Summary The distribution and abundance of orchid populations depend on a suite of biological and ecological factors, including seed production and dispersal, availability of mycorrhizal fungi and appropriate environmental conditions, with the weighting of these factors depending on the spatial scale considered. Disentangling the factors determining successful orchid establishment represents a major challenge, involving seed germination experiments, molecular techniques and assessment of environmental conditions. Identification of fungi from large‐scale surveys of mycorrhizal associations in a range of orchid species has shown that mycorrhizal fungi may be widespread and occur in varied habitats. Further, a meta‐analysis of seed introduction experiments revealed similar seed germination in occupied and unoccupied habitat patches. Orchid rarity was also unrelated to mycorrhizal specificity. Nonetheless, seed germination within sites appears to depend on both biotic and abiotic conditions. In the few cases that have been examined, coexisting orchids have distinct mycorrhizal communities and show strong spatial segregation, suggesting that mycorrhizal fungi are important drivers of niche partitioning and contribute to orchid coexistence. A broader investigation of orchid mycorrhizal fungus distribution in the soil, coupled with fungus and recruitment mapping, is needed to translate fungal abundance to orchid population dynamics and may lead to better orchid conservation.

Summary• Specific orchid-fungal associations are known for nonphotosynthetic orchids but fungal diversity in photosynthetic orchids is thought to be quite broad. Specific fungal associations will figure prominently in conservation efforts, while diverse associations may require less attention. We combined culture techniques with ITS and mtLSU sequences and phylogenetic analysis to determine the genetic diversity of mycorrhizal fungi associated with an evergreen, a spring-green, and a winter-green orchid and compared this diversity with that published for a nonphotosynthetic orchid.• Mycorrhizal diversity in two of the three photosynthetic orchids was lower than for the nonphotosynthetic orchid. Mycorrhizal diversity in protocorms of the third species was also equal to, or less than, the fungal diversity associated with the nonphotosynthetic species, but adult fungal diversity was greater.• We found that photosynthetic orchids do not necessarily have more diverse mycorrhizal associations than nonphotosynthetic orchids. Similarly, evergreen orchids do not necessarily have greater mycorrhizal diversity than seasonally green orchids. Thus, orchid mycorrhizal diversity may not be determined by adult photosynthetic capacity.

Summary• Despite advances owing to molecular approaches, several hurdles still obstruct the identification of fungi forming orchid mycorrhizas. The Tulasnellaceae exhibit accelerated evolution of the nuclear ribosomal operon, causing most standard primers to fail in polymerase chain reaction (PCR) trials. Insufficient sequences are available from well characterized isolates and fruitbodies. Lastly, taxon-specific PCR primers are needed in order to explore the ecology of the fungi outside of the orchid root. Here, progress in overcoming these hurdles is reported.• Broad-spectrum basidiomycete internal transcribed spacer (ITS) primers that do not exclude most known Tulasnellaceae are presented. BLAST searches and empirical PCR tests support their wide utility within the Basidiomycota.• Taxon-specific ITS primers are presented targeted to orchid-associated Tulasnella, and a core component of the Thelephora-Tomentella complex. The efficiency and selectivity of these primer sets are again supported by BLAST searches and empirical tests.• Lastly, ITS DNA sequences are presented from several strains of Epulorhiza, Ceratorhiza, Ceratobasidium, Sistotrema, Thanatephorus and Tulasnella that were originally described in the landmark mycorrhizal studies of Currah and Warcup. Detailed phylogenetic analyses reveal some inconsistencies in species concepts in these taxonomically challenging resupinate basidiomycetes, but also help to place several sequences from environmental samples.

The characteristics of plant-mycorrhizae associations are known to vary in both time and space, but the ecological consequences of variation in the dynamics of plant-fungus interactions are poorly understood. For example, do plants associate with single fungi or multiple fungi simultaneously, and do the associations persist through a plant's lifetime or do plants support a succession of different fungi? We investigated these and other questions related to plant-fungus interactions in Goodyera pubescens, an evergreen terrestrial orchid of the eastern United States, that interacts with closely related fungi in the genus Tulasnella. Unlike the mycorrhizal associations of other plants, orchid-mycorrhizal associations only benefit the orchid, based on current evidence. Many terrestrial orchids have been found to associate with specific groups of fungi. This characteristic could potentially limit orchids to relatively narrow ranges of environmental conditions and may be a contributing factor in the decline of many orchids in the face of changing environmental conditions. We found that G. pubescens protocorms (developing embryos prior to leaf production) and adults associated with only one fungal individual at a time. The orchid-fungus association persists for years, but during a drought period that was associated with the death of many plants, surviving plants were able to switch to new fungal individuals. These results suggest that G. pubescens interacts with the same fungal partner during periods of modest environmental variation but is able to switch to a different fungal partner. We hypothesize that the ability to switch fungi allows G. pubescens to survive more extreme environmental perturbations. However, laboratory experiments suggest that switching fungi has potential costs, as it increases the risk of mortality, especially for smaller individuals. Our findings indicate that it is unlikely that switching fungi is a common way to improve tolerance of less severe environmental fluctuations and disturbances. These findings may have important implications for plant responses to severe climatic events or to more gradual environmental changes such as global warming.

Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species-to-species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the lady's slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with

Mycorrhizal fungi have substantial potential to influence plant distribution, especially in specialized orchids and mycoheterotrophic plants. However, little is known about environmental factors that influence the distribution of mycorrhizal fungi. Previous studies using seed packets have been unable to distinguish whether germination patterns resulted from the distribution of appropriate edaphic conditions or the distribution of host fungi, as these cannot be separated using seed packets alone. We used a combination of organic amendments, seed packets and molecular assessment of soil fungi required by three terrestrial orchid species to separate direct and indirect effects of fungi and environmental conditions on both seed germination and subsequent protocorm development. We found that locations with abundant mycorrhizal fungi were most likely to support seed germination and greater growth for all three orchids. Organic amendments affected germination primarily by affecting the abundance of appropriate mycorrhizal fungi. However, fungi associated with the three orchid species were affected differently by the organic amendments and by forest successional stage. The results of this study help contextualize the importance of fungal distribution and abundance to the population dynamics of plants with specific mycorrhizal requirements. Such phenomena may also be important for plants with more general mycorrhizal associations.

Summary 1.A fundamental challenge to invasion ecology is to determine what factors cause an exotic species to spread rapidly long after the initial introduction. The increase of a resource (e.g. nitrogen) could trigger an expansion, but in other instances, species have overcome biological limitations (e.g. an Allee effect) like accumulating sufficient genetic diversity for successful reproduction. Understanding the ecological mechanisms governing plant invasions, such as nutrient enrichment or Allee effects, can be used to improve invasive plant management. 2. We used the invasive, introduced grass Phragmites australis as a model to evaluate the role of nutrient enrichment and Allee effects in invasion. Based on recent studies that demonstrated the importance of sexual reproduction in this plant's spread, we chose to focus our efforts on reproductive output. We examined the effects of patch-level genetic diversity on viable seed production across watersheds of the Chesapeake Bay, USA, with differing levels of anthropogenic development (a proxy for nutrient enrichment). In an outdoor mesocosm experiment, we treated Phragmites plants originating from forested and developed watersheds with elevated vs. ambient nutrients and cross vs. self-pollination and determined the effects on viable seed, floret and inflorescence production. 3. The proportion of viable seeds produced at field sites varied widely and was not directly related to watershed development. Instead, seed viability was positively related to patch-level genetic diversity, and patches in more developed watersheds had higher genetic diversity. Also, plants in larger patches produced a higher proportion of viable seeds. In the mesocosm experiment, seed viability was substantially higher for out-crossed plants. Elevated nutrients resulted in greater floret and inflorescence production, particularly for plants originating from developed vs. forested watersheds. 4. These findings have important management implications: small populations should be controlled before they accumulate sufficient genetic variation for prolific viable seed production, and landscape-scale nutrient management could further limit reproductive output. 5. Synthesis and applications. Our research shows how nutrient enrichment and a weak Allee effect can interact across multiple scales to impact invasion success and how understanding the ecological mechanisms governing plant invasions can be used to better inform invasive plant management.

Symbioses are ubiquitous in nature and influence individual plants and populations. Orchids have life history stages that depend fully or partially on fungi for carbon and other essential resources. As a result, orchid populations depend on the distribution of orchid mycorrhizal fungi (OMFs). We focused on evidence that local-scale distribution and population dynamics of orchids can be limited by the patchy distribution and abundance of OMFs, after an update of an earlier review confirmed that orchids are rarely limited by OMF distribution at geographic scales. Recent evidence points to a relationship between OMF abundance and orchid density and dormancy, which results in apparent density differences. Orchids were more abundant, less likely to enter dormancy, and more likely to re-emerge when OMF were abundant. We highlight the need for additional studies on OMF quantity, more emphasis on tropical species, and development and application of next-generation sequencing techniques to quantify OMF abundance in substrates and determine their function in association with orchids. Research is also needed to distinguish between OMFs and endophytic fungi and to determine the function of nonmycorrhizal endophytes in orchid roots. These studies will be especially important if we are to link orchids and OMFs in efforts to inform conservation.