Review

Research review

Mycorrhizal fungi affect orchid distribution and population dynamics

Author for correspondence: Melissa K. McCormick Melissa K. McCormick 1,2 Canchani-Viruet 1 2 Tel: +1443 4822433 Email: [email protected]

Received: 10 April 2018 Smithsonian Environmental Research Center, 647 Contees Wharf Rd, Edgewater, MD 21037, USA; Universidad Accepted: 17 April 2018 Metropolitana, Escuela de Ciencias y Tecnologıa, 1399 Avenida Ana G. Mendez, San Juan 00926, Puerto Rico

1 1 , Dennis F. Whigham and Armando

Summary New Phytologist (2018) doi: Symbioses are ubiquitous in nature and influence individual and populations. Orchids have 10.1111/nph.15223 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 Key words: fungal distribution, focused on evidence that local-scale distribution and population dynamics of orchids can be abundance, mycorrhizae, orchid distribution, limited by the patchy distribution and abundance of OMFs, after an update of an earlier review orchid mycorrhizal fungi, orchid performance, 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 abundanceinsubstratesanddeterminetheirfunctioninassociationwithorchids.Researchisalso needed to distinguish between OMFs and endophytic fungi and to determine the function of nonmycorrhizalendophytesin orchidroots.These studieswillbeespecially importantifwe areto link orchids and OMFs in efforts to inform conservation.

Introduction Roger et al., 2013; Minton et al., 2016), the distribution of nonnative (Menzel et al., 2017) and native species, and population ecological and evolutionary dynamics are allowing growth on nutrient-poor substrates (e.g. Denison & determined by many factors, including distances between Kiers, 2011). Most studies of symbiotic interactions have been individuals and local variation in population density, that primarily concerned with whether plants had a mycorrhizal together determine propagule production and dispersal and the association or not, rather than whether the distribution, identity, behavior of pollinators, and thus gene flow (Kunin & Iwasa, or abundance of mycorrhizal partners affected characteristics of 1996; Colas et al., 1997; Matsumura & Wahitani, 2000; plants and plant populations, such as frequency of emergence Goverde et al., 2002; Grindeland et al., 2005; Pierce et al., or density of plants. There is evidence, however, that the patchy 2006). Plant population dynamics are also influenced by distribution of mycorrhizal fungi affects local plant distribution, symbioses. Symbioses are ubiquitous in nature and are key by altering pollen flow and subsequent seed production and interactions in structuring ecological communities (Bronstein, dispersal (Carvalho et al., 2003). 1994; Stachowicz, 2001). This is especially true for Ecologically, fungi can limit the size of plant populations mycorrhizal associations (Brundrett, 2009), which are known to because patchy distribution of mycorrhizal fungi can affect key affect plant and population characteristics as diverse as processes such as seed germination and plant growth and flowering time (Wagner et al., 2014; Panke-Buisse et al., 2015), survival. For example, Jacquemyn et al. (2012) found that local resistance to and (e.g. Vicari et al., 2002; spatial segregation of orchids was determined by the

No claim to original US Government works New Phytologist (2018) New Phytologist 2018 New Phytologist Trust 1 www.newphytologist.com 2 Review New Research review Phytologist distribution of the distinct mycorrhizal fungi needed by the carbon from orchids under some conditions, the fungi grow well species. The patchy distribution of plants and fungi can also without orchids and are likely distributed independently. contribute to diversification, because limited gene flow among As already indicated, the distribution of OMFs is potentially populations and small population sizes create ideal conditions a critical factor in determining the distribution and fate of for speciation, which is particularly relevant for the species-rich orchids. From a conservation perspective, orchids that are orchid family (Tremblay et al., 2005). grown in laboratory cultures and transplanted to nature need to Orchid mycorrhizae be already inoculated with an appropriate OMF or the OMFs need to be present at the transplantation site to increase the Association with mycorrhizal fungi is a crucial that probability that mediatesgrowth, competitive interactions,and theorchidwillsurvive.McCormick&Jacquemyn(2014)reviewed protection for the vast majority of terrestrial plants, but the the literature on whether the distribution of OMFs limited ability of fungi to drive plant dynamics is perhaps most likely orchid distribution. They concluded that orchid distribution was to be seen in orchid mycorrhizal associations (Swarts & Dixon, not limited by OMFs at geographic scales, but it could be 2017). Orchid mycorrhizaehavebeenstudiedfor morethan a locally limited. Some of the apparent lack of limitation and wide century(Rasmussen,2002) geographic distribution of OMFs, as well as many arbuscular and,unlikemanyothertypesofmycorrhizae,theorchidmycorrhiza and ectomycorrhizal fungi, may be a result of lumping fungi l association is only obligate for the orchids, with the fungi into ‘species’ based on inappropriate DNA sequence similarity maintaining independent distributions. Orchids form (Bruns & Taylor, 2016). The goal of this review is to expand mycorrhizal associations with phylogenetically and beyond the conclusions of McCormick and Jacquemyn by ecologically diverse fungi. The mostcommonlyorchid- examining more associatedfungalgeneraare Tulasnella, Ceratobasidium, and recentresearchresultsandtheevidenceforthewaysinwhichOMFs Serendipita, which include saprotrophs, ectomycorrhizal fungi, can be limiting at local scales, including evidence for the effects endophytes, and some parasites and plant pathogens of OMFs abundance and identity. We also present results of (Dearnaley et al., 2012). Some orchids associate with other recent research that demonstrate a relationship between the basidiomycete ectomycorrhizal fungi, ascomycetes quantity of OMF and orchid density, further demonstrating the (Bidartondo et al., 2004; Selosse et al., 2004; Dearnaley, 2007), potential importance of OMFs on population dynamics. wood decomposers (Kinoshita et al., 2016), and other saprotrophic basidiomycetes (Ogura-Tsujita & Yukawa, 2008; Martos et al., 2009; Kottke et al., 2010; Leeet al., Effects of mycorrhizae on orchid distribution 2015).Mycorrhizalinteractions are especially critical in plants McCormick & Jacquemyn (2014) found that orchids were that are partially (Roy et al., 2013) or fully mycoheterotrophic rarely limited by the availability of appropriate OMFs at a (Leake, 1994; Smith & Read, 2008; geographic scale, but there were some orchids that were limited Hynson&Bruns,2009;Hynsonet al.,2016).Allorchidsareatleast by availability of OMFs at a local scale. We examined the initially mycoheterotrophic (Leake, 1994; Rasmussen, 2002) studies used in McCormick & Jacquemyn (2014) and those and are fully dependent on orchid mycorrhizal fungi (OMFs) at published since that time (details in Supporting Information the Tables S1, S2) to determine whether any further distinctions protocormstage,butalsointeractwithfungitovaryingdegreesatthe have emerged about OMF limitations of orchid distribution and seedling stage (Whigham et al., 2008) and beyond (Girlanda et also whether there was evidence for an effect of OMF al., 2011; Selosse & Martos, 2014; St€ockel et al., 2014). Fully abundance on orchid ecology. mycoheterotrophic orchids are dependent on OMFs throughout As concluded by McCormick & Jacquemyn (2014), the their lives (Rasmussen, 2002; McCormick et al., 2012). Some majority of studies found that OMFs did not limit orchid partially and most fully mycoheterotrophicorchids have distribution (Table 1). With additional studies, however, a beenfound to associate with Agaricomycetes (e.g. Taylor et al., pattern is now emerging about when OMFs do limit orchid 2002; Bidartondo et al., 2004; McCormick et al., 2009; Roy et distribution. al., 2009) and some ascomycetes (Selosse et al., 2004) that also AllbutoneofthestudiesthatfoundaroleforOMFsindetermining form ectomycorrhizae with other plants. orchid distribution involved specialist orchids (13/151 orchids Fungi associated with orchids have diverse nutritional needs in 9/22 studies), and all limitations involved local distributions. (Nurfadilah et al., 2013), but it is not currently thought that they OMFs were patchy in distribution and/or abundance in the soil, needtoassociatewithorarenecessarilyco-distributedwithorchids. andorchiddistributionreflectedthatpatchiness.Anadditionalline A few studies have demonstrated or intimated that orchids may of evidence has been provided by OMF inoculation studies. contribute carbon to their fungi (Cameron et al., 2006, 2008; Hollick et al. (2007) and McCormick et al. (2012) found that Hynson et al., 2009; Ogura-Tsujita et al., 2009; Liebel et al., introducing fungi to unoccupied areas adjacent to existing 2015),suggestingthatassociationwiththeorchidmightbenefitthe orchid populations induced orchid seeds to germinate. fungus. However, even when fungi are expected to obtain New Phytologist (2018) No claim to original US Government works www.newphytologist.com New Phytologist 2018 New Phytologist Trust New Review 3 Phytologist Research review Table 1 The number of studies, with number of orchid species in parentheses, 2009, 2012, 2016), but whether abundant fungi were equally that found a role for fungus distribution or abundance in limiting (or not important for other orchid lifehistorystageswas not limiting) orchid distribution investigated(McCormick & Jacquemyn, 2014). Fungal Fungus To the extent that microsites with more abundant fungi distribution abundance Orchid fungus limitation limitation No distribution support greater seed germination and those seedlings grow into specificity Scale limitation mature plants, areas with more abundant fungi will also support Specific Local 5 (5) 5 (6) 7 (30) more abundant orchids, but this has very rarely been examined. Geographic 0 – 10 (132) McCormick et al. (2009) found that there were more odontorhiza plants in areas with more abundant General Local 1 (1) – 8 (10) Tomentella fungi. Importantly, C. odontorhiza is a Geographic 0 – 5 (5) mycoheterotrophic orchid, so there was little possibility that it could positively affect OMF abundance, clearly indicating that Studies were separated by the specificity of the orchid’s fungal requirements and the scale they examined, whether local (within and near existing the OMFs were influencing orchid abundance. In recent studies, populations) or geographic (considering areas distant from existing conspecific we used specific PCR primers and quantitative real-time PCR orchids) and whether they concluded that the orchids were limited by the (qPCR) to quantify the soil abundance of the Ceratobasidium distribution or abundance of the fungi they needed. Note that studies may be clade that associated with spectabilis within 28 long- represented in more than one category; that is, some studies assayed fungus term monitoring plots that had varied density of G. spectabilis distribution and abundance or assessed fungus limitation at multiple scales. (Methods S1). When two soil samples were collected Effects of orchid mycorrhizal fungi abundance on fromareasineachplotthatwerenotimmediatelyadjacenttoorchids orchids and two soil samples were collected from within 10 cm of orchids that were growing in the plot, the number of G. Abundance effects on orchid distribution spectabilis was positively related to the quantity of the target Because no study has found that orchid geographic distribution Ceratobasidium in the soils (Fig. 1, P < 0.001, F-ratio 50.953, is limited by availability of appropriate OMFs, this also r2= 0.654). Critically, the suggests that OMFs have the potential to colonize all local sites within existing populations, where studies have nevertheless found that orchids lacksuitablefungi.Thisindicatesthatsomethingotherthanstrictly OMF distribution is limiting orchid distribution. Fungi can be present in very small amounts as spores or diffuse hyphae and be undetectable using many techniques. The dynamics of the quantitative relationships between symbiotic partners can have important ecological and evolutionary consequences (Bruna et al., 2014), but very few studies have quantified how the abundance of mycorrhizal fungi influences plant population dynamics (but see Lovelock&Miller,2002;McCormicket al.,2009,2012;Vannette & Hunter, 2013). Only five studies that we are aware of have examined the relationship between orchid distribution and OMFs abundance. Abundance in these studies Fig.1 Relationships between the abundance of the Galearis is expected to be mostly attributable to active fungal hyphae, spectabilisassociated clade of Ceratobasidium (mean nanograms of DNA per 2 based on rare sporulation and lack of hyphal pigmentation that gram dry soil) and G.spectabilis density (number of plants per 1 m subplot) in longterm monitoring plots. might indicate resistance to decay in the predominant OMF genera, but it may also include spores and/or dead hyphae. For assessed soil had to include samples collected adjacent to all of these studies, where seed germination was greater near existing orchids, as a previous study where soils were only mature orchids, OMF abundance was also greater there. When collected from unoccupied microsites within the same subplots germination occurred distant from conspecific plants, those (Methods S1) found no relationship between orchid density and were locations where appropriate OMFs were abundant (e.g. OMF abundance (P = 0.90, F-ratio 0.016, r2= 0.001). These McCormick et al., 2009, 2016). Similarly, when McCormick et studies indicated that orchid abundance was related to OMFs al. (2012) added OMFs to locations where orchid seed packets abundance, but the patches where OMFs were abundant were were placed, seeds of the three species only germinated when small and closely tied to the OMFs became abundant. Greater abundance of OMFs was locationsofindividualorchids.Hence,scaleisimportantinassessin associated with increased seed germination and protocorm g how OMF abundance contributes to orchid population development of four terrestrial orchids (McCormick et al., No claim to original US Government works New Phytologist (2018) New Phytologist 2018 New Phytologist Trust www.newphytologist.com 4 Review New Research review Phytologist dynamics. Unlike C. odontorhiza, G. spectabilis is a green, especially reproductive success. To the extent that OMF affect photosynthetic orchid and so it is possible that the abundance plant carbon provisioning, fungi can affect the likelihood of of OMFs may be affected by the density of orchids, rather than orchids becoming vegetatively dormant. Vegetative dormancy the reverse. As described later, there is clearly a need for is atimewhen orchidsare thoughtto relyexclusively on additional research on factors that control OMFs abundance and mycorrhizalfungifor nutrition, soemergence from dormancyis the resultant impacts on individual orchids and orchid also likely to be affected by the abundance of appropriate fungi. populations. Rock-Blake et al. (2017) found that abundant OMFs were Concerns about the unknown temporal consistency of OMF associated with locations where Isotria medeoloides emerged distribution patterns (e.g. Ercole et al., 2015) are even more above ground, while the orchid was either vegetatively dormant important to abundance. For example, temporarily abundant or absent in locations with less abundant OMFs (Fig. 2). OMFs may promote seed germination in locations that currently Further, they demonstrated that the probability of future lack conspecific orchids, but if the abundance of those fungi emergence from dormancy also depended on the local fluctuates in subsequent years or seasons then orchids may not abundance of appropriate OMFs, indicating that the direction of survive to maturity in those locations. Such temporal fluctuation causation was from the fungus to the orchid. If OMF abundance may explain sporadic germination that is detected in habitats affects orchid emergence or dormancy duration, then, by that do not normally support orchids (e.g. Jersakova & extension, it will also likely affect rates of reproduction and Malinova, 2007). Mature orchids will be indicative of locations patterns of outcrossing and gene flow. where OMFs are consistently abundant. A handful of studies Vegetative dormancy is a fairly obvious place to look for have now documented changes in OMF communities in orchid evidence of OMF abundance on orchid population dynamics, with season and habitat (Kohout et al., 2013; Ercole et al., but not all plants, or even all orchids, engage in vegetative 2015; Oja et al., 2016; Han et al., 2017), but understanding how dormancy. With mutualistic mycorrhizal associations, such as these changes impact orchid distribution and population most arbuscular and ectomycorrhizal associations, it is nearly dynamics awaits further study. impossible to separate the interactive effects of plants and fungi as drivers of abundance, and plant growth and mycorrhizal fungus abundance are frequently correlated (e.g. Brundrett, Abundance effects on orchid population dynamics 2004). Nevertheless, studies of invasive species have provided The extent to which OMFs contribute to the support of mature some insights. Grove et al. (2017) reviewed 112 studies that photosynthetic orchids is still an open question and likely varies examined the impact of invasive species on mycorrhizal fungi among species and habitats (Girlanda et al., 2011; Sommer et and found that 58% of invading plants decreased the abundance al., 2012; Selosse & Martos, 2014; McCormick & Jacquemyn, of the mycorrhizal fungi used by the native plant community 2014; Liebel et al., 2015; Hynson et al., 2016). Fungi almost and that impacted the growth and competitive ability of native certainly play an important role during periods of vegetative plants. Similarly, Vannette & Hunter (2013) demonstrated that dormancy, a widespread phenomenon where plants, including the abundance of arbuscular mycorrhizal fungi impacted plant many orchids, biomass and defense against herbivory in Asclepias syriaca, but failtoproduceanyabovegroundtissues,butremainphysiologically few studies have examined plant performance as a function of active,duringoneormoregrowingseasons(Sheffersonet al.,2003, separately manipulated mycorrhizal fungus abundance. That 2011, 2014, 2018; Shefferson, 2009). Vegetative dormancy is mature orchid performance can also be impacted by abundance associated with plant stress, often carbon limitation, and often of appropriate OMFs is suggested by the link between carbon has negative consequences on survival (Shefferson & Tali, supply and entering vegetative dormancy (Shefferson et al., 2007; Gremer et al., 2010, 2012; Shefferson et al., 2018). 2018). Studies demonstrating that the manipulation of OMF Because vegetative dormancy alters the number of apparent abundance leads to greater seed germination (McCormick et al., plants, it also affects population dynamics, such as 2012) and that OMF abundance in one year predicts emergence crosspollination, seed production, herbivory rates, and so on in the following year (Rock-Blake et al., 2017) suggest that the (e.g. Tali & Kull, 2001). There have been few studies that directionality of causation is from the OMFs to the orchid, more demonstrate clear evidence that OMF distribution influences so than the reverse. However, in light of studies now showing orchid population dynamics, but Machaka-Houri et al. (2012) that nutrients can also flow from orchids to fungi (e.g. Cameron found that greater floral display and conspecific density et al., 2006, 2008; Hynson et al., 2009), the directionality of increased fruit set in Orchis galilaea, suggesting that, like for benefit may differ for other photosynthetic orchids, and more many other plants, the apparent density of orchid populations is research is needed on the functioning and an important determinant of their population dynamics,

New Phytologist (2018) No claim to original US Government works www.newphytologist.com New Phytologist 2018 New Phytologist Trust New Review 5 Phytologist Research review Johnson et al., 2012; Hazard et al., 2017b; Shi et al., 2017). Orchid distribution and performance can also be influenced by the identity of potentially suitable OMFs in different locations or by the interaction of OMF identity and environment. It is possible, and even likely, that not all potential OMFs are suitable in all habitats. For example, some orchids may associate with a mix of fungi that form ectomycorrhizae with nearby trees and those that function as saprotrophs. Logically, ectomycorrhizal fungi would be far less beneficial, as well as less abundant, in an environment without ectomycorrhizal hosts. Few studies have examined whether the OMFs used by orchids differ among habitats (but see Bonnardeaux et al., 2007; Bunch et al., 2013; Mujica et al., 2016), and even less is known about whether their ability to support orchids differs as a

function of edaphic or climatic conditions. Fig.2 The abundance of in the soil by years since an Isotria Fungal communities are well known to differ among habitats medeoloides last emerged at that location. ‘Never’ indicates locations where I. medeoloideshasneverbeenobservedinover9 yrofdetailedobservations. Zero and to be patchily distributed (e.g. Grau et al., 2017; Maghnia indicates locations where I. medeoloides plants are currently emergent. Data et al., 2017; Chaudhary et al., 2018), but OMFs have received presented are means of nanograms of DNA per gram dry soil, normalized to little attention. Bunch et al. (2013) found that Cypripedium place very different abundances across three distant sites on the same scale; acaule associated with different OMFs in locations with modified from Rock-Blake et al. (2017). different soil chemistry. Similarly, Mujica et al. (2016) found nutrient exchanges in orchid mycorrhizae. The application of that two species of Bipinnula associated with different OMF transcriptomic studies to identifying changes in expression of taxa and different diversities of OMFs as a function of soil nutrient transfer genes may shed light on whether OMFs are nutrients. Weedy orchids that associated with a wide range of driving orchid abundance or orchids are driving OMFs. fungi also associated with different fungi among habitats It is worth noting again that the few studies that have (Bonnardeaux et al., 2007). Furthermore, the fungi that orchids examined OMF abundance, rather than just presence, were also associated with had consequences for their population studies with dynamics. McCormick et al. (2009) found that though the C. orchidsthatassociatedwithspecificfungiandhadlocaldistribution odontorhiza within one population associated with a range of s that were limited by OMFs. qPCR based on well-tested, Tomentella spp., only those that associated with a single specific primers works well to quantify OMFs associated with operational taxonomic unit were able to orchids that require specific fungi,anda broader application maintaingrowthandfloweringduringdroughtconditions(Fig. 3). ofthis techniquefor those orchids would greatly expand our Takentogether,thisevidencesuggeststhatOMFspeciesdifferin understanding of how OMFs are distributed, independent of ability to support orchids among habitats and climatic orchid germination. However, qPCR does not work well for conditions (McCormick et al., 2009; Kohler et al., 2015; Fochi orchids that associate with many fungi, because it is not possible et al., 2016; Mujica et al., 2016). Genotypic and epigenetic to design primers that amplify all of the OMFs that can support differences among strains of OMFs may also affect their ability generalist orchids while excluding those that cannot. Thelack of to support orchids. Such evidence is still limited, but the studies that directlytested OMFabundance for generalist orchids suggestion that OMFs differ in mycorrhizal ability is (Table 1) reflects this difficulty. unsurprising. Arbuscular and ectomycorrhizal fungi andgenotypes have been shown to differ in their ability to Orchid mycorrhizal fungus identity and orchid support their plant hosts (e.g. Johnson et al., 2012; Roger et al., 2013; Hazard et al., 2017a,b; Shi et al., 2017), and OMFs have distribution and population dynamics been shown to differ in their ability to access distinct nitrogen Of course, which OMFs an orchid associates with also matters. sources (Nurfadilah et al., 2013) and to support orchids in vitro There are abundant references demonstrating fungal identity (e.g. Warcup, 1973; Masuhara & Katsuya, 1994; Rasmussen & effects in arbuscular and ectomycorrhizal associations (e.g. Whigham, 1998; McCormick et al., 2006). In the years before Kiers et al., 2000; Roger et al., 2013; Hazard et al., 2017a). For sequencing-based fungal identification, the diagnostic to example, Klironomos (2003) found that plant growth, distinguish Epulorhiza and Ceratorhiza strains was a test for flowering, and competitive ability differed depending on which production of laccase (e.g. Currah et al., 1987). Clearly, these mycorrhizal fungi they associated with. Similarly, it has been taxa would differ in ability to access lignin and phenolic found that plant growth and competitive ability depended on the compounds in the substrate, yet many generalist orchids identity and genotype of their ectomycorrhizal partner (e.g.

No claim to original US Government works New Phytologist (2018) New Phytologist 2018 New Phytologist Trust www.newphytologist.com 6 Review New Research review Phytologist associate with strains of both groups of fungi. The importance woodlandorchids,andthereremainsadesperateneedtodetermine of OMF identity has been less commonly whether these patterns also hold for OMFs in both terrestrial and epiphytic habitats in the tropics. Very little is known about how fungi are distributed in the tropics, but there are some suggestions that patterns may differ. Martos et al. (2012) found that epiphytic orchids were more specific than their terrestrial counterparts and that tropical terrestrial mycoheterotrophic orchids associated with saprotrophs, rather than fungi that were supported by ectomycorrhizal trees, as has been found in temperate regions. To understand how OMFs affect local orchid abundance across diverse systems, it will be important to develop better primers for next-generation sequencing (NGS). NGS studies have needed multiple sets of primers, with correspondingly Fig.3 Map of Corallorhiza odontorhiza in 2004, showing the locations of higher sequencing costs, to consistently detect OMFs in soil plants that associated with a single Tomentella operational taxonomic unit (e.g. Oja et al., 2016; Voyron et al., 2017). Furthermore, (OTU) (OTU 28). All plants that emerged following a severe drought were because of primer biases and varying copy numbers of plants that associated with OTU 28. Plants associated with other OTUs within ribosomal genes, it is not currently possible to interpret NGS Tomentella were either dormant or died; modified from McCormick et al. (2009). metabarcode data as reflecting abundance. The rapid increase in reference genomes is expected to enable metagenomic studies that, because they do not involve PCR amplification, will studied in situ, but the evidence that some orchids associate with alleviate some of these problems and facilitate the use of NGS different fungi in different habitats (detailed earlier) presents for OMF quantification in the future. the potential for orchids to switch among differently beneficial The increased number of fungal genomes is also poised to fungi (e.g. McCormick et al., 2006). shed light on how different OMFs function in their association AdditionaldifferencesamongOMFshavebeensuggestedbyrec with orchids (e.g. Kohler et al., 2015). While the ‘symbiosis ent genome (Zuccaro et al., 2011; Kohler et al., 2015) and toolkit’ for arbuscular and ectomycorrhizal plants has received transcriptome (Fochi et al., 2016, 2017; M. K. McCormick, considerable attention in the last decade (e.g. Delaux et al., unpublished data) studies demonstrating that OMFs differed 2013), even more recent studies involving fungal genomes and genetically in their ability to access nutrient and carbon sources. transcriptomes are beginning to reveal how fungi contribute to In particular, fungi in the genus Tulasnella and some the symbiosis (e.g. Fochi et al., 2016). It is also becoming clear Serendipita lacked the genes to access nitrate and nitrite, while that the contributions and genetic capabilities ofOMF taxaare fungi belonging to Ceratobasidium have multiple genes for not equalin thesupportof orchid germination and growth. accessing nitrate and nitrite. Tulasnella, Ceratobasidium, and A further critical direction will be distinguishing between Serendipita fungi also had different genes for accessing carbon fungi that are mycorrhizal associates and those that are present substrates (Kohler et al., 2015). This reinforces the nutrient in roots as nonmycorrhizal endophytes. Currently, the only way access studies by Nurfadilah et al. (2013) and clearly raises the to tell if a fungus is mycorrhizal is to culture it and demonstrate prediction that some orchid–OMF combinations will be more that it can support protocorm development. However, OMFs beneficial than others and that it will be habitat dependent. with adult orchids may nevertheless not support seed germination, as fungal specificity may change with life stage Conclusions and future directions (Bidartondo & Read, 2008). All of these approaches will shed more light on how habitat and climatic conditions affect We provided evidence that the abundance, rather than solely contributions by distinct OMF strains to orchid performance. distribution, of OMFs is an important driver of orchid Technical advancements in our ability to study interactions population dynamics, especially orchid density and apparent between habitat conditions, OMFs, and orchids will eventually density that results from vegetative dormancy. Our review of also benefit efforts to restore orchid populations. recent publications supported the conclusions reached by McCormick & Jacquemyn (2014) that orchid distribution at large scales is rarely limited by the availability of appropriate Acknowledgements OMFs, but found that local OMF distribution and, particularly, We wish to acknowledge Brianna Glase for data from an earlier abundance are heterogeneous and contribute to patchy orchid study on that led to the study presented here distributions. However, all of the current studies have been in and Simone Evans, John O’Neill, Emma Thrift and Eve Bahler temperate, terrestrial for help collecting and processing samples. M.K.M. wishes to

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