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BACKGROUND: Conservation through reserves alone is now considered unlikely to achieve protection of plant species necessary to mitigate direct losses of habitat and the pervasive impact of global climate change. Assisted translocation/migration represent new challenges in the face of climate change; species, particularly orchids, will need artificial assistance to migrate from hostile environments, across ecological barriers (alienated lands such as farmlands and built infrastructure) to new climatically buffered sites. The technology and science to underpin assisted migration concepts are in their infancy for plants in general, and orchids, with their high degree of rarity, represent a particularly challenging group for which these principles need to be developed. It is likely that orchids, more than any other plant family, will be in the front-line of species to suffer large-scale extinction events as a result of climate change. SCOPE: The South West Australian Floristic Region (SWAFR) is the only global biodiversity hotspot in Australia and represents an ideal test-bed for development of orchid conservation principles. Orchids comprise 6 % of all threatened vascular plants in the SWAFR, with 76 out of the 407 species known for the region having a high level of conservation risk. The situation in the SWAFR is a portent of the global crisis in terrestrial orchid conservation, and it is a region where innovative conservation solutions will be required if the impending wave of extinction is to be averted. Major threatening processes are varied, and include land clearance, salinity, burning, weed encroachment, disease and pests. This is compounded by highly specialized pollinators (locally endemic native invertebrates) and, in the most threatened groups such as hammer orchids (Drakaea) and spider orchids (Caladenia), high levels of mycorrhizal specialization. Management and development of effective conservation strategies for SWAFR orchids require a wide range of integrated scientific approaches to mitigate impacts that directly influence ecological traits critical for survival. CONCLUSIONS: In response to threats to orchid species, integrated conservation approaches have been adopted (including ex situ and translocation principles) in the SWAFR with the result that a significant, multidisciplinary approach is under development to facilitate conservation of some of the most threatened taxa and build expertise to carry out assisted migration to new sites. Here the past two decades of orchid conservation research in the SWAFR and the role of research-based approaches for managing effective orchid conservation in a global biodiversity hotspot are reviewed.

Critical for conserving endangered orchids is identifying their pollinators and their distribution. Caladenia xanthochila is an endangered orchid that has floral traits characteristic of pollination by food foraging insects. We identified the pollinator(s), mechanisms of attraction and the presence of pollinators at natural, existing and potential translocation sites. Furthermore, we quantified pollination success at translocation sites and investigated the effect of rainfall on pollination success over 19 years at a natural site. We clarify if sharing of pollinators occurs with closely related species by comparing the CO1 barcoding region of the pollinators' DNA. Caladenia xanthochila was pollinated by a single species of thynnine wasp, Phymatothynnus aff. nitidus. Caladenia xanthochila produced 27.0 µg ± 7.1 sucrose on the labellum, while pollinators vigorously copulated with glandular clubs on the sepal tips, suggestive of a mixed pollination system. Pollination success of C. xanthochila was 7.6 ± 1.5% SE at the natural site and 16.1 ± 3.6% SE across the translocation sites. Furthermore, hand pollinations demonstrated that pollination was pollen limited. Pollination success was significantly related to average rainfall during the growth phase of the orchid (P < 0.001). Potential translocation sites for C. xanthochila were limited, with four of six surveyed lacking the pollinator. We found evidence for cryptic species of Phymatothynnus, with C. xanthochila pollinators being unique amongst the orchids studied. We recommend hand pollinations at translocated and remnant wild populations to boost initial recruitment. The evidence for cryptic species of pollinators further highlights the need for accurate identification of pollinators.

Innate colour preferences promote the capacity of pollinators to find flowers, although currently there is a paucity of data on how preferences apply to real flowers. The Australian sugarbag bee (Tetragonula carbonaria Sm.) has innate preferences for colours, including UV-absorbing white. Sugarbag bees are pollinators of the terrestrial orchid Caladenia carnea R.Br., which has both white and pink morphs. In laboratory conditions, we tested flower-naïve bees with the white and pink flower morphs revealing a significant preference for the white morph, consistent with experiments using artificial stimuli. In experiments to understand how bees may select food-deceptive orchids following habituation to a particular colour morph, we observed a significant increase in choices towards novel white flowers. We also observed that the presence of a UV-reflecting dorsal sepal signal significantly increased bee choices compared to flowers that had the UV signal blocked. Our findings demonstrate that innate preference testing of insect pollinators with artificial stimuli is replicated in a biologically significant scenario with flowers. The findings also underscore how food-deceptive orchids can receive sufficient pollinator visits to ensure pollination by having different morphs that draw on the innate preferences of bees and their ability to make decisions in a complex ecological setting.

Background and AimsMany terrestrial orchids have an obligate requirement for mycorrhizal associations to provide nutritional support from germination to establishment. This study will investigate the ability of orchid mycorrhizal fungi (OMF) to utilize a variety of nutrient sources in the nutrient-impoverished (low organic) soils of the Southwest Australian Floristic Region (SWAFR) in order to effectively compete, survive and sustain the orchid host.MethodsMycorrhizal fungi representing key OMF genera were isolated from three common and widespread species: Pterostylis recurva, Caladenia flava and Diuris corymbosa, and one rare and restricted species: Drakaea elastica. The accessibility of specific nutrients was assessed by comparing growth including dry biomass of OMF in vitro on basal CN MMN liquid media.Key ResultsEach of the OMF accessed and effectively utilized a wide variety of nutrient compounds, including carbon (C) sources, inorganic and organic nitrogen (N) and inorganic and organic phosphorus (P). The nutrient compounds utilized varied between the genera of OMF, most notably sources of N.ConclusionsThese results suggest that OMF can differentiate between niches (micro-niche specialization) in a constrained, highly resource-limited environment such as the SWAFR. Phosphorus is the most limited macronutrient in SWAFR soils and the ability to access phytate by OMF indicates a characterizing functional capacity of OMF from the SWAFR. Furthermore, compared with OMF isolated from the rare D. elastica, OMF associating with the common P. recurva produced far greater biomass over a wider variety of nutritional sources. This suggests a broader tolerance for habitat variation providing more opportunities for the common orchid for recruitment and establishment at a site.

BACKGROUND AND AIMS: Mycorrhizal associations are essential to the plant kingdom. The largest flowering plant family, the Orchidaceae, relies on mycorrhizal fungi for germination, growth and survival. Evidence suggests varying degrees of fungal-host specificity based on a single fungal isolate from a single plant. This paper shows for the first time the diversity of endophytes colonizing in a single plant over consecutive years and the functional significance of this diversity. METHODS: Stem-collars of Caladenia formosa were collected in different seasons and years. Mycorrhizal fungi isolated were tested for their efficacy to induce leafing and genetically determined using ITS-RFLP and sequencing. RESULTS: Multiple mycorrhizal fungi were repeatedly isolated from a single collar that displayed varying effectiveness in germination percentages and adult leaf length. Additional factors contributed to the isolation of effective mycorrhizal fungi; fungal collection season, year of collection and individual isolates. Surface sterilization only improved the number of isolated mycorrhizal fungi. Dual inoculation did not increase germination. All 59 mycorrhizal fungi effective in germinating seed belonged to one clearly defined ITS (internal transcribed spacer) clade and clustered close to Sebacina vermifera (79-89 % homology). Isolates resulting in the greatest germination were not necessarily those resulting in the greatest survival and growth 1 year after germination. CONCLUSION: Single orchid plants contained multiple mycorrhizal fungal strains of one species that had diverse functional differences. These results suggest that our current knowledge of fungal-host specificity may be incomplete due to experimental and analytical limitations. It also suggests that the long-term effectiveness of a mycorrhizal fungus or fungi could only be found by germination and longer-term growth tests rather than genetically.

In a bid to better conserve endangered terrestrial orchids, we detail cryogenic research using a widely distributed terrestrial orchid, Caladenia latifolia, as a model species for development of cryopreservation for primary (seed generated) and secondary (adventitious) protocorms. Primary protocorm cryopreservation (using droplet vitrification) involved a number of experimental lines of inquiry: investigation of a suitable plant vitrification solution (PVS) by comparing three variants of a standard PVS (2, 3 and 4), determining the most suitable primary protocorm developmental stage for successful cryopreservation, testing the effectiveness of a preculture medium treatment prior to cryopreservation, and investigating temperature preconditioning at the preculture stage as well as different components of the recovery medium. Primary protocorms were generated using asymbiotic in vitro germination media developed by the authors specifically for the test species (half-strength MS macroelements and microelements with 5% (v/v) fresh filter sterilized coconut water). Secondary protocorms were propagated using an in vitro proliferation medium (½ MS with 5 μM α-naphthaleneacetic acid+2 μM 6-benzylaminopurine). A modified preconditioning step was developed, involving incubation on ½ MS with 0.2 M raffinose for 48 h at 15°C instead of 20°C. The standard recovery medium (½ MS 1 μM zeatin+0.5 μM gibberellic acid) was replaced after the first week following warming from liquid nitrogen (LN), with asymbiotic germination medium (½ MS+5% (v/v) coconut water) for the remainder of the recovery phase. This new step increased the survival of primary protocorms from 68 to 85%. The average postcryostorage regeneration of plants from primary protocorms increased from 17 to 48% after a 6-wk incubation. A similar protocol increased the survival of secondary protocorms from 63 to 84%. Regeneration of plants from secondary cryostored protocorms increased from 11 to 26% after 14 wk. The protocols developed here provide a useful template for advancing cryoconservation of other orchid taxa, particularly rare and threatened species.

• The dependence of seeds of terrestrial orchids on specific fungi for germination provides a means of locating these fungi in the wild and to investigate the role of appropriate fungi in the germination of orchid seed and development of seedlings under natural field conditions. • Seed baits, comprising orchid (Caladenia arenicola) seed enclosed in fine nylon mesh, were placed at sample points along four transects through two orchid populations in bushland in Western Australia. Seed germination was scored and compared with adult orchid plant distribution and soil factors. • A small fraction of available seed (< 1%) germinated to a stage of tuber formation where survival over the subsequent dry season would have been possible. Germination increased in the vicinity of adult C. arenicola plants, but other factors, such as soil potassium levels and presence of leaf litter, were also correlated with seed germination. • The measurement of the spatial variability in germination events within an orchid habitat demonstrated the availability of new recruitment sites. This information is required to assess the natural recruitment capacity and the potential for orchid reintroduction in natural habitats.

Sexually deceptive orchids attract specific pollinators by mimicking insect sex pheromones. Normally this mimicry is very specific and identical compounds have been identified from orchids and matching females of the pollinators. In this study, we conduct a detailed structure-activity investigation on isomers of the semiochemicals involved in the sexual attraction of the male pollinator of the spider orchid Caladenia plicata. This orchid employs an unusual blend of two biosynthetically unrelated compounds, (S)-β-citronellol and 2-hydroxy-6-methylacetophenone, to lure its Zeleboria sp. thynnine wasp pollinator. We show that the blend is barely attractive when (S)-β-citronellol is substituted with its enantiomer, (R)-β-citronellol. Furthermore, none of the nine-possible alternative hydroxy-methylacetophenone regioisomers of the natural semiochemical are active when substituted for the natural 2-hydroxy-6-methylacetophenone. Our results were surprising given the structural similarity between the active compound and some of the analogues tested, and results from previous studies in other sexually deceptive orchid/wasp systems where substitution with analogues was possible. Interestingly, high-level ab initio and density functional theory calculations of the hydroxy-methylacetophenones revealed that the active natural isomer, 2-hydroxy-6-methylacetophenone, has the strongest intramolecular hydrogen bond of all regioisomers, which at least in part may explain the specific activity.

The Orchidaceae is characterised by the repeated evolution of sexual deception, one of the most specialised pollination strategies. In orchids, sexual deception involves long-range pollinator attraction via mimicry of female insect sex pheromones. At close range, visual signals involving colour mimicry, contrast to the background, and exploitation of pollinator sensory biases could attract pollinators, but remain largely untested. Here we focus on a remarkable system in which species from two only distantly related sexually deceptive orchid genera with strikingly different flowers ( Drakaea livida and three species of Caladenia ) share the same pollinator, males of the thynnine wasp Zaspilothynnus nigripes . We used spectral reflectance measurements and modelling to investigate pollinator perception of colour, including the first examination of overall colour patterns in flowers via colour pattern geometry analyses. Rather than closely matching the colours of female Z. nigripes , these orchids had strong chromatic and achromatic contrast against their backgrounds. For Caladenia , the sepals and petals show high contrast, while in D. livida , which has diminutive petals and sepals, it is the labellum that contrasts strongly against the background. Despite varying in colour, the Caladenia species all had strong within-flower contrast between a UV-bright central target (column and labellum) and a corolla of radiating stripes (petals and sepals). The colour pattern geometry analyses also indicated that the orchids’ overall colour patterns are highly conspicuous against their backgrounds. Contrast, UV, and target patterns could all enhance detection, and exploit pollinators’ innate preferences. Since colour contrast may function with a range of colours and floral forms, attracting pollinators via contrast rather than visual mimicry may be a critical but previously overlooked process facilitating the evolution of sexual deception.

Terrestrial orchids depend on orchid mycorrhizal fungi (OMF) as symbionts for their survival, growth and nutrition. The ability of OMF from endangered orchid species to compete for available resources with OMF from common species may affect the distribution, abundance and therefore conservation status of their orchid hosts. Eight symbiotically effective OMF from endangered and more common Caladenia species were tested for their ability to utilise complex insoluble and simple soluble carbon sources produced during litter degradation by growth with different carbon sources in liquid medium to measure the degree of OMF variation with host conservation status or taxonomy. On simple carbon sources, fungal growth was assessed by biomass. On insoluble substrates, ergosterol content was assessed using ultra-performance liquid chromatography (UPLC). The OMF grew on all natural materials and complex carbon sources, but produced the greatest biomass on xylan and starch and the least on bark and chitin. On simple carbon sources, the greatest OMF biomass was measured on most hexoses and disaccharides and the least on galactose and arabinose. Only some OMF used sucrose, the most common sugar in green plants, with possible implications for symbiosis. OMF from common orchids produced more ergosterol and biomass than those from endangered orchids in the Dilatata and Reticulata groups but not in the Patersonii and Finger orchids. This suggests that differences in carbon source utilisation may contribute to differences in the distribution of some orchids, if these differences are retained on site.