Academic literature on the topic 'Orchids – Western Australia'

Few plants are so cryptic as the underground orchids, Rhizanthella Rogers (1928: 1), of Australia. Unlike the species on the eastern seaboard of Australia, the Western Australian species spend their entire life cycle, including flowering, below the soil surface (only rarely with the tips of the bracts showing), making them unique among orchids and indeed, among flowering plants generally (Brown et al. 2013). Discovery in 1928 of the first underground orchid in Western Australia was an international sensation where the plant was described as ‘a remarkable subterranean orchid’ (Wilson 1929). The new taxon described in this paper resolves the enigmatic, disjunct distribution of Rhizanthella in Western Australia, where there was thought to be a central and southern node of a single species, R. gardneri Rogers (1928: 1).

Twenty-three orchid species were recorded in Alcoa's permanent vegetation-monitoring plots in unmined and rehabilitated jarrah forest. Of these, 22 were identified in the unmined jarrah forest and 20 were recorded in rehabilitated areas of between 1 and 31 years old. Three species (Cyrtostylis ovata, Lyperanthus serratus and Prasophyllum elatum) were only recorded in the unmined forest and one species was only recorded in rehabilitated areas (Diuris carinata). The overall density of native orchids in the forest was 13 755 plants ha–1, 10 times greater than the density in rehabilitated areas (1381 plants ha–1). The most abundant species in the forest were Cyrtostylis robusta, Caladenia flava, Pterostylis nana and Thelymitra crinita, all with densities greater than 1000 plants ha–1. The most abundant species in the rehabilitated areas were Microtis media, Disa bracteata (an introduced species), Caladenia flava, Pterostylis nana, Diuris longifolia and Pterostylis vittata, all with densities greater than 60 plants ha–1. In rehabilitation older than 10 years, the density of orchids increased to 2685 plants ha–1. Burning in rehabilitated areas resulted in large increases in orchid densities. It is believed that orchid colonisation of rehabilitated bauxite mines is dependent on symbiotic mycorrhiza, which are in turn dependent on development of an organic litter component in the soil.

Recent data on monocotyledonous geophytes from south-western Australia are compiled and compared with those from other areas of mainly mediterranean climate, especially California, Chile and Victoria, Australia. South-western Australia has a high monocot geophyte diversity of 496 species (7% of an estimated native flora of 7100 vascular species), like Victoria (12%) and the Cape region (14%). As in Victoria, orchids are by far the most important group, with c. 400 species, including those likely to be described once ongoing taxonomic research is completed. South-western Australia has higher geophyte numbers than Victoria in all families considered, but a lower geophyte percentage because of a much higher vascular species total. Among south-western Australian non-orchid geophytes, as in Victoria, the most common storage organ is tuberous roots, followed by corms then bulbs and rhizomes, whereas in California bulbs are by far the most common. The presence of microgeophytes with seed-like storage organs is a special feature seen in several phylogenetically unrelated lineages in the south-western Australian and Victorian geophytic flora, especially on granite outcrops. Divergent phylogenetic history is undoubtedly a major factor underlying striking differences between the monocot geophytes of mediterranean Australia, California, Chile, South Africa and Mediterranean countries. Further studies, particularly on the last two regions, will enable better elucidation of these patterns.

This review summarises scientific knowledge concerning the mycorrhizal associations, pollination, demographics, genetics and evolution of Australian terrestrial orchids relevant to conservation. The orchid family is highly diverse in Western Australia (WA), with over 400 recognised taxa of which 76 are Declared Rare or Priority Flora. Major threats to rare orchids in WA include habitat loss, salinity, feral animals and drought. These threats require science-based recovery actions resulting from collaborations between universities, government agencies and community groups. Fungal identification by DNA-based methods in combination with compatibility testing by germination assays has revealed a complex picture of orchid–fungus diversity and specificity. The majority of rare and common WA orchids studied have highly specific mycorrhizal associations with fungi in the Rhizoctonia alliance, but some associate with a wider diversity of fungi. These fungi may be a key factor influencing the distribution of orchids and their presence can be tested by orchid seed bait bioassays. These bioassays show that mycorrhizal fungi are concentrated in coarse organic matter that may be depleted in some habitats (e.g. by frequent fire). Mycorrhizal fungi also allow efficient propagation of terrestrial orchids for reintroduction into natural habitats and for bioassays to test habitat quality. Four categories of WA orchids are defined by the following pollination strategies: (i) nectar-producing flowers with diverse pollinators, (ii) non-rewarding flowers that mimic other plants, (iii) winter-flowering orchids that attract fungus-feeding insects and (iv) sexually deceptive orchids with relatively specific pollinators. An exceptionally high proportion of WA orchids have specific insect pollinators. Bioassays testing orchid-pollinator specificity can define habitats and separate closely related species. Other research has revealed the chemical basis for insect attraction to orchids and the ecological consequences of deceptive pollination. Genetic studies have revealed that the structure of orchid populations is influenced by pollination, seed dispersal, reproductive isolation and hybridisation. Long-term demographic studies determine the viability of orchid populations, estimate rates of transition between seedling, flowering, non-flowering and dormant states and reveal factors, such as grazing and competition, that result in declining populations. It is difficult to define potential new habitats for rare orchids because of their specific relationships with fungi and insects. An understanding of all three dimensions of orchid habitat requirements can be provided by bioassays with seed baits for fungi, flowers for insects and transplanted seedlings for orchid demography. The majority of both rare and common WA orchids have highly specific associations with pollinating insects and mycorrhizal fungi, suggesting that evolution has favoured increasing specificity in these relationships in the ancient landscapes of WA.

Vital-statistics data concerning population viability were gathered for four of the rarest orchids in Western Australia using surveys to define population sizes and habitat areas and annual measurements of plant demographics. These orchids were Caladenia melanema, C. graniticola, C. williamsiae and Drakaea isolata from the wheatbelt of Western Australia. This agricultural area has a Mediterranean climate with unreliable rainfall, and is >80% cleared of native vegetation. Surveys with 10–30 volunteers increased population-size estimates by up to 10 times and provided spatial data to define core habitat areas. These areas included most of the individuals of a species, but were only 2–10 ha in size. Within these areas, orchids were often highly aggregated in patches a few metres wide, potentially resulting in a high degree of intraspecific competition. Vital statistics were obtained using 4-m wide and 30–50-m-long transects to measure rates of emergence, flowering, grazing and seed-set for each orchid. Plants emerging at the same position in different years were considered to be the same individual, but most emerged in new positions. Many plants emerged just once in 4 years, and 2–3 years of dormancy was common. Emergence frequencies were used to provide estimates of population sizes that were two or three times larger than suggested by data from a single year. Seed production was typically very low. Grazing by kangaroos and rabbits was most severe for C. melanema, but was greatly reduced by fencing. Severe drought prevented flowering of C. graniticola in the driest year, whereas other species were more resilient. These orchids are likely to persist as long as there are some years where rainfall is sufficient for flowering and seed set followed by a year with adequate rain for seed germination. Populations of all these orchids were stable or increasing, but they are still at high risk of extinction because of the impacts of increasing soil salinity or fire on their habitats. These species are unlikely to spread elsewhere in the highly cleared and fragmented wheatbelt. Intervention by hand-pollination, grazing protection and translocation to new locations is required to mitigate these risks. Results were summarised in vital statistics report cards with thresholds set to inform conservation management for these species. Core habitat maps and vital-statistics report cards should also be valuable new tools for terrestrial-orchid conservation in other biomes.

The pollination of 20 common terrestrial orchids was studied in a 60-ha urban banksia and eucalypt dominated woodland in Western Australia. Five years of data (24,000 flowers, 6800 plants) measured fruit set relative to floral areas, capsule volumes, climate, phenology, pollination mechanisms, disturbance tolerance and demography. Pollination varied from 0–95% of flowers, floral displays from 90–3300 mm2 and capsules from 15–1300 mm3 per spike. Pollination traits strongly influenced outcomes, with self-pollination highest (59—95%), followed by sexually deceptive autumn or winter-flowering (18–39%), visual deception (0–48%) and sexually deceptive spring-flowering (13–16%). Pollination was limited by drought in autumn or spring and cool winter temperatures. Some orchids were resilient to drought and one formed seed after the leaves withered. Plant density had the greatest impact on fruit set for orchids forming large groups, especially for sexually deceptive pollination. Consequently, small group average (SGA) pollination was up to 4× greater than overall averages and peak seed production occurred in the best locations for genetic exchange and dispersal. SGA rates and seedpod volumes were strongly linked to clonality, but not to demographic trends. Resource competition limited flowering at higher plant densities and competition within spikes resulted in smaller, later-forming seedpods. Pollination data from co-occurring common orchids identified five evolutionary trade-offs linked to pollination, provided baseline data for rare species and revealed impacts of changing climate.

Data on monocotyledonous geophytes from a recent Victorian flora are compiled and compared with those from California and some other areas of mainly mediterranean climate. Victoria's monocot geophyte diversity of 9% places it with parts of South Africa and Western Australia in a group of much higher diversity than California and Chile. The Victorian list is dominated by orchids (all with tuberous roots) and that from California by Alliaceae, Calochortaceae and Liliaceae, with bulbs being the predominant storage organ. Only four families of the 17 involved have native species in both California and Victoria. Most taxa in both areas are dormant in summer and grow during the cool season. However, the Amaryllidaceae found in the Sonoran Desert and the driest parts of Victoria are able to grow in the warm season in response to summer rain.

Orchids require mycorrhizal fungi for germination of seed and growth of seedlings. The colonisation of bauxite-mine rehabilitation areas by orchids is therefore dependent on the availability of both seed and mycorrhizal fungi. Orchid mycorrhizal fungi baiting trials were carried out in rehabilitation areas that were 1, 10 and 26 years old (established in 2001, 1992 and 1976) and adjacent unmined jarrah forest areas at Jarrahdale, Western Australia. Fungal baits consisted of buried six-chambered nylon-mesh packets containing seed of six jarrah forest orchid taxa, Caladenia flava subsp. flava R.Br., Disa bracteata Sw., Microtis media subsp. media R.Br., Pterostylis recurva Benth., Pyrorchis nigricans (R.Br.) D.L.Jones & M.A.Clem. and Thelymitra crinita Lindl. Detection of orchid mycorrhizal fungi was infrequent, especially at the youngest rehabilitation sites 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 subsp. flava and T. crinita mycorrhizal fungi were the most frequently detected. The presence of C. flava mycorrhizal fungi was correlated with leafy litter cover and maximum depth, and soil moisture at the vegetation type scale (50 × 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 both at rehabilitated sites (15–25% of baits) and in 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, since the infrequent detection of fungi suggests that they occur in isolated patches of soil, the majority of dispersed orchid seeds are likely to perish, especially in recently disturbed habitats.

The Orchidaceae is characterized by a remarkably diverse range of life forms and some of the most highly specialized interactions with soil fungi and insect pollinators found in the flowering plants. Many species are rare or threatened with extinction either directly through loss of habitat or over-collection or, indirectly through debilitation or loss of mycorrhizal association or pollinator capacity. Australian temperate terrestrial orchids represent one of the most threatened groups in the Australian flora with many taxa clinging to existence in urban and rural bushland remnants, road verges and unprotected bushland. The aim of this study is to research and develop integrated conservation based on critical aspects of terrestrial orchid biology and ecology, towards the recovery of the rare and endangered Western Australian terrestrial orchid Caladenia huegelii. This study identified key aspects involved in an integrated conservation approach and research focused on conservation genetics, mycorrhizal interactions and in situ and ex situ conservation strategies for this species. Using polymorphic microsatellite molecular markers, high levels of genetic diversity were found within remnant populations of C. huegelii, while weak differentiation was observed among populations over the species geographic range. These results indicate historic genetic exchange between C. huegelii populations, a possible consequence of the sexually deceptive pollination strategy and the capacity for widespread seed dispersal. Symbiotic germination studies revealed compatibility barriers to C. huegelii germination with the orchid possessing a highly specific orchid-mycorrhizal association relative to common sympatric congeners. These results were reflected in a phylogenetic analysis of DNA sequences, revealing C. huegelii associates with only one endophyte species within the fungal family Sebacinaceae across its geographic range. Large scale in situ seed baiting demonstrated that endophytes compatible with C. huegelii were limited in distribution relative to common and widespread orchid species, a feature for C. huegelii that may be a major contributing factor in limiting the distributional range of the species. Detailed, within site seed baiting methods identified hotspots for mycorrhizal fungus compatible with C. huegelii that were unoccupied by the orchid. These mycorrhizal hotspots where used to investigate the effect of endophyte presence on survival of transplanted mature plants and seedling outplants. The in situ survival of glasshouse propagated seedlings was further optimized by incubating seedlings in growth containers before transfer to soil and outplanting seedlings in their second growing season. The findings of this study will substantially advance the recovery of C. huegelii and provide benchmark knowledge for similar projects with other rare and threatened terrestrial orchid species.

[Truncated 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. ... 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. 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.

Many orchids require mycorrhizal symbioses with fungi for their development and survival. Rhizanthella gardneri the Western Australian underground orchid is associated with the companion plant Melaleuca uncinata and its ectomycorrhizal fungus symbiont. Much less is known about the habitat requirements of its sister species, R. slateri, which occurs in Eastern Australia. The absence of chlorophyll from Rhizanthella gardneri and R. slateri results in total dependency on associations with fungal symbionts. Many ecological and biological aspects of these fascinating orchids remained poorly known, including the identity of the fungal associates and the nature of their tripartite associations with Rhizanthella and Melaleuca. Extremely high specificity of these mycorrhizal relationships is likely to be the most important factor explaining the highly specific habitat requirements of underground orchids. The purpose of this study was to conduct further investigations of the role of the mycorrhizal associations of Australian underground orchids by identifying the fungi involved in these associations, optimising their growth in sterile culture and devising efficient means for synthesising their tripartite associations with R. gardneri and M. uncinata. In total, 16 isolates of fungi were successfully obtained from the two underground orchids and used in a series of experiments to understand both the nature of the fungi and their relationship with orchids. The identity of these fungi was established by using conventional morphological and molecular methods. Cultural and morphological studies revealed that all isolates from R. gardneri and R. slateri were binucleate rhizoctonias with affinities to members of the genus Ceratobasidium. However, the teleomorph state that was observed from the R. slateri symbiont during this study more closely resembled a Thanatephorus species. Further identification using ITS sequence comparisons confirmed that mycorrhizal fungi of Rhizanthella belonged to the Rhizoctonia alliance with relatives that include Thanatephorus, Ceratobasidium, or Rhizoctonia from other continents with over 90% similarity. Most of these related fungi are known as plant pathogens, but some were orchid mycorrhizal fungi. However, the isolates from the two underground orchids were most closely related to each other and formed a discrete group relative to other known members of the Rhizoctonia alliance. Sterile culture experiments determined culture media preferences for mycorrhizal fungi from Rhizanthella and other orchids. A fully defined sterile culture medium designed to more closely resemble Australian soil conditions was formulated. This new medium was compared to undefined media containing oats or yeast extract and recommendations for growth of these fungi are provided. The undefined media based on oats provided the best growth of most fungi, but the new Australian soil media was also effective at growing most orchid mycorrhizal fungi and this fully defined media was less prone to contamination and should provide more reproducible results. A comparison of three methods for inoculating M. uncinata with the underground orchid fungi resulted in the production and characterisation of ectomycorrhizal roots and hyphae formed by fungi isolated from R. gardneri and R. slateri. These underground orchid fungi could easily be distinguished from other mycorrhizal fungi (caused by airborne contamination) by the characteristic appearance of these roots and hyphae. A new system for growing and observing tripartite mycorrhizal associations was devised using pots with side viewing windows and the use of transparent seed packets to contain Rhizanthella seeds. This method allowed all the stages of seed germination to be observed in the glasshouse, culminating in the production of underground orchid rhizomes. Seed germination was only successful when seed was placed directly over active M. uncinata ectomycorrhizas confirmed to belong to the correct fungus by microscopic observations through the side of window pots. The importance of these new scientific discoveries concerning the biology and ecology of the underground orchids and their associated fungi for the recovery of these critically endangered orchids are discussed.

The specificity of fungal isolates from endemic Western Australian orchid species and hybrids in the tribe Diurideae was investigated using symbiotic seed germination and analysis of the fungal DNA by amplified fragment length polymorphism (AFLP). The distribution of the fungal isolates in the field was also assessed using two different seed baiting techniques. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. Two groups of orchids in the tribe Diurideae were studied. Firstly, a number of Caladenia species, their natural hybrids and close relatives from the southwest of Western Australia were selected because orchid species from the genus Caladenia are considered to have among the most specific mycorrhizal relationships known in the orchid family ? an ideal situation for the investigation of mycorrhizal specificity. Secondly, species of Drakaea and close relatives, from the southwest of Western Australia and elsewhere in Australia, which are never common in nature and occur in highly specialised habitats, were selected to investigate the influence of habitat on specificity. Seed from the common species Caladenia arenicola germinated on fungal isolates from adult plants of both C. arenicola and its rare and endangered relative C. huegelii, while seed from C. huegelii only germinated on its own fungal isolates. The AFLP analysis grouped the fungal isolates into three categories: nonefficaceous fungi, C. huegelii type fungi, and C. arenicola type fungi. The group of C. huegelii type fungi included some fungal isolates from C. arenicola. An analysis of the AFLP fingerprints of C. arenicola fungal isolates from different collection locations showed that some, but not all, populations were genetically distinct, and that one population in particular was very variable. Despite being thought to have very specific mycorrhizal relationships, Caladenia species hybridise frequently and prolifically in nature, often forming self-perpetuating hybrid lineages. Five natural hybrids within Caladenia and its closest relatives were investigated. Symbiotic cross-germination studies of parental and hybrid seed on fungi from the species and the naturally occurring hybrids were compared with AFLP analyses of the fungal isolates to answer the question of which fungi the hybrids use. The germination study found that, while hybrid seeds can utilise the fungi from either parental species under laboratory conditions, it is likely that the natural hybrids in situ utilise the fungus of only one parental species. Supporting these observations, the AFLP analyses indicated that while the parental species always possessed genetically distinct fungal strains, the hybrids may share the mycorrhizal fungus of one parental species or possess a genetically distinct fungal strain which is more closely related to the fungus of one parental species than the other. The work on Caladenia hybrids revealed that C. falcata has a broadly compatible fungus that germinated seeds of C. falcata, the hybrid C. falcata x longicauda, and species with different degrees of taxonomic affinity to C. falcata. In general, germination was greater from species that were more closely related to C. falcata: seeds from Caladenia species generally germinated well on most C. falcata isolates; species from same subtribe (Caladeniinae) germinated well to the stage of trichome development on only some of the fungal isolates and rarely developed further; and seeds from species from different subtribes (Diuridinae, Prasophyllinae, Thelymitrinae) or tribes (Orchideae, Cranichideae) either germinated well to the stage of trichome development but did not develop further, or did not germinate at all. The AFLP analysis of the fungal isolates revealed that the fungi from each location were genetically distinct. In situ seed baiting was used to study the introduction, growth and persistence of orchid mycorrhizal fungi. A mycorrhizal fungus from Caladenia arenicola was introduced to sites within an area from which the orchid and fungus were absent, adjacent to a natural population of C. arenicola. In the first growing season, the fungus grew up to 50 cm from its introduction point, usually persisted over the summer drought into the second season and even into the third season, stimulating germination and growth to tuber formation of the seeds in the baits. Watering the inoculated areas significantly increased seed germination. Mycorrhizal relationships in Drakaeinae were less specific than in Caladeniinae. A study of the species Spiculaea ciliata revealed that this species, when germinated symbiotically, develops very rapidly and has photosynthetic protocorms, unlike all other members of the Drakaeinae. An AFLP analysis of the fungal isolates of this species grouped the isolates according to whether they had been isolated from adult plants or reisolated from protocorms produced in vitro. Isolates were genetically distinct when compared before germination and after reisolation. A cross-species symbiotic germination study of seeds of three Drakaea species and one Paracaleana species against fungal isolates from the same species and several other Drakaeinae species revealed lower specificity in this group than previously thought. A number of fungal isolates from Drakaea and Paracaleana species germinated two or more seed types, while all seed types germinated on fungal isolates from other species and the seed of Drakaea thynniphila germinated to some extent on every fungal isolate tested. An AFLP analysis of the Drakaeinae fungal isolates supported this information, revealing little genetic differentiation between the fungi of different orchid species. An ex situ seed baiting technique was used to examine the role of mycorrhizal fungi in microniche specialisation in the narrow endemic Drakaea. Soil samples from within and outside two Drakaea populations were tested for germination of the relevant seed types. In both cases, germination was significantly higher on soil samples from within than outside the populations, suggesting that the relevant mycorrhizal fungi may be restricted to the same microniches as the Drakaea species. The presence of similar fungi at distant, disjunct locations may be related to the extreme age and geological stability of the Western Australian landscape. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. It appears that the mycorrhizal relationships in these groups of orchids are not as specific as was previously thought. For reintroduction work, a broad sampling strategy is necessary, as it cannot be assumed that the same orchid species has the same fungus at different locations. A broadly compatible fungus may be of considerable utility in conservation work, such as in situations where a specific fungus appears to have poor saprophytic competence or where soil conditions have been altered. Seed baiting studies provide additional data on fungal distribution in situ. In general, molecular data do not provide information about efficacy or fungal distribution, so research programs that combine symbiotic germination studies with seed baiting investigations and genetic analyses of the fungi will provide the maximum benefit for designing more effective conservation programs.

The specificity of fungal isolates from endemic Western Australian orchid species and hybrids in the tribe Diurideae was investigated using symbiotic seed germination and analysis of the fungal DNA by amplified fragment length polymorphism (AFLP). The distribution of the fungal isolates in the field was also assessed using two different seed baiting techniques. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. Two groups of orchids in the tribe Diurideae were studied. Firstly, a number of Caladenia species, their natural hybrids and close relatives from the southwest of Western Australia were selected because orchid species from the genus Caladenia are considered to have among the most specific mycorrhizal relationships known in the orchid family – an ideal situation for the investigation of mycorrhizal specificity. Secondly, species of Drakaea and close relatives, from the southwest of Western Australia and elsewhere in Australia, which are never common in nature and occur in highly specialised habitats, were selected to investigate the influence of habitat on specificity. Seed from the common species Caladenia arenicola germinated on fungal isolates from adult plants of both C. arenicola and its rare and endangered relative C. huegelii, while seed from C. huegelii only germinated on its own fungal isolates. The AFLP analysis grouped the fungal isolates into three categories: nonefficaceous fungi, C. huegelii type fungi, and C. arenicola type fungi. The group of C. huegelii type fungi included some fungal isolates from C. arenicola. An analysis of the AFLP fingerprints of C. arenicola fungal isolates from different collection locations showed that some, but not all, populations were genetically distinct, and that one population in particular was very variable. Despite being thought to have very specific mycorrhizal relationships, Caladenia species hybridise frequently and prolifically in nature, often forming self-perpetuating hybrid lineages. Five natural hybrids within Caladenia and its closest relatives were investigated. Symbiotic cross-germination studies of parental and hybrid seed on fungi from the species and the naturally occurring hybrids were compared with AFLP analyses of the fungal isolates to answer the question of which fungi the hybrids use. The germination study found that, while hybrid seeds can utilise the fungi from either parental species under laboratory conditions, it is likely that the natural hybrids in situ utilise the fungus of only one parental species. Supporting these observations, the AFLP analyses indicated that while the parental species always possessed genetically distinct fungal strains, the hybrids may share the mycorrhizal fungus of one parental species or possess a genetically distinct fungal strain which is more closely related to the fungus of one parental species than the other. The work on Caladenia hybrids revealed that C. falcata has a broadly compatible fungus that germinated seeds of C. falcata, the hybrid C. falcata x longicauda, and species with different degrees of taxonomic affinity to C. falcata. In general, germination was greater from species that were more closely related to C. falcata: seeds from Caladenia species generally germinated well on most C. falcata isolates; species from same subtribe (Caladeniinae) germinated well to the stage of trichome development on only some of the fungal isolates and rarely developed further; and seeds from species from different subtribes (Diuridinae, Prasophyllinae, Thelymitrinae) or tribes (Orchideae, Cranichideae) either germinated well to the stage of trichome development but did not develop further, or did not germinate at all. The AFLP analysis of the fungal isolates revealed that the fungi from each location were genetically distinct. In situ seed baiting was used to study the introduction, growth and persistence of orchid mycorrhizal fungi. A mycorrhizal fungus from Caladenia arenicola was introduced to sites within an area from which the orchid and fungus were absent, adjacent to a natural population of C. arenicola. In the first growing season, the fungus grew up to 50 cm from its introduction point, usually persisted over the summer drought into the second season and even into the third season, stimulating germination and growth to tuber formation of the seeds in the baits. Watering the inoculated areas significantly increased seed germination. Mycorrhizal relationships in Drakaeinae were less specific than in Caladeniinae. A study of the species Spiculaea ciliata revealed that this species, when germinated symbiotically, develops very rapidly and has photosynthetic protocorms, unlike all other members of the Drakaeinae. An AFLP analysis of the fungal isolates of this species grouped the isolates according to whether they had been isolated from adult plants or reisolated from protocorms produced in vitro. Isolates were genetically distinct when compared before germination and after reisolation. A cross-species symbiotic germination study of seeds of three Drakaea species and one Paracaleana species against fungal isolates from the same species and several other Drakaeinae species revealed lower specificity in this group than previously thought. A number of fungal isolates from Drakaea and Paracaleana species germinated two or more seed types, while all seed types germinated on fungal isolates from other species and the seed of Drakaea thynniphila germinated to some extent on every fungal isolate tested. An AFLP analysis of the Drakaeinae fungal isolates supported this information, revealing little genetic differentiation between the fungi of different orchid species. An ex situ seed baiting technique was used to examine the role of mycorrhizal fungi in microniche specialisation in the narrow endemic Drakaea. Soil samples from within and outside two Drakaea populations were tested for germination of the relevant seed types. In both cases, germination was significantly higher on soil samples from within than outside the populations, suggesting that the relevant mycorrhizal fungi may be restricted to the same microniches as the Drakaea species. The presence of similar fungi at distant, disjunct locations may be related to the extreme age and geological stabilityof the Western Australian landscape. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. It appears that the mycorrhizal relationships in these groups of orchids are not as specific as was previously thought. For reintroduction work, a broad sampling strategy is necessary, as it cannot be assumed that the same orchid species has the same fungus at different locations. A broadly compatible fungus may be of considerable utility in conservation work, such as in situations where a specific fungus appears to have poor saprophytic competence or where soil conditions have been altered. Seed baiting studies provide additional data on fungal distribution in situ. In general, molecular data do not provide information about efficacy or fungal distribution, so research programs that combine symbiotic germination studies with seed baiting investigations and genetic analyses of the fungi will provide the maximum benefit for designing more effective conservation programs.

Rhizanthella gardneri (Rogers) is a critically endangered orchid restricted to two isolated regions of south-western Australia. Rhizanthella gardneri is an entirely subterranean mycoheterotrophic species that purportedly forms a tripartite relationship with a mycorrhizal fungus (Ceratobasidiales) that links with an autotrophic shrub of the Melaleuca uncinata complex to acquire nutrients. Whether the rarity of R. gardneri is intrinsic is overshadowed by the recent effect of extrinsic factors that means R. gardneri requires some form of conservation and may also be a viable candidate for restoration. To create an integrated conservation strategy for R. gardneri, reasons for its decline and knowledge of its biological and ecological functioning must be elucidated. This thesis focuses on three key questions; 1) what are the habitat requirements and limitations to R. gardneri survival; 2) what is the identity and specificity of the fungus R. gardneri forms mycorrhizas with; and 3) does R. gardneri form a nutrient-sharing tripartite relationship with a mycorrhizal fungus and autotrophic shrub. Key climate, soil and vegetation characteristics of known R. gardneri habitats were quantified to provide baseline data for monitoring known R. gardneri populations, to better understand how R. gardneri interacts with its habitat, and to identify possible new sites for R. gardneri introduction. Habitats of the two known R. gardneri populations differed considerably in soil chemistry, Melaleuca structure and Melaleuca productivity. Individual sites within populations were relatively similar in all attributes measured while overall Northern and Southern habitats were distinct from each other. These results suggest that R. gardneri can tolerate a range of conditions and may be more widespread than previously thought, given that there are extensive areas of Melaleuca thickets with similar habitat characteristics across south-western Australia. The fungus forming mycorrhizas with R. gardneri was identified, using nuclear ribosomal DNA sequences, as a Rhizoctonia-type fungus within the Ceratobasidiales. All fungi isolated from R. gardneri individuals representative of its currently known distribution were genetically similar, suggesting R. gardneri is highly dependent on this specific fungal species. Given that R. gardneri appears to exclusively associate with a specific fungal species, species-specific molecular primers were designed and used to analyse the fungi’s presence in known and potential R. gardneri habitats. These results 6 suggest that the fungus exists beyond the known R. gardneri habitats and gives hope to finding new populations.