what constrains the distribution of orchid populations? · to associate with orchids. although...

Research review

What constrains the distribution of orchid populations?

Author for correspondence:Melissa K. McCormickTel: +1 443 482 2433

Email: [email protected]

Received: 16 September 2013Accepted: 14 November 2013

Melissa K. McCormick1* and Hans Jacquemyn2*1Smithsonian Environmental Research Center, PO Box 28, Edgewater, MD 20137, USA; 2KU Leuven, Department of Biology,

Laboratory of Plant Population and Conservation Biology, B-3001 Leuven, Belgium

New Phytologist (2014) 202: 392–400doi: 10.1111/nph.12639

Key words: mycorrhiza, orchid, rarity,recruitment limitation, specificity.

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 raritywas also unrelated tomycorrhizal specificity. Nonetheless, seedgerminationwithin

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.

Introduction

Recruitment from seeds is a primary determinant of the distribu-tion of nearly all plant populations (Clark et al., 2007). The twomajor determinants of where plants recruit are seed and micrositelimitation (Eriksson & Ehrl�en, 1992). When suitable habitatsremain uncolonized because seeds do not reach them, plants areconsidered, depending on the scale, dispersal or seed limited.Whenseeds are widely dispersed but fail to recruit because habitats areunsuitable, plants are considered habitat- or microsite-limited(M€unzbergov�a & Herben, 2005; Johansson & Eriksson, 2013).While most plants are now thought to be at least somewhat seed-limited (Eriksson & Ehrl�en, 1992), the extent to which seed andmicrosite limitation dominate in determining plant distributiondepends to some extent on the types of seeds producedby a plant. Innature there is a general tradeoff between the number of seedsproduced and seed mass (Leishman et al., 2000). With a given

amount of resources available for seed production, a plant will beable to produce more small seeds than larger seeds (Henery &Westoby, 2001). On the other hand, seedlings from small-seededspecies generally have a lower survival rate than seedlings fromlarge-seeded species (Moles & Westoby, 2004). As a result, small-seeded species may be more likely to disperse long distances tocolonize vacant sites and therefore be less dispersal-limited, butmore habitat-limited than large-seeded species (Venable&Brown,1988; Greene & Johnson, 1993). In the most extreme case, seedsmay be so small that they contain almost no resources and have torely on external sources for resource acquisition, often mycorrhizalfungi (Eriksson & Kainulainen, 2011). In this case, habitat ormicrosite limitation may result from the absence of suitablemycorrhizal fungi.

Seeds that are extremely small have been called dust seeds andhave evolved independently in at least 12plant families (Eriksson&Kainulainen, 2011), including the Orchidaceae. Despite the vastnumbers of dust-like seeds that can be easily dispersed across largedistances (reviewed in Arditti &Ghani, 2000), the exceedingly low*These authors contributed equally to this work

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rates of orchid recruitment have astounded researchers forcenturies. Charles Darwin, being among the first to count thenumber of seeds within a single orchid fruit, found that a capsule ofOrchis (Dactylorhiza)maculata contained c. 6200 seeds. Given thatan individual plant can produce > 30 capsules within 1 yr, Darwincalculated that a single plant would produce 186 300 seeds(Darwin, 1862). To put these figures in perspective, he alsocalculated possible rates of increase (Darwin, 1862):

An acre of land would hold 172 240 plants, each having a space of six

inches square, and this would be just sufficient for their growth; so that,

making the fair allowance of 400 bad seeds in each capsule, an acre would

be thickly clothed by the progeny of a single plant. At the same rate of

increase, the grandchildren would cover a space slightly exceeding the

Island of Anglesea; and the great grand children of a single plant would

nearly clothe with one uniform green carpet the entire surface of the land

throughout the globe.

These calculations are in sharp contrast with the generalobservation thatmany orchid species tend to have small populationsizes and that populations occur scattered across the landscape,suggesting that important factors are limiting the settlement oforchid populations. Darwin himself was puzzled by this observa-tion when he wrote: ‘What checks this unlimited multiplicationcannot be told.’ It was not until the pioneering works of No€elBernard on orchid seed germination (Boullard, 1985; Selosse et al.,2011) that the idea arose that mycorrhizal fungi may be involved indetermining the abundance anddistribution of orchid populations.Because of their minute size, orchid seeds lack an endosperm andrely on fungal colonization for germination and growth into anunderground heterotrophic achlorophyllous stage called a proto-corm (Rasmussen, 1995; Smith & Read, 2008). Because thepresence of fungi is indispensable, at least for germination and earlygrowth, this suggests that mycorrhizal fungi can be a strong factorlimiting the distribution of orchid populations.

However, compelling evidence for the availability ofmycorrhizalfungi alone affecting orchid population dynamics is still lackingand it is likely that other factors are of comparable or greaterimportance in determining orchid establishment success. Seeddispersal andmicrosite characteristics such as soil moisture contentor pH can also be expected to have a profound impact onestablishment success at both the landscape and local scales, and thebalance between the factors affecting orchid distribution maydepend on the scale considered. When multiple orchid speciesco-occur at a site, local competition for resources, possiblymediated by the availability of or competition between differentfungi, may further contribute to the abundance and spatialdistribution of orchids in natural environments.

The aim of this paper is to provide an overview of the potentialedaphic and mycorrhizal factors affecting the abundance andspatial distribution of orchid species at different scales in naturalenvironments. Given thatmany orchids are currently threatened orendangered, a better understanding of the factors governing orchidpopulation dynamics may be critical to their long-term conserva-tion. We first give a brief overview of the main fungal speciesassociating with orchids and highlight what is known of theirecological characteristics. We then investigate the roles of seed and

recruitment limitation and distribution of mycorrhizal fungi indetermining orchid abundance at the landscape and local scales.Finally, we highlight avenues for future research.

Orchid mycorrhizal fungi

Orchid mycorrhizas have been studied for more than a century(Rasmussen, 2002), yet identification of associated fungi wasseverely limited until DNA sequencingwas applied to the task. Themajority of fungal taxa found associated with both terrestrial andepiphytic green orchids are Basidiomycetes in the Ceratobasidia-ceae, Sebacinales, and Tulasnellaceae and association with fungi inthese taxa is thought to be the ancestral state for the Orchidaceae(Rasmussen, 2002; Taylor et al., 2002; Dearnaley, 2007). Most ofthese are resupinate fungi that produce morphologically depau-perate fruiting structures, which has hindered their taxonomicresolution. Orchid fungal associates in these groups includesaprotrophs, ectomycorrhizal fungi, and some parasites and plantpathogens (Dearnaley et al., 2012). There have also been a fewexamples of orchids associating with the Ascomycetes Tuber,Wilcoxina, Tricharina, Peziza, and Phialophora (Bidartondo et al.,2004; Selosse et al., 2004; Dearnaley, 2007) and, in the tropics,with Basidiomycetes in the Atractiellomycetes (Kottke et al., 2010;Dearnaley et al., 2012).

Most fully and some partially mycoheterotrophic orchids haveprimarily been found to associate with ectomycorrhizal Agarico-mycetes. These include many members of the Russulales (Tayloret al., 2004), Thelephorales (McCormick et al., 2009), and Seb-acinales (Roy et al., 2009), and some associatewith ectomycorrhizalAscomycetes (Selosse et al., 2004). In addition to these ectomy-corrhizal fungi, recent studies have also identified a diverse group ofsaprotrophic fungi forming mycorrhizas with mycoheterotrophicorchids in tropical forests (Martos et al., 2009;Ogura-Tsujita et al.,2009).

Fungi associated with orchids have diverse ecological strategiesand nutritional needs, but it is not currently thought that they needto associate with orchids. Although Cameron et al. (2006, 2008)found that Goodyera repens contributed carbon to its mycorrhizalfungus, suggesting that association with the orchid might benefitthe fungus, even in this case, the fungi grow well without orchidsand are probably distributed independently.

Landscape-scale dynamics

Despite the production ofmany seeds with the potential to disperseacross long distances (Arditti & Ghani, 2000) and, for somespecies, the potential to survive formany years in the soil (Whighamet al., 2006), many orchid species show highly scattered distribu-tion patterns at the landscape scale. This suggests thatmany sites arenot suitable for germination and establishment of seedlings (habitatlimitation sensu M€unzbergov�a & Herben, 2005). One mightsuppose that this is because orchid mycorrhizal fungi, like mostectomycorrhizal fungi, are uncommon even within suitablehabitats and are restricted to small areas (Villeneuve et al., 1989;Horton & Bruns, 2001; Taylor et al., 2002). Yet, identification offungi from surveys of mycorrhizal associations in a wide range of

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orchid species and populations across large scales has shown thatmany orchid mycorrhizal fungi are widespread and occur in a widevariety of habitats. For example, themostwidespread fungal lineageassociating with species from the genus Orchis was found from theMediterranean up to northern Belgium and was retrieved from drycalcareous grasslands, mesic grasslands, and both pine andtemperate deciduous forests (Jacquemyn et al., 2011). Similarly,the mycorrhizal fungi associating with several species of Australianorchids also have very wide distribution ranges (Swarts et al., 2010;Phillips et al., 2011). InCypripedium, closely related fungal lineageswere even found across different continents (Shefferson et al.,2007). An alternative hypothesis for the scattered distribution oforchid populations is that orchid populations are absent fromseemingly suitable habitat patches because they are sufficiently farfrom existing orchid populations that seeds rarely encounter them(dispersal limitation sensu M€unzbergov�a & Herben, 2005). Seedand fungal limitation may also act in concert, such that if the localdistribution of fungi is patchy then correspondingly more seedswould need to arrive in an uncolonized habitat to have a highprobability of encountering locations with appropriate fungi.

The most straightforward way to unravel the importance ofhabitat vs dispersal limitation is to perform seed introductionexperiments, in which germination of seeds is compared betweensites where the species is absent and present (Turnbull et al., 2000).Because of the minute size of orchid seeds, most seed introductionexperiments are conducted using seed packets (Rasmussen &Whigham, 1993). These are small packets that consist mostly offine nylon netting, such as is used in phytoplankton research, eitherimposed within a 35 mm slide mount or heat-sealed into discretemesh packets (Brundrett et al., 2003). The mesh size of thephytoplankton netting is small enough to retain the seeds in thepackets, but large enough to allow hyphae to reach the seeds andpromote germination.

The few studies that have compared seed germination betweenoccupied and unoccupied habitat patches have shown that in allinvestigated species at least some seed packets that were deployed inunoccupied habitat patches produced protocorms (Table 1).Comparing the proportion of seed packets with and withoutprotocorms showed no significant difference in seed germinationbetween occupied and unoccupied habitat patches (Table 1).However, nine of the 12 (germinating) species germinated more atthe occupied sites, so the resultsmay be biased by the lownumber ofstudies available. It should also be noted that four of the five studiesdeployed seed packets only in sites that were potentially suitable forsustaining orchid growth (but see T�e�sitelov�a et al., 2012). Adifferent picture would emerge if less suitable habitats were alsoconsidered. Nonetheless, these results suggest that dispersallimitation is an important factor determining the distribution oforchids at the landscape scale. These results are also supported bythe fact that many studies have found little relationship betweenorchid specificity for mycorrhizal fungi and rarity (Table 2). It hasbeen hypothesized that low specificity evolved to counteractlimitations resulting from a rare fungal associate. However, anorchid that required a specific but widespread fungus could evenencounter greater opportunities for germination than an orchidthat associated with many fungi, each of which was very narrowly

distributed. A comparison of very rare, rare, and common orchids(note that the rarity designation is also positively correlatedwith thedegree of habitat specificity) revealed no statistical difference in thenumber of fungal operational taxonomic units (OTUs) theyassociated with (Table 2), regardless of how rarity was partitioned(i.e. whether uncommon was counted as rare or common, whetherrare and restricted were counted as very rare or only rare). Theseresults challenge the general assumption that orchid seeds are easilycapable of traveling long distances and so are able to colonize allsuitable habitat patches. However, it is worth noting that none ofthese studies monitored subsequent growth to the adult stage, so itremains unclear whether some habitat patches may be onlytemporarily suitable or whether introduced seeds germinating inseed packets would establish and form self-sustaining populations.

Local-scale dynamics

One interesting observation from the seed introduction experi-ments is that, in most species, a substantial proportion of the seedpackets deployed, evenwithin existing orchid populations, failed todevelop protocorms (Jacquemyn et al., 2007, 2012a,b), suggestingthat suitable microsites are not evenly distributed (i.e. micrositelimitation sensu M€unzbergov�a & Herben, 2005). Nonrandomdistribution of mycorrhizal fungi, spatial restrictions in theavailability of suitable environmental conditions, or a combinationof the two can be expected to translate into nonrandom above-ground distributions of orchid individuals. Such a nonrandomdistribution was evident in a comparison of the spatial distributionof seedlings and adults in two populations of the Lady orchid(Orchis purpurea) in Belgium. This study showed that seedlingestablishment was mainly restricted to areas where adults occurred(Jacquemyn et al., 2007) (Fig. 1), suggesting that germination andseedling establishment were strongly dependent on biotic and

Table 1 The proportion of seed packets showing germination (protocormformation) of several orchid species at sites with and without the orchidspecies present

Study Species Occupied Unoccupied

De hert et al. (2013) Dactylorhiza fuchsii 0.43 0.25Dactylorhiza

praetermissa

0.22 0.35

Herminium monorchis 0.06 0.09Phillips et al. (2011) Drakaea elastica 0.10 0.08

Drakaea glyptodont 0.17 0.04Drakaea livida 0.08 0.04Drakaea micrantha 0.08 0.05

McKendrick et al.(2002)

Neottia nidus-avis 0.71 0.01

T�e�sitelov�a et al. (2012) Epipactis albensis 0.00 0.00Epipactis atrorubens 0.08 0.47Epipactis helleborine 0.55 0.40Epipactis purpurata 0.09 0.00

McKendrick et al.(2000)

Corallorhiza trifida 0.58 0.61Mean 0.24 0.18

A paired t-test with subsequent bootstrapping showed no significantdifference (t12 = 0.40; P > 0.05) in seed germination between occupied andunoccupied habitat patches.

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abiotic environmental conditions reflected by the presence of adultplants. Similar results have also been obtained for other species (e.g.Caladenia arenicola (Batty et al., 2001), Goodyera pubescens (Diez,2007),Anacamptis morio,Orchis mascula andGymnadenia conopsea(Jacquemyn et al., 2012a)).

What defines suitable microsites for different orchid species willlikely reflect a combination of how specific orchid fungusrequirements are and the distribution of abiotic conditions, factorswe have just begun to tease apart. Appropriatemycorrhizal fungi areunquestionably a component of any suitable microsite, and manyresearchers hypothesized that an orchid that required specific fungiat any life stage would be more likely to be restricted by amycorrhizal fungus than one that could associate with manydifferent fungi (Brundrett et al., 2003; Bonnardeaux et al., 2007;Dearnaley, 2007;Ogura-Tsujita et al., 2009).Molecular tools havebeen used to document varying degrees of specificity in orchid–fungal associations (reviewed in Taylor et al., 2002). Photosyn-thetic (McCormick et al., 2004; Phillips et al., 2011) andmycoheterotrophic orchids (Taylor & Bruns, 1999; Roy et al.,2009), and both terrestrial and epiphytic (Su�arez et al., 2006)orchidsmay associate with a very narrow range ofmycorrhizal fungi(e.g.Liparis liliifolia;McCormick et al., 2004) or a very broad range(e.g. Microtis media; Bonnardeaux et al., 2007; De Long et al.,

2013). Another possibly widespread but rarely assessed specificitypattern is associating with a wide range of fungi as mature,photosynthetic plants, yet requiring a very narrow range of fungi tosupport germination and protocorm development (e.g. Tipulariadiscolor (McCormick et al., 2004)).

Some rare orchids do require specific fungi and may be limitedby their availability (e.g. Swarts et al., 2010; Graham&Dearnaley,2012), yet others associate with diverse fungi (Pecoraro et al., 2012;Pandey et al., 2013) andmay be limited by other factors. However,separating the components of a suitable microsite requiresindependent assessment of abiotic conditions, mycorrhizal fungusdistribution, and ability to support orchids. Batty et al. (2001) andDiez (2007) were the first to directly assess germination andenvironmental conditions independently by combining seedpacket experiments and environmentalmeasurements. They foundthat, beyond spatial clustering near conspecific plants, seedgermination was also affected by soil moisture, pH, and organiccontent.However, whether these factors affected orchids directly orwhether seed germination was affected indirectly through mycor-rhizal fungi was not examined.

Additional advances have been made by researchers usingmolecular techniques to assess the distribution of fungi in the soilindependent of seed germination. McCormick et al. (2012)

Table 2 Studies comparing the mycorrhizal specificity of rare and common orchids

Orchid (no. of populations, no. of plants) Rarity No. of fungal OTUs

Jacquemyn et al. (2012c) Dactylorhiza fuchsii (5, 16) R 5Dactylorhiza incarnata (5, 20) VR 6Dactylorhiza maculata (6, 23) C 6Dactylorhiza majalis (6, 25) C 9Dactylorhiza praetermissa (3, 7) U 6

Phillips et al. (2011) Drakea elastica (3, 6) VR 1Drakea glyptodon (6, 14) C 1Drakea livida (2, 13) C 1Drakea micrantha (2, 6) VR 1Drakea thynniphila (1, 9) C 1

Swarts et al. (2010) Caladenia huegelii (9, 9) R, R 1Caladenia arenicola (1, 3) C, R 1Caladenia longicauda (1, 3) C, R 2Caladenia discoidea (1, 3) C 3Caladenia flava (1, 3) C 5

Wright et al. (2010) Caladenia audasii (1, 5) VR 1Caladenia amoena (1, 1) VR 1Caladenia sp. aff. fragrantissima (1, 2) VR 1Caladenia sp. aff. patersonii (1, 2) VR 1Caladenia rosella (1, 1) VR 1Caladenia orientalis (1, 1) VR 1Caladenia tentacula (6, 36) C 6

Shefferson et al. (2005) Cypripedium calceolus (2, 2) C 2Cypripedium californicum (5, 10) R, R 5 (3 genera)Cypripedium candidum (3, 6) C, R 1Cypripedium fasciculatum (7, 16) C, R 1Cypripedium guttatum (2, 2) C 1Cypripedium montanum (8, 12) C, R 5Cypripedium parviflorum (7, 11) C 3

Orchid rarity is classified as very rare (VR), rare and geographically restricted (R, R), rare (R), uncommon (U), common but geographically restricted (C, R), orcommon (C), in order of decreasing rarity. Numbers in parentheses after each orchid name are (number of populations sampled, number of individual plantssampled). A linear mixed-effects model with study as a random effect and rarity as a fixed effect revealed no significant relationship between rarity andmycorrhizal specificity (df = 1, 23; F = 2.99; P > 0.05; SYSTAT 12 for Windows, Systat Software Inc., San Jose, CA, USA). OTUs, operational taxonomic units.





combined seed packets, manipulations of environmental condi-tions, and direct quantification of mycorrhizal fungi in the soilusing specific primers and quantitative real-time PCR (qPCR) todemonstrate that organic amendments affected the germination oforchid seeds by altering the abundance of mycorrhizal fungi. Thissuggested that areas with existing orchids might be ‘hot spots’ ofabundant mycorrhizal fungi (Otero & Flanagan, 2006;Waterman& Bidartondo, 2008; McCormick et al., 2009; M. K. McCormicket al. unpublished). Combined, these studies suggest that theabundance of orchid mycorrhizal fungi, and hence their ability tosupport orchid germination and growth, may depend on edaphicconditions. However, this has only been investigated in a feworchids, and edaphic conditions may also affect orchids directly.Additional experimental manipulation of environments andmycorrhizal fungi will provide further insight into the mechanismsbehind observed patterns of orchid and fungus distribution.

The distribution of mycorrhizal fungi associated with epiphyticorchids is less well studied, perhaps because early seed packettechniques using slidemounts were less successful on branches thanin the ground (Zettler et al., 2011). Otero et al. (2002) suggestedthat epiphytic orchids may also be affected by the distribution andabundance of their mycorrhizal fungi, but research is onlybeginning on mycorrhizal fungus distribution in epiphytes(Kartzinel et al., 2013). Assessment of how often orchids arelimited bymycorrhizal fungus abundance, particularly in epiphyticorchids, and clear separation of environmental effects on mycor-rhizal fungi from those on orchids will be a fruitful topic for futureresearch.

The need for mycorrhizal fungi to be abundant to either providesufficient nutrients or a high probability of the fungus encounteringseeds suggests that locations where orchids can reach maturity maybe sites with persistently abundant fungi. Just as at larger scales,fungi or environmental conditions in microsites without existing

orchids may be ephemeral (Wright et al., 2010), or may representdistinct, possibly less favorable, species or genotypes of fungi thanthose at occupied microsites (McCormick et al., 2009). Forexample, McCormick et al. (2006) found that Goodyera pubescensin sites that supported diverse mycorrhizal fungi recovered after adrought by switching to associations with new mycorrhizal fungiand McCormick et al. (2009) suggested that the dynamics of apopulation of Corallorhiza odontorhiza might be driven by thedistribution of differently drought-resistant fungi.

Assessing survival to maturity awaits future research, butunderstanding how edaphic conditions affect the distributionand abundance of orchid mycorrhizal fungi may improve orchidconservation and restoration success by identifying conditions thatfavor persistent growth of mycorrhizal fungi (Gale et al., 2010).The first step in this direction has been taken by Nurfadilah et al.(2013), who examined the nutrient acquisition abilities of fungiassociated with several species of Australian orchids. They foundthat fungi that associated with rare, habitat-specialist orchids wereable to utilize a narrower range of nutrient sources than those thatassociated with more widespread orchids, suggesting a possiblemechanism for the connection between mycorrhizal fungusdistribution and abundance and orchid rarity. Additional researchinto the ecology of orchidmycorrhizal fungi will no doubt improveour understanding of fungal habitat requirements.

Heterogeneous environments and coexistence oforchid species

Factors affecting the distribution of orchids at regional and localscales also impact which orchid species co-occur (Waterman &Bidartondo, 2008).When two ormore orchid species co-occur at asite, they are likely to compete for the same set of resources.Classical ecological theory predicts that two species competing for

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Fig. 1 Visualization of the spatial clustering ofadult and seedling plants in two populations ofthe Lady orchid (Orchis purpurea) in Belgium(from Jacquemyn et al., 2007). Differentcolors distinguish areas within the populationswith high and low abundances of orchids.Circles represent the area where c. 86% ofadults (a, c) or seedlings (b, d) are expected tobe located.




the same resources cannot coexist stably. This raises the question ofwhy there are numerous examples of natural systems where a largenumber of orchid species co-occur. Spatially explicit models haveshown that localized dispersal and interactions may lead to stablecoexistence of species because of strong interspecific spatialsegregation (Pacala & Levin, 1997). In this case, one would expectcoexisting orchid species to show strong spatial clustering andsegregation. Recent research investigating the fine-scale spatialdistribution of seven different orchid species in Mediterraneangrasslands in southern Italy has indeed shown that at smallneighborhood sizes the proportion of conspecifics was high in allspecies (Fig. 2, Jacquemyn et al., 2013). This high local dominancewasmainly caused by the strong clustering of individual species andlittle overlap among species.

However, similar patterns of spatial segregation could alsoemerge from small-scale habitat heterogeneity. In this case, co-occurring species segregate and coexist, not because of finitedispersal and local interactions alone, but because each species has acompetitive advantage in, or is specifically adapted to, a differenthabitat type (Pacala&Levin, 1997). It is plausible that competitiverankings among orchids depend on the availability and abundanceof suitable mycorrhizal fungi. Coexistence may then depend ondifferences in mycorrhizal fungi that allow each orchid species togerminate and establish, making fungal diversity crucial tocoexistence. Recent research has shown that coexisting orchidspecies of the subtribe Coryciinae associated with differentmycorrhizal fungi (Waterman et al., 2011). Similarly, coexistenceof diploid and tetraploid cytotypes of the terrestrial orchidGymnadenia conopsea (T�e�sitelov�a et al., 2013) and pure and hybridplants in three species of the genusOrchis (Jacquemyn et al., 2012b)could be explained by association with distinct mycorrhizalcommunities. In the latter case, both the dominant fungal lineageand the total fungal community differed significantly between pureandhybrid species (Jacquemyn et al., 2012b). Further evidence that

mycorrhizal fungi were involved in determining the spatialdistribution of co-occurring orchid species was provided byJacquemyn et al. (2012a). Using seed germination experiments ina site where three orchid species that associated with differentmycorrhizal fungi co-occurred, they showed that spatial variationin seed germination ofAnacamptismorio,Gymnadenia conopsea andOrchis mascula was the limiting factor determining the above-ground spatial distribution of the orchids. Altogether these resultssuggest that mycorrhizal fungi are important factors driving nichepartitioning in terrestrial orchids and may therefore contribute toorchid coexistence. However, this has only been investigated in afew terrestrial orchids, so how broadly applicable these results areremains to be seen. Determining whether species segregation isexplicitly attributable to competition between or for fungi orwhether it can be attributed to orchid or fungal adaptation tospecific microhabitats will require experiments specificallydesigned to discriminate between these options. One possibilitycould be to add seeds of different orchid species in seed packets andto compare seed germination at different locations in thepopulation.

Future directions

In addition to the increased use of experimental manipulation ofenvironmental conditions and fungal availability and explicit testsof the competition hypothesis that were called for in the text, severaltracks of future research are poised to transform our understandingof orchidmycorrhizas dramatically. First, a broader investigation ofthe distribution of orchid mycorrhizal fungi in the soil and on hostphorophytes through the use of qPCR and next-generationsequencing will better define the link between mycorrhizal fungusdistribution and abundance and successful association withorchids. The distribution of mycorrhizal fungi has only beeninvestigated for a few temperate terrestrial orchids (M. K.McCormick et al., unpublished) and even less is currently knownabout the distribution of epiphytic orchid mycorrhizal fungi andtheir distribution on host trees. Kartzinel et al. (2013) have begunto investigate mycorrhizal fungus distribution on different phoro-phyte species and at a range of distances from existing orchids.However, they focused on using seed packets to define fungusdistribution and did not examine fungus distribution independentof orchid germination. Coupling such fungus mapping withassessment of edaphic or phorophyte conditions and fungalnutrient utilization abilities will allow translation of fungalcharacteristics to fungus and orchid distribution.

Secondly, Nurfadilah et al. (2013) investigated the nutrientaccess abilities of several Australian orchids and found that fungiassociated with habitat specialist orchids were less able to accessdiverse nutrient sources than those associated with widespreadorchids, suggesting that mycorrhizal fungus ability to accessnutrients may be an important driver of orchid distribution. Theability to leverage such research on a few fungi to predict conditionsneeded by many others will rely on understanding how fungalnutrient access abilities are constrained by evolution and whetherthey can be predicted based on their relatedness to other fungi thathave been studied. However, this will depend critically on

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Fig. 2 Spatial distribution of seven orchid species in a 259 25m plot insouthern Italy (from Jacquemyn et al., 2013). Colors indicate differentspecies.





development of an accurate phylogeny for orchid mycorrhizalfungi. Phylogenetic relationships of fungi in general are in a state offlux and those for the fungi that most commonly formmycorrhizaswith orchids are especially poorly understood. However, Lindeet al. (2013) recently identified new nuclear loci that are useful fordelimiting species in these difficult groups of fungi.

Thirdly, application of transcriptomics to elucidate how therelationship between fungi and orchids is established and affectedby environmental conditions promises to provide an unprece-dented window into the functioning of this symbiosis. This will beenabled by annotating and comparing the genomes of manymycorrhizal fungi, including some orchidmycorrhizal fungi, beingsequenced as part of the 1000 Fungal Genomes Project (Marmeisseet al., 2013). Zhao et al. (2013) have begun research in thisdirection by comparing genes expressed in germinating symbioticand asymbiotic orchid seeds. However, additional research will berequired to identify the functions of fungal gene products and toseparate genes involved in growth from those involved inestablishment of the symbiosis. When combined with extremelyhigh-resolution microscopy, which can reveal the timing andlocations of nutrient transfers (as per Yukari Kuga, pers. comm.),such transcriptomic studies promise to reveal the dynamics of howorchid mycorrhizal associations function.

Many of the unique characteristics of orchid mycorrhizalassociations, such as their wide range of specificities and degree ofdependence on fungi, make this a particularly powerful system inwhich to study the evolution and functioning of symbioticassociations. Yet, our understanding of these mycorrhizal associ-ations is still strongly limited by our restricted knowledge of theecology and spatial distribution of fungi. Many of the advances inunderstanding the correspondence between orchids and theirmycorrhizal fungi have not yet been addressed in arbuscularmycorrhizas or ectomycorrhizas, although a similar study has beencarried out with one species of Pyrola (Hashimoto et al., 2012). Yetother aspects of the functioning of mycorrhizas, such as thedynamics and mechanisms of nutrient exchange, are much betterstudied in those systems. We feel that similar studies conducted inorchid mycorrhizas will be extremely powerful ways to advanceunderstanding of the evolution and functioning of symbioticassociations. Such studies will also transform orchid conservationfrom mere land preservation to more targeted, active conservationand restoration.

Acknowledgements

The authors would like to thank the New Phytologist Trust forinviting them to the 31st New Phytologist Symposium, Orchidsymbioses: models for evolutionary ecology, where the idea for thispaper was generated and some of the new directions for futureresearch were presented. Three anonymous reviewers andMarc-Andr�e Selosse provided helpful comments on an earlierdraft.Wewould like to thank Ryan Phillips andTamara T�e�sitelov�afor providing data on seed germination. This project wassupported by the European Research Council (ERC starting grant260601 – MYCASOR) and the US National Park Service (PMIS#144281).

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