phosphorus1 - 0.791 - 0.250 - 0.784 - 0.612 Potassium 0.832 ✝ - 0.081 0.681 - 0.180 Sulphur - 0.750 0.211 - 0.884 * - 0.481 Sodium 0.383 0.533 0.165 - 0.515 Calcium - 0.120 - 0.590 - 0.101 - 0.301 Magnesium - 0.159 - 0.755 0.212 0.498 Copper - 0.818 ✝ - 0.276 - 0.742 - 0.160 Zinc - 0.818 ✝ 0.082 - 0.929 * - 0.507 Manganese -0.014 - 0.585 0.221 0.740 Iron -0.834 ✝ - 0.283 - 0.753 - 0.182 C:N ratio 0.541 - 0.121 0.722 0.752 Litter load (t/ha) 0.944 * 0.254 0.793 0.147
Composted Litter: Phosphorus - 0.476 0.027 - 0.631 - 0.917 * Potassium 0.626 0.203 0.374 - 0.495 Sulphur - 0.651 0.193 - 0.813 - 0.870 ✝ Sodium 0.985 ** 0.106 0.906 * 0.437 Calcium - 0.438 - 0.243 - 0.507 - 0.892 * Magnesium 0.423 - 0.868 ✝ 0.598 0.145 Copper - 0.348 0.283 - 0.307 0.667 Zinc - 0.783 0.214 - 0.920 * - 0.671 Manganese 0.556 - 0.436 0.744 0.884 * Iron 0.003 0.318 0.055 0.860 C:N ratio 0.688 - 0.200 0.839 ✝ 0.860 ✝ Litter load (t/ha) 0.922 * - 0.019 0.793 - 0.059
Total Litter (t/ha) 0.940 * 0.046 0.795 - 0.010 1 P, K, S, Na, Ca, Mg and Cl were measured in percentage and Cu, Mn, Fe as ppm.
80 DISCUSSION
Detection of Orchid Mycorrhizal Fungi There was no correlation between the percentage of germinating seeds reaching stage 2 (and higher) and the percentage reaching stage 4 (and higher) suggesting cracking of testa is a poor indicator of successful germination. The initiation of shoot or leaf growth has been recognised as an indication of successful germination and formation of a mycorrhiza for many years; however, earlier development stages are still commonly used by many researchers as indicators of germination (Clements 1981; Warcup 1983; Ramsey et al. 1986; Zettler and Hofer 1998; Otero et al. 2004; Hollick et al. 2005). The lack of correlation found here supports the use of stage 4 (initiation of leaf primordia) or stage 5 as more reliable indicators of successful germination and establishment of a mycorrhizal association.
Baiting with orchid seed packets showed orchid mycorrhizal fungi (OMF) to be widespread, but infrequently detected, in soils of both rehabilitation and forest sites for both years of baiting. The frequency of a positive bait (OMF of all orchid species combined) in the rehabilitation sites and forest sites was similar to a previous study undertaken on a single orchid species, but much lower than a study of seven species on the sandy soils of the Swan coastal plain in Western Australia (Batty et al. 2001a; Brundrett et al. 2003). Batty et al. (2001a) detected the OMF of Caladenia arenicola Hopper & A.P.Br. in 14.2% of OMF detection baits placed along transects through populations of that species in bushland at Kings Park and Botanic Gardens, Perth, Western Australia. Brundrett et al. (2003) detected OMF at higher frequencies using multi-species OMF detection baits in a study comparing sites with ‘few’ or ‘many’ weeds in bushland at Bold Park, Perth, Western Australia. Frequency of detection of OMF in the ‘few weeds’ bushland was between 33.3 and 100% of baits, and in ‘many weeds’ bushland between 53.3 and 93.3% of baits. However, Batty et al. (2001a) defined successful germination as development of first trichomes, an earlier developmental stage than is used here to define successful germination (stage 4) (Table 1), and therefore could have over-estimated successful germination. Brundrett et al. (2003) do not indicate the growth stage used to indicate successful germination so the results from the two studies may also not be directly comparable.
81 This low frequency of detection of OMF in jarrah forest for most of the species examined may be a characteristic of the fungal associates of these orchid taxa in lateritic soils, of limited sensitivity of the technique, or merely be reflective of a natural variability of population size and species richness. In a previous study, repeated annual baiting of a bushland site at Kings Park and Botanic Gardens (Hollick 2004), demonstrated that the OMF of C. arenicola did not necessarily re-occur in the same locations, nor with the same frequency, along a transect in consecutive years. Similar observations were made during this study (Fig. 3.3).
In our study, 2004 baits were established approximately 6 weeks later in the growing season than in 2002, and a lower frequency of OMF detection observed. It has not been determined if the later establishment time and resultant exposure to different temperature and rainfall patterns was responsible for this reduction in frequency of OMF detection. Rainfall recorded at Karnet, 16.25 km south of the Jarrahdale mine site reveals a very dry summer and an unusual rainfall pattern for 2004 compared with mean rainfall data (Fig. 3.2). There is no evidence of water availability problems during the baiting period and water holding capacity of lateritic soils is known to be high (Hingston et al. 1988). However, temperature has been shown to affect the symbiotic germination of some Western Australian terrestrial orchids in vitro (M.T. Collins, unpubl. data) and low temperatures could have inhibited germination especially in mid- wet season (Fig. 3.2).
Vegetation and litter associations of Orchid Mycorrhizal Fungi Vegetation data, collected in 2002, was used to examine correlations between vegetation structural and soil surface cover characteristics, and frequency of a positive bait, OMF diversity and the presence of the two most frequently detected OMF in 2002 (those of C. flava and T. crinita) at both vegetation type (transect) and microhabitat (bait quadrat) scale. At the vegetation type scale, the correlation of the frequency of a positive bait with minimum litter depth indicates a possible link between the fungi and their nutrient source. A previous Western Australian study using ex-situ baiting techniques to detect OMF has shown that these are most common in surface litter and the coarse organic fraction (>2.4 mm) of sandy soils in south west Western Australia (Brundrett et al. 2003). The importance of woody debris as a source of fungal inoculum in the in situ germination of orchid seed has also been demonstrated with the European terrestrial species Tipularia discolor (Pursh) Nuttall (Rasmussen and Whigham 1998). 82 Although an orchid and its OMF are expected to occur in close proximity, this was not necessarily the case in our study, where there was no correlation between the presence of mature plants of an individual orchid taxon and the detection of its OMF. Previous studies on Australian terrestrial orchids (Perkins et al. 1995; Batty et al. 2001a) have found that orchid seed germinates better in close proximity to orchid plants of the same species, linking the presence of plants with OMF. The baiting frequency used in this study was probably not adequate to detect small patches of OMF close to individual plants, especially if inoculum potential was low. However, the presence of mature plants of T. crinita was correlated with the frequency of a positive bait and the number of orchid species OMF detected. In this study, this orchid species was found only in unmined forest transects (Chapter 5), so the presence of T. crinita seems to be a good indicator of undisturbed vegetation.
The detection of OMF for C. flava was correlated with soil surface cover characteristics associated with high tree and litter cover and high soil moisture at both the vegetation type scale and at the microhabitat scale in rehabilitation areas (Table 3.5 and 3.6). However, at the forest microhabitat scale, high litter depth was negatively correlated with detection of C. flava OMF (Table 3.6). Vegetation characteristics that provide high litter cover in forest may also be associated with other characteristics, such as excessive shade, that are not conducive to the survival of these fungi and/or orchids whereas in rehabilitation areas much of the litter comes from the death of leguminous shrubs. The detection of T. crinita OMF was only correlated with soil moisture in rehabilitation areas and low shrub cover in forest areas at the microhabitat scale (Table 3.6). As some fungi capable of forming mycorrhizas with orchids are thought to form ectomycorrhizas with tree species these vegetation associations warrant further investigation (Selosse et al. 2002a; Selosse et al. 2002b).
Rasmussen (2002) has suggested that sympatric orchid species may avoid competition for nutrients by employing different fungal species in mycorrhizal associations. Both Caladenia flava and Thelymitra crinita are very common orchids in the jarrah forest, often growing together in the same habitat and may employ a similar non-competitive nutrient acquisition strategy. C. flava has no roots, is a clonal species and can produce extensive colonies by means of underground stems that extend horizontally from the dropper (vertical underground stem) and form new tubers at their ends (Hoffman and Brown 1998). T. crinita, however, has an extensive root system and is known to 83 reproduce primarily from seed. These different growth habits and reproductive strategies may place different nutrient demands on the orchid-fungus association. The mycorrhizal endophytes of the orchid genera Caladenia and Thelymitra are known to belong to different fungal genera within the Rhizoctonia alliance (Warcup 1971; 1981). The observed difference in vegetation and litter characteristics associated with the OMF of each of these orchids may therefore reflect the use of fungi in mycorrhizal associations that are non-competitive with respect to nutrient sources.
Litter composition and distribution Release of nutrient elements from litter is dependent on their solubility, thus, N, P and Ca accumulate in the composted litter while the more soluble nutrients, S, Mg, K and Na, decrease rapidly over the first 6 months (O'Connell and Menage 1983). Differences in chemical composition of litter with age reflect these different solubilities, but observed high Fe concentrations may also reflect contamination of litter with iron rich soil particles. The presence and nature of the understorey vegetation also affects nutrient loss from litter with dense legume understorey known to be associated with reduced loss and/or greater accumulation of nutrients in decomposing litter (O'Connell 1986). The current study has found a positive correlation between detection of C. flava OMF and the amount of both loose and composted litter and the sodium content of the composted litter layer. Combined with our findings of a correlation with litter cover and depth at both vegetation type and microhabitat scales (Tables 3.4 and 3.5), this appears to indicate a strong relationship between the detection of OMF of C. flava and the quantity of litter.
Litter and other soil organic matter is used by saprophytic root-infecting fungi as a nutrient source which may explain this relationship (Garrett 1970). Sodium in litter is predominantly derived from sodium chloride in rainwater throughfall, therefore this correlation is likely to be with localised drainage areas such as the bottom of rip lines (Hingston et al. 1988). Soil potassium and organic carbon content have been shown to be negatively correlated, and the amount of litter positively correlated, with OMF detection by germination of C. arenicola in sandy soils of Kings Park and Botanic Gardens, Western Australia (Batty et al. 2001a). Batty et al. (2001a) postulated that both K and organic C would affect soil moisture content and that the correlation with OMF may be indirectly related to the water-holding capacity of the soil. The negative correlation between OMF of C. flava and zinc is interesting. Zinc sufficiency has been 84 shown to decrease disease severity in root rots caused by Rhizoctonia solani Kühn, and elevated zinc concentration to suppress its growth rate in vitro (Babich and Stotzky 1978; Thongbai et al. 1993a; Thongbai et al. 1993b; Streeter et al. 2001; Siddiqui et al. 2002). Orchid mycorrhizal fungi belong predominantly to the form-genus Rhizoctonia and if elevated zinc is similarly associated with reduced vigour in these fungi, the ability to germinate orchid seed and establish mycorrhizal associations may possibly be affected.
The observation that D. bracteata rapidly invades the highly disturbed rehabilitation areas coupled with failure to detect its OMF in rehabilitation areas may indicate inadequate sampling frequency or poor sensitivity of the baiting technique in situ. However, it also suggests that disturbance of the jarrah forest will allow invasion by this species. The orchid P. nigricans was absent from all rehabilitation areas examined in this study but its OMF was detected in the 1992 rehabilitation areas in both sampling years. This orchid is a common jarrah forest species (Table 2.3) but is dependent on fire for flowering and hence seed production. Management burns did not occur during the time the mine site was actively managed by Alcoa World Alumina Australia Limited and wild fires were extinguished quickly due to the risk to staff and infrastructure (J. Koch pers. comm. 2002), therefore little seed was produced. This orchid’s failure to invade rehabilitation areas is therefore probably not due to scarcity of its OMF.
Rehabilitation areas within the bauxite mined forest are undergoing a successional process that includes re-establishment of vegetation cover and species composition, soil microbial population size and diversity, and soil development (Dansereau 1974; Krebs 1985; Koch and Ward 1994; Dix and Webster 1995; Ward 2000; Grant and Loneragan 2001) . The correlation of OMF detection rates with litter measurements and other environmental factors that increase with time indicates that OMF are likely to follow the same successional pathways. A similar pattern was seen in ectomycorrhizal fungi in rehabilitated bauxite mines (Gardner and Malajczuk 1988). Provided there is continued vegetation development jarrah forest OMF will re-establish in the rehabilitated sites.
85 CONCLUSIONS
This study was the first to examine the recovery of orchid mycorrhizal fungi in the post- bauxite mining landscape of southwest western Australia. The major findings are as follows: (i) the frequency of detection of OMF for each orchid species was generally low in both in rehabilitation areas (0-20%) and in unmined forest sites (0- 25%), (ii) the frequency of detection of OMF varied spatially and temporally for all orchid species studied, (iii) litter quality and quantity change with age in rehabilitation areas, (iv) detection of OMF was generally associated with greater litter depth and cover in the rehabilitation areas, (v) detection of the OMF of two common jarrah forest orchids, C. flava and T. crinita were correlated with different vegetation structural and soil surface cover characteristics, (vi) the diversity of OMF detected was lowest in the youngest rehabilitation area surveyed for both sampling years, but in older rehabilitation areas was similar to unmined forest, (vii) there is not enough evidence to determine if a succession of OMF occurs.
Therefore, the absence of the appropriate orchid mycorrhizal fungi is unlikely to be the main cause of failure of some orchids to invade rehabilitation areas. However, the low frequency of detection of OMF suggests that they occur in isolated patches of soil therefore the majority of dispersed orchid seeds is likely to perish, especially in recently disturbed habitats.
86 CHAPTER 4
DIVERSITY OF ORCHID MYCORRHIZAL ENDOPHYTES
INTRODUCTION
‘Specificity’ is a characteristic of the orchid-fungus mycorrhizal association and is a measure of the number of different fungi that form mycorrhiza with a particular orchid taxon. Taylor and Bruns (1999) considered that specificity existed in the relationship between an orchid taxon and fungal endophyte if there was some limitation on the phylogenetic breadth of that association. Previous studies of mycorrhizal associations of Australian terrestrial orchids have found specificity with particular groups of basidiomycete fungi at various taxonomic levels (Table 1.4, Chapter 1). Terrestrial orchids are dependant on mycorrhizal fungi for germination of their seeds, nutrition of protocorms and the early growth of seedlings (Rasmussen 1995; Peterson et al. 1998). An understanding of the specificity of the orchid-fungus mycorrhizal associations is therefore essential to evaluate the significance of these fungi in the establishment of native terrestrial orchids in the post-mining landscape. Mycorrhizal specificity may account for the rapid invasion by disturbance opportunist orchids, for differences in the timing of invasion by individual orchid taxa, and/or for rarity or abundance of particular orchid taxa in unmined forest (see Chapter 2).
The mycorrhizal endophytes associated with orchids are generally members of the form-genus Rhizoctonia. This is a taxonomically diverse assemblage of fungi belonging to several different orders of basidiomycetes (Sneh et al. 1991; Rasmussen 1995; Roberts 1999). Mycorrhizal Rhizoctonia are anamorphic, that is the sexual phase is not present or at least is very difficult to induce; therefore, the features traditionally used to identify fungi are usually absent. Early studies of the mycorrhizal endophytes of Australian terrestrial orchids identified these organisms using classical morphological techniques (Warcup and Talbot 1967; 1971). However, anamorph morphology can be variable and unreliable for identification below the generic level (Currah et al. 1997; Rasmussen 2002; Zettler et al. 2003).
In recent studies, molecular techniques using PCR amplification, RFLP, and sequencing of the ITS region, LSU rDNA and/or SSU rDNA, have been adopted for the 87 identification of orchid endophytes (Pope and Carter 2001; Bougoure et al. 2005; Bonnardeaux et al. 2007). Sequence variability differs at different genetic regions, so they can be used for resolving phylogenetic relationships at different taxonomic levels. For example: the nuclear small subunit rRNA gene is highly conserved and so is unlikely to be useful beyond the family level; the nuclear large subunit rRNA gene can discriminate at the genus level; while the ITS region is most variable and is useful at the species or strain level (Bruns et al. 1991; Mitchell and Zuccaro 2006). Sequencing of the rDNA ITS region, in particular, is widely used for the identification of orchid mycorrhiza fungi at the species or strain level (Bruns et al. 1991; Rasmussen 2002; Zettler et al. 2003; Bougoure et al. 2005). This technique has been used successfully in studies of the mycorrhizal fungi associated with Australian terrestrial orchids; to discriminate between fungi, to assign putative identities, and in the examination of phylogenetic relationships (Perkins and McGee 1995; Carling et al. 1999; Pope and Carter 2001; Hollick 2004; Bougoure et al. 2005; Bonnardeaux et al. 2007).
In this study, the ecological specificity of the four Jarrah forest orchid taxa; Caladenia flava subsp. flava R.Br, Cryptostylis ovata R.Br, Disa bracteata Sw, and Thelymitra crinita Lindl., was examined by isolation and identification of mycorrhizal endophytes associated with plants collected from the Jarrahdale mine site. ‘Ecological specificity’ is the specificity of the orchid-fungus association occurring under natural field conditions (Batty et al. 2001a). These four taxa were selected for study as they represent different combinations of the following characteristics: clonal and non-clonal growth habit; rare and common within jarrah forest; and present and absent from minesite rehabilitation areas (Table 4.1). These differences in growth habit and population densities may be associated with differences in ecological specificity that will provide insight into the recruitment of orchids in the post-mining landscape.
TABLE 4.1. Growth habits and frequency of occurrence of orchid species selected for isolation of mycorrhizal endophytes.
Orchid species Clonal Disturbance Forest Rehabilitation Opportunist Areas Caladenia flava yes no common common Disa bracteata* no yes rare common Cryptostylis ovata yes no rare** absent Thelymitra crinita no no common rare * Disa bracteata is an exotic species that has naturalised in Western Australia. ** C. ovata is at the northern limit of its habitat and found at only a few sites within the Jarrahdale area.
88 The aims of this study were to determine: a) the identity of mycorrhizal fungi associated with, and ecological specificity of orchid mycorrhizal associations for the orchid taxa; Caladenia flava subsp. flava, Cryptostylis ovata, Disa bracteata, and Thelymitra crinita; b) if fungi found in soil surrounding adult orchid plants that are able to germinate seed of that orchid taxa are the same fungi that form persistent mycorrhizal associations with those adult plants; c) if the disturbance opportunist D. bracteata was ‘promiscuous’ in its use of fungi as mycorrhizal associates; d) if the sympatric orchid taxa C. flava and T. crinita form mycorrhizas with similar or different fungi; e) if multiple mycorrhizal endophytes occur in the same plant; f) the phylogenetic relationships between mycorrhizal endophytes of the study taxa.
89 MATERIALS AND METHODS
Selection of study species and collection of plant material The orchid species: C. flava, D. bracteata, C. ovata and T. crinita, were selected for study on the basis of field observations in 2001 and 2002, and an examination of Alcoa’s long term vegetation monitoring data (Grant and Koch 2003). The growth habits and frequency of occurrence in rehabilitation areas and unmined forest of the study species are listed in Table 4.1.
Plants of orchid species used in this study were collected from rehabilitation and forest areas adjacent to belt transects at Alcoa’s Jarrahdale mine-site during the peak of the 2003 growing season (August – October). Additional C. ovata and T. crinita plants were collected in July or October 2004. Details of collection site and the number of replicate plants collected are given in Table 4.2. Entire plants with surrounding surface soil and litter (approx. dimensions 20 x 20 x 5 cm) were collected and transported to the laboratory in plastic bags. Soil was then carefully loosened and separated from the underground parts of the plants to minimise damage to tissues. Roots and underground stems were washed in tap water and adhering soil particles and organic material removed by gentle brushing with an artist’s bristle paint brush.
Isolation of Fungal Endophytes Fungal endophytes were isolated using a modified version of Clements and Ellyard’s (1979) technique (Rasmussen 1995; Batty et al. 2001b). Infected roots, droppers or collars were excised from plants and surface sterilised by washing with vigorous shaking in sterile deionised water (five times for five minutes). Fungal coils (pelotons) were excised from the infected tissue by scraping the cortex with a sterile scalpel blade in 0.5 – 1 ml sterile deionised water (SDI H2O) to produce a suspension of pelotons and cell debris. Pelotons were removed from the suspension using a finely drawn Pasteur pipette and washed by serial dilution in sterile deionised water (5 x). Droplets of the final suspension of pelotons were then placed on both Soil Solution Extract (SSE) (Mursidawati 2003) and Fungal Isolation Medium (FIM) (Clements and Ellyard 1979) agar plates and the water allowed to evaporate in a sterile air-flow in a laminar flow cabinet. (Note: Formulae for all isolation media are provided in Appendix 3.) Plates were then incubated in the dark at 20oC and examined daily for signs of hyphal growth. Pelotons exhibiting hyphal growth were subcultured on to SSE or FIM plates. Repeated
90 subculture from the active growing edge of cultures yielded axenic cultures derived from a single peloton. Hyphal tips were subcultured when possible. However, pelotons can contain more than one fungus and this may be a source of mixed cultures. Potential mycorrhizal isolates were then tested for the capacity to form mycorrhiza by incubation with orchid seed. (Note: fungi forming mycorrhiza with orchids rarely sporulate on agar, and most of the sporulating fungi isolated were common soil saprophytes, therefore only non-sporulating fungi were retained and tested.)
TABLE 4.2. The number of orchid plants collected from Alcoa’s Jarrahdale mine site for isolation of mycorrhizal fungi in 2003 and 2004. Collection sites are coded as follows; age of rehabilitation area in years, R or F (where R = rehabilitation site, F = forest site adjacent to rehabilitation site), – replicate number (1 to 4). Cov-1 and Cov-2 were two additional transects placed through populations of C. ovata. Rehabilitation areas 26 or 27 years old were combined in the age category 25+. Plants were numbered to enable fungal isolates from the same plant to be identified.
Collection year and Orchid species Collection site Plants collected Plant numbers 2003 Caladenia flava subsp.flava* F15-4 5 Cf1-5 F25+-2 5 Cf6-10 F1-1 5 Cf11-15 F10-3 5 Cf16-20
R25+-1 10 CfR1-10 R25+-2 10 CfR11-20
Cryptostylis ovata Cov-1 10 Cov1-10 Cov-2 10 Cov11-20
Disa bracteata R15-4 6 Db1-5, 21 R15-1 5 Db6-10 R10-1 5 Db11-15 R10-4 5 Db16-20
Thelymitra crinita F15-4 6 Tc1-5, 21 F10-1 5 Tc6-10 F1-1 5 Tc11-15 F5-2 5 Tc16-20 July 2004 Cryptostylis ovata Cov-1 5 JCov1-5 Cov-2 5 JCov6-10
Thelymitra crinita F15-4 5 JTc1-5 F10-1 5 JTc6-10
October 2004 Cryptostylis ovata Cov-1 5 OCov1-5 Cov-2 5 OCov6-10
Thelymitra crinita F15-4 5 OTc1-5 F10-1 5 OTc6-10 * Hereafter Caladenia flava subsp. flava is referred to as C. flava.
91 Ex situ baiting Ex situ baits were established in 10 cm square plates as described by Brundrett et al. (2003), using wet sieved coarse organic material collected from soil surrounding adult orchid plants. Soil samples (15 x 15 x 3 cm) were collected from the base of each orchid plant taken for endophyte isolation (Table 4.2). Seed from the following nine jarrah forest orchid species: D. bracteata; M. media subsp. media; T. crinita; C. flava subsp. flava; Pterostylis recurva; C. ovata; P. nigricans; Thelymitra macrophylla; and Pterostylis sp. crinkled leaf, were used to screen for the presence of orchid mycorrhizal fungi. For all species, except P. nigricans and C. ovata, local provenance orchid seed collected from the Jarrahdale area in 2002 was used. P. nigricans seed obtained from the Kings Park and Botanic Gardens seed collection was collected in 2001 from jarrah forest at Forestdale, 50 km north of Jarrahdale. C. ovata seed was obtained from a population 5 km west of the study site near Kwinana. Ex situ baits were examined weekly for germination until the first seeds reached stage 2, then at fortnightly intervals. After 20 weeks incubation baits were scored for successful germination of each orchid species’ seed. The presence of a mycorrhizal fungus was confirmed by protocorm development to ≥ stage 4 (initiation of shoot growth) (see Table 1.3).
Isolation of Fungi from Ex-situ Baits Protocorms that had reached development stage 4 or 5 were carefully removed from ex- situ baits and washed by shaking in a 0.2% aqueous solution of Tween 20, then rinsed in tap water. The protocorms were then surface sterilised by immersion in a 30% w/w solution of hydrogen peroxide for 30 seconds and rinsed five times in SDI H2O (Wright 2002). Protocorms were then placed on SSE agar plates and incubated at in the dark at 20oC. Plates were examined daily for signs of hyphal growth. Plates exhibiting growth were subcultured on to SSE plates. Repeated subculture from the active growing edge of cultures yielded axenic cultures derived from a single protocorm. All fungi isolated by this method were fast growing, sporulating fungi.
Clearing and Staining of Orchid Roots and Root tubers Roots and root-tubers of T. crinita and C. ovata plants were cleared and stained to determine if poor isolation rates were due to absence of intact pelotons. A photograph taken prior to clearing and staining shows the differences in root systems of the two taxa (Fig. 4.1). The underground parts of T. crinita and C. ovata plants collected from the
Jarrahdale study site were washed in deionised water (DI H2O), and a bristle artist’s
92 paint brush used to remove adhering soil and organic material. Root-tubers of C. ovata, approximately 5 mm thick, were cut in two longitudinally for clearing, and underground stems were cleared intact. Tubers were removed from the T. crinita plant and the intact root systems cleared. Orchid tissues were then cleared by incubating at 65oC in 10% KOH for 60 minutes. Cleared roots and root tubers were washed in 3% HCl (to neutralise the KOH), rinsed in DI H2O, and stained in a solution of Trypan Blue (0.05% Trypan blue in 1:1:1 lactic acid/glycerol/water) (Brundrett et al. 1996). The tissues were stained overnight then transferred to 1:1:1 lactic acid/glycerol/water and stored at ambient temperature before examination by light microscopy. Thin transverse and longtudinal sections of C. ovata root-tubers were prepared by hand using a razor blade, cleared for 30 min at 60oC and stained as described above.
Orchid Seed Orchid seed was collected from wild, naturally and hand pollinated plants in spring/summer of 2001, 2002, 2003 and 2004 just prior to dehiscence. The seed was cleaned by passing through a fine sieve, dried over silica gel for 24 h at ambient temperature and stored in Nunc© tubes at 4oC. Local provenance orchid seed collected from the Jarrahdale area, was used whenever possible. When local provenance seed was unavailable seed of known provenance and age from either the Kings Park and Botanic Gardens seed collection or the collected from nearest population was used. The seed collected for use in ex situ baiting and testing endophytes for mycorrhizal capacity is listed in Table 4.3. (A complete list of orchid seed collected during the project is provided in Table A1.5, Appendix 1)
Confirmation of Mycorrhizal Capacity Mycorrhizal capacity of axenic fungal cultures was examined by co-culture on Oatmeal agar (Clements and Ellyard 1979) (see formula for Oatmeal agar, Appendix 3) with local provenance seed of the orchid taxa from which the fungus was isolated. Confirmation of mycorrhizal capacity was obtained by development of ≥ stage 4 protocorms (initiation of shoot growth) (Collins 2005) within 20 weeks.
93 2.5 cm
A B
FIGURE 4.1 The underground parts of A. Cryptostylis ovata and B. Thelymitra crinita. Arrows indicate areas that are infected by mycorrhizal fungi. Pelotons of OMF are found in root tubers and the dropper (underground stem) of C. ovata and in the apogeotropic (upward growing) roots of T. crinita.
94 TABLE 4.3. Provenance of orchid seed used in ex situ seed baits for detecting mycorrhizal fungi and for testing orchid endophytes for mycorrhizal capacity. All orchid seed used in the baits was collected from the Jarrahdale study site in the previous two growing seasons (2001 and 2002) with the exception of P. nigricans seed, which was obtained from the seed collection of Kings Park and Botanic Gardens, Perth, Western Australia. Seed collected less than one year old was used in preference to older seed whenever possible. (Note: collection year and seed weights are in the same chronological order.)
Orchid species Collection Site Collection year/s Weight of seed (g) Caladenia flava Jarrahdale 2001, 2002, 2003, 0.015, 0.071, 0.201, 2004 0.085 Cryptostylis ovata Jarrahdale 2003 0.002 Kwinana* 2002 0.166 Disa bracteata Jarrahdale 2001, 2002, 2003 0.272, 1.041, 0.821 Microtis media subsp. Jarrahdale 2001, 2002, 2003, 0.106, 0.313, 1.533, media 2004 0.012 Pterostylis aff nana Jarrahdale 2001, 2002, 2003, 0.016, 0.044, 0.086, 2004 0.030 Pterostylis recurva Jarrahdale 2001, 2002, 2003, 0.227, 0.238, 0.104, 2004 0.074 Pyrochis nigricans Forestdale** 2001 0.107 Jarrahdale 2003 0.174 Thelymitra crinita Jarrahdale 2001, 2002, 2003, 0.258, 0.256, 0.163, 2004 0.244 Thelymitra macrophylla Jarrahdale 2002, 2003 0.439, 0.048 * The Kwinana site is approximately 5 km west of the Jarrahdale study site ** Forestdale is approximately 50 km north of the Jarrahdale study site
Growth of Mycorrhizal Isolates for DNA Extraction For each mycorrhizal isolate, a 50 ml nutrient broth culture was initiated using a small agar cube (2-4 mm3) cut from an actively growing agar culture. Cultures were grown in 250 ml polycarbonate jars containing 50ml of sterile nutrient broth (Difco). Cultures were incubated at ambient temperature with gentle shaking until hyphal growth had extended 1-2 cm from inoculum block (2-6 months). Mycelia were removed from the culture vessels aseptically and washed (1 x) in sterile deionised water (SDI H2O). Hyphae were then dissected from inoculum blocks and rinsed twice in sterile deionised water. Excess water was removed by blotting dry on sterile filter paper (Whatman No. 1) and each sample frozen at –80oC prior to lyophilization. Lyophilised samples were stored at RT over silica gel.
Isolation of DNA DNA isolated using the modified technique of Cenis (1992). Extraction buffer (300 µl) was added to each tube and the frozen lyophilised hyphae were crushed by hand using a conical grinder fitting exactly the base of the Eppendorf tube. Then, 150 µl of 3M 95 sodium acetate (pH 5.2) was added and the tubes placed at –20 oC for 10 minutes. Tubes were centrifuged in a microfuge for 2 minutes at 14000 rpm and the supernatant transferred to another tube. Then an equal volume of isopropanol was added and the tubes allowed to stand at ambient temperature for 10 minutes and the precipitated DNA pelleted by centrifugation in a microfuge for 20 minutes at 14000 rpm. The pellet was washed with 70% ethanol, dried in a sterile air-flow (laminar flow hood) and resuspended in 50 µl of TE buffer. The DNA extracts were stored at –20oC.
ITS Amplification The Polymerase Chain Reaction (PCR) technique was used to amplify the ITS region of each mycorrhizal isolate in 50 µl reaction volumes containing 21.5 µl sterile UltraPure water (Fisher Biotec, Australia), 10 µl 5 x PCR Polymerisation Buffer (335 mM Tris-
HCl, 83 mM (NH4)2SO4, 2.25% Triton X-100, 1 mg/ml Gelatin, 1 mM dNTP’s; Fisher
Biotec, Australia), 3 µl 25 mm MgCl2, 5 µl of each of the universal fungal primers ITS1 and ITS4 (10 pg/µl) (White et al. 1990), 0.5 µl of Taq DNA polymerase (10 U/µl, Geneworks) and 5 µl of extracted genomic DNA. PCR amplifications were performed in a FTS-960 Thermal Sequencer (Corbett Research, Mortlake, NSW, Australia) with an initial denaturing cycle of 95 oC for 2 min followed by 35 cycles of 55oC for 1 min, 72oC for 2 min and 95 oC for 1 min, followed by a final incubation at 72o for 10 min. Negative controls containing no DNA were included in each reaction run. The resulting amplification products were electrophoresed in 1% agarose gels containing ethidium bromide and visualised under UV light.
ITS Sequencing Analysis Prior to sequencing ITS products were purified using a UltraClean PCR Clean-up Kit (Mo Bio Laboratories Inc., Solana Beach, CA, USA) following the manufacturers protocol. Clean ITS products were quantified using a Fluorometer (Hoefer DyNA Quant 200, Amersham Biosciences). Sequencing reactions were performed in 10 µl volumes using 4 µl of Big Dye version 3.1 terminator mix, 20 ng of ITS product, 3.2 pmoles of primer (either ITS1 or ITS4) and enough sterile UltraPure water (Fischer Biotec, Australia) to make the final volume of the reaction 10 µl. Reactions were performed in a FTS-960 Thermal Sequencer (Corbett Research, Mortlake, NSW, Australia) with an initial denaturing cycle of 96oC for 2 min followed by 25 cycles of 96oC for 10 seconds, 50oC for 5 seconds and 60oC for 4 min. Reaction products were cleaned up as follows; the entire reaction product was added to a 0.5 ml tube containing 25 µl 100% ethanol, 96 1 µl 3M sodium acetate pH5.2 and 1µl 125 mM EDTA, and mixed by pipetting. Tubes were allowed to stand at ambient temperature for 20 min then centrifuged at 14000 rpm for 30 min, The supernatant was removed carefully using a micropipette and the tubes allowed to drain inverted. The pellet was washed by addition of 125 µl of 70% ethanol and centrifugation at 14000 rpm for 5 min. As much supernatant as possible was removed using a micropipette and the tubes left inverted in a laminar flow hood to drain and dry. Samples were submitted to SABC, Murdoch University, Western Australia, for sequencing on a ABI 3730 48-capillary machine (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404 USA)
Sequence Comparison with Data Bank Sequences BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/) were conducted on all sequences to identify the closest match and to confirm that the ITS region of nuclear rDNA had been sequenced. Sequences for well-characterised orchid mycorrhizal fungi in Tulasnellales and Sebacinaceae, and for mycorrhizal endophytes of Australian terrestrial orchids from other studies were obtained from GenBank for inclusion in the alignment and analysis (Table 4.4). Multiple sequence alignment was carried out using the AlignX component of the computer program VectorNTI Suite 7 (http://www.invitrogen.com/content.cfm?pageid=10373). The sequence alignment was manually trimmed using MacClade 4.05 (Maddison and Maddison 2002). The matrix was analysed with PAUP 4.0b10 (Swofford 2002) using parsimony methods with the ITS sequences of an ericoid mycorrhiza (GenBank code AF072293) and the isolate OTc8-4-1 (closest match AF072293) defined as the outgroup. A heuristic search was performed using 100 random sequence addition with tree bisection-reconnection (TBR) branch swapping, saving 100 trees per replicate. Branch reliability was based on Boot Strap (BS) analysis with 100 simple sequence replicates and TBR branch swapping.
Problems Associated with Isolation of DNA and PCR Amplification of Orchid Mycorrhizal Fungi, Laboratory infestation by fungus mites during the isolation and purification of cultures resulted in the contamination of many of the isolates with fast growing sporulating fungi in genera Trichoderma, Penicillium, Acremonium and Fusarium. Orchid mycorrhizal fungi were very slow growing compared to these contaminants. Attempts to recover axenic cultures of orchid mycorrhizal fungi from contaminated cultures by repeated subculture from the growing edges generally failed. DNA was unable to be extracted 97 from some isolates and PCR amplification of the ITS regions of some other isolates failed despite repeated attempts. Lyophilised hyphae of isolates from which either DNA could not be extracted, or which did not amplify were sent to the Australian Genome Research Facility (AGRF) Australian Genome Research Facility Ltd, Plant Genomics Centre, Waite Campus PMB1, Glen Osmond S.A. 5064, for DNA extraction, amplification and sequencing. However, PCR amplification of the ITS regions of these isolates was not generally successful. Contamination with fast growing fungi, failure of DNA extraction and failure of PCR amplification resulted in the loss of information from 141 (75%) mycorrhizal endophytes, including all of the isolates from Caladenia flava plants collected from rehabilitation areas (61 isolates).
Fungal Nomenclature Orchid mycorrhizal fungi belong to three polyphyletic clades of ‘Rhizoctonia’ fungi: the Sebacinaceae; the Tulasnellales; and the Ceratobasidiales (Taylor et al. 2003). Weiss et al. (2004) have recently erected the new order Sebacinales to replace the polyphyletic clade previously identified as the family Sebacinaceae. The taxonomy of the mycorrhizal Rhizoctonias is currently in a state of flux, and unless otherwise stated, the fungal nomenclature used in this study is that used by the cited references.
TABLE 4.4 Source of fungal isolates and GenBank codes for fungi included in the alignment and phylogenetic analysis. All fungal and orchid nomenclature is as used in cited references. (Note: C. carnea, C. catenata, D. bracteata, E. gigantea, H. spicata and D. revolutum (syn. Pterostylis revoluta) are all terrestrial orchids.)
Fungal Identity Source Country GenBank Reference of Origin Code Ericoid mycorrhizal sp. Woollsia pungens Australia AF072293 Chambers et al., A49 unpublished Exidea pithya not indicated Germany AF291275 Weiss and Oberwinkler, 2001 Exidiopsis grisea not indicated Germany AF291281 Weiss and Oberwinkler, 2001
Sebacina vermifera Hexalectris spicata Australia AF202728 Taylor et al., unpublished Fungal sp. CC2 Caladenia carnea Australia AY643802 Bougoure et al. 2005 uncultured Sebacinaceae Epipactis gigantea USA AY634116 Bidartondo, unpublished Sebacina vermifera Caladenia catenata Australia DQ983814 Deshmukh et al. 2006
Tulasnella asymmetrica Thelymitra luteocilium Australia DQ388046 Suarez et al., 2006 Tulasnella calospora not indicated USA AY373298 McCormick et al., 2004 Tulasnella danica not indicated USA AY373297 McCormick et al., 2004 Tulasnella pruinosa not indicated USA AY373295 McCormick et al., 2004 Tulasnella pruinosa not indicated USA DQ457642 Nilsson et al., unpublished Tulasnella violea not indicated USA DQ457643 Nilsson et al., unpublished Tulasnella sp. JTO307 Diplodium revolutum Australia DQ061110 Otero, unpublished Epulorhiza sp. Disa bracteata Australia EF176482 Bonnardeaux et al., 2007* * Western Australian study
98 RESULTS
Isolation of mycorrhizal endophytes A total of 187 mycorrhizal endophytes were isolated from 102 orchid plants collected from the Jarrahdale mine-site (Table 4.5). Isolation of orchid mycorrhizal fungi (OMF) from individual plants was more frequently successful in C. flava (37/40 plants) and D. bracteata (20/21 plants) than in T. crinita (8/21 plants) and C. ovata (6/20 plants) (Table 4.5). The percentage of non-sporulating endophytes confirmed as mycorrhizal also varied widely between species and sites (i.e. between 0 and 55%) (Table 4.5). OMF were readily isolated from C. flava (21 - 55% of isolates) and D. bracteata (36 - 55% of isolates), but were more difficult to isolate from T. crinita (0 - 26% of isolates) and C. ovata (0 - 0.08% of isolates) (Table 4.5).
Seasonal isolations The poor recovery of mycorrhizal endophytes from T. crinita and C. ovata was further investigated to determine if the presence of viable OMF peletons was seasonal. Plants of these two taxa were re-collected in mid-winter (July) and mid-spring (October) 2004 and fungal endophytes isolated. Mycorrhizal endophytes were isolated from T. crinita at both sampling times, but success rates were low. Isolation of mycorrhizal endophytes was successful from 30% of orchid plants (3/10 plants) at both mid-winter and mid- spring sampling times (Table 4.5). Attempted seasonal isolations from C. ovata were all unsuccessful, with no mycorrhizal endophytes obtained in either mid-winter or mid- spring (Table 4.5).
Additional plants of C. ovata and T. crinita collected from the field in mid-spring and the underground parts were cleared and stained with Trypan blue to determine; the extent of infection and if intact pelotons were present. Root squashes of cleared and stained T. crinita roots showed sparse patches of infection along roots and a high proportion of ‘degraded’ pelotons, distinguished by pale staining, and fragmented hyphae when examined by light microscopy (Fig. 4.2A). Examination of root squashes of cleared and stained root-tubers of C. ovata found that strongly stained intact pelotons were extremely sparse (Fig. 4.2B).
99 TABLE 4.5 Species, source and number of orchid plants collected during the 2003 and 2004 for the isolation of mycorrhizal endophyte. 2004 sampling occurred during mid-winter (July) and mid-spring (October). The number of plants from which mycorrhizal endophytes were successfully isolated, the total number of endophytes isolated, and number confirmed as mycorrhizal are shown. The capacity to form mycorrhizal associations was regarded as confirmed by the ability of a fungus to germinate seed and support the development of protocorms to ≥ stage 4 (initiation of shoot growth) (Collins 2005).
Number of Plants Number of Endophytes Species Collection Total Successful Total Percent site* collected isolations isolated mycorrhizal 2003 Caladenia flava F15-4 5 3 34 52.2 F25+-2 5 5 27 33.3 F1-1 5 4 33 21.2 F10-3 5 5 34 38.2
R25+-1 10 10 61 49.2 R25+-2 10 10 57 54.9
Disa bracteata R15-4 6 5 29 55.2 R15-1 5 5 30 50.0 R10-1 5 5 29 51.7 R10-4 5 5 36 36.1
Cryptostylis ovata Cov-1 10 6 75 0.08 Cov-2 10 0 52 0.0
Thelymitra crinita F15-4 6 2 42 26.2 F10-1 5 3 38 18.4 F1-1 5 0 28 0.0 F5-2 5 2 56 16.1
Total 102 70 661 28.4 July 2004 Cryptostylis ovata Cov-1 5 0 52 0.0 Cov-2 5 0 33 0.0
Thelymitra crinita F15-4 5 2 51 25.5 F10-1 5 1 46 10.9
October 2004 Cryptostylis ovata Cov-1 5 0 51 0.0 Cov-2 5 0 44 0.0
Thelymitra crinita F15-4 5 1 48 6.2 F10-1 5 2 56 16.1
Total 40 6 381 7.9 * The site codes are described in Table 4.2.
100 A
B
FIGURE 4.2 Cleared and stained root or root-tuber squashes of A. Thelymitra crinita and B. Cryptostylis ovata showing the presence of intensely stained intact pelotons (filled arrows) and pale degraded pelotons with fragmented hyphae (outlined arrows).
101 Ex-situ baiting Ex-situ baiting using organic material wet sieved from soil collected from the base of adult plants was not generally successful in the detection of OMF for any of the study taxa. The percentage of baits where OMF were detected in soil surrounding adult plants were: Caladenia flava - 25% in forest soil and 20% in rehabilitation area soil; Cryptostylis ovata - 5%; Disa bracteata - 0%; and Thelymitra crinita - 5% (Table 4.6). OMF of non-target taxa, that is, orchid taxa other than that of the adult orchid plant from where the soil was collected, were also detected in many of the baits (Table 4.6). The fungal associates of C. flava (16 baits), Microtis media (7 baits) and T. crinita (6 baits) were the most frequently detected OMF (Table 4.6). This is a similar result to that obtained using in situ baiting to survey rehabilitation and unmined forest soils for OMF, where C. flava and T. crinita were the most frequently detected OMF (Chapter 4). Detection of fungal associates of the other orchid taxa included in the baits was generally less frequent: C. ovata (1 bait); D. bracteata (3 baits); Pterostylis aff nana (2 baits); Pyrorchis nigricans (3 baits) Pterostylis recurva (1 bait); and Thelymitra macrophylla (2 baits) (Table 4.6). Attempts to isolate mycorrhizal fungi from stage 4 and 5 protocorms produced in ex-situ baits were unsuccessful. Protocorms and their endophytes were either killed by the surface sterilisation technique, over-grown by bacterial and fungal contaminants that were not removed by the sterilisation procedure, or possibly the fungal pelotons may have been non-viable (i.e. not capable of growth).
TABLE 4.6 Comparison of frequency of detection of orchid mycorrhizal fungi (OMF) in soil collected from the base of adult orchid plants using an ex-situ baiting technique (Brundrett et al. 2003). Organic matter was wet sieved from soil collected from the base of C. flava, C. ovata, D. bracteata and T. crinita plants, then tested for the presence of OMF by incubating with the seeds of nine orchid species. Codes for orchid species are as follows: Cf = C. flava; Co = C. ovata; Db = D. bracteata; Mm = M. media; Pa = P. aff nana; Pn = P. nigricans; Pr = P. recurva; Tc = T. crinita; Tm = T. macrophylla. The column labelled Positive refers to the total number of baits where OMF of at least one orchid was detected. Mean refers to the mean number of OMF detected/bait for each litter source. Results for targeted species are in bold.
Litter source Site type Total Positive Cf Co Db Mm Pa Pn Pr Tc Tm Mean* Caladenia flava forest 20 10 5 0 2 0 0 1 0 3 1 0.65 rehabilitation 20 6 4 0 1 2 0 1 0 1 1 0.50
Cryptostylis ovata forest 20 7 1 1 0 0 2 1 1 1 0 0.35
Disa bracteata rehabilitation 21 5 3 0 0 2 0 0 0 0 0 0.24
Thelymitra crinita forest 21 7 3 0 0 3 0 0 0 1 0 0.33
Total 102 35 16 1 3 7 2 3 1 6 2 * Differences in the number of OMF detected/bait between different litter sources were not significant (P < 0.05). 102 Ecological specificity of orchid mycorrhizal associations of the study taxa DNA was successfully isolated, amplified and sequenced from 47 isolates: 34 from 18 plants collected in 2003; seven C. flava; six D. bracteata; and five T. crinita (Fig. 4.3) and 13 from three T. crinita plants collected in 2004. PCR amplification of ITS regions produced single bands for all isolates. Sequencing of ITS amplicons followed by BLAST search for closest related fungi found: i) Caladenia flava was associated with two fungi with close similarity to Sebacina spp. and an ectomycorrhizal fungus; ii) Disa bracteata was associated with seven fungi with close similarities to seven Tulasnella spp.; and iii) Thelymitra crinita was associated with fungi with close similarity to two Tulasnella spp. and an ericoid mycorrhizal fungus (Tables 4.7 and 4.8). Two mycorrhizal isolates were obtained from each of three C. flava plants, one D. bracteata plant and two T. crinita plants (Table 4.7 and 4.8). The closest identity for fungi associated with the two Thelymitra crinita plants collected in mid-winter (July) was T. pruinosa and for the plant collected in mid-spring (October) was both T. asymmetrica and an ericoid mycorrhiza.
A B C
FIGURE 4.3 The orchid taxa from which mycorrhizal fungi and fungal DNA were successfully isolated: A. Caladenia flava - flowering in unmined forest; B. Disa bracteata - a group of plants in bud (white arrows) in one of the 15 year old rehabilitation areas; and C. Thelymitra crinita - flowering in unmined forest.
103 Phylogenetic Analysis Most of the orchid mycorrhizal fungi isolated in this study were closely related to either Sebacina vermifera or a number of Tulasnella spp. (Table 4.7 & 5.8). Phylogenetic analysis of ITS sequences found that only one of the orchid mycorrhizal fungi that were closely identified with Sebacina vermifera grouped with other Sebacina spp. sequences obtained from the GenBank database (Sebacinoid Clade 1). The remaining sebacinoid mycorrhizal fungi were more closely grouped with the mycorrhizal fungi identified as Tulasnella spp. There was significant bootstrap support (100%) for distinguishing these mycorrhizal fungi as a separate group from the GenBank Sebacina spp. sequences (Fig. 4.4). The group containing the majority of orchid mycorrhizal fungi isolated in this study was further split into two polyphyletic clades: Sebacinales fungi isolated from C. flava (Sebacinoid Clade 2) (100% bootstrap support) and members of the Tulasnellales isolated from D. bracteata and T. crinita (94% bootstrap support) (Fig. 4.4). There was strong bootstrap support (100%) for grouping the D. bracteata mycorrhizal fungi (Tulasnelloid Clade 1) separately to the T. crinita endophytes (Tulasnelloid Clade 2) (Fig. 4.4).
104 TABLE 4.7 Closest matches from Blast searches of ITS sequences of mycorrhizal endophytes isolated from the orchids; C. flava, D. bracteata and T. crinita during the 2003 growing season. For each isolate: the name of the closest match from a Blast search; the E Value; percentage identity and the closest identities GenBank accession code are provided. The collection site is listed in the column labelled Origin, for explanation of site code see Table 4.2.
Isolate code* Closest Blast identity E Percentage GenBank Code Origin Value identity CfF1-1/1 Sebacina vermifera 0.0 96% DQ983814.1 F15-4
CfF3-3/1 fungal sp. 0.0 93% AY643802.2 F15-4 CfF3-3/2 Sebacina vermifera 0.0 92% DQ983814.1 F15-4
CfF5-1/2 Sebacina vermifera 0.0 94% DQ983814.1 F15-4 CfF5-2/1 Sebacina vermifera 0.0 92% DQ983814.1 F15-4
CfF7-2/2 Sebacina vermifera 0.0 94% DQ983814.1 F27-1 CfF7-3/2 Sebacina vermifera 0.0 96% DQ983814.1 F27-1 CfF7-4/1 Sebacina vermifera 0.0 90% DQ983814.1 F27-1
CfF15-3/2 Sebacina vermifera 0.0 95% DQ983814.1 F1-1 CfF15-4/2 uncultured mycorrhiza 0.0 98% DQ497937.1 F1-1
CfF16-1/1 Sebacina vermifera 0.0 95% DQ983814.1 F10-3 CfF16-1/2 fungal sp. 1e-151 95% AY643802.2 F10-3
CfF17-1/1 Sebacina vermifera 0.0 92% DQ983814.1 F10-3
Db5-1 Tulasnella sp. 241 2e-128 98% AY373270.1 R15-4
Db9-2 Tulasnella sp. JT0306 3e-130 98% DQ61110.1 R15-1
Db12-2 Tulasnella sp. 247 6e-27 95% AY373277.1 R10-1
Db17-3 Epulorhiza sp. 6e-50 100% EF176482.1 R10-4 Db17-4/1 Epulorhiza sp. 7e-139 100% EF176482.1 R10-4
Db20-2/1 Tulasnella asymmetrica 1e-126 98% DQ388046.1 R10-4
Db21-1/2 Tulasnella sp. 144 4e-70 96% AY373265.1 R15-4 Db21-2 Tulasnella sp. 149 9e-77 98% AY373273.1 R15-4 Db21-3/1 Tulasnella sp. 144 6e-141 98% AY373265.1 R15-4
Tc2-2/2 Tulasnella pruinosa 1e-87 94% DQ457642.1 F15-4
Tc9-4/3 Tulasnella asymmetrica 0.0 98% DQ388046.1 F10-1
Tc16-3/1 Tulasnella pruinosa 2e-87 94% DQ457642.1 F1-1 Tc16-3/2 Tulasnella pruinosa 2e-87 94% DQ457642.1 F1-1 Tc16-3/2a Tulasnella pruinosa 1e-87 94% DQ457642.1 F1-1 Tc16-4/2 Tulasnella asymmetrica 0.0 97% DQ388046.1 F1-1
Tc20-2/1 Tulasnella asymmetrica 0.0 97% DQ388046.1 F5-2
Tc21-1/1 Tulasnella asymmetrica 0.0 98% DQ388046.1 F15-4 Tc21-1/2 Tulasnella asymmetrica 0.0 97% DQ388046.1 F15-4 Tc21-2/1 Tulasnella asymmetrica 0.0 97% DQ388046.1 F15-4 Tc21-2/3 Tulasnella asymmetrica 0.0 98% DQ388046.1 F15-4 Tc21-3/2 Tulasnella asymmetrica 0.0 98% DQ388046.1 F15-4 * Isolates were coded as follows: species code; Cf = Caladenia flava, Db = Disa bracteata, Tc = Thelymitra crinita; plant number (1 to 21); number of isolation plate (1 to 4); and a number or number/letter code for the isolate. 105 TABLE 4.8 Closest matches from Blast searches of ITS sequences of mycorrhizal endophytes isolated from the orchids; C. ovata and T. crinita during the 2003 growing season. For each isolate: the name of the closest species from a Blast search; the E Value; percentage identity and the closest identities GenBank accession code are provided. The column labelled Origin lists the Jarrahdale site where the orchids were collected (for an explanation of site codes see Table 4.2).
Isolate Closest relative GenBank E Percentage Origin code* code Value identity July JTc4-4/1-1 Tulasnella pruinosa DQ457642.1 3e-92 97% F15-4
JTc10-2/1 Tulasnella pruinosa DQ457642.1 2e-87 94% F10-1 JTc10-2/2 Tulasnella pruinosa DQ457642.1 2e-87 94% F10-1 JTc10-2/4 Tulasnella pruinosa DQ457642.1 2e-87 94% F10-1 JTc10-3/2 Tulasnella pruinosa DQ457642.1 2e-87 94% F10-1 JTc10-4/2 Tulasnella pruinosa DQ457642.1 2e-87 94% F10-1
October OTc8-1/1 Tulasnella asymmetrica DQ388046.1 0.0 95% F10-1 OTc8-1/2a Tulasnella asymmetrica DQ388046.1 0.0 96% F10-1 OTc8-1/2b Tulasnella asymmetrica DQ388046.1 0.0 96% F10-1 OTc8-2/2 Tulasnella asymmetrica DQ388046.1 0.0 96% F10-1 OTc8-3/1 Tulasnella asymmetrica DQ388046.1 0.0 96% F10-1 OTc8-3/1a Tulasnella asymmetrica DQ388046.1 0.0 95% F10-1 OTc8-4/1 Ericoid mycorrhiza AF072293.1 0.0 98% F10-1
* Isolates were coded as follows: two letter code for orchid taxa; Cf = Caladenia flava, Db = Disa bracteata, Tc = Thelymitra crinita; plant number = 1 to 21; number of isolation plate = 1 to 4; and a number or number/letter code for the isolate from that plate.
106 Ericoid mycorrhiza AF072293 OTc8-4-1 100 Exidiopsis grisea AF291281 Auriculariales 70 Exidia pithya AF291275 uncultured Sebacinaceae AY634116 Sebacina vermifera DQ983814 Sebacinoid 100 Sebacina vermifera AF202728 Clade 1 Fungal sp. CC2 AY643802 Cf3-3-1 Cf15-4-2 Cf16-1-2 100 Cf1-1-1 Cf17-1-1 Sebacinoid 100 Cf5-2-1 100 Cf5-1-2 Clade 2 Cf16-1-1 Cf3-3-2 Cf15-3-2 Cf7-4-1 Cf7-3-2 Cf7-2-2 92 Tulasnella danica AY373297 Db17-3 Epulorhiza sp. EF176482 100 100 Db17-4-1 82 Tulasnella calospora AY373298 Tulasnelloid Tulasnella sp. JTO307 DQ061110 Clade 1 Db9-2 Db21-3-1 62 Db1-1 Db12-2 63 Db21-2 Db21-1-2 JTc4-4-2a 92 Tc2-2-2 Tulasnella violea DQ457643 94 Tulasnella pruinosa AY373295 70 Tulasnella pruinosa DQ457642 Tc16-4-2 Tc16-3-2a Tc16-3-1 100 JTc10-4-2 JTc10-3-2 JTc10-2-4 JTc10-2-2 89 JTc10-2-1 Db20-2-1 Tulasnelloid OTc8-3-1a Clade 2 OTc8-3-1 OTc8-2-2 OTc8-1-2b OTc8-1-2a OTc8-1-1 100 Tulasnella asymmetrica DQ388046 Tc9-4-3 Tc21-2-3 Tc21-2-2 Tc21-2-1 10 changes Tc21-1-2 Tc21-1-1 Tc20-2-1
FIGURE 4.4 Phylogenetic tree of ITS sequences of fungi isolated from Caladenia flava (Cf), Disa bracteata (Db), Thelymitra crinita (Tc) in this study. The fungal isolates that form mycorrhizas with each orchid taxa are roughly grouped into three polyphyletic clades: C. flava with Sebacinoid Clade 2; D. bracteata with Tullasnelloid Clade 1; and T. crinita with Tullasnelloid Clade 2. The tree resulted from a heuristic search performed using 100 random sequence addition with tree bisection-reconnection (TBR) branch swapping, saving 100 trees per replicate. Numbers above or below branches show reliability based on bootstrap analysis (percentage). Sequences of closely related well characterised fungi obtained from GenBank were included in the analysis. An ericoid mycorrhizal fungi (GenBank code AF072293) and isolate OTc8-4-1 (closest match AF072293) were used as the outgroup. Two fungi from the Auriculariales; Exidiopsis grisea and Exidia pithya, were included as a sister group to the mycorrhizal Rhizoctonias. See Table 4.4 for details of GenBank sequences selected for inclusion in the analysis. 107 DISCUSSION
Ecological Specificity of Orchid Mycorrhizal Associations In this study the ecological specificity of the mycorrhiza of the two common and widespread orchids, C. flava and T. crinita, was found to be narrow, whereas the disturbance opportunist D. bracteata had broad specificity (Note: Specificity is defined as follows: high = one fungal taxon/orchid taxon; narrow = 2-3 fungal taxa/orchid taxon; broad ≥ 4 fungal taxa/orchid taxon.). C. flava was associated with mycorrhizal fungi that had closest identities to two fungi in the Sebacina vermifera group, closest identities isolated from Caladenia catenata and C. carnea in Australia, and a putative ectomycorrhizal fungus isolated from Tsuga heterophylla (Western Hemlock) in Northern America. Thelymitra crinita was associated with two mycorrhizal fungi that had closest identities to Tulasnella asymmetrica, isolated from Thelymitra luteocilium in Australia, T. pruinosa (source unknown), and an ericoid mycorrhiza isolated from Woolsia pungens, in Australia (Chambers et al. 2000). Disa bracteata was associated with endophytic fungi that had closest identities to seven different Tulasnella spp. all isolated from terrestrial orchids. Unfortunately, identities of many of the mycorrhizal isolates were lost due to contamination, failure of DNA extraction or failure of ITS PCR amplification. Nonetheless, the results of this study indicate that the ecological specificities for C. flava, D. bracteata and T. crinita are similar to that observed in previous studies that have included these orchid species (Table 4.9).
TABLE 4.9 Ecological specificity of mycorrhizal associations in the study species; C. flava, C. ovata, D. bracteata and T. crinita, determined in this study. The results are compared with previously studies that examined the same orchid taxa. Specificity is defined as follows: high = one fungal taxon/orchid taxon; narrow = 2-3 fungal taxa/orchid taxon; broad ≥ 4 fungal taxa/orchid taxon. (Note: the acronym OMF is used here as an abbreviation for orchid mycorrhizal fungus/fungi).
Orchid species Growth Number of Specificity Literature Reference habit OMF (this study) specificity Caladenia flava clonal 3 narrow high Warcup 1971; 1981 Hollick 2004
Disa bracteata* non-clonal 7 broad broad Bonnardeaux et al. 2007
Thelymitra crinita non-clonal 3 narrow high** Bonnardeaux et al. 2007 * D. bracteata is a South African orchid that has naturalised in southern Australia ** Fungal endophyte not tested for the capacity to form mycorrhiza
108 Seasonal Effects There appeared to be seasonal changes in fungi forming mycorrhiza with T. crinita in 2004. Only fungi closely related to T. pruinosa were isolated in mid-winter, while fungi closely related to T. asymmetrica and an ericoid mycorrhiza were isolated in mid-spring (Table 4.8). Mycorrhizal fungi from T. crinita plants (five) collected in mid-spring, 2003, had closest affinities to either Tulasnella asymmetrica or T. pruinosa. One plant was infected with two fungi, one closely related each of the two fungi T. asymmetrica and T. pruinosa. However, DNA isolation and ITS sequencing was successful from mycorrhizal isolates of only three plants in 2004. It is therefore most likely that the variation in mycorrhizal fungal associate was not a seasonal effect but an artefact of low sample number in 2004, and poor success rates in DNA extraction, PCR amplification and sequencing. Further studies: optimising DNA extraction, PCR amplification and sequencing methods; more intensive sampling of plants; seasonal sampling of roots of individual plants; and/or sampling of protocorms, may reveal whether seasonal and/or sequential infections occur in T. crinita or other orchid taxa.
Do the fungi found in soil surrounding adult orchid plants form persistent mycorrhizal associations with those orchids? Failure to isolate pure cultures of fungi capable of germinating orchid seed from litter surrounding plants meant that was not possible to determine if the identities of these were the same as fungi that formed persistent mycorrhizal associations. Further studies using both in situ and ex situ baiting are required to determine if a sequential infection process occurs during growth and development in Australian terrestrial orchids and to optimise the use of the ex-situ baiting technique in lateritic soils.
Is the disturbance opportunist Disa bracteata promiscuous in its mycorrhizal associations? This study found all mycorrhizal fungi associated with the disturbance opportunist Disa bracteata belonged to the same group of fungi, the Tulasnellales. Similar results were found in an earlier study of endophytes isolated from D. bracteata collected over a wide range of habitats and sites; from South Africa, through Western Australia to Victoria in eastern Australia, (Bonnardeaux et al. 2007). All of the mycorrhizal fungi isolated from D. bracteata by Bonnardeaux et al. (2007) were closely related to fungi belonging to several clades of the Tulasnalles. Bonnardeaux et al. (2007) postulated that disturbance opportunist or invasive orchid taxa, such as D. bracteata, would utilise widespread 109 fungal taxa in their mycorrhiza and that these would be less sensitive to disturbance than fungal taxa associated with orchids with high specificity mycorrhizal associations. In the current study, one of the mycorrhizal fungi associated with D. bracteata was most closely related to Tulasnella asymmetrica. Fungi closely related to this fungus were identified as frequent mycorrhizal associates of T. crinita, a common forest orchid with narrow specificity. A soil baiting survey of rehabilitation areas and unmined areas of jarrah forest carried out as part of this project found that OMF associated with T. crinita were among the most frequently detected OMF (Chapter 4). This provides evidence in support of the suggestion that the disturbance opportunist D. bracteata forms mycorrhiza with widespread fungi. However, OMF associated with both T. crinita and D. bracteata were rare in the rehabilitation areas, so there is no evidence for a link between narrow specificity and sensitivity to disturbance. The evidence indicates that Disa bracteata forms mycorrhiza with a genetically diverse suite of fungi from the Tulasnellales, suggesting that it is promiscuous in its mycorrhizal associations.
Comparison of mycorrhizal fungi of the sympatric orchid taxa Caladenia flava and Thelymitra crinita Earlier in situ baiting surveys found different vegetation structure, litter cover and litter depth characteristics were associated with the presence of OMF for each of the two orchids Caladenia flava and Thelymitra crinita (Chapter 3). These two orchid taxa have very different reproductive and nutrient gathering strategies that will make very different nutritional demands on the their mycorrhiza (Chapter 3). Rasmussen (2002) has suggested that the use of different fungi in mycorrhizal associations may be a means for sympatric orchid taxa to avoid competition for nutrients. Identification of mycorrhizal associates of Caladenia flava and T. crinita in this study found OMF of each orchid confined to one of two different clades of mycorrhizal Rhizoctonias. Thus, C. flava and T. crinita appear to be avoiding competition for limited resources by using fungi from different groups within the mycorrhizal Rhizoctonias that have different microhabitat requirements.
Multiple Mycorrhizal Fungi in the Same Plant Two mycorrhizal fungi were isolated from the underground parts of each of six of the 21 individual plants examined; three C. flava plants, one D. bracteata plant and two T. crinita plants. In most cases, these fungi were very closely related. For example, in two of the C. flava plants the two mycorrhizal fungi were both very closely related to
110 Sebacina vermifera (Tables 5.7 & 5.8). Multiple endophytes have previously been found in association with Australian terrestrial orchids. However, often these fungi have not been tested to confirm their ability to form mycorrhiza, and/or were identified only by morphology or sequencing of PCR products of total DNA extracts of root fragments (Huynh et al. 2004; Bougoure et al. 2005; Dearnaley 2006; Dearnaley and Le Brocque 2006). Absence of confirmation of mycorrhizal capacity means that these endophytes cannot be described with certainty as orchid mycorrhizal fungi.
In this study, an ericoid mycorrhizal fungus and an ectomycorrhizal fungus were identified among the OMF associated with multiple infections of orchids. Fungi that are closely related to ericoid mycorrhiza and ectomycorrhiza are often identified amongst orchid endophytes but are not usually regarded as mycorrhizal in photosynthetic orchids (Currah et al. 1997; Bayman and Otero 2006; Bonnardeaux et al. 2007). It is possible that the ericoid and ectomycorrhizal fungi were identified as OMF in this study because the criterion used to define mycorrhizal capacity was not rigorous enough. However, there is considerable evidence that ericoid and ectomycorrhizal fungi can form mycorrhizal associations with both chlorophyllous and achlorophyllous orchids, and that they may even be implicated in tripartite relationships with tree species (Currah et al. 1987; Taylor and Bruns 1997; Selosse et al. 2002a; Julou et al. 2005; Shefferson et al. 2005). Selosse et al. (2004) even regarded the use of ectomycorrhizal symbionts rather than Rhizoctonias in Epipactis microphylla, as evidence of a predisposition towards achlorophylly in Neottieae (E. microphylla occasionally produces achlorophyllous specimens). However, achlorophylly has not been observed in either C. flava or T. crinita. Thus, it appears that colonisation of an individual orchid with more than one mycorrhizal fungus can occur, and that occasionally fungi normally associated with ericoid and ectomycorrhizal fungi will form mycorrhiza with C. flava and T. crinita. Longer term seed germination and seedling growth experiments, using these fungi and the orchid from which they were isolated, would determine if this was a long lasting association capable of supporting adult plants.
Phylogenetic relationships of orchid mycorrhizal fungi Orchids in the genus Caladenia often have mycorrhizal associations with fungi with close affinities to Sebacina vermifera (Table 1.4, Chapter 1). However, the Sebacina spp. found associated with C. flava in this study do not appear to be as closely related to S. vermifera as might be expected. Bougoure (2005) using LSU sequences, found a
111 fungal endophyte from Caladenia carnea was most closely grouped with Sebacina vermifera sensu Oberwinkler (originally isolated from C. dilatata) rather than S. vermifera sensu Warcup and Talbot (1967) (originally isolated from Phyllanthus calycinus) and a similar result was expected in this study. However, although a Blast search indicated that 12 of the 13 mycorrhizal fungi isolated from C. flava were closely related to Sebacina vermifera, phylogenetic analysis of ITS sequences grouped only one of these fungi with Sebacina spp. sequences obtained from GenBank (Fig. 4.4). The four GenBank Sebacina spp. ITS sequences that were included in this analysis were all obtained from fungi originally isolated from terrestrial orchids and three had very close similarities to the orchid mycorrhizal fungi isolated in this study (Table 4.7). The separate grouping of C. flava mycorrhizal fungi from other Caladenia OMF was therefore unexpected.
The ITS region can be extremely variable in the mycorrhizal Rhizoctonias leading to problems in determining phylogenetic relationships (McCormick et al. 2004). Mitchell and Zuccaro (2006) state that the presence of multiple indels in the ITS region rather than base substitutions can make alignments and phylogenetic analysis difficult in orchid mycorrhizal fungi. Sequences of nLSU and 5 S rRNA regions of Tulasnella spp. are also particularly difficult to align unambiguously (Weiss and Oberwinkler 2001). It is possible that similar alignment difficulties in the ITS region sequences were responsible for what appears to be an anomalous grouping of these Sebacinoid fungi, or that trimming of aligned sequences prior to phylogenetic analysis removed sequence information that would have identified these fungi more closely with other OMF in the Sebacinales.
Sebacina spp. are common endophytes of orchids. Many form ectomycorrhizal and ericoid mycorrhizal associations, and are even involved in tripartite relationships between orchids and tree species (Selosse et al. 2002b; Selosse et al. 2007). However, the taxonomy of the Sebacinaceae is currently unclear. Weiss and Oberwinkler (2001) state that: ‘there is evidence that: (1) Sebacina vermifera in its original sense is not a member of the Sebacinaceae’ and ‘(2) Sebacina vermifera sensu Warcup and Talbot (1967) possibly represents a new species or an assemblage of closely related new species’. Taylor et al. (2003) found Sebacina-like fungi were the primary mycorrhizal fungi of orchids in the Hexalectris complex and that these were phylogenetically mixed with
112 ectomycorrhizal taxa. Phylogenetic analysis of nLSU (nuclear Large SubUnit) sequence data from a broad selection of Basidiomycota included in this study grouped the orchid mycorrhizal fungi into three polyphyletic ‘Rhizoctonia’ clades: Ceratobasidiales; Sebacinaceae; and Tulasnellales (Taylor et al. 2003). A phylogenetic analysis of four rDNA regions has found that which form mycorrhiza with orchids, and were usually regarded as Heterobasidiomycetes: Ceratobasidiales; Sebacinaceae; and Tulasnellales (Roberts 1999) were included in the the cantharelloid clade of the Homobasidiomycetes (Binder et al. 2005). It is evident that the taxonomy of the fungi that form mycorrhiza with orchids is poorly resolved and requires further investigation.
113 CONCLUSIONS
Many studies of orchid mycorrhiza (including this one) examine only culturable endophytes while others amplify target genes directly from infected orchid tissues. Identification of ericoid mycorrhizal fungi from DNA and culture-based detection methods has shown a ‘culturing detection-bias’ (Allen et al. 2003) and it is likely that different ecological specificities for orchid mycorrhiza will be obtained from each of these practices. Not all fungal endophytes in orchids are partners in mycorrhizal associations, nor is there any evidence that only culturable fungi are capable of forming mycorrhizal associations with orchids. However, while examination of culturable fungi will potentially reduce the number of mycorrhizal fungi identified in an individual plant, only these fungi can be tested in vitro for the capacity to form mycorrhiza. The results of this study provide information about fungal endophytes of orchids that are known to have mycorrhizal capacity.
The aims of this study have substantially been fulfilled. The ecological specificity of mycorrhizal associations of the three orchid taxa: Caladenia flava subsp. flava, Thelymitra crinita and Disa bracteata have been determined. The disturbance opportunist D. bracteata appears to be promiscuous in its mycorrhizal associations. The two common sympatric orchids: C. flava and T. crinita, used fungi from different taxonomic groups as mycorrhizal fungi, which suggests that they are not competing directly for nutrient resources. Several orchid plants contained more than one mycorrhizal fungus, and in two orchids the second endophyte was a fungus more commonly associated with an ericoid mycorrhiza or ectomycorrhiza. Phylogenetic analysis using ITS rDNA region sequences of OMF isolated in this study were in agreement with previous studies: C. flava was associated with fungi from the Sebacinales; and T. crinita and D. bracteata were associated with fungi from the Tulasnellales. Further studies are required to: optimise molecular techniques for use with OMF; determine the ecological specificity of Cryptostylis ovata mycorrhiza; and determine if promiscuous mycorrhizal associations are common amongst other disturbance opportunist orchids. It is evident that there is currently no clear concept of a species or genus within the ‘Rhizoctonia’ complex. Further more extensive and intensive phylogenetic studies using molecular techniques, multiple target genes, and evidence from classical techniques, are required to clarify the taxonomy of orchid mycorrhizal fungi.
114 CHAPTER 5
THE RELATIONSHIP BETWEEN ORCHID DISTRIBUTION,
VEGETATION STRUCTURE AND PLANT SPECIES ASSEMBLAGES OF
BAUXITE MINE REHABILITATION AREAS IN SOUTH-WEST
WESTERN AUSTRALIA
INTRODUCTION
Orchid colonisation of the post-mining landscape is dependent on seed production in the surrounding unmined forest, its dispersal and germination, and the presence of suitable microhabitats (Brundrett 2007). Annual seed production is affected by multiple factors. These include: whether an orchid can produce viable seed by self-pollination or requires out-crossing; the distribution and abundance of plants and pollinators; the specificity of the plant-pollinator relationship; the distance pollinators travel; the nutritional status of plants; climatic and environmental factors that affect flowering and fruiting; and seed loss by predation. Flowering, pollination, fruit set and seed dispersal were not examined in this study. The germination of orchid seed in soils of rehabilitation areas requires the presence of the appropriate mycorrhizal fungi. The recovery of these fungi is examined in more detail in Chapter 4. Microhabitats suitable for orchid seedling establishment, growth and survival are expected to develop over time as vegetation structure and species diversity characteristics of this new habitat develop.
In classical ecological theory, vegetation establishment on soil not previously colonised by plants is believed to undergo a primary ecological succession process where a series of plant associations progressively occupy the site until a final self-sustaining plant community develops (Clements 1916; Whittaker 1953). However, the recovery of jarrah (Eucalyptus marginata) forest vegetation following natural (endogenous) disturbances is a secondary succession process where there is no successive replacement of plant communities. Instead the species present in the climax community are present in the initial species mix. This is known as the ‘initial floristic composition’ model of succession (Egler 1954). The post-bauxite mining landscape has not previously been colonised by plants but contains plant propagules from three main sources: the soil seed
115 bank in respread topsoil; sown seed; and re-planted recalcitrant species (Willyams 2005; Grant and Koch 2007). Thus, despite being a new landscape, vegetation establishment on rehabilitated mine-sites is not a primary succession but is believed to follow a secondary succession process (Grant and Loneragan 2001; Norman et al. 2006).
The recovery of vegetation following bauxite mining differs from that following an endogenous disturbance, as few resprouter species are present (Bell and Koch 1980). Resprouting is one of three broad fire response strategies used to categorise elements of the Australian flora, the other two categories are ephemerals and seeders (Gill 1981; Bell et al. 1984; Burrows and Wardell-Johnson 2003). The traits defining these categories and the approximate proportions of plant taxa exhibiting these characteristics in south-west Western Australian forest ecosystems are listed in Table 5.1. The resprouter category of plant taxa represents approximately two thirds of the flora of these forest ecosystems (Table 5.1). The process of topsoil stripping before and respreading after bauxite mining causes severe soil disturbance that results in the death of lignotubers, rhizomes, tubers and corms, the organs that enable resprouters to survive fire (Grant and Loneragan 2001). High seed production by seeder species means that the soil seed bank in respread topsoil is dominated by their seed (Vlahos and Bell 1986). Seed mixes used in rehabilitation programs are designed to provide rapid vegetation cover, reduce erosion and fix nitrogen, and therefore also contain a high proportion of fast growing seeder species, especially legumes (Grant and Loneragan 2003). Thus two
TABLE 5.1 Fire response categories of Australian plant taxa with the proportion of species in each category for the forest ecosystems of south-west Western Australian (after Burrows and Wardell-Johnson (2003) with additional information from Bell et al. (1984)).
Fire Response Category Life Span Proportion of Species Ephemerals – avoid fire, regenerate from seed 1-3 yrs post 5-10 % fire Obligate seeders - reproductively mature plants die following 100% leaf > 10 yrs scorch or stem girdling, regenerate from seed - seed stored on plant (serotinous) 5-10 % - seed stored in soil 20-25 % - seed dispersed from unburnt areas ? Resprouters - reproductively mature plants that survive 100% leaf >50 yrs scorch or stem girdling - fleshy below ground storage organs (includes geophytes) 25-30 % - lignotuber or rhizome 25-30 % - epicormic buds 3-5 % - apical buds 3-5 %
116 of the three main sources of plant propagules in the post-mining landscape (i.e. the soil seed bank in respread topsoil; sown seed; and re-planted recalcitrant species) are dominated by seeder species with the result that post-mining vegetation that is also dominated by these species (Bell and Koch 1980).
Orchids are resprouters that produce seed capsules with many thousands of seeds. In indigenous Western Australian terrestrial orchids this prolific seed production is coupled with very low natural recruitment (Batty et al. 2001). Batty et al. (2001) estimated that annual recruitment of Caladenia arenicola seedlings was approximately 0.4 new plants per plant per year. In part, low recruitment is due to the dependence on orchid mycorrhizal fungi for successful germination of seed. Baiting experiments carried out during this study suggest that orchid mycorrhizal fungi occur infrequently in jarrah forest and rehabilitation area soils (see Chapter 4). A preliminary orchid survey found that orchid numbers and species diversity in a temporal sequence of rehabilitation areas were similar to that of adjacent unmined forest transects in all except 1 year old rehabilitation areas (see Chapter 2). However, in some sites orchid numbers were dominated by high populations of a few species of disturbance opportunists, some orchid species were found only in either forest or rehabilitation areas, and populations of individual species were generally very low compared with natural forest populations (see Chapter 2, Table 2.3).
Completion criteria for the rehabilitation of mined landscapes are a set of site-specific objectives determined by the joint stakeholders (Ward 1998; Grant 2006; Grant and Koch 2007). Alcoa World Alumina Australia aims to establish a self-sustaining forest ecosystem that retains or enhances the original water, timber, conservation and recreation values on land mined for bauxite before relinquishing tenement (Elliott et al. 1996). There is a need for indicators that measure progress towards fulfilling these completion criteria (Thompson and Thompson 2004; Anonymous 2006). Currently, there is limited information about the recovery of terrestrial orchids in relationship to establishment of vegetation structural and species diversity characteristics in the post- bauxite mining landscape of south-west Western Australia. Such information is essential for the identification of suitable orchid habitats and to gain an understanding of the factors determining the success of seedling establishment in this habitat. The relationships between orchids, vegetation structural and species diversity characteristics
117 may also reveal whether or not terrestrial orchids can be useful as indicators of the successful establishment of a jarrah forest ecosystem in the post-mining landscape. This chapter examines changes in vegetation structural, soil surface cover and species richness characteristics of a temporal sequence of post-bauxite mining areas as a replacement jarrah forest ecosystem develops. The aims of the study were:
a) to compare the vegetation structure and species richness in rehabilitation areas with that of the unmined jarrah forest; b) to determine if particular orchid species associate with particular plant species assemblages, and if these associations are similar in rehabilitation areas and unmined forest; and c) to evaluate the potential of native terrestrial orchids as indicators for completion of the rehabilitation process.
118 MATERIALS AND METHODS
Study Site The study site is described in materials and methods section of Chapter 2 (page 47) and the distribution of transects within the mine site is shown in Fig. 1.6 (page 44).
Vegetation Survey Single belt transects 5 m by 50 m, each consisting of ten contiguous 5 m x 5 m quadrats, were established in four replicates each of 1, 5, 10 and 15 year old rehabilitation areas (established in 2001, 1997, 1992 and 1987, respectively) and in adjacent unmined forest at Jarrahdale, Western Australia, in autumn/winter 2002 (May-July). A further two transects were established in 27 year old rehabilitation areas with an over-storey of marri and a single transect in a 26 year old rehabilitation area. Each was paired with transects in adjacent unmined forest. These three transects are identified as 25+ year old rehabilitation areas. Adjacent unmined forest was used as unmined controls in this study as there was no historical data on the pre-mining vegetation for specific study sites. Two additional transects, designated Cov-1 and Cov-2, were also placed through populations of Cryptostylis ovata giving a total of 40 transects.
Vegetation in each quadrat was scored for percentage cover provided by each of the following categories of vegetation structure: Trees > 30 m; Trees 10 – 30 m; Trees < 10 m; Shrubs > 2 m; Shrubs 1 – 2 m; Shrubs < 1 m and Herbs, sedges and grasses. Note: for analyses of vegetation structure the ground covering creepers Kennedia prostrata R.Br. and K. coccinea Vent. were included in the structural category ‘Herbs, sedge and grasses’. Vegetation structure categories are those used by the Wildflower Society of Western Australia (Keighery 1994) and the Department of Conservation and Land Management (CALM), Western Australia, (Specht 1970; Muir 1977). The structural category Trees < 10 m includes saplings as these provide much of the vegetation cover in younger rehabilitation areas. In addition, data were collected for soil surface cover in the following categories: Leafy litter (leaves and fine twigs); Litter - minimum depth; Litter - maximum depth; Woody litter (large branches > 2.5 cm diameter and logs); and Bare ground. Typical vegetation characteristics of a chrono-sequence of rehabilitation areas (1, 5, 10, 15 and 25+ years sold) and an unmined forest area are shown in Fig 5.1.
119 A B
C D
E F
FIGURE 5.1 Typical vegetation of a chrono-sequence of rehabilitation areas (A - E), and an area of unmined forest (F). Time since rehabilitation establishment is: A = 1 year; B = 5 years; C = 10 years; D = 15 years; and E = 25+ years. The white tipped post in D is approximately 1 m tall and marks one end of a transect.
Plant species present were scored for percentage cover in each quadrat. Plant species that were present but provided little cover were scored as 0.1% cover. The number of orchid species present and number of plants of each species in each quadrat was also recorded. In the examination of vegetation structure dead plants were regarded as standing litter and were included in either leafy or woody litter layer (definitions of categories are described above). For analysis of species assemblages dead plants were regarded as separate species from living specimens of the same species. Cover was defined as that area of each quadrat covered by foliage for a particular vegetation 120 category or species. For dead plants (standing litter) cover was the area delimited by fine twigs and dead leaves.
Species Identification Voucher specimens of plants were collected for identification. Plant identification was consistent with Florabase (http://www.florabase.calm.wa.gov.au, accessed February 2007). Voucher specimens of orchids were collected from each species for submission to the Western Australian Herbarium. Note: Eriochilus sp. was putatively identified as Eriochilus dilatatus subsp. multiflorus (Lindl.) Hopper and A.P.Br. ms, but this has not been confirmed as it was not observed flowering in any of the study transects over the period of the project.
Data Analysis a) Comparison of the vegetation structure and species richness in rehabilitation areas with that of the unmined jarrah forest. Vegetation structure and species richness of rehabilitation areas were compared with that of the unmined jarrah forest by ordination and principal coordinate analysis. Percentage cover data was fourth root transformed before ordination analysis by non- metric multi dimensional scaling (nMDS) and hierarchical cluster analysis using
PRIMER V6 (Primer-E Ltd., Plymouth Marine Laboratory). Clarke and Warick (1994) refer to use of the 4th root transform in the following statements; ‘which takes some account also of rarer species’; and ‘The practical choice is therefore often between a moderate (√) and fairly severe (√√ or log) transform, retaining the hard-won quantitative information but downplaying the species dominants; the 4th root transform is widely used in this manual’. Orchids provided little cover, even when numerous, and the 4th root transform was applied to percentage cover data this study to retain quantitative information for these species. Analysis of differences in vegetation structure and species assemblage between rehabilitation areas and unmined forest by principal coordinate analysis (PCoA) was carried out using PCO (Anderson 2003b). Temporal changes of vegetation structure and species assemblage differences were examined by permutational multivariate analysis of variance, using PERMANOVA v1.6
(Anderson 2001; McArdle and Anderson 2001; Anderson 2005). PERMDISP2 was used to compare multivariate dispersion of data groups on the basis of distance or similarity measures (Anderson 2004). Anderson (2004) states that this is done ‘in two steps: (1) calculation of the distances of observations from their centroids and (2) comparison of
121 the average distances among these groups using ANOVA’. ‘The rejection of the null hypothesis for PERMANOVA suggests that groups may differ because of their location, their relative dispersion or both. PERMDISP2 is used as a companion to PERMANOVA to assist in determining the reasons for the rejection of the null hypothesis’ (Anderson 2004).
Resemblance matrices were calculated using the Bray-Curtis similarity coefficient for vegetation strata and species cover data, and the Jaccard similarity coefficient for analysis of presence/absence data. Permutational multivariate analyses were performed using 9999 permutations and nMDS ordinations using 100 restarts. Plant species occurring only once were removed from the data before analysis unless otherwise indicated. Permutational and Monte Carlo P-values are designated P and P (MC) respectively. The Monte Carlo P-value has been used where the number of possible permutations is low (Anderson 2005).
PERMANOVA and PCO required balanced experimental designs (i.e. an equal number of observations per cell with no missing cells). To fulfil these design requirements both transects placed through Cryptostylis ovata populations (Cov-1 and Cov-2) were omitted from comparisons of plant assemblages of rehabilitation areas and unmined forest. In analyses of temporal changes, the two unpaired transects Cov-1 and Cov-2, the three 25+ year old rehabilitation transects and their adjacent forest transects were omitted from the analyses. The three 25+ year old rehabilitation transects were compared to their adjacent forest transects in separate analyses.
Univariate analysis of variance for individual vegetation strata cover characteristics was used to examine temporal changes between vegetation structural characteristics of all age rehabilitation areas and to compare rehabilitation areas with their adjacent unmined forest. Univariate statistical calculations for analysis of variance (ANOVA) and regression analyses were carried out using GenStat Version 8 (Lawes Agricultural Trust, Rothamsted Experimental Station). Percentage cover data were arcsine square root transformed and abundance data were log10 (1+n) transformed prior to analysis by
ANOVA.
122 b) Comparison of plant species assemblages.
The computer program TWINSPAN (Hill, 1979) was used to classify transects and species assemblages to determine if particular orchid species associated with particular plant species assemblages and if these associations were characteristic of either, rehabilitation areas or unmined forest. Species data were presence/absence transformed prior to analysis to retain the influence of orchid species, as although these species may be present in large numbers they produce very little cover. Species present in only one transect were removed from the data prior to analysis. To more closely examine the species assemblages associated with individual orchid taxa, TWINSPAN classification was repeated using the data for the ten contiguous quadrats that made up each belt transect. Rehabilitation area and unmined forest quadrat data were analysed separately. In this study analysis of transect vegetation data is termed ‘vegetation type scale’, and quadrat data is termed ‘microhabitat scale’. c) Potential of native terrestrial orchids as indicators for completion of the rehabilitation process.
Multivariate regression analysis carried out using DISTLM forward (McArdle and Anderson 2001; Anderson 2003a) was used to evaluate the potential of native terrestrial orchids and other vegetation characteristics as indicators for completion of the rehabilitation process. The analysis included data for: orchid species richness; the abundance of clonal, non-clonal and disturbance opportunist orchids; and abundance of 17 orchid species found in more than one transect (Table 5.2). A number of species diversity indices; non-orchid species counts; and plant category cover measures were also included in the analyses (Table 5.2). Categories of plant taxa were based on differences in structural characteristics of forest and rehabilitation areas, plant growth habits observed in the field, and the presence or absence of taxa in different age rehabilitation areas (Tables A2.1 and A2.2, Appendix 2). Separate analyses were made for plant cover categories and species richness categories.
The output from DISTLM forward contains two tables. Anderson (2003a) describes these as follows: the first table; (marginal tests), shows ‘the relationship of each variable with the response data independent of all other variables’; and the second table; (conditional (sequential) tests), shows the results of the ‘forward selection procedure with conditional tests (i.e. each variable is fitted one at a time, conditional on the variables that have already been included in the model)’. The latter procedure avoids the problem
123 of linear correlation of variables. The F-statistic calculated by DISTLM forward is referred to as the pseudo-F statistic. The pseudo-F statistic is calculated from the sums of squares of the similarity measure used to separate sites (transects) in ordinations (i.e. in this study from either Bray-Curtis similarity or Jaccard similarity) (Legendre and Anderson 1999, McArdle and Anderson 2001, Anderson 2001).
TABLE 5.2 Species diversity indices; species counts, orchid abundances; and plant category cover measures that were examined by multivariate regression analysis using DISTLM forward (Anderson 2003a). Note: older rehabilitation areas were planted with non-indigenous Eucalypt spp. as part of the rehabilitation process. The increase in Myrtaceae tree species with rehabilitation age is the result of trial planting of non-indigenous Eucalypts and not of recruitment and maturation of vegetation, therefore only Myrtaceae shrubs were examined in this analysis (see Appendix 2).
Diversity Indices* Species Categories Orchid Abundance Cover Category Categories Species richness Alien herbs and Clonal species Alien herbs and grasses grasses Maximum Alien Non-clonal species Alien diversity Shannon-Weiner Legumes (live) Disturbance Legumes (live) Evenness Legumes (dead) opportunist species Legumes (dead) Dominance Dead plants Caladenia flava Dead plants Orchid species Lomandra and Cyanicula sericea Lomandra and richness sedge-like Cryptostylis ovata sedge-like Epacridaceae Cyrtostylis huegelii Epacridaceae Proteaceae Disa bracteata Proteaceae Myrtaceae (shrubs) Diuris brumalis Myrtaceae (shrubs) Eriochilus sp. Total cover Microtis media Total litter cover Pterostylis sp. crinkled leaf Woody litter Pterostylis barbata Pterostylis recurva Pterostylis vittata Pterostylis sanguinea Pyrorchis nigricans Thelymitra macrophylla Thelymitra benthamiana Thelymitra crinita * Species richness = number of taxa (S); Maximum diversity = Hmax ; Shannon-Weiner = H'; Evenness = E 2 (H'/Hmax); Simpson’s Dominance = D (Σpi ); Orchid species richness = number of orchid taxa.
124 RESULTS
Vegetation structure and species richness Ordination of transect vegetation strata and soil surface cover data by Non-metric Multi-Dimensional Scaling (nMDS) reveals that the structural characteristics of the rehabilitation area vegetation were becoming more like those of unmined forest with time (Fig. 5.2). Circles representing clusters of transects with 85% similarity have been overlain on the nMDS ordination (Fig. 5.2). The oldest transects examined (labelled R25+) are closely grouped with unmined forest transects while younger rehabilitation transects, with the exception of the 10 year old rehabilitation transects, are grouped by age. Two of the four 10 year old rehabilitation transects are clustered with each of the five and fifteen year old rehabilitation transects groups.
Figure 5.2 Ordination of transect vegetation strata and soil surface cover data by non-metric multi-dimensional scaling (nMDS). Data were fourth root transformed prior to analysis and comparisons made using Bray-Curtis similarities. nMDS was calculated from 100 restarts, 2D stress = 0.09. Circles group transects with 85% similarity determined from hierarchical cluster analysis using Bray-Curtis similarities. SIMPROF test of similarity found that grouping of transects by cluster analysis was significant at P < 0.05. The dotted line indicates the trajectory of vegetation recovery. Data were obtained from a vegetation survey of a temporal sequence of rehabilitation areas and from unmined forest at Alcoa’s Jarrahdale mine site. Legend: R = rehabilitation; F = unmined forest. Rehabilitation areas are colour coded by age. The number following the letter R refers to the rehabilitation areas age in years at the time of sampling, (i.e. one, five, ten, fifteen and over 25 years old (25+)). The two transects labelled Cov were additional transects placed through two populations of the orchid Cryptostylis ovata.
125 120 A Trees 10-30 m Trees < 10 m 100 Shrubs > 2 m Shrubs 1-2 m Shrubs < 1 m 80 Herbs, sedges and grasses
60 % Cover
40
20
140 B Bare ground Woody litter 120 Leafy litter
100
80
% Cover 60
40
20
0 1 5 10 15 >25 Forest Time since establishment (years)
FIGURE 5.3 Temporal changes in cover characteristics in rehabilitation areas compared with mean values for unmined forest; A) Vegetation structure strata and B) Soil surface cover categories. Values in the column labelled “Forest’ are the mean of all the adjacent unmined forest transects. Vegetation structure strata are based on height strata used to classify Western Australian vegetation by the Wildflower Society of Western Australia (Keighery 1994) and the Department of Conservation and Land Management (CALM), Western Australia, (Specht 1970; Muir 1977). The category ‘Herbs/sedges/grasses’ includes all low growing cormatous, rhizomatous and tuberous geophytes.
Figure 5.3 provides a clear graphical illustration of the physical changes in vegetation structure and soil surface cover characteristics that have occurred with increasing site age. These changes include: increasing tree height, the rapid increase then decrease in
126 shrub cover, and increasing litter cover, but also show reduced cover of ‘Herbs, sedges and grasses’ strata in all but one-year old rehabilitation vegetation (Fig. 5.3).
Comparison of Plant Species Cover and Species Assemblages
Principal coordinate analysis (PCOA) was performed separately for species cover (percentage cover for each plant species/transect) and species assemblage (presence/absence) data to examine clustering of rehabilitation area and forest transects. Species cover ordinations clustered rehabilitation transects and forest transects separately (Fig 5.4A). Species assemblage analysis found a single 25+ year old rehabilitation transect grouped with the forest transects indicating a slight overlap of rehabilitation and forest transects clusters (Fig 5.4B). No obvious trends were evident within rehabilitation data in either analysis (Fig 5.4A and B). Only paired rehabilitation area and unmined forest transects were included in the analyses.
Multivariate ANOVA using permutations (PERMANOVA) was then used to Comparison of plant species cover and plant species assemblage (presence or absence) of the rehabilitated landscape and unmined forest showed significant differences between rehabilitation area and unmined forest transects in both species cover and plant species assemblage data (P < 0.001) (Table 5.3). There were no significant differences in data homogeneity for either analysis (Table 5.3).
TABLE 5.3 Multivariate ANOVA table ( analysed using PERMANOVA) for comparisons of rehabilitation area and unmined forest transects. Plant species cover and plant species assemblage (presence or absence) were compared using data for all paired transects (n = 38). Rare plant species were omitted from analysis (N.B. non-rare orchids were included in the species total). Orchid species abundance1 was compared for all paired transects where both transects had a population of orchids (i.e. population was > 0) (n = 28). Data homogeneity for each analysis was compared using PERMDISP. (Anderson 2004). Note: PERMDISP. calculates an ANOVA on distances or similarities from points to their centroids.
Species Measure df SS MS F-value P P (MC)
Multivariate ANOVA Plant species cover1 1 17089.6204 17089.6204 9.2932 0.0001*** 0.0001*** Species assemblage2 1 1.5246 1.5246 5.6922 0.0001*** 0.0001*** Orchid species abundance1 1 18439.25 18439.25 7.2928 0.0001*** 0.0001***
Tests of heterogeneity in average similarities of points from their centroid Plant species cover1 1 0.7789 0.7789 0.0146 0.9063 - Species assemblage2 1 0.0014 0.0014 0.3938 0.5377 - Orchid species abundance1 1 285.3413 285.3413 2.2683 0.1428 -
1 2 ANOVA calculated on either Bray-Curtis or Jaccard similarities * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 127 A Forest R1 40 R5 R10 R15 30 R25+ Centroids
20 2 10 Axis
-30 -20 -10 10 20 30 40
-10
-20
B 0.4
0.3
0.2
0.1 2 Axis
-0.3 -0.2 -0.1 0.1 0.2 0.3 0.4
-0.1
-0.2
-0.3
Axis 1
FIGURE 5.4 Ordination by principal coordinates analysis (PCoA) of plant species data for rehabilitation area transects and their adjacent unmined forest transects. A) Species cover (percentage cover for each species/transect). Data were fourth root transformed prior to analysis and the resemblance matrix calculated using Bray-Curtis similarities. Percentage variation accounted for by first four axes are; Axis 1 = 13.75%, Axis 2 = 7.17%, Axis 3 = 6.68% and Axis 4 = 6.13%. B) Species assemblage. Data were presence/absence transformed and the resemblance matrix calculated using Jaccard similarities. Percentage variation accounted for by first four axes are; Axis 1 = 16.65%, Axis 2 = 7.93%, Axis 3 = 6.43% and Axis 4 = 6.04%. Centroids for rehabilitation and unmined forest data are indicated by crosses. Rehabilitation area transects are colour coded by age at sampling time; Legend R = rehabilitation area followed by a number representing age in years. 128 Comparison of orchid assemblages
Principal coordinate analysis (PCOA) was then performed using orchid species population data to determine if there were significant differences in orchid assemblages between the vegetation of rehabilitation areas and unmined forest sites. PCoA analyses of orchid population data and orchid species assemblage (presence/absence) data gave very similar results therefore only the population ordination is presented (Fig. 5.5). PCoA clustered rehabilitation transects and forest transects separately into two overlapping habitat-based groups (Fig. 5.5).
Multivariate ANOVA using permutations was then used to compare orchid species abundance for all paired transects where both transects had a population of orchids. A highly significant difference was found between rehabilitation area transects and unmined forest (P < 0.001) (Table 5.3). There was no significant difference in data homogeneity for orchid abundance analysis (P > 0.05) (Table 5.3). Therefore, the disturbance due to mining and the rehabilitation process has had a significant effect on orchid population size and composition.
Temporal Changes in Rehabilitation Vegetation
Multivariate ANOVA using permutations (PERMANOVA) was used to examine the temporal changes in vegetation structure (vegetation strata and soil surface cover), species cover and plant species assemblage (presence or absence) of rehabilitation transects compared to a random group of four unmined forest transects. First, a permutational multivariate ANOVA compared each group of forest transects (i.e. groups of forest transects adjacent to 1, 5, 10 or 15 year old rehabilitation). This was repeated for each set of data: that is; vegetation structure (vegetation strata and soil surface cover), species cover and plant species assemblage (presence or absence). No significant differences were found between vegetation structure, species cover or plant species assemblage (presence or absence) of the groups of forest transects (P > 0.05) (Table 5.4). Data homogeneity was not significantly different between groups of forest transects for vegetation structure (P > 0.05), but there were significant differences for species cover and plant species assemblage data (0.05 < P <0.01) (Table 5.4).
129 60 R F Centroids 40
20 2 0 -80 -60 -40 -20 0 20 40 60 Axis
-20
-40
-60 Axis 1
FIGURE 5.5 Ordination by principal coordinates analysis (PCoA) of orchid species abundance (i.e. number of plants per species per transect) for rehabilitation area transects and their adjacent unmined forest transects. Transects with no orchids (i.e. number of plants/transect = 0) and their adjacent forest or rehabilitation area transect were removed from the data before analysis. Data were log10(1+ n) transformed prior to analysis and the resemblance matrix calculated using Bray-Curtis similarities. Percentage variation accounted for by first four axes are; Axis 1 = 33.39%, Axis 2 = 16.48%, Axis 3 = 14.05% and Axis 4 = 12.70%. Centroids for rehabilitation and forest data are represented by crosses. Legend: R = rehabilitation; F = unmined forest.
Multivariate ANOVA using permutations (PERMANOVA) was carried out on: i) vegetation structure (vegetation strata and soil surface cover) data; ii) species cover data; and iii) plant species assemblage (presence or absence) data, for the temporal sequence of rehabilitation transects and a randomly selected group of unmined forest transects. There were highly significant differences associated with rehabilitation area age in each of the analyses: vegetation structure (vegetation strata and soil surface cover) (P < 0.001); species cover (P < 0.001) and plant species assemblage (presence or absence) (P < 0.001) (Table 5.4). However, there were also significant differences in data homogeneity for each of the analyses (0.01 < P < 0.05) (Table 5.4). Analysis of species cover and plant species assemblage (presence or absence) was repeated with each group of unmined forest transects because of the significant differences between the groups (0.01 < P <0.05). All analyses had highly significant differences associated with age rehabilitation areas (P < 0.001) (Table 5.4).
130 TABLE 5.4 Multivariate ANOVA (using PERMANOVA) table for comparisons of temporal changes in rehabilitation areas. Vegetation structural data1 (percentage cover of vegetation height categories), plant species cover1 (percentage cover) and plant species assemblage2 (presence or absence) of 1, 5, 10 and 15 year old rehabilitation area transects (for each age n = 4) were compared to a randomly selected group of forest transects (n = 4). Multivariate ANOVA was used to compare the groups of unmined forest transects paired with each age of rehabilitation area. No significant difference was found between groups of forest transects (P > 0.1). Percentage cover data were fourth root transformed prior to analysis. PERMANOVA required a balanced experimental design therefore 25+ year old rehabilitation area and adjacent unmined forest transects were compared in a separate analysis. Data homogeneity for each analysis was compared using PERMDISP. (Anderson 2004). (Note: for each multivariate ANOVA the analysis with the lowest F-value is presented)
Species Measure df SS MS F-value P P (MC) Temporal sequence of rehabilitation area and unmined forest transects
Multivariate ANOVA Vegetation structure 4 4501.9690 1125.4923 20.1928 0.0001*** 0.0001*** Plant species cover 4 17966.1038 4491.5260 3.7862 0.0001*** 0.0001*** Species assemblage 4 1.0859 0.2715 2.5661 0.0001*** 0.1452
Tests of heterogeneity in average similarities of points from their centroid Vegetation structure 4 54.1821 13.5455 5.0337 0.0104 - Plant species cover 4 360.6755 90.1689 4.5668 0.0172 - Species assemblage 4 0.0243 0.0061 3.1772 0.0429 -
25+ year old rehabilitation and adjacent unmined forest transects
Multivariate ANOVA Vegetation structure 1 113.3113 113.3113 1.8660 0.2928 0.1968✝ Plant species cover 1 2544.6581 2544.6581 1.7503 0.0983 0.1924✝ Species assemblage 1 0.1531 0.1531 1.2974 0.2053 0.3338✝
Tests of heterogeneity in average similarities of points from their centroid Vegetation structure 1 1.0382 1.0382 0.2643 0.5039 - Plant species cover 1 105.3858 105.3858 10.7378 0.0983 - Species assemblage 1 84.0120 84.0120 35.1285 0.0983 -
Groups of unmined forest transects3
Multivariate ANOVA Vegetation structure 3 124.1303 41.3768 0.5730 0.8493 0.8221 Plant species cover 3 4389.8698 1463.2899 0.8227 0.7374 0.6743 Species assemblage 3 3946.7888 1315.5963 0.8276 0.7097 0.6563
Tests of heterogeneity in average similarities of points from their centroid Vegetation structure 3 13.2496 4.4165 1.9650 0.1779 - Plant species cover 3 408.3708 136.1236 5.1783 0.0137 - Species assemblage 3 518.3824 172.7941 5.3627 0.0145 -
1 2 ANOVA calculated on either Bray-Curtis or Jaccard similarities; * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, ✝ for this analysis use P(MC) (the number of permutations is low (10)) 3 Note: The unmined forest transects were grouped based on the age of their adjacent rehabilitation areas.
131 The multivariate analysis comparison of the three 25+ year old rehabilitation area transects and their adjacent forest transects (omitted from previous analyses because of the requirements of PERMANOVA for a balanced design) showed no significant differences in: vegetation structure (vegetation strata and soil surface cover); species cover; or plant species assemblage (presence or absence) (P (MC) > 0.05) of the three 25+ year old rehabilitation area transects and their adjacent forest transects (Table 5.4). There were no significant differences in data homogeneity for 25+ year old rehabilitation area transects and their adjacent forest transects in any of the three analyses (Table 5.4).
Plant Species Richness A total of 246 plant taxa were recorded from the 40 transects, representing 51 families and 90 genera. Of these, 240 taxa were identified to species and six unidentified taxa to genus. A total of 198 and 177 taxa were identified in unmined forest transects and rehabilitation area transects respectively. Plant taxa were grouped into growth habit categories to examine differences in the prevalence of species with particular growth forms in rehabilitation areas and unmined forest. The most species-rich families in the growth habit categories: Herbs and grasses; Ferns; Cycads; Trailing and climbing shrubs; Shrubs; Grass trees; Trees; Alien herbs and grasses; and Alien trees and shrubs are summarised in Table 5.5. The greatest difference between rehabilitation areas and unmined forest are in the lower number of taxa in the category ‘Herbs and grasses’ and the higher numbers of indigenous and alien trees taxa in rehabilitation areas (Table 5.5). The most species-rich families amongst the indigenous flora were, Papilionaceae (19 spp), Orchidaceae (18 spp), Proteaceae (18 spp), Myrtaceae (17 spp), Mimosaceae (15 spp) and Asteraceae (13 spp). Complete lists of indigenous and alien taxa identified in survey transects are contained in Appendix 2, Tables A2.1 and A2.2 respectively.
132 TABLE 5.5 Comparison of the most species-rich plant families in unmined forest, rehabilitation areas (combined) and across a temporal sequence of rehabilitation areas. Data are from surveys undertaken at the Jarrahdale bauxite-mine site during autumn and spring 2002. Plants are listed by growth form category, then by most species-rich family in unmined forest. The growth form category ‘Herbs and grasses’ includes all low growing cormatous, rhizomatous and tuberous geophytes. ‘Common’ refers to taxa present in both unmined forest and rehabilitation areas. The category ‘Alien’ refers to taxa not indigenous to Western Australia.
Growth Form Total Number of Taxa Age of Rehabilitation Area (years) Major Plant Families Forest Rehab. Common 1 5 10 15 >25
Indigenous Taxa Herbs and grasses Orchidaceae 13 12 7 0 5 6 8 8 Asteraceae 12 8 7 6 3 5 2 3 Dasypogonaceae 10 5 5 1 4 4 3 5 Poaceae 9 7 7 4 4 0 4 4 Cyperaceae 6 4 4 1 4 1 4 2 Restionaceae 3 1 1 1 0 0 0 0 Other Families 46 36 30 17 19 15 17 15 Total 99 73 61 30 39 31 38 37 Ferns Dennstaedtiaceae 1 0 0 0 0 0 0 0 Total 1 0 0 0 0 0 0 0 Cycads Macrozamiaceae 1 1 1 1 1 1 1 0 Total 1 1 1 1 1 1 1 0 Trailing and Climbing Shrubs Papilionaceae 3 3 3 2 3 3 2 2 Ranunculaceae 1 1 1 0 0 0 0 1 Total 4 4 4 2 3 3 2 3 Shrubs Papilionaceae 13 12 10 5 10 8 8 7 Mimosaceae 9 11 7 7 7 6 8 3 Proteaceae 9 9 4 3 3 2 6 1 Dilleniaceae 7 8 7 1 4 5 5 3 Epacridaceae 6 5 5 0 1 1 3 3 Myrtaceae 4 8 2 1 0 3 6 0 Other Families 20 14 12 9 9 7 9 7 Total 68 67 47 26 34 32 45 24 Grass trees Xanthorrhoeaceae 2 1 1 0 1 0 1 0 Total 2 1 1 0 1 0 1 0 Trees Myrtaceae 3 6 2 3 3 4 4 3 Proteaceae 3 3 2 1 2 1 1 2 Casuarinaceae 1 1 1 0 1 1 1 0 Mimosaceae 1 2 1 2 1 1 1 0 Total 8 12 6 6 7 7 7 5 Total Number of Indigenous Taxa 183 158 120 65 85 74 94 69
Alien Taxa Herbs and grasses Poaceae 5 3 3 1 1 2 1 2 Asteraceae 4 5 2 4 4 1 1 2 Orchidaceae 0 1 0 0 1 1 1 0 Other Families 4 5 2 4 0 1 2 1 Total 13 14 7 9 6 5 5 5 Trees and shrubs Myrtaceae 1 3 1 0 0 0 3 1 Mimosaceae 1 1 1 1 0 0 1 1 Total 2 5 2 0 0 0 4 2 Total Alien Taxa 15 19 9 10 6 5 9 7
Total Number of Plant Taxa 198 177 129 75 91 79 103 76
133 Comparison of Plant Species Assemblages
TWINSPAN classification of transect sites was carried out on presence/absence data for
201 plant species (see Appendix 2.3). The first TWINSPAN division separated transects into a group consisting of rehabilitation area transects ≤ 15 years old and another of all forest and 25+ year old rehabilitation transects. The second division of the ≤ 15 year old rehabilitation group separated all 15 year old transects and one 10 year old transect from the remaining 1, 5 and 10 year old rehabilitation area transects. In the forest group, four forest transects containing plant species consistent with recent disturbance were separated at the second division. Transects were classified into six major site groups at the third division. These terminal groups are described briefly in Fig. 5.6. Group 1 contains 25+ year old rehabilitation transects, the adjacent forest transects and one of the C. ovata transects (Cov-2), Group 2 unmined forest transects and Group 3 the disturbed forest transects. Groups 4, 5 and 6 contain only rehabilitation area transects and are divided roughly by age with the exception of 10 year old transects which are divided amongst the three groups (Fig. 5.6).
Plant species were grouped into six species assemblages at the third TWINSPAN division. These are described briefly in Table 5.6. The column labelled Transect Groups lists those site groups where a particular species assemblage predominates. Species assemblages in Groups 1 and 2 were associated exclusively with transects grouped into the first site division, that is unmined forest and 25+ year old rehabilitation transects, and Group 6 exclusively with rehabilitation area transects ≤ 15 years. The other species assemblages are spread across both rehabilitation and forest site groups. Characteristic orchid species for the six species assemblages are also listed. Five orchid species previously found to be absent from the rehabilitation areas; Cryptostylis ovata, Cyrtostylis huegelii, Eriochilus sp., Pyrorchis nigricans and Thelymitra crinita, are present in species assemblage designated Group 1 (Chapter 2, Table 2.3). This species assemblage is associated exclusively with site Groups 1, 2 and 3, although the indicator species are absent from the three 25+ year old rehabilitation transects in site Group 1. The disturbance opportunists Disa bracteata and Microtis media are associated with species Group 6, a species assemblage associated exclusively with rehabilitation areas > 1 year old and ≤ 15 years old.
To examine more closely the relationship between species assemblages and individual orchid taxa the TWINSPAN classification was repeated using data for the ten contiguous
134 quadrats that made up each belt transect (i.e. the ‘microhabitat’ scale). Rehabilitation areas and unmined forest were analysed separately to determine if the relationship between orchids and plant species assemblages were similar in both habitats. The resulting species assemblages are described in Tables 5.7 and 5.8 respectively. Classification of rehabilitation vegetation was stopped at the second division. Further divisions separated groups of plants containing few species and with few occurrences from the main species assemblages, which provide little useful information.
Rehabilitation areas and unmined forest contained three and five species assemblages respectively. Rehabilitation species assemblages were well defined with indicator species present in almost all sites (quadrats) that grouped together. Management practices at the time of establishment had a strong impact on species assemblages. The first species assemblage consisted of a group of taxa that were unique to a single 25+ year old transect. This site was revegetated mainly with non-indigenous tree species but was also one of the earliest rehabilitation sites to have a substantial understorey (Koch pers. comm. 2002). The second species assemblage was present only in 15 year old sites, reflecting changes Alcoa made to the species mix used in its rehabilitation processes in 1988 (15 year old sites were established in 1987) (Grant and Koch 2007). The third species assemblage was found in the remaining rehabilitation areas and was characterised by the indigenous disturbance opportunist species Acacia extensa Lindl., A. lateriticola Maslin, A. pulchella R.Br and Mirbelia dilatata R.Br. Alien herbs and grasses (i.e. weeds) were also common in quadrats containing this species assemblage indicating that the disturbance due to mining facilitates invasion by weed species.
Forest species assemblages were generally less strongly defined than rehabilitation species assemblages as none of the species identified as a potential indicator species was present in all sites that grouped together. Often transects contained more than one species assemblage, and roughly 20% of quadrats contained none of the identified assemblages. Rehabilitation vegetation thus appears to be far more homogeneous than that of unmined forest when examined at this ‘microhabitat scale’.
Rare species (i.e. species present in only one transect) were excluded from analyses of species number and cover. There were 54 rare species observed in survey transects, 29 in rehabilitation areas and 25 in unmined forest. Two-way ANOVA was performed on the number of rare species/transect to compare paired rehabilitation areas and unmined
135 136
40
Bossaea aquafolia 24 16 Acacia extensa Acacia lateriticola Thelymitra crinita (-)
Hibbertia amplexicaulis Kennedia coccinea 20 4 5 11 Aira cupaniana (-) Stylidium hispidum (-)
Lechenaultia biloba (-) Platysace tenuissima 7 13 6 5 Loxocarya cinerea (-) dead Acacia lateriticola (-) Stypandra glauca (-) dead Acacia drummondii (-)
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 F25+-1, -2, -3 F1-1, -2, -3 F1-4 R10-3 R5-1, -2, -3, -4 R1-1, -2, -3, -4 R25+-1, -2, -3 F5-1, -2, -3, -4 F10-1, -3 R15-1, -2, -3, -4 R10-1, 2 R10-4 Cov-2 F10-2, -4 F15-1 F15-2, -3, -4 Cov-1
FIGURE 5.6 Dendrogram of TWINSPAN site classification of 40 transects x 201 plant species using presence/absence data for vegetation of Alcoa’s Jarrahdale mine- site (i.e. ‘vegetation type’ scale classification). Living and dead plants of short-lived leguminous species were recorded as separate species. Rare species were removed from the data before analysis. Indicator species and the number of transects are shown at each division. Transects are listed below group numbers. Transects are labelled either R for rehabilitation area or F for unmined forest, followed by the age of the rehabilitation area in years (1, 5, 10, 15 or 25+) and replicate number (1-4), for example F1-4 is a transect in unmined forest adjacent to the fourth replicate site of 1 year old rehabilitation. Cov-1 and Cov-2 are two transects placed through populations of Cryptostylis ovata. (see Appendix 2; Fig. A2.3 for TWINSPAN output matrix) TABLE 5.6 TWINSPAN classification of plant species assemblages at Alcoa’s Jarrahdale mine-site using presence/absence data for 40 transects x 201 plant species (i.e. ‘vegetation type scale’ classification). Living and dead plants of short-lived leguminous species were recorded as separate species. Rare species were removed from the data before analysis. Indicator species are in bold. Site groups (Fig 3.5) that contain the particular plant assemblages are listed in the column labelled Transect Groups.
TWINSPAN Dendrogram** Group*** Plant Species Present Transect Groups Orchid Species Present
Group 1 Astroloma ciliatum, Burchardia sp. Clematis pubescens, Dryandra lindleyana, 1, 2 and 3 Cryptostylis ovata, Cyrtostylis huegelii, Eriochilus 0.185 n = 50 Eriochilus sp., Isotoma hypercrateriformis, Lechenaultia biloba, Leucopogon (forest and rehab. sp., Pterostylis vittata, Pyrorchis nigricans, capitellatus, L. propinquus, L. verticillatus, Patersonia babianoides, Thelymitra 25+years old) Thelymitra crinita crinita, Trichocline spathulata, Xanthorrhoea gracilis Group 2 Adenanthos barbigerus, Astroloma pallidum, Austrodanthonia pilosa, 1 and 2 Thelymitra macrophylla 0.376 n = 28 Conostylis serrulata, C. setosa, Dampiera linearis, Dianella revoluta var. (forest) divaricata, Dichelachne crinita, Hibbertia commutata, Hovea chorizemifolia, Lagenifera heugelii, Lepidoserma squamatum, Levenhookia preissii, Lomandra hermaphrodita, Lomandra spartea. Loxocarya cinerea, Patersonia occidentalis, Persoonia longifolia, Stylidium amoenum, Stypandra glauca, Tetraria capillaris, Tetrarrhena laevis, Xanthorrhoea preissii Group 3 Boronia fastigata, Cyanicula sericea, Lomandra micrantha, Lomandra sonderi, 1, 2 and 5 Caladenia flava, Cyanicula sericea, Diuris n = 35 Tetratheca setigera, Thysanotus thyrsoideus, Acacia alata, Amphipogon (forest, 5 and 10 year brumalis, Pterostylis barbata amphipogonoides, Banksia grandis, Caladenia flava, Cyathochaeta avenacea, old rehab. ) Diuris brumalis, Gompholobium polymorphum, G. preissii, Hibbertia acerosa, 0.784 H. amplexicaulis, Lepidosperma leptostachyum, Leucopogon nutans, Macrozamia riedlyei, Pentapeltis peltigera, Phyllanthus calycinus, Platysace compressa, Scaevola calliptera, Stylidium hispidum, Trachymene pilosa, Xanthosia candida Group 4 Acacia pulchella (dead), Corymbia calophylla, Hyalosperma cotula, 1, 2, 5 and 6 Pterostylis sp. crinkled leaf, P. recurva, Thelymitra n = 28 Opercularia echinocephala, Senecio quadridentus, Trymalium ledifolium var (forest, 1, 5 and 10 year benthamiana rosmarinifolium, Allocasuarina fraserii, Bossiaea ornata, Eucalyptus old rehab. ) marginata, Lasiopetalum floribunda, Pterostylis sp. crinkled leaf, P. recurva, 0.432 Thysanotus manglesianus, T. multiflorus Group 5 *Aira cupaniana, *Hypochaeris glabra, Acacia pulchella, A. saligna, 3, 5 and 6 Orchids absent n = 23 Gnaphalium sphaericium, Gompholobium knightianum, G. marginatum, Hakea (5, 10 year old rehab. lissocarpha, Hypocalymma angustifolium, Kennedia coccinea, K. prostrata, and degraded forest) 0.198 Mirbelia dilatata, Group 6 Acacia celastrifolia, Acacia drummondii, Acacia extensa, A. extensa (dead), 4, 5 and 6 Disa bracteata, Microtis media, Pterostylis n = 38 Acacia lateriticola, Acacia urophylla, Bossiaea aquifolium, Disa bracteata, (rehab. ≤ 15 years old) sanguinea Eucalyptus patens, Sollya heterophylla, Viminaria juncea 137 * Indicates a taxa not indigenous to Western Australia; ** Numbers on TWINSPAN dendrogram are Eigenvalues for each division.; *** n = the number of plant species in each group. 138 TABLE 5.7 TWINSPAN classification of plant species assemblages in rehabilitation areas at Alcoa’s Jarrahdale mine-site using presence/absence data for 190 quadrats x 154 plant species (i.e. ‘microhabitat scale’ classification). Living and dead plants of short-lived leguminous species were recorded as separate species. Rare species were removed from the data before analysis. Indicator species are in bold. Transects containing the particular plant assemblages are listed in the column labelled Occurrence.
TWINSPAN Dendrogram ** Group*** Plant Species Present Occurrence Orchid Species Present
Group 1 Adenanthos barbigerus, *Acacia decurrens, Austrodanthonia pilosa, Bossiaea R25+-1 Caladenia flava, Diuris brumalis, Microtis media, n = 22 aquifolium, Caladenia flava, Diuris brumalis, *Eucalyptus resinifera, Pterostylis vittata Lepidosperma squamatum, Lomandra micrantha, Lomandra, sonderi, Lomandra spartea. Microtis media, Persoonia longifolia, Platysace compressa, Tetraria capillaries
0.372 Group 2 *Aira cupaniana, *Lotus angustissimus, Acacia celastrifolia, A. celastrifolia R15-1, 2, 3 and 4 Caladenia macrostylis, Cyanicula sericea, Disa n = 59 (dead), A. drummondii, *A. longifolia, A. saligna, A. urophylla, Allocasuarina R10-1 and 2 bracteata, Pterostylis sp. crinkled leaf, P. barbata, fraserii, Amphipogon amphipogonoides, Boronia fastigata, Calothamnus P. recurva, P. sanguinea rupestris, Disa bracteata, Corymbia calophylla, E. diversifolia, Eucalyptus marginata, *E. muelleriana, E. patens, Hardenbergia comptoniana, Hibbertia acerosa, H. amplexicaulis, Hyalosperma cotula, Hypocalymma angustifolium, Kennedia prostrata, Lagenifera heugelii, Leucopogon nutans, Levenhookia pusilla, Macrozamia riedlyei, Opercularia echinocephala, Pentapeltis peltigera, 0.167 Phyllanthus calycinus, Pterostylis recurva, Sollya heterophylla, Stylidium hispidum, Stypandra glauca, Thomasia glutinosa, Xanthosia candida
Group 3 *Conyza bonariensis, *Dittrichia graveolens, *Hypochaeris glabra, *Juncus R1-1, 2, 3 and 4 Thelymitra macrophylla, T. benthamiana n = 73 bufonus, *Polycarpon tetraphyyllum, *Sonchus asper, *Vellerophyton R5-1, 2, 3 and 4 dealbatum, Acacia alata, A. drummondii (dead), A. extensa, A. extensa (dead), R10-1, 2, 3 and 4 A. lateriticola, A. lateriticola (dead), A. pulchella, A. pulchella (dead), Banksia R15-3 grandis, Bossiaea ornata, Conostylis setosa, Cyathachaeta avenacea, Daucus R25+-2 and 3 glochidiatus, Dichelachne crinita, Eucalyptus marginata subsp. thalassica, Gnaphalium sphaericium, Gompholobium knightianum, G. marginatum, Hakea lissocarpha, H. undulata, Hibbertia commutata, Hovea chorizemifolia, H. trisperma, Kennedia coccinea, Lasiopetalum floribunda, Lepidosperma leptostachyum, Lomandra integra, Mirbelia dilatata, Mirbelia dilatata (dead), Platysace tenuissima, Scaevola calliptera, Senecio quadridentus, Tetrarrhena laevis, Thelymitra macrophylla, T. benthamiana, Thysanotus multiflorus, T. patersonii, Trachymene pilosa, Trymalium ledifolium var rosmarinifolium, Viminaria juncea, Waitzia paniculata * Indicates a taxa not indigenous to Western Australia; ** Numbers on TWINSPAN dendrogram are Eigenvalues for each division.; *** n = the number of plant species in each group. TABLE 5.8 TWINSPAN classification of plant species assemblages in unmined forest areas at Alcoa’s Jarrahdale mine-site using presence/absence data for 210 quadrats x 166 plant species (i.e. ‘microhabitat scale’ classification). Living and dead plants of short-lived leguminous species were recorded as separate species. Rare species were removed from the data before analysis. Indicator species are in bold. Transects containing the particular plant assemblages are listed in the column labelled Occurrence.
TWINSPAN Dendrogram ** Group*** Plant Species Present Occurrence Orchid Species Present
Group 1 Conostylis serrulata, Cyathochaeta avenacea, Dampiera linearis, Loxocarya F1-1, 3, 4 Pyrorchis nigricans, Thelymitra crinita n = 13 cinerea, Pyrorchis nigricans, Stylidium hispidum, Thelymitra crinita, Waitzia F10 -1, 3 0.460 paniculata, Cov-1 0.372 *Aira cupaniana, *Hypochaeris glabra, Acacia pulchella, A. pulchella (dead), F1-1, 2 Caladenia flava Baeckea camphorosmae, Caladenia flava, Dryandra lindleyana, Corymbia F5-1, 2, 4 calophylla, Gompholobium marginatum, Grevillea wilsonii, Hakea lissocarpha, F10-2 Group 2 Hibbertia acerosa, H. hypericoides, Hyalosperma cotula, Hypocalymma F15-2, 3, 4 n = 59 angustifolium, Hypolaena exsulca, Isotoma hypocrateriformis, Levenhookia Cov-2 preissii, Lepidoserma squamatum, Leucopogon propinquus, Mesomelaena tetragona, Mirbelia dilatata, Phyllanthus calycinus, Ptilotus manglesii, Senecio 0.733 quadridentus, Stylidium repens, Trachymene pilosa, Trymalium ledifolium var 0.372 rosmarinifolium, Xanthorrhoea preissii Group 3 Allocasuarina fraserii, Bossiaea ornata, Eriochilus sp.,Conostylis setosa, F1-3 Eriochilus sp., Pterostylis recurva n = 18 Gompholobium preissii, Lagenifera heugelii Lepidosperma leptostachyum, F5-1, 3 Leucopogon capitallis. L. nutans, Lomandra hermaphrodita, Opercularia F10 -1, 4 echinocephala, Patersonia occidentalis, Pterostylis recurva, Thomasia glutinosa, F25+-1 Trichocline spathulata, Thysanotus manglesianus, Xanthorrhoea gracilis Cov1 Acacia alata, Adenanthos barbigerus, Amphipogon amphipogonoides, Cyanicula F1-3 Cyanicula sericea, Cryptostylis ovata, Pterostylis Group 4 sericea, Cryptosylis ovata, Banksia grandis, Boronia fastigata, Gompholobium F5-1, 4 barbata 0.204 polymorphum, Hibbertia commutata, Hovea chorizemifolia, Labichea punctata, F10-1, 3 0.372 n = 44 Lechenaultia biloba, Lomandra drummondii, L. sonderi, L. spartea. Patersonia F15-1 babianoides, Pentapeltis peltigera, Persoonia longifolia, Poranthera heugelii, F25+ -1, 2 Stylidium amoenum, Stypandra glauca, Tetraria capillaris, Tetrarrhena laevis Tetratheca setigera, Thysanotus multiflorus, T. patersonii, T. thyrsoides, 0.164 0.372 Group 5 Burchardia sp., Clematis pubescens, Cyrtostylis huegelii, Dichelachne crinita, F1 –1, 2, 4 Cyrtostylis huegelii, Pterostylis sp. crinkled leaf, P. n = 32 Eucalyptus marginata, Hibbertia amplexicaulis, H. perfoliata, Kennedia F10 -2 vittata, Thelymitra macrophylla coccinea, Lasiopetalum floribunda, Leucopogon verticillatus, Logania F25+ -1, 2, 3 serpyllifolia, Lomandra micrantha, Macrozamia riedlyei Platysace compressa, Cov-2 Pteridium esculentum, P. esculentum (dead), Pterostylis vittata, Scaevola calliptera, Xanthosia candida
139 * Indicates a taxa not indigenous to Western Australia; ** Numbers on the TWINSPAN dendrogram are Eigenvalues for each division.; *** n = the number of plant species in each group. forest sites (Appendix 2, Figure A2.2). No significant differences were found (P > 0.05). Though not statistically significant the number of rare species/transect was highest in 15 year old rehabilitation areas and the forest adjacent to 25+ rehabilitation sites. Only eight of these rare species were also alien species (i.e. weeds); six in rehabilitation areas and two in unmined forest.
Examination of Predictors of Variation in Multivariate Vegetation Data Most of the indigenous plant taxa associated with the differences in structural characteristics between forest and rehabilitation areas were either, short-lived leguminous shrubs belonging to Papilionaceae and Mimosaceae, rhizomatous and tufted herbs in the Cyperaceae, Dasypogonaceae, Restionaceae or low growing shrubs in Epacridaceae and Proteaceae (see Table 5.4 and Appendix 2; Table A2.2 and A2.2).
DISTLM forward (Anderson 2003) was used to carry out multivariate regression analysis to determine if particular categories of plant taxa could be useful in explaining temporal changes in transect structural cover data. The cover categories: Alien herbs and grasses, Legumes (living), Legumes (dead), Lomandra and sedge-like, Epacridaceae and Total (all species) were all highly significant (P < 0.01) predictors of variation in the vegetation structural data (Table 5.9). The species richness categories: Alien herbs and grasses, Legumes (living), Legumes (dead), Lomandra and sedge-like, and Epacridaceae were also all highly significant (P < 0.01) as predictors of vegetation structural data. The cumulative cover categories and species richness categories account for over 58 % and 54 %, respectively, of the variability in the transect vegetation strata cover data (Table 5.9).
Orchid species richness; the number of clonal orchids present, and one orchid species Eriochilus sp., also have potential as predictors of vegetation cover (P < 0.05) (Table 5.10). The cumulative contribution for orchid species richness and growth habit categories accounted for 24% of the variability in the transect vegetation cover data. The cumulative contribution for the abundance of all orchid species populations was 45% (Table 5.10).
140 TABLE 5.9 Selected plant cover and species richness categories as predictors of variability of transect multivariate cover data used in ordination by nMDS (see Fig.3.1). The ‘Legume’ categories include all taxa in Caesalpiniaceae, Mimosaceae and Papilionaceae. ‘Lomandra and sedge-like’ category includes all taxa in Centrolepidaceae, Cyperaceae, Dasypogonaceae and Restionaceae. Numbers are pseudo F-values with associated P-value and percentage of variation in multivariate cover data attributable to each plant category (Note: the pseudo F-value is a multivariate analogue to the F-value). Only plant categories and species richness indices that had significant correlations with either cover or species richness data are shown.
Cover Category pseudo-F P Proportion
Marginal tests Alien herbs and grasses1 4.7654 0.0038** 0.1114 Legumes (living) 9.9564 0.0001*** 0.2076 Legumes (dead) 10.5904 0.0001*** 0.2180 Lomandra and sedge-like 8.2092 0.0002*** 0.1777 Epacridaceae 5.5149 0.0027** 0.1267 Total (all species) 4.7125 0.0049** 0.1103
Conditional (sequential) tests Cumulative proportion of variation in data accounted for: 0.5865
Species Richness Category pseudo-F P Proportion
Marginal tests Alien herbs and grasses1 6.6167 0.0015** 0.1483 Legumes (living) 20.7124 0.0001*** 0.3528 Legumes (dead) 11.697 0.0001*** 0.2354 Lomandra and sedge-like 4.6904 0.0078** 0.1099 Epacridaceae 9.0668 0.0002*** 0.1926 Total 0.2248 0.8691 0.0059
Conditional (sequential) tests Cumulative proportion of variation in data accounted for: 0.5490
1 ‘Alien’ refers to plants that are not indigenous to Western Australia. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001
141 TABLE 5.10 Orchid species richness, abundance of orchids in the growth habit categories: clonal; non-clonal; and disturbance opportunists, and abundance of individual species as predictors of variability of multivariate cover data used in ordination by nMDS (see Fig.3.1). Numbers are pseudo F-values with associated P-value and percentage of variation in multivariate cover data attributable to each plant category. Orchid growth habit categories are described in Chapter 2.
Orchid Category pseudo-F P Proportion
Marginal tests Orchid species richness 3.7180 0.0192* 0.0891 Clonal 2.9993 0.0426* 0.0732 Non-clonal 1.0224 0.3770 0.0262 Disturbance opportunists 2.1664 0.1196 0.0539
Conditional (sequential) tests Cumulative proportion of variation in data accounted for: 0.2469
Orchid Species Abundance pseudo-F P Proportion
Marginal tests Caladenia flava 1.6642 0.1928 0.0420 Cyanicula sericea 2.3785 0.0880 0.0589 Cryptostylis ovata 1.9937 0.1412 0.0499 Cyrtostylis huegelii 1.1437 0.3228 0.0292 Disa bracteata 2.0311 0.1359 0.0507 Diuris brumalis 0.4452 0.6796 0.0116 Eriochilus sp. 2.9441 0.0473* 0.0719 Microtis media 1.9817 0.1199 0.0496 Pterostylis sp. crinkled leaf 0.3785 0.7749 0.0099 Pterostylis barbata 0.8746 0.4326 0.0225 Pterostylis recurva 0.4085 0.7398 0.0106 Pterostylis vittata 2.646 0.0617 0.0651 Pterostylis sanguinea 2.1917 0.0918 0.0545 Pyrorchis nigricans 1.2279 0.2993 0.0313 Thelymitra macrophylla 0.2057 0.9111 0.0054 Thelymitra crinita 2.1368 0.1097 0.0532 Thelymitra benthamiana 0.3257 0.7625 0.0085
Conditional (sequential) tests Cumulative proportion of variation in data accounted for: 0.4521
* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001
142 Multivariate regression analysis was then carried out using those plant cover and species richness categories that were significant predictors of transect multivariate vegetation cover to determine if they were also useful as predictors of orchid species richness and the populations of orchid growth habit categories. Total litter cover, that is the sum of leafy and woody litter cover, was included in the analysis. The two cover categories; Alien herbs and grasses (P < 0.05) and Total litter (P < 0.001) appear to have the greatest potential as predictors of orchid species richness and orchid growth habit categories data (Table 5.11). The species richness categories; Legumes (living), Lomandra and sedge-like, and Epacridaceae were all significant (P < 0.05), and Alien herbs and grasses was highly significant (P < 0.01), and therefore appear to have potential as predictors of multivariate orchid category population size data (Table 5.11). The combined cover categories and species richness categories accounted for 50% and 35% respectively, of the variability in orchid species richness and orchid growth habit categories data (Table 5.11). A single cover category, Total litter accounted for almost 37% of the variability in orchid abundance data.
143 TABLE 5.11 Selected plant cover and species richness categories as predictors of variability of transect multivariate orchid category data. The ‘Legume’ categories include all taxa in Caesalpiniaceae, Mimosaceae and Papilionaceae. ‘Lomandra and sedge-like’ category includes all taxa in Centrolepidaceae, Cyperaceae, Dasypogonaceae and Restionaceae. Total litter is the sum of al leafy litter (leaves and small twigs) and woody litter (branches > 2.5cm diameter) and cover can be > 100%. Numbers are pseudo F-values with associated P-value and percentage of variation in multivariate cover data attributable to each plant category. Note: only total litter cover and those plant cover or species richness categories with significant correlations with transect cover data were examined (Table 3.9).
Cover Category pseudo-F P Proportion
Marginal tests Alien herbs and grasses1 4.4697 0.0353* 0.1052 Legumes (living) 2.2568 0.0821 0.0561 Legumes (dead) 1.8624 0.1022 0.0467 Lomandra and sedge-like 1.7211 0.1530 0.0433 Epacridaceae 2.0637 0.0988 0.0515 Total cover 1.7889 0.1459 0.0450 Total litter 22.13 0.0002*** 0.3680
Conditional (sequential) tests Cumulative proportion of variation in data accounted for: 0.5013
Species Richness Category pseudo-F P Proportion
Marginal tests Alien herbs and grasses1 5.2443 0.0074** 0.1213 Legumes (living) 2.8638 0.0464* 0.0701 Legumes (dead) 2.2434 0.0802 0.0557 Lomandra and sedge-like 3.1913 0.0348* 0.0775 Epacridaceae 3.6596 0.0223* 0.0878 Total species richness 0.7008 0.5352 0.0181
Conditional (sequential) tests Cumulative proportion of variation in data accounted for: 0.3514
1 ‘Alien’ refers to plants that are not indigenous to Western Australia. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001
144 Visualization of Plant Cover and Species Richness Categories Values for significant plant cover and species richness categories were superimposed over transect vegetation strata and soil surface cover nMDS ordination to visualise trends and the examine the potential of the predictor variables as indicators of recovery (Figs 5.7A - F and Figs 5.8A - E). Cover from alien herbs and grasses is highest in 1 and 5 year old rehabilitation areas but the number of alien species present does not appear to be related to rehabilitation age (Fig. 5.7A). It is evident that high cover by living legumes separates rehabilitation areas from unmined forest, while dead legume cover is highest in 5 to 15 year old rehabilitation areas (Figs 5.7B and C). The number of legume species is also higher in high legume cover areas. Both legume cover and species number were good indicators of recent disturbance (Figs 5.8B and C). The amount of cover from ‘Lomandra and sedge-like species’ appears to be an indicator of lack of disturbance as it is highest in unmined forest. The number of ‘Lomandra and sedge-like species’ is lowest in two transect clusters; that containing only 1 year old transects and that containing 10 and 15 year old transects (Figs 5.7D and 5.8D). Cover from Epacridaceae is generally low at all sites but the number of species is higher in unmined forest and older rehabilitation sites (Figs 5.7E and 5.8E).
Visualization of Distribution of Orchid Populations Orchid population sizes for the categories, Orchid species richness, Clonal, Non-clonal and Disturbance opportunists were superimposed over vegetation strata cover nMDS ordination to visualise possible trends and their potential value as predictor variables (Figs 5.9A - D). It is evident that the largest populations of clonal species are present in 25+ year old rehabilitation areas and unmined forest transects (Fig. 5.9B), and that disturbance opportunists are present only in rehabilitation areas that have been established for five years or more (Fig. 5.9D). Orchid population sizes for species that were observed in more than six transects (Caladenia flava, Cyanicula sericea, Disa bracteata, Eriochilus sp., Microtis media, Pterostylis sp. crinkled leaf, Pterostylis recurva and Thelymitra crinita) were also superimposed over vegetation strata cover nMDS ordination (Fig. 5.10A - H). These superimpositions reveal that D. bracteata was found exclusively in rehabilitation area transects between 5 and 15 years old (Fig. 5.10C), M. media in rehabilitation area transects between 5 and 25 years old (Fig. 5.10E) and Eriochilus sp. and T. crinita exclusively in unmined forest transects (Figs 5.10D and H). The remaining four species were distributed across both rehabilitation
145 and unmined forest sites (Figs 5.10A, B, F and G). There was also a relationship between litter and the presence of orchids (Fig. 5.11).
Potential Indicators
Two-way ANOVA was performed on data for cover and species richness categories that were significant predictors of data dispersion and appeared to change with rehabilitation area age. These were the cover and species richness categories: Legumes (living) and Lomandra and sedge-like. All rehabilitation areas had significantly higher ‘Legumes (living)’ cover than adjacent forest (Fig. 5.12A) but, both 10 year old and 25+ year old rehabilitation areas were significantly lower than other rehabilitation areas. Cover for ‘Lomandra and sedge-like’ species increased very slowly with time, and was very low in all rehabilitation areas (Fig. 5.12B), and was always significantly lower than adjacent unmined forest. Regression analysis gave an estimate of 297 years for recovery of ‘Lomandra and sedge-like’ cover in rehabilitation areas (see Appendix 2; Fig. A2.3).
Species richness for the Legumes (living) category decreased in rehabilitation areas over 10 years old, with the value for 25+ year old rehabilitation area not significantly different to for unmined forest (Fig. 5.12C). Legumes (living) species richness of all rehabilitation areas except 5 year old and 25+ year old rehabilitation areas were significantly different to the adjacent forest. The number of species in the category ‘Lomandra and sedge-like’ generally increased with time and all rehabilitation areas except 5 year old and 25+ year old rehabilitation areas were significantly different to unmined forest (Fig. 5.12D).
146 % Cover R1 A. Alien herbs & 6 R1 grasses R1R1
24
F 42 F F Cov F F F F FFF F F F F F F 60 F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
B. Legumes (living) R1
R1 R1 R1
F F F Cov F F F F FFF F F F F F F F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
C. Legumes (dead) R1 R1 R1R1
F F F Cov F F F F FFF F F F F F F F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5
R15 R15 R10 R15
FIGURE 5.7 Values for plant cover categories superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). Only cover categories with potential as predictors of multivariate transect cover data (P < 0.05) are shown; A. Alien herbs and grasses, B. Legumes (living), C. Legumes (dead), D. Lomandra and sedge-like, E. Epacridaceae, and F. Total cover (sum of cover for all plant species). The ‘Legume’ categories include all taxa in Caesalpiniaceae, Mimosaceae and Papilionaceae. ‘Lomandra and sedge-like’ category includes all taxa in Centrolepidaceae, Cyperaceae, Dasypogonaceae and Restionaceae. The scale is from 0% to 60% cover for all maps except F. where the scale is 0% to 150%.
147 % Cover R1 D. Lomandra & 6 R1 sedge-like R1R1
24
F 42 F Cov F F F F F FFF F F F F F F 60 F CovR25+F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
E. Epacridaceae R1 R1 R1R1
F F F Cov F F F F FFF F F F F F F F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
% Cover F. Total cover R1 15 R1 R1R1
60
F 105 F F Cov F F F F FFF F F F F F F 150 F CovR25+F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
FIGURE 5.7 contd. Values for plant cover categories superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). D. Lomandra and sedge-like, E. Epacridaceae, and F. Total cover (sum of cover for all plant species). ‘Lomandra and sedge-like’ category includes all taxa in Centrolepidaceae, Cyperaceae, Dasypogonaceae and Restionaceae. The scale is from 0% to 60% cover for all maps except F. where the scale is 0% to 150%. Note: ‘Total cover’ is the sum of the cover for all plant species and includes plants of different heights and therefore can exceed 100%.
148 No. Species R1 A. Alien herbs & 2 R1 grasses R1R1
8
F F F Cov 14 F F F F FFF F F F F F F F R25+ Cov F 20 R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
B. Legumes (living) R1 R1 R1R1
F F F Cov F F F F FFF F F F F F F F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
C. Legumes (dead) R1 R1 R1R1
F F F Cov F F F F FFF F F F F F F F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
FIGURE 5.8 Values for plant species richness categories superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). Only species richness categories with potential as predictors of multivariate transect cover data (P < 0.05) are shown; A. Alien herbs and grasses, B. Legumes (living), C. Legumes (dead), D. Lomandra and sedge-like and E. Epacridaceae. The ‘Legume’ categories include all taxa in Caesalpiniaceae, Mimosaceae and Papilionaceae. ‘Lomandra and sedge-like’ category includes all taxa in Centrolepidaceae, Cyperaceae, Dasypogonaceae and Restionaceae. The scale is from 0 to 20 species for all maps.
149 No. Species R1 D. Lomandra & 2 R1 sedge-like R1R1
8
F 14 F F Cov F F F F FFF F F F F F F 20 F R25+ Cov F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
E. Epacridaceae R1 R1 R1 R1
F F F Cov F F F F FFF F F F F F F F CovR25+F R5 R25+ R10R10 F R10 R5 R25+ R15 R5R5 R15 R15 R10 R15
FIGURE 5.8 contd. Values for plant species richness categories superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). Only species richness categories with potential as predictors of multivariate transect cover data (P < 0.05) are shown; D. Lomandra and sedge-like and E. Epacridaceae.
150 Log10 (1+ n) A. Orchid species R1 B. Clonal orchids R1 0.3 R1 R1 richness R1R1 R1R1
1.2
F F F F Cov F Cov F F F F F F F F F 2.1 FFF FFF F F F F F F F F F F F F F R25+ F R25+ Cov F Co F R5 R5 R25+ R10R10 v R25+ R10R10 3 F F R10 R5 R10 R5 R25+ R25+ R15 R5R5 R15 R5R5 R15 R15 R15 R15 R10 R10 R15 R15
C. Non-clonal orchids R1 D. Disturbance R1 R1 R1 R1R1 opportunists R1R1
F F F F Cov F Cov F F F F F F F F F FFF FFF F F F F F F F F F F F F F R25+ F R25+ Cov F Cov F R5 R5 R25+ R10R10 R25+ R10R10 F F R10 R5 R10 R5 R25+ R25+ R15 R5R5 R15 R5R5 R15 R15 R15 R15 R10 R10 R15 R15
FIGURE 5.9 Orchid species richness and population sizes superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2); A. orchid species richness, B. clonal orchids; C. non-clonal orchids; and D. disturbance opportunists (combined populations of: D. bracteata; C. macrostylis and M. media). The 151 latter three categories represent the major growth habits of jarrah forest orchids. Note: clonal and non-clonal categories are mutually exclusive. Populations are mapped on a logarithmic scale, that is log10(1 + number of orchids per transect). 152
Log10(1+ No. Plants) A Caladenia flava R1 B Cyanicula sericea R1 0.3 R1 R1 R1 R1 R1R1
1.2
F F F F F Cov Cov F 2.1 F F F F F F F FFF F F FFF F F F F F F F F F F F F R25+ Cov F F R25+ R5 Cov F 3 R25+ R10 R5 R10 R25+ R10R10 F R10 F R5 R10 R5 R25+ R15 R5 R25+ R5 R15 R5R5 R15 R15 R15 R15 R10 R10 R15 R15
C Disa bracteata R1 D Eriochilus sp. R1
R1 R1 R1 R1 R1R1
F F F F F Cov Cov F F F F F F F F F FFF FFF F F F F F F F F F F F F F R25+ F R25+ Cov F Cov F R5 R5 R25+ R10R10 R25+ R10R10 F F R10 R5 R10 R5 R25+ R25+ R15 R5R5 R15 R5R5 R15 R15 R15 R15 R10 R10 R15 R15
FIGURE 5.10 Orchid population sizes for individual species superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). The species are; A. C. flava, B. C. sericea, C. D. bracteata, D. Eriochilus sp., E. M. media, F. P. sp. crinkled leaf, G. P. recurva and H. T. crinita. Populations have been mapped on a logarithmic scale, that is log10(1 + number of orchids per transect). Note: only orchid species found in more than six transects have been included. Log10(1+ No. Plants) E Microtis media R1 F Pterostylis nana R1 aff 0.3 R1 R1 R1R1 R1R1
1.2
F F F F Cov F Cov F 2.1 F F F F F F F F FFF FFF F F F F F F F F F F F F F R25+ F R25+ Cov F Cov F 3 R5 R5 R25+ R10R10 R25+ R10R10 F F R10 R5 R10 R5 R25+ R25+ R15 R5R5 R15 R5R5 R15 R15 R15 R15 R10 R10 R15 R15
G Pterostylis recurva R1 H Thelymitra crinita R1 R1 R1 R1R1 R1R1
F F F F Cov F Cov F F F F F F F F F FFF FFF F F F F F F F F F F F F F R25+ F R25+ Cov F Cov F R5 R5 R25+ R10R10 R25+ R10R10 F F R10 R5 R10 R5 R25+ R25+ R15 R5R5 R15 R5R5 R15 R15 R15 R15 R10 R10 R15 R15
FIGURE 5.10 contd. Orchid population size for the species; E. M. media, F. P. sp. crinkled leaf, G. P. recurva and H. T. crinita, superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). Populations have been mapped on a logarithmic scale, that is log10(1 + number of orchids per transect). 153 % Cover Total litter R1 15 R1 R R1 1
60
F 105 F F Cov F F F F FFF F F F F F F 150 F R25+ Cov F R5 R25+ R1R1 F 0 0 R1 R R25+ 0 5 R1 RR5 5 5 R1 R1 5 R1 5 0 R1 5
FIGURE 5.11 Values for total litter cover superimposed on the nMDS ordination of transect vegetation structure (Fig. 5.2). Note: total litter cover is the sum of cover for leafy litter (leaves and small twigs) and woody litter (branches and logs > 2.5 cm diameter). The scale is from 0% to 150% cover.
154 50 A a a 40 a
30
b 20 b 10
20 B
Percentage Cover 15
10
5 a a a a a 20 C a a 15 a a
10 b
5
20 D
15 Number of Species
10
b b 5 a a a
0 1 5 10 15 25+
Time since establishment (years)
FIGURE 5.12 Changes in values for cover and species richness categories identified as potential indicators of vegetation recovery. Mean percentage cover for; A. Legumes (living) and B. Lomandra and sedge-like species, and mean species richness for C. Legumes (living) and D. Lomandra and sedge-like species for a temporal sequence of transects in rehabilitation areas and adjacent unmined forest (for 1, 5, 10 and 15 year old rehabilitation areas n = 4, for >25 year old n = 3). Data are means of raw values. Transect area = 0.025 ha (5 m x 50 m). Vertical bars represent ± 1 s.e. Columns identified by the same letter are not significantly different by ANOVA (P > 0.05). The dashed line represents the mean for unmined forest and the broad coloured bar the mean ± 1 s.e.
155 DISCUSSION
Vegetation Structure This study found that the vegetation structure of the post-mining landscape was returning to a state similar, but not identical, to that of unmined jarrah forest as it ages (see Fig. 5.1). The main difference between mined and unmined landscapes was the lack of cover from the ‘Herb, sedge and grass’ strata and greater uniformity in species assemblages. Three major temporal changes occurred in the vegetation structure; the early reduction of cover of the ‘Herb, sedge and grass’ strata, rapid increase then decrease in shrub cover, and increasing tree height and cover (see Fig. 5.1). In 1 year old rehabilitation areas the ‘Herb, sedge and grass’ strata cover was predominantly from short-lived Kennedia species which die and decompose within five years of rehabilitation establishment. Thereafter, cover of this vegetation strata remains low so that even after in excess of 25 years it was still significantly lower than in adjacent unmined forest. The rapid increase then decrease in shrub cover is the result of the growth and death of short-lived leguminous shrubs (predominantly Acacia spp.) and was associated with increased litter depth and cover. Many of these structural changes are evident in Fig. 5.1.
Reduced cover for the ‘Herb, sedge and grass’ strata in rehabilitation areas in rehabilitation areas was associated with reduced species richness. Potential causes of low recruitment for species in this category include: low seed viability; absence or scarcity in the seed bank of respread topsoil or the seed mix used for revegetation; reduced emergence and establishment of seedlings; selective collection or grazing by invertebrate and vertebrate predators; or a combination of these factors. Rehabilitation practices have been found to affect seed viability and vegetation composition, and a number of studies have found that canopy cover, litter depth and type, and grazing by herbivores can affect emergence and establishment of some jarrah forest species (Koch and Ward 1994; Koch et al. 1996; Koch et al. 2004).
Early rehabilitation procedures involved seeding with an understorey seed mix composed primarily of fast growing legumes, and stockpiled topsoil until required for rehabilitation of mined areas (Tacey and Glossop 1980; Norman et al. 2006). Over the life of the mining operations at Jarrahdale both rehabilitation practices and seed mixes were modified to increase species diversity by including more long-lived indigenous
156 understorey taxa and a lower proportion of legumes (Nichols et al. 1991; Koch et al. 1994; Grant and Koch 2007). However, Bell et al. (1987) reported that seeds of long- lived resprouter species had a relatively low proportion of viable and germinable seed, and postulated that reproductive output for these taxa may not be important in natural forest. For example, in two jarrah forest understorey species: Lomandra sonderi (F.Muell.) Ewart and L. drummondii (F.Muell. ex Benth) Ewart; few seeds are produced; and germination is inhibited by the pericarp and embryo dormancy (Plummer et al. 1995). For these taxa and other resprouter species with similar characteristics, few new plants will be produced from either, the soil seed bank or seed mixes. Stockpiling of topsoil was found to be associated with a rapid loss of seed and seed viability, and direct return of topsoil is now practised whenever possible to maintain viability and diversity of the soil seed bank (Tacey and Glossop 1980; Nichols and Michaelsen 1986; Koch et al. 1996).
Few studies have examined emergence, establishment and predation of plant taxa that are absent from, or under-represented in the ‘Herb, sedge and grass’ strata in rehabilitation areas. However, some of the factors affecting the emergence and establishment of over-storey species (i.e. trees) and a few understorey shrubs have been studied. The absence of canopy cover has been found to have an adverse impact on the emergence and establishment of the four canopy species; Eucalyptus marginata, Corymbia calophylla (syn. E. calophylla), E. patens Benth. and E. diversicolor (Stoneman and Dell 1994; McChesney et al. 1995). White light (representing solar illumination) has been found in laboratory studies to reduce the percentage germination of the seed of the jarrah forest species, Acacia lateriticola, Bossiaea aquifolium Benth, E. marginata, C. calophylla, Gompholobium knightanum Lindl., Gompholobium marginatum R.Br., Gompholobium tomentosum Labill. and Sphaerolobium vimineum Sm., and presumably would have a similar effect in the field (Rokich and Bell 1995). Tacey and Glossop (1980) suspected canopy closure in young rehabilitation sites (6 - 7 years old) resulted in reduced recruitment of early coloniser species. However, there has been little in situ research into the effect of canopy cover and light on emergence and establishment of jarrah forest understorey species, other than through indirect means, for example through examination of the effect of canopy reduction due to the disease ‘Jarrah dieback’ caused by the pathogen Phytophthora cinnamomi (Mc Dougall et al. 2002).
157 Ecological factors that have been less frequently examined include litter depth and type, and predation of seed and seedlings. Litter depth and type were found to affect the germination and survival of four jarrah forest tree species; E. marginata, C. calophylla, Banksia grandis Willd. and Allocasuarina fraseriana (Miq,) L.A.S.Johnson, in virgin (unlogged) jarrah forest (Abbott 1984). However, little is known of the effect of litter depth and type on the emergence of understorey species. Many Australian plant taxa are known to be myrmecochorous and under natural conditions, a high proportion of seed of these taxa can be collected and stored by ants (Berg 1975; Wellington and Noble 1985). Myrmecochory has been observed in jarrah forest species and removal of the pericarp by ants was found to be associated with increased germination in Lomandra spp., but little is known of the effect on recruitment in rehabilitated mine sites (Plummer et al. 1995). Grazing by kangaroos reduces survival and growth of Xanthorrhoea gracilis Endl. and X. preissii Endl. in Lehm. in jarrah forest mine site rehabilitation areas, and was also associated with reduced frequency of occurrence of Lomandra filiformis Thunb. in grasslands of the Australian Capital Territory (Naeve and Tanton 1989; Koch et al. 2004). It is reasonable to assume that other palatable species will be similarly affected by herbivores. Thus, while the factors discussed in these studies will be influencing the recruitment of under-represented taxa of the ‘Herb, sedge and grass’ strata, the significance of each factor is not known for most of those taxa.
Despite the significantly lower cover from the ‘Herb, sedge and grass’ strata, there was no significant difference between the overall vegetation structural characteristics of the oldest rehabilitation areas examined (25+ years old) and adjacent unmined forest areas. Koch and Ward (1994) found that pre-mining vegetation had a strong influence on post- mining vegetation through the seed bank in returned topsoil, but it must be noted that direct return was not widely practised in the late 1970s when these sites were rehabilitated. Many of the low growing understorey species in unmined forest are resprouters, and have previously been identified as recalcitrant taxa in the recovery process (Koch et al. 1994; Bright 1996; Norman et al. 2006). Often these taxa are slow growing, rhizomatous and/or clump forming (e.g. Lomandra spp. and Restionaceae) or tuberous geophytes (e.g. Drosera spp). Thus, while the over storey and taller vegetation structural components of the rehabilitated landscape are gradually becoming similar to those of unmined jarrah forest, this study has found that an extended recovery period will be required for cover of the ‘Herb, sedge and grass’ strata to return to levels approaching those of the surrounding unmined forest. Alcoa World Alumina Australia
158 has ongoing propagation research programs directed at enhancing the recovery of these taxa in the post-mining landscape (Willyams 2005; Grant and Koch 2007).
Species richness The most species rich plant families at the study site were Papilionaceae, Orchidaceae, Proteaceae, Myrtaceae, Mimosaceae, Poaceae and Asteraceae. The Western Australian flora is species-rich in Papilionaceae, Proteaceae and Myrtaceae, with these three families representing 45.3% of the 993 jarrah forest taxa growing on lateritic soils and 25.2% of the total 15,830 Western Australian taxa (http://florabase.calm.wa.gov.au, accessed 4th February 2007). However, at the study site they represented only 23.2% of the taxa (Table 5.5). A similar result (25.6%) was reported by Bell and Heddle (1989) based on examination of a published description of the northern jarrah forest flora (Marchant et al. 1987). The study site at Jarrahdale is a small part of the jarrah forest habitat and bauxite mining takes place in particular vegetation types corresponding to laterite mantled uplands, Havels’s jarrah forest site-vegetation types P, S and T (Havel 1975a, 1975b). The lower species richness of Papilionaceae, Proteaceae and Myrtaceae, at the study site compared to Florabase records for jarrah forest may be characteristic of localised vegetation types, an artefact of limited sample size or the result of increased species richness of other families.
Factors affecting the species richness and composition of jarrah forest vegetation include fire frequency, the disease ‘Jarrah dieback’, as well as anthropogenic disturbances caused by mining and/or proximity to centres of human habitation. Management burns of the jarrah forest are carried out by CALM on a five to seven year rotation, therefore plant species that are killed by fire and are slow to mature may be adversely affected by this regime (Shea et al. 1981). In addition, many Proteaceae and Myrtaceae are susceptible to the disease ‘Jarrah dieback’ and increased species richness of annual herbs has been found in areas where the disease has reduced canopy cover (McDougall et al. 2002). The families: Asteraceae; Mimosaceae; and Poaceae contain many taxa that are annuals, fire-weeds or disturbance opportunists. These families had proportionally higher species richness at the Jarrahdale study site than in Florabase records for the northern jarrah forest. The increased species-richness for these families in the flora of the study site probably reflects the combined effects of disease, mining, logging, and other anthropogenic disturbances.
159 Overall species richness of rehabilitated and unmined forest transects were significantly different. This is not surprising as rehabilitated mine-site vegetation is dominated by legumes. The temporal changes in species composition and in the variability of composition data are probably a reflection of not only rehabilitation age, but also changes in procedures and seed mixes over the life of the mine. Grant and Loneragan (2001) believed that variability in species composition associated with pit age was due to either, differences in rehabilitation practices, or to particularly favourable environmental conditions at particular sites following establishment. For example: two of the 25+ year old rehabilitation study areas were sown with a Marri only over storey, but have since been invaded by Jarrah (the most probable source of these trees is seed rain from adjacent unmined forest) (Koch pers. comm. 2002). However, the similarity in species richness of the oldest rehabilitation areas examined (25+ years old) and their adjacent unmined forest sites may also be a result of the site selection procedure. These rehabilitation area transects were selected for incorporation into the study because they contained some indigenous tree species and understorey vegetation, but are not typical of early (pre-1976) rehabilitation areas, most of which were rehabilitated with non- indigenous tree species with little or no understorey (Grant and Koch 2007). Thus while the vegetation of these sites is not typical of older rehabilitation areas, the results of this study suggest that, given the appropriate initial species mix, rehabilitation areas will develop a substantial proportion of the species richness and abundance characteristics of unmined jarrah forest over time.
Plant Species Assemblages The distribution of jarrah forest plant communities has been extensively studied and mapped at the landscape scale, but the relationships between orchid species and vegetation assemblages has rarely been examined (Beard 1974; Havel 1975a; b; Gioia and Piggott 2000). TWINSPAN analysis of vegetation data was performed to determine if there were particular species assemblages associated with particular orchids at the ‘vegetation type’ and the ‘microhabitat’ scales in either rehabilitation areas or unmined forest. In this study ‘vegetation type’ was determined by analysis of transect vegetation data and ‘microhabitat’ by analysis of quadrat vegetation data.
At the ‘vegetation type scale’ the main TWINSPAN site classification division was between rehabilitation areas ≤ 15 year old and, unmined forest (including 25+ year old rehabilitation sites), indicating substantial differences in species composition of each
160 vegetation type. Bright (1996) found a similar TWINSPAN first division, separating forest and rehabilitated sites, when assessing vegetation development of the post bauxite mining landscape at Boddington, in the south west of WA (Boddington is approximately 65 km SSE of Jarrahdale). This study found only three species assemblages (c.f. the current study with six), one exclusive to each of the forest and rehabilitation sites, and another common to both. However, only young rehabilitation areas (up to nine years old) were examined. In the current study, group 5 species assemblage, characterised by the weed species Hypochaeris glabra L., and the disturbance opportunists Acacia pulchella and Mirbelia dilatata, was common in one, five and 10 year old rehabilitation areas and appears to be characteristic of young establishing vegetation. However, this species assemblage was also found in a group of four unmined forest sites transects, and may indicate these sites have been recently disturbed, or are degraded by disease, as increased species richness of annual herbs (e.g. H. glabra) is known to be associated with loss of canopy caused by ‘Jarrah dieback’ (McDougall et al. 2002).
Orchids present in each vegetation assemblage were generally not exclusive to these assemblages with the following exceptions: D. bracteata was found only in species assemblages associated with rehabilitation areas; and Eriochilus sp. and T. crinita were found only in species assemblages associated with unmined forest. Only two previous vegetation classification studies have mentioned orchids in relationship to plant species assemblages. Bright found Pterostylis vittata Lindl. and an unidentified Pterostylis sp. in association with a species assemblage common only in rehabilitation sites (Bright 1996). A more recent study of the effects of burning on species composition of 11-13 year old rehabilitation sites at the Jarrahdale mine site found no relationship between rehabilitation species assemblages and the presence of orchid species (Grant and Loneragan (2001). Thus, with the exception of D. bracteata, Eriochilus sp. and T. crinita, individual orchid species were not closely associated with plant species assemblages at the ‘vegetation type scale’.
Separate TWINSPAN classifications of rehabilitation and unmined forest quadrat species composition data found management practices at the time of establishment had a strong impact on species assemblages in rehabilitation areas. The vegetation of unmined forest was far more heterogeneous than that of rehabilitation areas. Five plant species assemblages were identified in unmined forest at the ‘microhabitat scale’ however,
161 there was poor support for these assemblages. The greater homogeneity of rehabilitation vegetation is not surprising as younger rehabilitation sites (i.e. those ≤ 15 years old) were dominated by the short-lived seeder species that dominate the seed mixes used for revegetation.
Examination of the distribution of orchids associated with each ‘microhabitat’ (i.e. quadrat) vegetation assemblage found that orchids were not exclusive to sites containing these assemblages in either rehabilitation areas or unmined forest sites. Orchid taxa that were observed infrequently (i.e. found in only a few sites) could be common at those sites but absent from other transects with the same species assemblage present. For example; each of the two clonal species, Cyrtostylis huegelii and Cyrtostylis ovata were found in only two forest transects, but were present in most quadrats of those transects. This suggests that orchids are relatively independent of the vegetation structural and species assemblage characteristics examined in this study and that there are other more significant factors affecting their distribution.
Orchid Abundance and Vegetation Characteristics as Potential Indicators of Recovery The current objective of Alcoa’s rehabilitation processes is to establish a self-sustaining jarrah forest ecosystem that retains or enhances the water, timber, conservation and recreation values of the original jarrah forest (Elliott et al. 1996). Site-specific completion criteria for the rehabilitated landscape need to be fulfilled before relinquishment of title (Ward 1998; Grant 2006). There is a need for indicators that measure progress towards fulfilling these completion criteria (Thompson and Thompson 2004; Anonymous 2006). Plant diversity (measured as species richness) is the most widely studied and the most commonly used indicator of ecosystem recovery in jarrah forest (Nichols and Michaelsen 1986; Ward et al. 1996). However, there are also many studies examining the temporal recovery of mammal, reptile, bird, invertebrate and fungal species richness of these disturbed habitats, which provide valuable additional information on ecosystem recovery (Majer et al. 1984; Gardner and Malajczuk 1988; Majer and Nichols 1998; Armstrong and Nichols 2000; Nichols and Nichols 2003; Majer et al. 2006).
Vegetation cover categories identified as predictors of data dispersion in this study appear to be good predictors of the stage of vegetation establishment when mapped onto the nMDS ordination of strata cover. Cover categories may be useful as indicators of:
162 initiation; transition state vegetation (i.e. rapidly changing rehabilitation vegetation); or stable state vegetation (i.e. unmined forest). For example: living legume cover is greatest in rehabilitation sites ≤ 15 years old and recently disturbed forest sites; there was little cover from dead legumes in rehabilitation areas < 5 years or ≥ 15 years old; and ‘Lomandra and sedge-like species’ provide little cover except in unmined forest.
The rapid growth and short life span of legumes, and low cover of resprouter species in rehabilitated mine sites is well known (Koch and Ward 1994; Bright 1996; Grant and Koch 1997; Norman et al. 2006; Grant and Koch 2007). The temporal changes in cover from dead legumes reflect the short lifespan of many leguminous taxa, for example; the fire weed Acacia pulchella is believed to have a maximum lifespan of < 15 years (Monk et al. 1981), and rapid decomposition of nitrogen-rich plant material. Many legumes are also disturbance opportunists and/or fire-weeds, therefore legume cover can be an indicator of recent disturbance in both rehabilitation areas and unmined forest (Monk et al. 1981; Shea et al. 1981). The reduction in living legume cover observed in 10 year old rehabilitation areas is probably associated with death of short-lived seeder species (e.g. A. pulchella). However, sustained high legume cover in rehabilitation areas at 15 and 25+ years after establishment is probably due to the presence of long-lived non- indigenous legumes (i.e. Acacia longifolia (Andrews) Willd. and Acacia decurrens Willd.) that were included in the seed mixes used for rehabilitation prior to 1988 (Grant and Koch 2007), because little seedling recruitment was observed.
The cover provided by the rhizomatous taxa of the ‘Lomandra and sedge-like’ category was very low in all rehabilitation areas compared to unmined forest, and the estimated time for return to the mean value for unmined forest was close to three centuries (see Appendix 2; Fig. A2.1). Lomandra species are difficult to propagate from seed, and many species in Restionaceae produce few seeds and/or have inordinately slow growing seedlings (Bell and Pate 1993; Plummer et al. 1995; Meney et al. 1999). Other species in the ‘Lomandra and sedge-like’ category may behave similarly and further study is required to determine if slow growth is an innate quality of these rhizomatous taxa, is caused by unidentified adverse characteristics of the rehabilitated landscape, or a combination of both.
Species richness categories may be more useful as indicators of vegetation recovery than cover categories. The number of species in the categories ‘Legumes (living)’ and
163 ‘Lomandra and sedge-like’ exhibited changes with increased age of the rehabilitation areas. Legume species richness decreased with time as short-lived seeder species senesced and died, and in 25+ year old rehabilitation areas had returned to a value within the range for unmined forest. The number of ‘Lomandra and sedge-like’ species also returned to values within the range for forest transects over the temporal period examined in this study. The changes in these species richness categories suggest that the process of establishing a jarrah forest-like ecosystem is underway.
Examination of orchid categories identified by DISTLM forward as predictors of data dispersion found that these were generally not the most effective predictors of the progress of vegetation establishment. Orchid species richness only separated very young rehabilitation areas (1 year old) from older sites. The population size of clonal orchids appeared to be connected to ecosystem development, which makes sense intuitively, but was also highly variable even between undisturbed forest sites (see Chapter 2). Therefore, the absence of clonal orchids alone cannot be used as an indicator of restoration failure. Individual orchid species may have more validity as indicators of habitat conditions. For example, Eriochilus sp. and Thelymitra crinita were found only in unmined forest transects and therefore may be indicators of stable state vegetation or of completion of the rehabilitation processes. Disa bracteata, found only in five to fifteen year old rehabilitation areas may be useful as an indicator of transition state vegetation. The relative abundance of other species such as: Caladenia flava; Pterostylis sp. crinkled leaf; and Pterostylis recurva; which return early to the recovering vegetation and persist, are probably better indicators of the progress of vegetation establishment (see Chapter 2). Initiation of populations of these species indicates that there had been at least a partial recovery of soil microflora, and their persistence that microhabitats suitable for long-term survival have developed (see Chapter 2). The orchids Eriochilus sp. and T. crinita were both observed in rehabilitation areas outside the study transects and their failure to colonise the post- mining landscape warrants further investigation. Thus, while no single orchid species appeared to be an indicator of rehabilitation completion, combinations of orchid taxa may be useful in this context.
164 CONCLUSIONS
Evidence from this study indicates that with increasing age the vegetation of rehabilitation areas develops structural and soil surface cover characteristics similar to that of unmined forest. Vegetation of rehabilitation areas was generally more homogeneous than that of unmined forest as there were fewer plant species and species assemblages present, and younger areas (≤ 15 years old) were characterised by high cover and species richness of short-lived seeder species (mostly Acacia spp.). The oldest rehabilitation areas examined were not significantly different to adjacent areas of unmined forest. The post-mining landscape is developing a ‘new’ jarrah forest ecosystem that is structurally similar to unmined jarrah forest but floristically appears less species rich and is more homogeneous (i.e. has fewer species assemblages). This lower species richness is in part due to the lower cover and species richness of tufted, rhizomatous and herbaceous species.
Orchids present in each vegetation assemblage were generally not exclusive to these assemblages with the following broad exclusions: D. bracteata was found only in species assemblages associated with rehabilitation areas; and Eriochilus sp. and T. crinita only in species assemblages associated with unmined forest. No single orchid species appears to be an indicator of ecosystem recovery. However, the presence of populations of C. flava, P. sp. crinkled leaf or P. recurva, in combination with the absence of D. bracteata appear to be a measure of the maturity of the rehabilitation vegetation.
165 166 CHAPTER 6
GENERAL DISCUSSION
INTRODUCTION
The Orchidaceae is an extremely species-rich plant family that is of interest ecologically because of their complex life cycles, bizarre pollination strategies and wide variety of life forms (Dressler 1981; Chase et al. 2003), however, orchids are under-represented in scientific literature when compared to other species rich plant families (Dearnaley 2007; Peakall 2007). The literature on terrestrial orchids generally focuses on either conservation issues such as: population monitoring; protecting and/or managing natural habitats; transplantation trials; in situ and ex situ propagation techniques or on the specificity of orchid associations with their mycorrhizal fungi (Reinhammar et al. 2002; Dixon et al. 2003; Janeckova et al. 2006; Brundrett 2007). Most long term demographic studies of terrestrial orchid populations have been limited to European and North American ecosystems (Willems 1982; Tamm 1991; Light and MacConaill 2006) but few of these have examined the recruitment of orchids in severely disturbed habitats. There are few Australian studies on the effect of disturbance on orchid populations (Coates et al. 2006) and the only study of the post-mining landscape is a retrospective examination of vegetation monitoring data by Alcoa World Alumina Australia (Alcoa) (Grant and Koch 2003). Terrestrial orchids are believed to be particularly sensitive to competition from weeds as well as disturbance (Scade et al. 2006), this combined with the obligate nature of the orchid-mycorrhizal fungus association suggested that orchids would colonise rehabilitation areas only when both microhabitat sites and soil microflora had established successfully. Orchids are therefore expected to be useful as indicators of the success of vegetation establishment, maturity of rehabilitation vegetation and the recovery of edaphic conditions suitable for orchid mycorrhizal fungi (OMF).
Objectives This study is the first to specifically examine the establishment of terrestrial orchids in a severely disturbed Australian landscape; the post-bauxite mined jarrah forest at Jarrahdale, Western Australian. The overall objectives were to determine:
167 • the patterns of terrestrial orchid colonisation across a chrono-sequence of rehabilitation areas and to compare these populations with those of adjacent unmined forest; • if colonisation was affected by the inoculum potential of OMF or mycorrhizal specificity; • if orchid presence was related to vegetation structure or the presence of particular plant species assemblages in rehabilitation areas and unmined forest; and • if the presence of a particular orchid taxon or group of taxa could be used as indicators of the successful establishment of a ‘new’ jarrah forest ecosystem.
To address these objectives a chrono-sequence of rehabilitation sites and areas of adjacent unmined forest were surveyed to: identify the orchid taxa present; determine orchid population sizes and species richness; identify the vegetation structural changes that occur in rehabilitation areas with time; and compare the species richness and species assemblages of rehabilitation areas with adjacent unmined forest. The diversity and frequency of occurrence of OMF were examined in a limited subset of these survey sites using a seed packet ‘baiting’ technique (see Chapter 3) (Brundrett et al. 2003). To determine if specificity of mycorrhizal association was an important factor in recruitment, the phylogenetic relationships of the fungi involved in these associations were then examined by isolation and identification of OMF from three orchid taxa with differing rehabilitation colonisation patterns; Caladenia flava, Disa bracteata and Thelymitra crinita. The outcomes of this work are discussed below.
ORCHID COLONISATION OF THE POST-MINING LANDSCAPE This study found that terrestrial orchids had colonised bauxite-mined areas within five years of rehabilitation establishment. Different orchid taxa were observed in different age rehabilitation areas, with the total number of species increasing with rehabilitation age suggesting colonisation by a ‘succession’ of orchid taxa (see Chapter 2). No relationship was evident between the age of rehabilitation area and the mean number of orchid taxa or mean population size, with the exception of 1 year old areas where orchids were consistently absent. Orchid populations were found to be highly variable in size, the species present, and species richness in rehabilitation areas, and unmined forest. Population densities of most orchid species were lower in rehabilitation areas than in unmined forest, often contained large numbers of disturbance opportunist
168 species (see Chapter 2). Some of the species found in unmined forest were absent from the rehabilitation areas (Table 6.1).
The most populous orchids in the unmined jarrah forest were the clonal species: Cyrtostylis huegelii, C. flava, Cryptostylis ovata, Pterostylis sp. crinkled leaf and Pyrorchis nigricans; and the non-clonal T. crinita. Four of these orchids belonged to the group of six taxa that either failed to colonise or were extremely rare in rehabilitation sites compared to unmined forest (Table 6.1). Failure to colonise rehabilitation sites may be accounted for by: the effects of disturbance; failure of detection (because of sparsity of individuals); or could be an artefact of experimental design (see Chapter 2).
The dominant disturbance opportunist orchid in rehabilitation areas was Disa bracteata, an invasive weedy species originating in South Africa (Table 6.1; Hoffman and Brown 1998). This orchid is short-lived (unpublished personal observations), appears to be self pollinating, produces large quantities of seed, and has ‘broad’ mycorrhizal associations, which are believed to have aided its widespread dissemination (Bonnardeaux et al. 2007). The appearance of D. bracteata has caused much consternation amongst local wildflower enthusiasts and orchidologists in the eastern Australian states because its weedy characteristics and sometimes, dense populations are perceived to be a threat to indigenous species (Bates 1997; Prescott 1997; Dempster 2004). However, the r- strategist traits of D. bracteata combined with its absence from undisturbed forest and 25+ year old rehabilitation areas may indicate that it is unable to compete with natural vegetation and has potential as an indicator of ecosystem disturbance.
Thelymitra crinita is one of the most common and widespread non-clonal orchids of the jarrah forest uplands but was conspicuously absent or rare in rehabilitation areas (Table 6.1). A single plant of this species was found adjacent to a transect in one of the rehabilitation sites, but was within a short distance of the edge of the rehabilitation area (~ 1.5 m) and may have been a remnant of the original flora rather than a new recruit. This orchid is very common in unmined forest where it readily flowers and produces seed, therefore its rarity was surprising. However, OMF of T. crinita were more sparsely distributed in rehabilitation areas when compared with unmined forest, and OMF detection appeared to be sensitive to sampling year (see Chapter 3). Recruitment success is the result of multiple factors which include: seed production and dispersal; the presence and persistence of OMF; the presence of suitable microhabitats; and
169 TABLE 6.1 Orchids found in very low abundance or absent from in either rehabilitation areas or unmined forest. Characteristics that may be affecting recruitment are listed in the column titled Characteristics. Numbers are mean densities of orchids (plants ha-1). Species lists have been separated on growth habit and listed in decreasing forest density. Note: Species observed in survey sites but not found inside transects have not been included because densities were unable to be calculated. All rehab = populations of all rehabilitation transects combined. (Adapted from Table 2.3, Chapter 2)
Orchid species Density (plants ha-1) Scientific Name Common Name Forest All Rehab Characteristics Clonal Species Cyrtostylis huegelii Mosquito orchid 2177.1 0.0 Pyrochis nigricans Red beaks 360.0 0.0 flowers after summer fire Cryptostylis ovata Slipper orchid 316.21 0.0 specific wasp. pollinator Thelymitra benthamiana Leopard orchid 0.0 65.3 Pterostylis sanguinea Dark banded greenhood 0.0 12.6 Microtis media Common mignonette orchid 0.0 6.1 disturbance opportunist Other Species Thelymitra crinita Blue lady orchid 817.1 0.02 Eriochilus dilatatus Common bunny orchid 53.3 0.0 Prasophyllum parvifolia Autumn leek orchid 1.8 0.0 Disa bracteata* South African orchid 0.0 595.8 disturbance opportunist Caladenia macrostylis Leaping spider orchid 0.0 4.2 disturbance opportunist Caladenia longiclavata Clubbed spider orchid 0.03 2.1 * Indicates an alien species; 1 value high due to biased sampling; 2 species found in rehabilitation area but outside survey transect; 3 species found in forest but outside survey transect.
competition with other vegetation; as well as climatic and edaphic conditions (Brundrett 2007). Colonisation by orchids that were rare in, or absent from rehabilitation areas could be limited by one or more of these factors, but for T. crinita the sparsity and variability of OMF distribution may be contributing factors.
The successful establishment of orchid populations in rehabilitation areas within five years indicates that seed dispersal is not the most important factor limiting orchid colonisation for most taxa. However, seed production and/or dispersal may be limited in those taxa that failed to establish. Examples of potential sources of limitations to seed production and dispersal are provided by two of the orchids that failed to colonise any of the rehabilitation areas examined; Pyrorchis nigricans and Cryptostylis ovata. Each of these orchid species has very particular requirements for seed production (Hoffman and Brown 1998).
Pyrorchis nigricans is a common and widespread orchid but does not usually flower unless there has been a hot summer bushfire (Hoffman and Brown 1998), therefore without fire stimulus this species will fail to produce seed. Wild fires were quickly extinguished and forest management burns normally carried out by Department of
170 Environment and Conservation (DEC) did not occur during the period that Alcoa World Alumina occupied the Jarrahdale mine site because of the need to protect personnel and infrastructure (J. Koch pers. comm. 2002). Forest management burns to reduce fuel load occur in spring, winter or autumn, and the timing of these burns does not stimulate flowering in P. nigricans (A. P. Brown pers. comm. 2007). Therefore, P. nigricans is unlikely to have flowered and produced seed in either the Jarrahdale mine site or the surrounding forested areas.
Cryptostylis ovata is pollinated as a result of pseudo-copulation with male ichneumon wasps of the taxon Lissopimpla semipunctata (Table 6.1) (Hoffman and Brown 1998). The Jarrahdale study site is close to the northern limit of the distribution of C. ovata, and personal observations of two populations within the mine site over three growing seasons found flowering and successful pollination were infrequent. Pollinators may be sparsely distributed at the extremes of this orchids habitat; fragmentation of the ecosystem by mining may have affected the viability of pollinator populations; or the ability of pollinators to find the orchids. However, this orchid is also a colony forming species and pollination incompatibility may occur within clonal populations derived from few individuals. Alternatively, pollination may be a naturally rare event because seed production is likely to be much less important to long term survival in vegetatively reproducing species than in other plants (Silvertown et al. 1993). If seed production is a naturally rare event in C. ovata, unaided colonisation by this species will be prolonged.
DETECTION OF ORCHID MYCORRHIZAL FUNGI Gardner and Malajczuk (1988) found that the abundance of spores of arbuscular (AM) and sporocarps of ectomycorrhizal fungi (ECM), and frequency of root colonisation by these fungi in rehabilitated bauxite mined jarrah forest returned to unmined forest levels within seven years, but that species richness was much lower, and less than 50% of species were common to both habitats. A similar trend was evident in this study; fewer orchid species OMF were found in rehabilitation areas and they were more sparsely distributed than in unmined forest. OMF of only one orchid taxon; P. recurva, was detected in 1 year old rehabilitation areas and this orchid may be considered as one of the ‘pioneer’ colonisers of rehabilitation areas (see Chapter 2). This suggests there may be a link between OMF colonisation and orchid colonisation, but more intensive sampling and repeated monitoring of sites is necessary to test this hypothesis.
171 Orchid mycorrhizal fungi (OMF) were sparsely and patchily dispersed across the landscape in a similar manner to orchids plants. The seed packet ‘baiting’ method used to detect OMF revealed a lower frequency of their occurrence in laterite soils than found in previous studies on sandy soils of the Swan Coastal plain (Batty et al. 2001; Brundrett et al. 2003). Detection of OMF for a particular orchid taxon was not always correlated with occurrence of that orchid at a particular site, and it was interesting to note that the OMF of P. nigricans was detected in one of the rehabilitation areas although the orchid was absent from all rehabilitation areas. Therefore, absence of the appropriate OMF does not seem to be the most important cause of this species’ failure to colonise the post-mining landscape. The distribution of OMF is similar to that of root disease pathogens in this Mediterranean region (Sivasithamparam 1993); that is OMF may be present without the orchid but orchids are present only where the fungi occur.
OMF abundance and diversity were expected to increase in rehabilitation areas with age. There are indications that this is occurring as the number of positive baits increased in older rehabilitation areas. Litter and other soil organic matter are major food substrates for saprophytic fungi and have a direct effect on fungal growth, vigour and survival. This study found litter cover and/or depth positively correlated with OMF occurrence (see Chapter 3), and orchid species richness and population size (see Chapter 4). Litter cover was found to increase in rehabilitation areas with temporal increases in vegetation cover and the death of short-lived disturbance opportunists (mostly legumes) (see Chapter 3).
It is not clear what OMF are present in jarrah forest or rehabilitated landscape on a large scale. More extensive and intensive sampling is required to understand fully the distribution of OMF in this habitat and the frequency of baiting required to detect total OMF richness, Presence or absence of OMF are clearly not only the only factors responsible for slow or failure of orchids to colonise rehabilitation areas. The evidence gathered in this study is not adequate to identify critical factors for recruitment of recalcitrant taxa but does suggest many profitable areas for future studies.
OMF IDENTIFICATION Phylogenetic analysis of rDNA ITS region sequences of OMF found narrower specificity associated with C. flava and T. crinita compared to the disturbance opportunist D. bracteata. OMF generally belonged to two of the three clades of
172 mycorrhizal Rhizoctonias: Sebacinales and Tulasnellales. These results were generally in agreement with previous specificity studies on Australian terrestrial orchids; C. flava was associated with Sebacina spp., and T. crinita and D. bracteata with Tulasnella spp. (see Chapter 4).
There did not appear to be a relationship between ease of orchid colonisation and fungal specificity as C. flava and D. bracteata both readily colonised rehabilitation sites, while T. crinita was a poor coloniser. Thelymitra crinita and D. bracteata OMF were more sparsely distributed in rehabilitation soils (measured by baiting) than C. flava OMF and this may indicate that Tulasnellales are more sensitive to disturbance and slower to recover than Sebacinales. However, the promiscuous nature of D. bracteata mycorrhizal associations may be an additional factor supporting its invasive properties.
VEGETATION ESTABLISHMENT Rehabilitation sites were not monitored continuously over the temporal range examined (approximately 27 years), therefore the data obtained on establishment of vegetation structure and species assemblages cannot directly be referred to as a succession or a recovery trend. However, the similarity of all sites of a particular age to one another, combined with the increasing structural similarity to unmined forest with increasing time since establishment (Fig. 6.1), suggests that a consistent ‘recovery’ process is occurring (see Chapter 5). The oldest rehabilitation sites examined had established a jarrah forest-like structure, dissimilar only in the lower cover provided by the ‘Herb, sedge and grass’ strata (Fig. 6.1). The number of orchid species and clonal orchid population size were correlated with changes in vegetation structure, but had no direct relationship with rehabilitation age. However, the sparse distribution of orchids and irregular colonisation patterns, combined with occasionally dense populations of clonal taxa made statistical analysis relating vegetation data to orchid population size and species richness difficult at the scales examined. The only individual species where presence had a significant correlation with vegetation structure was Eriochilus sp. This species was found in a subset of the unmined forest transect and it is not clear what structural characteristics were associated with its presence.
Early-growth phases of post-mining rehabilitation vegetation contained species assemblages that were dominated by disturbance opportunists and fast growing legumes. The high legume content of the early rehabilitation vegetation is a direct
173 A B
C D
E F
FIGURE 6.1 Vegetation of a chrono-sequence of rehabilitation areas (A – E) and an area of unmined forest (F) showing structural changes that occur with time. Age of rehabilitation area vegetation is as follows: A = 1 year; B = 5 years; C = 10 years; D = 15 years; and E = 25+ years. A dense understorey of large leguminous shrubs is evident in 5, 10 and 15 year old areas. The dead plant material visible in the 15 year old rehabilitation area (D) consists of ‘standing litter’ of short-lived leguminous shrubs. The structural similarity of the 25+ year old rehabilitation area (E) to the unmined forest is evident. Note: the reduced cover of low-growing plant species (i.e. the ‘Herb, sedge and grass’ category) in the 25+ year old rehabilitation area compared to the unmined forest understorey. (Figure taken from Chapter 5 for illustration of outcomes of vegetation analysis.)
consequence of the use of legume-rich seed mixes to initiate the rehabilitation process (Norman et al. 2006; Grant and Koch 2007). However, many of these legumes also dominate recently disturbed unmined areas (e.g. the fire weed A. pulchella), so while high legume content is not typical of unmined forest vegetation it is characteristic of the
174 early phase of natural recovery processes in recently disturbed forest (Dell et al. 1989; Grant and Loneragan 2001). Rehabilitation vegetation is more homogeneous than that of unmined forest with species assemblages related to rehabilitation site age, and the different rehabilitation practices and seed mixes used by Alcoa World Alumina at the time of establishment (see Chapter 5). Although orchids were present in many species assemblages the sparseness of their populations meant most species may not be useful as an indicator species for the establishment and maturation of rehabilitation vegetation assemblages. Only T. crinita and D. bracteata appeared to have potential as an indicator species; T. crinita as an indicator of undisturbed jarrah forest; and D. bracteata as an indicator of recent disturbance; neither species was useful as an indicator of trends in vegetation recovery.
LIMITATIONS OF STUDY Examination of successional trends for rehabilitation vegetation and orchids requires long-term monitoring of vegetation structure and species composition, with concurrent monitoring of orchid and OMF populations. Logistical constraints required the use of different age rehabilitation sites as ‘proxies’ for repeated long-term sampling. As a result, vegetation structure and species assemblages observed in this study cannot be directly ascribed to a successional process. However, the changes identified are consistent with long-term vegetation monitoring by Alcoa and others in earlier studies, suggesting that the observed physical and compositional changes in vegetation across the chrono-sequence examined are likely to occur in rehabilitation vegetation with increasing age (Bright 1996; Grant and Loneragan 2001; Norman et al. 2006).
Most orchid taxa and OMF found in rehabilitation areas and unmined forest were sparsely distributed across the landscape with occasional, localised, high-density populations of clonal orchid taxa. The sparse distribution of orchids suggested that more extensive sampling is needed to generate a complete species list and reduce intra-age- group variability for rehabilitation sites. Increased sampling would also assist in identifying if particular vegetation structural or species assemblages were associated with an individual orchid taxon or OMF and improve the statistical confidence in the outcomes.
Examination of mycorrhizal specificities of a wider range of orchid would have provided more support for generalisations suggested by this study. The rDNA ITS
175 region appears to be highly variable in the mycorrhizal Rhizoctonias, and the use of alternative gene sequences (e.g. rLSU) may have proved more useful in identification of each isolate and in the subsequent classification of the specificity of the mycorrhizal associations for each orchid taxon. The inclusion of classical taxonomic techniques for fungal identification would also have been useful in confirmation of fungal identities (Maclean 1993). The criteria used in this study for successful germination was the minimum required for confirmation of mycorrhizal capacity and may not indicate the ability to sustain the growth and development of plants to maturity (see Chapter 1). Therefore, the ability of ericoid mycorrhizal and ectomycorrhizal fungi, identified in the mycorrhizal associations with orchids, to maintain long-term associations with the taxa from which they were isolated needs to be verified.
FUTURE STUDIES This study aimed to provide some baseline information about orchid colonisation of the post-bauxite mining landscape, and to determine if these plants could be used as indicators of vegetation maturity and ecosystem establishment. Future studies could address some of the issues identified as limitations of this study and broaden the scope of the work. For example, establishing replicated long-term monitoring sites in jarrah forest that is to be mined, and following post-mining colonisation would provide information on before mining variability; post mining impact and the recovery process. Such data may provide supporting evidence for the putative ‘successional’ trends identified here.
Orchid species that were absent from rehabilitation areas or extremely rare (i.e. C. huegelii, C. ovata, Eriochilus dilatatus, P. parvifolia, P. nigricans and T. crinita) need to be investigated further. These orchids may be naturally rare, difficult to identify (especially when not flowering) or may not have produced seed in the surrounding unmined forest because of absence of flowering stimuli or pollinators. Reintroduction of appropriately timed management burns may enable colonisation by these taxa, and only long-term vegetation monitoring will indicate if this strategy is successful. There was a dramatic difference in recruitment of the two orchids; C. flava and T. crinita in rehabilitation areas despite both being very common in unmined forest. T. crinita was extremely rare in rehabilitated areas, while C. flava became more abundant with time. The abundance of OMF of C. flava and T. crinita were comparable in unmined forest but T. crinita OMF were less abundant than those of C. flava in
176 rehabilitation areas. Detection of C. flava OMF was also less sensitive to timing of baiting (see Chapter 3). These orchids form mycorrhiza with fungi from different clades within the mycorrhizal Rhizoctonias; C. flava with Sebacinales and T. crinita with Tulasnellales. These fungi may respond differently to disturbance and may have different microhabitat and edaphic requirements. Further comparative studies of these two orchids in relation to OMF, vegetation associations, and natural habitats may elucidate why C. flava is a successful coloniser of rehabilitation sites, while T. crinita is not, and if their mycorrhizal associations are a significant part of this difference.
The seed baiting technique has proved effective in sandy soils of the Swan coastal plain (Brundrett et al. 2003), but the low frequency of detection of OMF in lateritic soils may mean that the technique requires optimisation for other soil types. The effect of timing of bait establishment needs to be examined in greater detail to elucidate the effect of temperature and moisture on germination of orchid seeds in situ. Long term repeated annual studies using OMF detection baits could also be used to detect natural temporal and spatial fluctuations in the inoculum potential of OMF in natural habitats. Information gained through such studies, in conjunction with collection of soil, climate and vegetation data, may elucidate the cause of the spatial and temporal variability in OMF inoculum potential observed in this and other studies (Hollick 2004). In addition, further research is required to determine if the apparent relationship between litter and OMF detection exists for a more extensive range of orchid taxa, and if the type and nutrient status of litter affect inoculum potential of the OMF.
The association between C. flava, its OMF and tree cover, combined with the suggested ability of Sebacina spp. to form ectomycorrhizal and ericoid mycorrhizal associations requires further investigation (Glen et al. 2002; Selosse et al. 2007). Orchids associated with Sebacina spp. may be involved in tripartite relationships with other plant taxa via their OMF. A tripartite relationship has been shown to exist between the underground orchid Rhizanthella gardnerii, it’s OMF and the Broom myrtle, Melaleuca uncinata (Warcup 1991; Mursidawati 2003). Such relationships could be important in helping orchids to colonise disturbed areas where ericoid mycorrhizal or ectomycorrhizal plants had already established, and may explain the relative success of C. flava’s colonisation compared to T. crinita.
177 Examination of the mycorrhizal fungi associated with wider range of orchid taxa of similar growth habits to C. flava, D. bracteata and T. crinita may provide further evidence in support of generalisations made here from studies on relatively few taxa. There is a need to optimise techniques for the isolation of mycorrhizal fungi from recalcitrant taxa (e.g. C. ovata and T. crinita) and to improve techniques for the isolation of DNA from OMF. Alternatively, endophytic fungi can be identified from amplified rDNA ITS regions (and/or other target genes) of total DNA extracted from infected tissues. Most recent studies using molecular techniques to identify OMF have assumed that any fungal endophyte that forms pelotons within orchid tissues is mycorrhizal without undertaking germination and growth studies to confirm mycorrhizal capacity. Comparison of ericoid mycorrhizal fungi identified from culture- based techniques and total DNA extracted from mycorrhizal roots of Gaultheria shallon Pursh found a systematic bias from culture-based detection (Allen et al. 2003). Similar studies comparing the results from traditional isolation techniques and identification of orchid mycorrhizal endophytes from total DNA extracts from the same plant are required to determine if isolation is necessary to identity OMF and/or if there is a bias resulting from culture-based techniques. Phylogenetic congruence between Chiloglottis species and their wasp. pollinators has been demonstrated, providing evidence in support of the pollinator driven evolution (Mant et al. 2002). Similar molecular studies comparing phylogenies of orchids with those of their mycorrhizal partners could provide valuable insights into the co-evolution of orchids and their mycorrhizal fungi.
It is clear that molecular techniques have revealed diversity within the mycorrhizal Rhizoctonias that was not evident using classical techniques. However, there is currently no clear concept of a species or genus within the Rhizoctonia complex, and there is a need for more extensive and intensive phylogenetic studies using multiple target genes to clarify the taxonomy of orchid mycorrhizal fungi and to identify the target genes appropriate for identification of these fungi at different taxonomic levels.
MANAGEMENT RECOMMENDATIONS The results of this study suggest that the unassisted colonisation of rehabilitation sites by recalcitrant orchid species may be a prolonged process. Alcoa World Alumina aims to establish a self-sustaining jarrah forest ecosystem that maintains the functions of the previous landscape on areas that have been mined for bauxite. Maintenance of biodiversity is an important aspect of this process. Improved post-mining land
178 management could include in vitro propagation and transplantation of recalcitrant orchids (Batty et al. 2006) or sowing rehabilitated areas with mycorrhizal inoculum mixed with seed.
Although orchid species richness and clonal orchid population size were correlated with changes in vegetation structure, these characteristics of orchid populations did not show any direct relationship with rehabilitation age or vegetation maturity. Only two orchid taxa appeared to have potential as an indicator species; T. crinita as an indicator of undisturbed jarrah forest; and D. bracteata as an indicator of recent ecosystem disturbance. The results of this work will have applications not only in the management of post-mining landscapes but also in vegetation monitoring and conservation work in
Western Australia and elsewhere.
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202 APPENDIX 1: ORCHIDS OF THE JARRAH FOREST
TABLE A1.1 A complete list of the indigenous orchid taxa of the Northern Jarrah Forest. Taxa listed in bold were observed at the Jarrahdale study site. This list has been compiled from personal observations, information provided by A.P. Brown (pers. comm., 2005), and data accessed on FloraBase (http://florabase.calm.wa.gov.au/). Taxonomy is consistent with Florabase (http://florabase.calm.wa.gov.au/, accessed July 2007).
Orchid Taxa Recent Synonyms Comments Caladenia arrecta Caladenia attingens found near Collie Caladenia brownii Caladenia cairnsiana found near Collie Caladenia christineae Caladenia denticulata Caladenia falcata Caladenia ferruginea Caladenia flava subsp. flava Caladenia hirta subsp. hirta Caladenia lobata Caladenia longicauda subsp. longicauda Caladenia longicauda subsp. redacta Caladenia longiclavata Caladenia macrostylis Caladenia magniclavata Caladenia marginata Caladenia nana subsp.nana Caladenia nana subsp. nana Caladenia pectinata Caladenia pholcoidea Caladenia reptans subsp.reptans Caladenia xantha Cryptostylis ovata Cyanicula gemmata Caladenia gemmata Cyanicula sericea Caladenia sericea Cyrtostylis heugelii Cyrtostylis robusta Diuris brumalis Diuris longifolia Diuris setacea Diuris laxiflora Diuris aff. !corymbosa Elythranthera brunonis Elythranthera emarginata Ericksonella saccharata Caladenia saccharata Eriochilus dilatatus subsp.multiflorus Leporella fimbriata Leptoceras menziesii Microtis media subsp. media Microtis atrata Microtis orbicularis Microtis rara Paracaleana nigrita Paracaleana granitica Paracaleana gracilicordata Paracaleana brockmanii Pheladenia deformis Caladenia deformis Cyanicula deformis
203 TABLE A1.1 contd.
Orchis Taxa Recent Synonyms Comments Prasophyllum brownii Prasophyllum elatum Prasophyllum fimbria Prasophyllum giganteum Prasophyllum hians Prasophyllum parvifolium Prasophyllum regium Prasophylum plumiforme Pterostylis barbata Plumatichilos barbata Pterostylis recurva Pterostylis sp.crinkled leaf (G.J.Keighery 13426) Pterostylis sp.extended dorsal sepal (G.Paull 731) Pterostylis sp.inland (A.C.Beauglehole 11880) Pterostylis sp. Karri forest (W.Jackson BJ270) Pterostylis sp.red flowered (W.Jackson BJ269) Pterostylis sp.short sepals (W.Jackson BJ259) Pterostylis sp.Slender Snail Orchid (G.J.Keighery 14516) Pterostylis sanguinea Pterostylis vittata Oligochaetochilus vittatus Pyrorchis nigricans Spiculaea ciliata Thelymitra flexuosa Thelymitra benthamiana Thelymitra crinita Thelymitra fuscolutea Thelymitra sp.Brookton (A.S.George 11631) T. luteola Thelymitra variegata Thelymitra antennifera Thelymitra macrophylla Thelymitra villosa
204 TABLE A1.2 Descriptions of orchid taxa used as study species. Current name and authority were obtained from FloraBase (http://florabase.calm.wa.gov.au/). Descriptions and habitat are based on information from Hoffman and Brown (1998) and Marchant et al. (1987).
Taxon Status Description Flowering period Distribution and Habitat Caladenia flava R.Br subsp. Indigenous Habit: clonal, tuberous, perennial herb, July - December Widespread in South West Botanic Province, coastal flava 0.10 – 0.25 m high, woodlands, winter-wet swamps, forests and inland granite Cowslip Orchid Leaves: 1, basal, glandular hairy, broadly outcrops linear, length 50-15 mm, width 5-20 mm, acute or obtuse, abaxial surface red Flowers: 1-5 on flexuous stem, golden yellow with prominent bright red lines, spots or irregular markings centred on a strong median line on dorsal sepal and petals, length 20-50 mm, width 20-40 mm
Cryptostylis ovata R.Br. Indigenous Habit: clonal, tuberous, perennial herb, November - April Throughout lower south-west, more common near south Slipper Orchid 0.25 - 0.70m high, coast. Grows in large colonies in Jarrah and Karri forest, Leaves: 1-2, basal, erect, petiolate, blade Peppermint woodland and low coastal scrub. ovate, length 35-200 mm, width 22- 70mm, purplish on abaxial surface Flowers: 4-15+, green and red, flowers reversed so that labellum is above the column , length 20-30 mm, width ±8 mm
Disa bracteata Sw. Naturalised Habit: tuberous, perennial herb, 0.20 – October - November Alien species, endemic in South Africa, naturalised in South African Orchid 0.40 m high southern Australian. Widespread in South West Botanic Leaves: numerous cauline, linear, Province, prefers open areas and will grow in competition decreasing in size up stem, lower leaves, with introduced grasses. length 20-150 mm, channelled, acuminate apex Flowers: numerous, small, green and brown, spurred, spike thick, fleshy, cylindrical, flower bracts leaf-like, length ±5 mm, width ±5 mm TABLE A1.2 contd.
Orchid Name Status Description Flowering period Distribution and Habitat Microtis media subsp. media Indigenous Habit: clonal, tuberous, perennial herb, September - January Widespread in South West Botanic Province. Prefers R.Br. 0.20 – 0.60 m high winter-wet swamps, creeklines, shallow pockets on granite Common Mignonette Orchid Leaves: 1, glabrous, sheathing, terete, outcrops and open bushland in higher rainfall areas. Often hollow, succulent, length 25-65 mm found growing as a weed in suburban gardens. Flowers: numerous, minute green, length ±3 mm, width ±3 mm, dense raceme
Pterostylis sp.crinkled leaf Indigenous Habit: clonal, tuberous, perennial herb, Late May - Under low shrubs, blackboys and macrozamia palms (G.J.Keighery 13426) 0.05 – 0.25 m high September throughout the Darling Range and coastal plain Slender Snail Orchid Leaves: numerous, cauline, sheathing, lanceolate, length 5-10 mm, small basal rosette of crinkly leaves on both flowering and non-flowering plants Flowers: 1, rarely 2, pale green, length 30- 40 mm, width ±5 mm, sometimes tinged with brown
Pterostylis recurva Benth. Indigenous Habit: tuberous, perennial herb, 0.30 – August - October Widespread throughout South West Botanic Province, Jug Orchid 0.60 m high often in competition with other plants. Leaves: numerous, cauline, sheathing, lanceolate, length 10-50 mm, non flowering plants produce a rosette of ovate leaves Flower: 1, rarely 2, green and white striped, occasionally tinged red, jug-like with conspicuously recurved lateral sepals, length 30-35 mm, width 25-30 mm TABLE A1.2 contd.
Orchid Name Status Description Flowering period Distribution Pyrochis nigricans (R.Br.) Indigenous Habit: clonal, tuberous, perennial herb, August – October, Widespread throughout South West Botanic Province, D.L.Jones & M.A.Clem. 0.05 – 0.30 m high (extremely rare inland in moist sites around granite outcrops, elsewhere a Red Beaks, Elephant’s Ears Leaves: 1, fleshy, orbicular-cordate, except after summer range of habitat and soil types. appressed to soil surface, length 20-140 bushfire) mm, width 30-80 mm Flowers: 2-8, crimson and white, length 20-30 mm, width 20-30 mm, pressed specimens dry black
Thelymitra crinita Lindl. Indigenous Habit: robust, tuberous, perennial herb, September - Widespread throughout South West Botanic Province. Blue Lady Orchid 0.20 – 0.70 m high November Common in lateritic and loamy-clay soils in forest areas, Leaves: 1, broad, oval, length 50-150 mm but also found in swamps and sandy Banksia woodland width 20-30 mm nearer to coast Flowers: 4-15+, pale to brilliant blue, 40 mm diameter, labellum scarcely distinguishable from other 5 segments, open only in strong sunshine
Thelymitra macrophylla Lindl. Indigenous Habit: robust, tuberous, perennial herb, August - November Widespread throughout South West Botanic Province. 0.30 – 0.50 m high Open shrubland, low swampy heath, Wandoo woodland, Leaves: 1, channelled, broadly linear, Jarrah forest and granite outcrops length 100-250 mm, width 2-5 mm Flowers: 2-15+, fragrant, blue, purple and occasionally white, 30-40 mm diameter TABLE A1.3 Descriptions of orchid taxa identified within survey transects at the study site during the course of the project. Current name and authority were obtained from FloraBase 2.0.24 (http://florabase.calm.wa.gov.au/). Descriptions and habitat are based on information from Hoffman and Brown (1998) and Marchant et al (1987).
Taxon Status Description Flowering period Distribution Caladenia longiclavata Indigenous Habit: tuberous, perennial herb, 0.20 – September - October Widespread and common in open Banksia and Sheoak E.Coleman 0.35 m high woodlands near coast and lateritic loams of Jarrah and Clubbed Spider Orchid Leaves: 1, basal, hairy, 9 - 18 mm long Karri forest between Perth and Albany. Flowers: 1-2, maroon and yellow, petals and sepals with broad red stripes down centre, length 50-70mm, width 40-50 mm
Caladenia macrostylis Fitzg. Indigenous Habit: tuberous, perennial herb, 0.16 – August - November Uncommon species, found in lateritic soils of Jarrah forest, Leaping Spider Orchid 0.25 m high deep sands of coastal Banksia and Sheoak woodlands and Leaves:1, basal, hairy, length 120-160 rich loams of Karri forest between Bindoon and Albany. mm, width 6-10 mm Flowers: 1-3, pale yellow, petals and sepals with 1-3 red stripes down centre, broad band of crowded dark red or black calli on labellum, length 40-60 mm, width 30-60mm
Cyanicula sericea (Lindl.) Indigenous Habit: tuberous, perennial herb, 0.15 – August - October Common in laterite soils of the Jarrah forest, coastal Hopper & A.P.Br. 0.40 m high Banksia woodland and occasionally granite outcrops. Silky Blue Orchid Leaf: 1, basal, hairy, broadly linear, length Widespread from just north of Perth to Fitzgerald River 50-120 mm, width ± 20 mm National Park with a disjunct occurrence near Esperance. Flowers: 1-4, pale blue-purple, length 30- 50 mm, width 30-40 mm TABLE A1.3 contd.
Taxon Status Description Flowering period Distribution Cyrtostylis heugelii Endl. Indigenous Habit: tuberous, perennial herb, 0.10 – July - September Moist shaded situations in a wide range of habitats Midge Orchid 0.30 m high including coastal heath, margins of winter-wet swamps, Leaves: 1, basal, circular-cordate, length sheltered sites in Jarrah and karri forest, and inland granite 30-70 mm, width 20-50 mm outcrops. Found between Kalbarri in north and Condingup Flowers: 2-15+, reddish brown, length 10- 60 km east of Esperence. Forms large colonies. 20 mm, width ± 5 mm
Diuris brumalis D.L.Jones Indigenous Habit: tuberous, perennial herb, 0.20 – June - August Common on lateritic and granitic soils of Darling Range Winter Donkey Orchid 0.50 m high also found to north in hills and breakaways near Mt Leaves: 2or 3, unequal length, channelled, Lesueur. length 150-200 mm, width 8-10 mm Flowers: 3-15+, yellow and brown, length 20-40 mm, width 20-30 mm
Eriochilus dilatatus subsp. Indigenous Habit: tuberous, perennial herb, 0.25 – March - June Common sub-species, widespread in Jarrah forest and multiflorus (Lindl.) Hopper & 0.45 m high Banksia woodland between Perth and Albany. A.P.Br. ms Leaves: length 10 - 40 mm, width 5-11 Common Bunny Orchid mm Flowers: 1-20, white with yellow and reddish markings length 10-15 mm, width 9-12 mm
Prasophyllum parvifolium Lindl. Indigenous Habit: tuberous, perennial herb, 0. 15– June - August Found between Eneabba and Manjimup. Prefers growing Autumn Leek Orchid 0.40 m high under Sheoaks, occurs in coastal heath, Banksia woodlands Leaves: 1, basal, sheathing for > half and Jarrah forest. length, blade terete, hollow, length 100 - 350 mm, width ± 2 mm Flowers: 6-18+, green with white labellum, length ± 8, width ±10 mm TABLE A1.3 contd.
Taxon Status Description Flowering period Distribution Pterostylis barbata Lindl. Indigenous Habit: tuberous, perennial herb, 0.20 – July – early October Common in Jarrah forest from Bindoon to Albany. Prefers Bird Orchid 0.30 m high thick leaf litter under Sheoaks. Leaves: numerous, crowded at base, intergrading into stem leaves, length 20 - 30 mm, width 10-12 mm Flowers: 1, rarely 2, green with fine reticulate veins, length 20-50 mm, width 20-30 mm
Pterostylis vittata Lindl. Indigenous Habit: tuberous, perennial herb, 0.20 – May - September Widespread throughout higher rainfall areas of lower Banded Greenhood 0.45 m high south-west. Common between Perth and Albany but Leaves: basal leaves absent in flowering extends sporadically from Dongara to Esperance. plants, stem leaves well developed, length 20 - 80 mm width 4-15 mm Flowers: 2-25, green with whitish stripes, length 12-15mm, width 8-10 mm
Pterostylis sanguinea D.L. Jones Indigenous Habit: tuberous, perennial herb, 0.05 – June – early Widespread in inland parts of the South West Botanic & M. 0.30 m high September Province from north of Kalbarri to Balladonia, and east to Dark Banded Greenhood Leaves: basal leaves absent in flowering South Australia. Does not occur in higher rainfall areas. plants, stem leaves well developed, length 20 - 50 mm, width 6-12 mm Flowers: 2-10, , length 15-25 mm, width 12-20 mm
Thelymitra benthamiana R.Br. Indigenous Habit: tuberous, perennial herb, 0.25 – September - Widespread in the South West Botanic Province from Leopard Orchid 0.40 m high November Northhampton to Israelite bay. In inland areas occurs in Leaves: broad, pale green smooth, length moist run-off areas around granite outcrops, elsewhere in 50 -150 mm, width 20-30 mm winter-wet swamps and creek margins. Flowers: 2-10+, yellow with brown blotches, collumn with deeply fringed wings, ±40 mm diameter TABLE A1.4 Descriptions of orchid taxa observed in forest at the study site during the course of the project but not found within survey transects. Current name and authority were obtained from FloraBase (http://florabase.calm.wa.gov.au/). Descriptions and habitat are based on information from Hoffman and Brown (1998) and Marchant et al (1987).
Orchid Name Status Description Flowering period Distribution Caladenia reptans subsp. reptans Indigenous Habit: clonal, tuberous, perennial herb, July - September Widely distributed in western regions of South West Lindl. 0.05 – 0.15 m high Botanic province from Kalbarri to east of Esperance. Little Pink Fairy Orchid Leaves: 1, hairy, length 40 - 80 mm, width Common in open Jarrah, Wandoo woodland along western ±8 mm edge of wheatbelt. Flowers: 1-3, pink, usually paler on abaxial surface, length 10-20 mm, width 10-20 mm Elythranthers brunonis (Endl.) Indigenous Habit: tuberous, perennial herb, 0.15 – August- October Widespread on Coastal Plain and darling Range. Occurs in A.S. George 0.40 m high near-coastal areas from Port Gregory to Israelite Bay, Purple Enamel Orchid Leaves: 1, narrow ovate, length 30 - 100 extending inland to Cunderdin. mm, width 4 - 10 mm Flowers: 1 – 4, purple, shining above, white with purple blotches on abaxial surface, length 11 - 12 mm, width 5 – 7.5 mm Leptoceras menzesii (R.Br.) Indigenous Habit: clonal, tuberous, perennial herb, September - October Widespread throughout South West Botanic Province. Lindl. 0.10 – 0.30 m high (rare except after Favours winter-wet areas often forming large colonies. Rabbit Orchid Leaves: 1 or 2, oblong, succulent , disturbance or a Also found in South Australia, Victoria, New South Wales glabrous or nearly so; length 60 - 100 mm, summer bushfire) and Tasmania. width ±30 mm Flowers: 1-5, white and pink length 10-20 mm, width 10 mm Prasophyllum brownii Reichb. Indigenous Habit: tuberous, perennial herb, 0.40 – November - January Widely distributed in high rainfall Jarrah and Karri forests Christmas Leek Orchid 1.30 m high of lower south -west. To the north of Perth it is confined to Leaves: 1, sheathing for > half length, swampy sites. blade terete, hollow, length 60 - 100 mm, width 5 - 15 mm Flowers: 30 – 80+, green, cream and brown, length ±15 mm, width ±12 mm TABLE A1.4 contd.
Orchid Name Status Description Flowering period Distribution Prasophyllum hians Reichb. Indigenous Habit: tuberous, perennial herb, 0.15 – September - Widespread throughout sandy soils of coastal plain, Yawning Leek Orchid 0.50 m high November associated with granite or laterite on Darling Scarp. Grows Leaves: 1, sheathing for > half length, (only after a summer in both low-lying winter wet areas and well drained hill blade terete, hollow, length 15 - 30 mm, bushfire) slopes. Extends from Dondara to Israelite Bay. width 2 - 4 mm Flowers: 20 – 50+, white, length ±8 mm, width ±8 mm TABLE A1.5 Source and weights of orchid seed collected for use in experimental work and in confirmation of mycorrhizal capacity of putative mycorrhizal fungi over the period of the study. Disa bracteata and Microtis media subsp. media seed collected from Booragoon, Melville or Nedlands were from plants found growing as weeds in gardens. Taxonomy is consistent with Florabase (http://florabase.calm.wa.gov.au/, accessed July 2007).
Orchid species Collection Site Collection year/s Weigh of seed (g)
Caladenia flava subsp. Jarrahdale 2001, 2002, 2003, 0.015, 0.071, 0.201, flava 2004 0.085 Caladenia latifolia Jarrahdale 2001 0.067 Caladenia macrostylis Jarrahdale 2002, 2003, 2004 0.008, 0.149, 0.013 Cryptostylis ovata Jarrahdale 2003 0.002 Kwinana 2002 0.166 Cyrtostylis heugelii Jarrahdale 2002 0.015 Cyanicula sericea Jarrahdale 2003, 2004 0.032, 0.037 Disa bracteata Jarrahdale 2001, 2002, 2003 0.272, 1.041, 0.821 Melville 2001, 2002 0.138, 0.299 Diuris brumalis Jarrahdale 2003 0.140 Eriochilus multiflorus Jarrahdale 2002 0.077 Microtis media subsp. Jarrahdale 2001, 2002, 2003, 0.106, 0.313, 1.533, media 2004 0.012 Booragoon 2001 0.132 Melville 2001, 2002 1.095, 0.192 Nedlands 2004 0.019 Pterostylis barbata Jarrahdale 2003 0.031 Pterostylis sp. Slender Jarrahdale 2001, 2002, 2003, 0.016, 0.044, 0.086, Snail Orchid 2004 0.030 Pterostylis recurva Jarrahdale 2001, 2002, 2003, 0.227, 0.238, 0.104, 2004 0.074 Pterostylis vittata Jarrahdale 2001, 2002, 2003, 0.095, 0.017, 0.012, 2004 0.055 Pyrochis nigricans Forestdale 2001 0.107 Jarrahdale 2003 0.174 Thelymitra crinita Jarrahdale 2001, 2002, 2003, 0.258, 0.256, 0.163, 2004 0.244 Thelymitra macrophylla Jarrahdale 2001,2002, 2003, 0.112, 0.439, 0.048, 2004 0.040
213 TABLE A1.6 Specimens of Disa bracteata Sw. (syn. Monadenia bracteata, M. micrantha, M. australis) in the Western Australian Herbarium, Department of Conservation and Land Management, South Perth, W.A. (Date accessed May 2005) The specimen listed in bold is the first record of this taxon near the Jarrahdale mine site. Note: Admiral Road is on the north- western limit of the mine site.
Herbarium Ref Site Description Latitude Longitude Collection No Date 279382 Youngs Siding, near Denmark ?/11/1945 336866 Denmark 34o56' 117o22' ?/11/1945 577936 Youngs Siding ?/11/1945 279390 Youngs Siding ?/12/1945 278939 Youngs Siding ?/11/1946 278947 Youngs Siding ?/11/1946 278955 Albany 35o00' 117o53' 20/11/50 279358 Mt Barker ?/2/1952 925446 Marine Drive, Albany 35o00' 117o53' ?/1/1954 279412 Albany 30/10/60 279404 1.5m from Nannarup ?/10/1961 279331 Denmark 9/11/62 279366 Manjimup ?/11/1968 279323 Kent River, W of Denmark 34o58' 117o03' 12/10/69 279803 1m N Boyanup SW Hwy. 120m S Perth 11/11/69 278912 0.5mE of Cape Riche 24/10/71 280291 Walpole 34o59' 116o44' 25/10/71 279838 Caravan Park, Bremer Bay 29/10/72 1185535 4m S of Mt Barker townsite (property of 20/10/73 C. Milton) 278904 Bridgetown 25/10/76 278920 Mt Clarence, Albany 35o00' 117o53' 10/11/76 279374 Rd. verge, Dalyup, W of Esperance 1977 279846 S of Jingalup Townsite 1/11/77 280283 Boya 1977 336874 white sand near Albany 35o00' 117o53' 25/10/78 279862 Capel to Dunsborough Rd. 1/11/78 279854 Bickley Reservoir, top of hill N side 3/11/78 279870 Collie at Ewington 20/6/79 279897 35km N Nicholson Rd and Rivervale 32o55' 115o53' 25/10/79 Rd., Yarloop 279439 1km along Admiral Rd off Nettleton 32o15' 116o10' 7/11/79 Rd 4514068 Spencer Park 14/11/79 4514076 N Northumberand Rd off Rocky Gully 2/11/80 Rd. 278890 1km E Waterloo 17/11/80 278882 1.5km from Boyanup on Dardanup- 33o28' 115o44' 30/11/80 Boyanup Rd. 279420 15 - 20 km W Albany along Rd to 25/10/81 Denmark 279889 S margin L. Quarderwardup, Stirling 19/10/82 Range 280313 24km W of Bremer Bay along Bremer 34o23' 119o09' 24/10/82 Bay Rd. 279900 1km S of Tambellup to Cranbrook 20/10/83 278874 Tunney 21/10/83 280305 2.3km W Pinjarra on Mandurah Rd. 32o37' 115o22' 10/10/84 279919 19km NE Gingin 19/11/84 5415314 Kemerton 30o11'20" 115o44'15" 14/10/85
214 TABLE A1.6 Specimens of Disa bracteata contd.
Herbarium Ref Site Description Latitude Longitude Collection No Date 3704076 Boondalup R. crosses #2 Rabbit Proof 34o11'15" 119o30'59" 23/10/85 Fence 404101 Reserve C8220, Coulstan Rd, Boya 3/11/85 793787 Pioneer Park, Walpole 34o59' 116o44' 5/11/86 855642 Millinup Pass, Porongorup Range, 35km 20/11/86 NE Albany 4449975 2km from Dunsborough on Cape 27/11/86 Naturalist Rd. 855138 L. William, West Cape Howe, 30km W 9/11/87 of Albany 278866 Boyup Brook 19/11/87 855650 Camel L., Bold Park 8km W of Perth 20/11/87 2657333 13 km WSW of Walpole, Walpole - 35o01'50" 116o35'30" 14/12/88 Nornalup National Park 3313964 Boonanarring reserve, Wannamal W Rd, 20/10/90 20km NNE GinGin 4342887 off Rd. to Peaceful Bay, Irwin Inlet, 14/10/91 Walpole - Nornalup N. P. 4382714 4km E L. Clifton 32o 49' 11" 115o 44' 12/10/92 59" 4437853 Manjimup, Pt GA116 34o08'37" 115o11'06" 21/10/92 4514084 Kalgan River 6147500 592250 21/10/92 northing easting 4342895 Poison Hole, Walpole - Nornalup 27/10/92 National Park 5754283 34o22' 118o07' 2/11/93 4278925 30 Lakes Way, Jandakot 32o05'0" 115o48'30" 13/10/95 4136691 Mt Lindesay 34o50'30" 117o18'30" 26/10/95 4288882 16.5km WNW of Albany Hwy on Rd to 33o45'12" 116o59'18" 13/11/95 Moodiarrup 4289080 15.2km E of Cranbrook on Salt River Rd. 34o18'33" 117o42'00" 14/11/95 4629418 near Dardanup, Boyanup 33o55'0" 115o41'0" 14/11/95 4273761 Nancy Peak, Porongorup Range 34o40'59" 117o51'40" 22/11/95 4528077 Simmonds Block, Tuart Forest 33o37'30" 115o27'42" 30/11/95 5912628 Capercup property, W end of river 33o38'00" 116o45'00" 26/10/96 paddock, Arthur R.- Boyup Brook Rd. 5889979 E end Saunders St. Henley Brook 31o48'00" 115o59'00" 9/11/96 4707516 Site 13, Ridley Rd. 1.2km SW Mt. Billy 31o57'0" 116o26'0" 13/11/96 4786130 Collie - Preston Rd. 33o27'52" 116o07'40" 11/8/97 4773063 Trigwell Bridge Rd. 33o27'11" 116o29'11" 12/8/97 4774639 6km SW Wilga 33o42'26" 116o10'17" 14/9/97 5737818 Nature Reserve, Frankland Rd, W 34o18'12" 117o29'11" 2/10/97 Albany, 6Km W Cranbrook 5123062 Julimar Rd. 1.6km E of Chittering Rd 31o28'35" 116o07'38" 1/11/97 5526027 L. Matilda Reserve, N of Kendenup 34o25'35" 117o33'10" 13/10/98 5285771 20.6km from Ravensthorpe on Hopetoun 33o42'10" 120o11'16" 31/10/98 Rd. 5792342 road verge Redgum Pass Rd. Stirling 34o27'00" 117o40'00" 1998 National Park 5585201 Drummond Nature Reserve, 10km W 31o19'0" 116o24'0" 16/11/99 Bolgart 5836611 sandpit Old Bunbury Coast Rd. 3km N 33o09'15" 115o43'10" 27/10/00 Bunbury 1124366 1km W along Syred Rd. from 34o36'08" 118o05'05" 25/20/85 intersection with Palmdale Rd.
215 APPENDIX 2: VEGETATION OF THE JARRAHDALE MINE SITE
TABLE A2.1 A cumulative list of Western Australian indigenous plant taxa identified in forest and rehabilitation area transects during vegetation surveys at the Jarrahdale bauxite-mine site in 2002. Taxa are listed alphabetically: by family then species. + = taxon present, - = absent, * = Western Australian taxon not indigenous to the jarrah forest. Note: taxa observed outside survey transects have not been included in the list. Taxonomy is consistent with Florabase (http://florabase.calm.wa.gov.au/, accessed July 2007).
Rehabilitation Age (years) Family Taxon Forest 1 5 10 15 > 25
Amaranthaceae Ptilotus drummondii + - - - - - Amaranthaceae Ptilotus manglesii + - - - - - Anthericaceae Laxmannia sessiliflora + - - - - - Anthericaceae Thysanotus manglesianus + - + - + - Anthericaceae Thysanotus multiflorus + + + - - - Anthericaceae Thysanotus patersonii + - + - - - Anthericaceae Thysanotus sp. - - - - - + Anthericaceae Thysanotus sparteus + - - - - - Anthericaceae Thysanotus thyrsoideus + + + + + + Anthericaceae Tricoryne elatior + - - - - - Apiaceae Daucus glochidiatus + - - + - + Apiaceae Hydrocotyle callicarpa + - - - - - Apiaceae Pentapeltis peltigera + + + + + + Apiaceae Platysace compressa + + - + + + Apiaceae Platysace tenuissima - + - - - - Apiaceae Trachymeme pilosa + + + + + + Apiaceae Xanthosia candida + + + + + + Apiaceae Xanthosia heugelii + + - - - - Apiaceae Xanthosia pusilla - - + - - - Asteraceae Brachycome sp. - + - - - - Asteraceae Craspedia variabilis + - - - - - Asteraceae Hyalosperma cotula + + + + + + Asteraceae Lagenifera heugelii + - - + + + Asteraceae Olearia paucidentata + - - - - - Asteraceae Podotheca angustifolia + + - - - - Asteraceae Senecio diaschides + - + - - - Asteraceae Senecio leucoglossus + - - - - - Asteraceae Senecio quadridentus + + + + - + Asteraceae Siloxerus filifolius + - - - - - Asteraceae Trichocline spathulata + - - - - - Asteraceae Waitzia aurea + + - + - - Asteraceae Waitzia paniculata + + - + - - Caesalpiniaceae Labichea punctata + - - - - - Casuarinaceae Allocasuarina fraserii + - + + + - Centrolepidaceae Centrolepis alepyroides + - - - - - Colchicaceae Burchardia sp. + - - - - - Cyperaceae Cyathochaeta avenacea + + + - + - Cyperaceae Lepidosperma leptostachyum + - + + + - Cyperaceae Lepidosperma squamatum + - + - + + Cyperaceae Mesomelaena tetragona + - - - - - Cyperaceae Tetraria capillaris + - + - + + Cyperaceae Tetraria octandra + - - - - - Dasypogonaceae Lomandra brittanii + - - - - - Dasypogonaceae Lomandra caespitosa + - - - - - Dasypogonaceae Lomandra drummondii + - - - - - Dasypogonaceae Lomandra hermaphrodita + - + - + + Dasypogonaceae Lomandra integra + - + + - + Dasypogonaceae Lomandra micrantha + + - + + + Dasypogonaceae Lomandra odora + - - - - - Dasypogonaceae Lomandra preissii + - - - - - Dasypogonaceae Lomandra sonderi + - + + + + Dasypogonaceae Lomandra spartea + - + + - + Dennstaedtiaceae Pteridium esculentum + - - - - - 216 TABLE A2.1 contd. A cumulative list of Western Australian indigenous plant taxa identified in this study. Rehabilitation Age (years) Family Taxon Forest 1 5 10 15 > 25 Dilleniaceae Hibbertia acerosa + - + + + - Dilleniaceae Hibbertia aff. helianthemoides + - - + - - Dilleniaceae Hibbertia amplexicaulis + + + + + + Dilleniaceae Hibbertia commutata + - + + - + Dilleniaceae Hibbertia commutata sl. variant + - - - + - Dilleniaceae Hibbertia hypericoides + - - + + - Dilleniaceae Hibbertia pachyrrhiza - - - - + - Dilleniaceae Hibbertia perfoliata + - + - - + Droseraceae Drosera sp. + - - - - - Epacridaceae Astroloma ciliatum + - - - - - Epacridaceae Astroloma pallidum + - - - + - Epacridaceae Leucopogon capitellatus + - - - - + Epacridaceae Leucopogon nutans + - + + + + Epacridaceae Leucopogon propinquus + - - - + - Epacridaceae Leucopogon verticillatus + - - - - + Epacridaceae Styphelia tenuifolia + - - - + - Euphorbiaceae Phyllanthus calycinus + - + + + + Euphorbiaceae Poranthera huegelii + - - - - - Goodeniaceae Dampiera linearis + - + - - - Goodeniaceae Goodenia pulchella + + - - - - Goodeniaceae Lechenaultia biloba + - - - - - Goodeniaceae Scaevola calliptera + - + + + + Haemodoraceae Conostylis aculeata + - - - - - Haemodoraceae Conostylis serrulata + - + - - - Haemodoraceae Conostylis setigera + - - - + - Haemodoraceae Conostylis setosa + - + + - + Haemodoraceae Haemodorum paniculatum + - - - + - Iridaceae Patersonia babianoides + - - - - - Iridaceae Patersonia occidentalis + - + - - - Iridaceae Patersonia pygmaea + - - - - - Iridaceae Patersonia umbrosa - - + - - - Juncaceae Juncus pallidus - + + - - - Lamiaceae Hemigenia ramosissima + + + - - - Lobeliaceae Isotoma hypocrateriformis + - - - - - Lobeliaceae Lobelia gibbosa + - - - - - Loganiaceae Logania serpyllifolia + - - - - + Mimosaceae Acacia alata + + + - - - Mimosaceae Acacia barbinervis + - - - - - Mimosaceae Acacia celastrifolia* + + + + + - Mimosaceae Acacia cyclops + - - - - - Mimosaceae Acacia drummondii + + + + + - Mimosaceae Acacia drummondii subsp. affinis - - - - + - Mimosaceae Acacia empelioclada* - - - - + - Mimosaceae Acacia extensa + + + + + + Mimosaceae Acacia horridula - - + - - - Mimosaceae Acacia lateriticola + + + + + - Mimosaceae Acacia myrtifolia - - - - + - Mimosaceae Acacia pulchella + + + + - + Mimosaceae Acacia saligna + + + - + - Mimosaceae Acacia urophylla + + + + + + Mimosaceae Paraserianthes lophantha - + - + - - Myrtaceae Baeckea camphorosmae + - - - - - Myrtaceae Calothamnus graniticus subsp. - - - - + - leptophyllus Myrtaceae Calothamnus quadrifidus - - - - + - Myrtaceae Calothamnus rupestris - - - - + - Myrtaceae Eucalyptus calophylla + + + + + + Myrtaceae Eucalyptus diversifoilia* - - - - + - Myrtaceae Eucalyptus marginata + + + + + + Myrtaceae Eucalyptus marginata subsp. - + + + - - thalassica Myrtaceae Eucalyptus megacornuta* - - - - - + Myrtaceae Eucalyptus patens - - - + + - 217 TABLE A2.1 contd. A cumulative list of Western Australian indigenous plant taxa identified in this study. Rehabilitation Age (years) Family Taxon Forest 1 5 10 15 > 25 Myrtaceae Eucalyptus wandoo + - - - - - Myrtaceae Hypocalymma angustifolium + + - + + - Myrtaceae Kunzea recurva + - - - + - Myrtaceae Leptospermum erubescens + - - - - - Myrtaceae Melaleuca incana subsp. incana - - - + - - Myrtaceae Melaleuca viminea - - - + - - Myrtaceae Regelia ciliata* - - - - + - Onagraceae Epilobium billardierianum - + - - - - Orchidaceae Caladenia flava + - + - + + Orchidaceae Caladenia longiclavata - - - - - + Orchidaceae Caladenia macrostylis - - - - + - Orchidaceae Cryptostylis ovata + - - - - - Orchidaceae Cyanicula sericea + - - - + - Orchidaceae Cyrtostylis heugelii + - - - - - Orchidaceae Eriochilus sp. + - - - - - Orchidaceae Microtis media - - + + + + Orchidaceae Prasophyllum parvifolia + - - - - - Orchidaceae Pterostylis aff. nana + - + + + + Orchidaceae Pterostylis barbata + - - + + - Orchidaceae Pterostylis recurva + - + + + + Orchidaceae Pterostylis sanguinea - - + - + - Orchidaceae Pterostylis vittata + - - - - + Orchidaceae Pyrorchis nigricans + - - - - - Orchidaceae Thelymitra aff. macrophylla + - - + - + Orchidaceae Thelymitra benthamiana - - - + - + Orchidaceae Thelymitra crinita + - - - - - Papilionaceae Bossiaea aquafolia + + + + + + Papilionaceae Bossiaea ornata + + + + + + Papilionaceae Bossiaea pulchella - - - - + + Papilionaceae Davesia decurrens + - - - - - Papilionaceae Davesia ramosissima* - - - + + - Papilionaceae Davesia rhombifolia + - - - - - Papilionaceae Gnaphalium sphaericum + + + + - + Papilionaceae Gompholobium knightianum + + + + + - Papilionaceae Gompholobium marginatum + - + + - + Papilionaceae Gompholobium polymorphum + - + - + - Papilionaceae Gompholobium preissii + - + - - - Papilionaceae Hardenbergia comptoniana + - + + + - Papilionaceae Hovea chorizemifolia + + + - - + Papilionaceae Hovea trisperma + - + + - + Papilionaceae Kennedia coccinea + + + + - + Papilionaceae Kennedia prostrata + + + + + + Papilionaceae Mirbelia dilatata + + + + + + Papilionaceae Sphaerolobium linophyllum + - - - - - Papillionaceae Viminaria juncea + - + + + - Phormiaceae Dianella revoluta var. divaricata + - - - + + Phormiaceae Stypandra glauca + + - + + - Pittosporaceae Sollya heterophylla + - + + + - Poaceae Amphipogon amphipogonoides + + + - + + Poaceae Austrodanthonia caespitosa + - - - - - Poaceae Austrodanthonia pilosa + - - - + + Poaceae Austrodanthonia sp. + + - - - - Poaceae Austrostipa semibarbata + - - - + - Poaceae Dichelachne crinita + + + - - + Poaceae Microlaena stipoides var. stipoides + - - - - - Poaceae Neurachne alopecuroidea + - + - - - Poaceae Tetrarrhena laevis + + + - + + Polygalaceae Comesperma confertum + - - - - - Polygalaceae Comesperma virgatum + + - - - - Proteaceae Adenanthos barbigerus + - + + + + Proteaceae Banksia grandis + + + + - + Proteaceae Dryandra carduacea - - - - + - Proteaceae Dryandra formosa* - - - - + - 218 TABLE A2.1 contd. A cumulative list of Western Australian indigenous plant taxa identified in this study. Rehabilitation Age (years) Family Taxon Forest 1 5 10 15 > 25 Proteaceae Dryandra fraserii - - - - + - Proteaceae Dryandra lindleyana + - - - - - Proteaceae Dryandra nivea - - - - + - Proteaceae Dryandra sessilis + - - - - - Proteaceae Grevillea diversifolia - - - - + - Proteaceae Grevillea synapheae + - - - - - Proteaceae Grevillea wilsonii + - - - - - Proteaceae Hakea lissocarpha + + + + + - Proteaceae Hakea ruscifolia + + - - - - Proteaceae Hakea undulata + + + - - - Proteaceae Persoonia elliptica + - - - - - Proteaceae Persoonia longifolia + - + - - + Proteaceae Synaphea petiolaris subsp. petiolaris + - - - - - Proteaceae Xylomelum occidentale - - - - + - Ranunculaceae Clematis pubescens + - - - - + Restionaceae Desmocladus fasciculatus + - - - - - Restionaceae Hypolaena exsulca + - - - - - Restionaceae Loxocarya cinerea + + - - - - Rhamnaceae Trymalium floribundum subsp. + + - + - - floribundum Rhamnaceae Trymalium floribundum subsp. - + - - + - trifidum Rhamnaceae Trymalium ledifolium + + + + + + var.rosmarinifolium Rubiaceae Opercularia echinocephala + + + + + + Rubiaceae Opercularia hispidula + + + + + + Rutacaea Boronia fastigata + + + + + + Rutaceae Boronia crenulata subsp. viminea + - - - - - Rutaceae Philotheca nodiflora + - - - - - Santalaceae Leptomeria cunninghamii + - - - - - Sapindaceae Dodonaea viscosa subsp. angustissima - - - - - + Stackhousiaceae Tripterococcus brunonis + - - - - - Sterculiaceae Lasiopetalum floribunda + + + + + + Sterculiaceae Thomasia glutinosa + + - - + - Stylidiaceae Levenhookia preissii + + - - + + Stylidiaceae Levenhookia pusilla + + - + + - Stylidiaceae Stylidium amoenum + - + - - - Stylidiaceae Stylidium hispidum + - + + + + Stylidiaceae Stylidium repens + - - - - - Thymelaeaceae Pimelea lanata + - - - - - Thymelaeaceae Pimelea lehmanniana + - + - - - Thymelaeaceae Pimelea suaveolens + + - - - - Tremandraceae Tetratheca setigera + - + + - - Xanthorrhoeaceae Xanthorrhoea gracilis + - - - - - Xanthorrhoeaceae Xanthorrhoea preissii + - + - + - Zamiaceae Macrozamia riedlyei + + + + + -
219 TABLE A2.2 A cumulative list of alien plant taxa identified during vegetation surveys of forest and rehabilitation area transects at the Jarrahdale bauxite-mine site in 2002. Taxa are listed alphabetically by family then species. + = taxon present, - = absent. Author names for all species are available on Florabase at http://florabase.calm.wa.gov.au/.
Rehabilitation Age Family Taxon Forest 1 yrs 5 yrs 10 yrs 15 yrs >25 yrs
Asteraceae Conyza bonariensis + + - - - - Asteraceae Dittrichia graveolens + - - - - - Asteraceae Hypochaeris glabra + + + + + + Asteraceae Senecio vulgaris + - - - - - Asteraceae Sonchus asper - + + - - - Asteraceae Sonchus oleraceus - - + - - - Asteraceae Vellereophyton dealbatum - + + - - + Caryophyllaceae Polycarpon tetraphyllum - + - - - - Gentianaceae Centaurium erythraea + - - + + + Juncaceae Juncus bufonus - + - - - - Mimosaceae Acacia decurrens - - - - - + Mimosaceae Acacia longifolia + + - - + - Myrtaceae Eucalyptus maculata - - - - + - Myrtaceae Eucalyptus muelleriana - - - - + - Myrtaceae Eucalyptus resinifera + - - - + + Orchidaceae Disa bracteata - - + + + - Oxalidaceae Oxalis corniculata + - - - - - Papilionaceae Lotus angustissimus + + - - + - Phytolaccaceae Phytolacca octandra - + - - - - Poaceae Aira cupaniana + + + + + + Poaceae Briza maxima + - - - - + Poaceae Briza minor + - - - - - Poaceae Vulpia bromoides + - - - - - Poaceae Vulpia myuros var.myuros + - - + - - Primulaceae Anagalis arvensis var.arvensis + - - - - -
220 223322222223333341123334 11 111111 4913123567802456089078916789012345324567 34 Astr cili 1---1---1--1--1-----1--1------000 46 Burc sp. -----111---1--1------1------000 51 Clem pube 1---11---1-1-----111111------000 71 Drya lind -111-11111--1--1----1-1------000 72 Erio sp. ----1-11--111-11------000 105 Isot hypo 1-11------1------111------000 115 Lech bilo -1---1-1111-111-1------000 118 Leuc capi --1-11-1111---1----111-1------000 Group 1 120 Leuc prop 111111--1--1-1-1----11------1--- 000 121 Leuc vert 1---11--1-11-11--1--11-1------000 144 Pate babi -----11-111--111------11------000 183 Thel crin 1111111111111-111----111------000 191 Tric spat ------1-111-11-1------1------000 199 Xant grac 111--11-1111111-1----111------000 31 Aden barb 11--111-1111111111-11---1--11------1--- 001 35 Astr pall -11-1111-111------1--- 001 37 Aust pilo -11---11-1------1-1-11------1--- 001 54 Cono serr ---111--11--11------1------001 56 Cono seto -11-111111--11--11----1-1---1------1---- 001 61 Damp line -11-1-111------11------1------001 66 Dian revo -1--1--1-----1---1-11------1--- 001 67 Dich crin 1-----111------1-1-1---1----1------001 95 Hibb comm ----11-11111111-1--1-1111-111------001 100 Hove chor -----1-1111-11111-11-1---1-1-----1------001 112 Lage heug 1-11---11--111--1-1--1-1------11-1- 001 117 Lepi squa -111-11-11--11-1-1--1------1------1-- 001 122 Leve prei 1-1------1--1-----1--11------1--1--- 001 Group 2 128 Loma herm -11-111--111111--1------1------1 001 132 Loma spar -1---111----1-1--1------1------1---- 001 133 Loxo cine ---11-1-111-11-11------1------001 145 Pate occi ------1--1--1--1-----111---1------001 149 Pers long -----1--1-1--11-11-1-1-11------001 173 Styl amoe -1---1-1111-11------11---1------001 176 Styp glau -1--11-1111111------1----1--1--- 001 178 Tetr capi --1-11111-11111--11-1--1-1------1-- 001 179 Tetr laev -----1-11111111-1-11-1111--1----1-----1- 001 200 Xant prei 1111-111-1111-11-----1-11--1------1-- 001 42 Boro fast ----1-1-1111111-1-11-11-11-1-1-1----1--- 010 58 Cyan seri ----1--11-11--11------1------11-1 010 130 Loma micr -1--11-1-1-11-11-1-11111-----1--1--11--1 010 131 Loma sond ----111-111-111-11-1--111111------1--1-- 010 180 Tetr seti -----1--111--1-11----1---1-11------010 189 Thys thyr -----11-1-1111111-1------1-----1--1-1- 010 14 Acac alat ------1111-1------1-1---1------011 33 Amph amph -1---1-1--1-111-1--11-1-1-11----1---111- 011 41 Bank gran ----11--1111-1111--1-1-1-11-1-111------011 47 Cala flav -11111---1111-11111111-1-111------1-11 011 59 Cyat aven 111-1-11111--11-1-----1---11----1------1 011 69 Diur brum ------1----111------1----1 011 81 Gomp poly ------11------1----1----1------1 011 82 Gomp prei ------1111------1-1------011 Group 3 92 Hibb acer -11-1-11-----11------1---1------11--- 011 94 Hibb ampl ----11-11-1111111111-1111-111--11---1--1 011 116 Lepi lept 11--11-11111---1------11-1------11--- 011 119 Leuc nuta ---1-111111111-1---11-1-11-11------11-11 011 134 Macr ried -1--11-11-1111111---1111--11--11--1-11-- 011 147 Pent pelt 111-1111111111111--111111-111--11-1-1-1- 011 150 Phyl caly 11111-11------1---11-1-1------1-1-1- 011 154 Plat comp ----11-----111--1111-11------1-1-1111-1 011 168 Scae call 111-11111111-1--1111-11111--11-----1---1 011 174 Styl hisp. 1111-11111-111----1----11---1------11111 011 190 Trac pilo 1111-1-1-----1-1--11111-1---1----1111--- 011 201 Xant cand -1---11111-11111--1111--1111111--1-11--1 011 27 Acac dpul -11------11-11-1-1-1-----11---- 100 73 Cory calo 111111111-111-11-11111111111111111111111 100 102 Hyal cotu -111--1------11-----1---1-1-11-1-11--- 100 141 Oper echi --11-11111-1------11----1--1111-11-111-1 100 170 Sene quad --11---1-----1----11-11---11-1-111-1---- 100 195 Trym ledi 1111---1-11-11--1-11111-1-111--1111-11-- 100 32 Allo fras --1---1-11--11111------11111------11111 101 44 Boss orna -1----11111-11111--1-1--111111-11111-1-1 101 Group 4 74 Euca marg 11--1111111111111-1111111111111111111111 101 113 Lasi flor -1--1-111111-11-1-11111-11111111111111-1 101 160 Pter aff. ----1--1---1--1------1-1-1------1-1--1 101 162 Pter recu ------11111-1--1-1------111-1-----1--1- 101 185 Thys mang 1-----1------1--1------1-1------1-- 101 186 Thys mult -----1--1---1---1------1-1--1-11-1------101 2 *Air cupa 1111------1--1-11---11---1111-11 110 7 *Hyp glab 1-11--1------1----1--1--1111111111-1--11 110 26 Acac pulc -111------1-1-1---11111-111111111111---- 110 28 Acac sali -11------1-11---11 110 78 Gnap spha ------1--1----1-----11-1---- 110 79 Gomp knig --1----11------1--11----1-1-1-- 110 80 Gomp marg --11--1-----1-----1------11-1----1----- 110 Group 5 87 Hake liss -1-1---1---1---1------1111111-111----1 110 103 Hypo angu -1-1-----1-----1------1-----1--111--111- 110 108 Kenn cocc ------1---11----1-1-11-111111111----- 110 109 Kenn pros --1--1------1----1----1-----1111-11--1 110 138 Mirb dila 111---1------11--11111111111111111- 110 16 Acac cela -1------1----11---111-1-1111 111 18 Acac drum ------1---1------1-11--11111-1111 111 20 Acac exte ---1------1----1----1111111111111-11 111 21 Acac dext ------1-11-1----11-1-- 111 22 Acac late ------1------11111-11111111-- 111 29 Acac urop ------1---1------1--1111--11111---11 111 43 Boss aqua ------1--1-1-1111111111111111 111 Group 6 68 Disa brac ------1---11----111-11 111 76 Euca pate ------11-----111-1 111 171 Soll hete -----1------11---1-----11--1 111 196 Vimi junc ---1------1111------1-1--- 111 0000000000000000000000001111111111111111 0000111111111111111111110000000000011111 0111000000000000011111110000001111100111
FIGURE A2.1 Output matrix from TWINSPAN analysis of transect species presence/absence data. Taxa identified as indicator species are listed in bold. Codes for main characteristic species are the first four letters of genus and species names. Number across the top of the matrix are codes for transects. Transects numbered 2-20 are in rehabilitation areas listed by age then replicate; transects numbered 21-39 are the adjacent unmined forest transects adjacent; and 40 and 41 are transects Cov-1 and Cov-2. 221 1.00 lsd = 0.374 Rehabilitation Forest 0.80
a a 0.60 a
a a a 0.40 a a a a log10 (1+ # plants)
0.20
0.00 1 5 10 15 25+ Time since establishment (years)
FIGURE A2.2 Prevalence of rare species (number/transect) in rehabilitation areas and unmined forest. Vertical bars represent ± 1s.e. Data are means of log10(1+n) transformed values for transects. ANOVA found no significant differences between a temporal sequence of rehabilitation area and adjacent forest transects (F = 0.419; P > 0.05)
15
10 Cover %
5 y = 0.0363x + 0.1447 R2 = 0.7368
0 0 5 10 15 20 25 30 Time since establishment (years)
FIGURE A2.3 Changes in mean percentage cover for ‘Lomandra and sedge-like’ species over a chrono-sequence of rehabilitation areas compared to the mean value for unmined forest. The time required for cover of ‘Lomandra and sedge-like’ species cover to achieve the mean forest value was estimated by substituting y = 10.93 (mean forest value) into the equation for the regression line through rehabilitation area cover values. For 1, 5, 10 and 15 year old rehabilitation areas; n = 4, for 25+ year old; n = 3. Note: 26.67 years (the mean age for the three 25+ year old transects) has been used to make the horizontal scale linear. Data are means of raw values. Transect area = 0.025ha (5m x 50m). Vertical bars represent ± 1 s.e. The dashed line is the mean cover value for ‘Lomandra and sedge-like’ species for all forest transects and the broad coloured bar represents ± 1 s.e.
222 APPENDIX 3: FORMULAE FOR MEDIA
SEED GERMINATION MEDIUM
Oatmeal Agar (Clements and Ellyard 1979; Dixon 1989)
Rolled Oats 2.5 g Agar 7.0 g pH 5.5
Oatmeal was homogenised with 750 ml of deionised water (DIH2O) in a blender for a five minutes, then transferred to a 1 l jug, made up to volume with deionised water, and the pH adjusted to 5.5. The oatmeal suspension was transferred to a 1 l Schott® bottle containing 7.0 g agar and autoclaved for 20 mins at 121oC. The hot solution was mixed by repeated inversion of the bottle to disperse dissolved agar, then allowed to cool to 60oC. Plates were poured in a sterile airflow in a laminar flow hood and allowed to dry before use or storage.
ISOLATION AND GROWTH MEDIA
Soil Solution Equivalent Agar (SSE) (Mursidawati 2003; Hollick et al. 2004)
Stock A NH4NO3 0.40 g KH2PO4 0.0136 g MgCl.6H2O 0.61 g NaCl 0.058 g Dissolve in 1 litre of deionised H2O
Stock B CaSO4.2H2O 0.861 g/l
Stock C FeNaEDTA 0.073 g/l
MES buffer 0.2 g Sucrose 2.5 g Agar 8.0 g pH 5.5
MES and sucrose were dissolved with stirring in 650 ml of DI H2O in a 1 l heat proof jug. 100 ml of each stock solution is added in the order A, C, B to the stirred solution, NB Calcium salts may precipitate if added before solutions A and B. Adjust the pH to 6.8 and the solution made up to volume with deionised H2O. The solution was transferred to a 1 l Schott® bottle containing 10.0 g agar and autoclaved for 20 mins at 121oC. The hot solution was mixed by repeated inversion of the bottle to disperse dissolved agar, then allowed to cool to 60oC. Plates were poured in a sterile airflow in a laminar flow hood and allowed to dry before use or storage.
223 Fungal Isolation medium (FIM) (Clements and Ellyard 1979)
NaNO3 0.3 g K2HPO4 0.2 g MgSO4.7H2O 0.1 g KCl 0.1 g Yeast Extract 0.1 g Sucrose 2.5 g Agar 7.0 g Streptomycin sulphate 0.05g pH 6.8
Ingredients were added to 990 ml of DI H2O in a 1 l jug, dissolved and the pH adjusted to 6.8. The solution was transferred to a 1 l Schott® bottle containing 7.0 g agar, mixed and autoclave for 20 mins at 121oC. After autoclaving, the hot solution was mixed by repeated inversion of the bottle to disperse dissolved agar, then allowed to cool to 60oC. Streptomycin sulphate (0.05 g) was dissolved in 10ml of DI H2O. The antibiotic solution was sterilised by filtration through a sterile 0.2 µm Whatman Puradisc 25 AS syringe filter into the cooled media and mixed by repeated inversion of the bottle before dispensing. Plates were poured in a sterile airflow in a laminar flow hood and allowed to dry before use or storage.
Potato Dextrose Agar (PDA) PDA powder 39 g/l
Merck Potato Dextrose Agar (39 g) was suspended in 1 l of DI H2O in a 1 l Schott® bottle and autoclaved for 20 mins at 121oC. After autoclaving, the hot solution was mixed by repeated inversion of the bottle to disperse dissolved agar, then allowed to cool to 60oC. Plates were poured in a sterile airflow in a laminar flow hood and allowed to dry before use or storage.
Nutrient Broth NB powder 8 g/l
Bacto® Nutrient Broth powder (8 g) was dissolved in 1 l of DI H2O in a 1 l Schott® bottle and autoclaved for 20 mins at 121oC. Then allowed to cool and dispensed aseptically into sterile 250 ml polycarbonate jars at 50 ml per jar.
SSE Broth
SSE broth was prepared as for SSE Agar (above) omitting the agar. The broth dispensed aseptically into sterile 250 ml polycarbonate jars at 50 ml per jar.
224 Modified Melin Norkans (MMN) (Brundrett et al. 1996)
(NH4)2HPO4 0.25 g KH2PO4 0.50 g MgSO4.7H2O 0.15 g CaCl2.2H2O 0.05 g NaCl 0.025 g Fe2EDTA 0.02 g Glucose 10.0 g (5.0 g) Malt extract 3.0 g Thiamine HCl 0.1 µg pH 5.8
Ingredients were dissolved in 900 ml of DIH2O, mixed well then made up to 1 l with o DIH2O. The broth was autoclaved for 20 mins at 121 C. Then allowed to cool and dispensed aseptically into sterile 250 ml polycarbonate jars at 50 ml per jar.
10% V8/50% SSE Broth (Hollick 2004)
V8 vegetable juice 100 ml SSE broth 500 ml pH 5.5
Solutions were combined and mixed then made up to 1 l with DIH2O. The broth was autoclaved for 20 mins at 121oC. Then allowed to cool and dispensed aseptically into sterile 250 ml polycarbonate jars at 50 ml per jar.
225 226 Appendix 4: Publications
227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 Erratum: Cyrtostylis huegelii was misidentified as C. robusta on page 261 of this paper.
248 249 250 251 252 253 254 255 256 257 258 259 260 The Cat only grinned when it saw Margaret. It looked good natured, she thought: still it had very long claws and a great many teeth, so she felt it ought to be treated with respect.
“Cheshire–Puss,” she began rather timidly, as she did not at all know whether it would like the name: however, it only grinned a little wider.
“Come, it’s pleased so far,” thought Margaret, and she went on, “Would you tell me please, which way I ought to go from here?”
“That depends a good deal on where you want to get to,” said the Cat.
“I don’t care much where___” said Margaret.
“Then it doesn’t matter much which way you go,” said the Cat.
“___ so long as I get somewhere,” Margaret added as an explanation. (Apologies to Lewis Carroll and John Tenniel)
The End
261 262