Where the wild things are: looking for unculturedGlomeromycota

Brian M. Ohsowski1, P. Dylan Zaitsoff1, Maarja €Opik2 and Miranda M. Hart1

1Department of Biology, University of British Columbia, Okanagan Campus, 3333 University Way, Kelowna, BC, Canada; 2Department of Botany, Institute of Ecology and Earth Sciences,

University of Tartu, 40 Lai St, 51005 Tartu, Estonia

Author for correspondence:Miranda M. Hart

Tel: +1 250 807 9398Email: Miranda.hart@ubc.ca

Received: 24 February 2014

Accepted: 13 May 2014

New Phytologist (2014) 204: 171–179doi: 10.1111/nph.12894

Key words: arbuscular mycorrhizal (AM)fungi, disturbance, Diversisporales,Glomerales, Glomeromycota, host specificity,meta-analysis, virtual taxa.

Summary

� Our knowledge of Glomeromycotan fungi rests largely on studies of cultured isolates. How-

ever, these isolates probably comprise one life-history strategy – ruderal. Consequently, our

knowledge of arbuscular mycorrhizal (AM) fungi may be biased towards fungi that occur pri-

marily in disturbed habitats and associate with disturbance-tolerant host plants. We can

expect to see a signal for this in DNA-based community surveys: human-impacted habitats

and cultivated plants should yield a higher proportion of AM fungal species that have been

cultured compared with natural habitats and wild plants.� Using the MaarjAM database (a curated open-access database of Glomeromycotan

sequences), we performed a meta-analysis on studies that described AM fungal communities

from a variety of habitats and host plants.� We found a greater proportion of cultured AM fungal taxa in human-impacted habitats. In

particular, undisturbed forests and grasslands/savannahs contained significantly fewer cul-

tured taxa than human-impacted sites. We also found that wild plants hosted fewer cultured

fungal taxa than cultivated plants.� Our data show that natural communities of AM fungi are composed largely of uncultured

taxa, and this is particularly pronounced in natural habitats and wild plants. We are better

poised to understand the functioning of AM symbioses associated with cultivated plants and

human-impacted habitats.

Introduction

Arbuscular mycorrhizas (AMs) are ancient and essential mutual-isms between plants and fungi that have allowed plants to inhabita wide variety of environments. These fungi (Phylum Glomer-omycota) receive their entire carbon provisions from the hostplant while imparting several benefits to the plant: most notablyenhanced nutrient uptake, and increased abiotic and biotic stresstolerance (Smith & Read, 2008). Although there is evidence thatthese fungi associated with land plants from their inception(c. 460 million yr ago) (Pyrozinski & Malloch, 1975; Redeckeret al., 2000; Strullu-Derrien et al., 2014), they show a remarkablydepauparate species richness, with only 244 currentlydescribed taxa (Sch€ußler, 2013). This is significantly fewer thanthe numbers of taxa in other, less ancient fungal phyla: there are30 000 species in Basidiomycota and 64 000 in Ascomycota,and 900 and 700 species, respectively, in the closest relativesZygomycota and Chytridiomycota (all values from Kirk et al.,2008).

Such low species richness is difficult to understand, given thatAM fungi associate with a large diversity of plant taxa (estimated

74% of plant species) (Brundrett, 2009). Recently, however, asurfeit of metagenomic studies from natural ecosystems hasrevealed greater species numbers at both local and global scalesthan previously thought (Moora et al., 2011; €Opik et al., 2013).Concomitant with this burgeoning diversity is the number ofundescribed Glomeromycotan taxa – those known on the basis ofDNA sequences only, with no known counterparts among mor-phologically described species (€Opik et al., 2014). Given ourknowledge about the Glomeromycota, it is difficult to know whatthese ‘undescribed taxa’ represent. Do they represent sequencesfrom groups that have been already encountered, or are these newlineages? Would these undescribed taxa show novel functionsamong AM fungi? How many AM fungal species are actually outthere? Answers to these questions may change the way we thinkabout the Glomeromycota.

Although we are learning more about AM fungi in naturalcommunities, a more complete understanding of this phylumand the full taxonomic and functional diversity therein may beimpeded by certain biases inherent in the scientific culture. Habi-tats and hosts that are either economically important or easy towork with dominate the existing literature. Thus, the number,

� 2014 The Authors

New Phytologist� 2014 New Phytologist TrustNew Phytologist (2014) 204: 171–179 171

www.newphytologist.com

Research

identity and functioning of fungi that are described may under-represent the full biological complexity within this group.

The habitat bias

Molecular descriptions of AM fungal communities are providinggreater sensitivity of detection and increased resolution of speciescomposition compared with the earlier prevailing spore morphol-ogy-based surveys. Based on morphologically described speciesalone, estimates of global species richness in the Glomeromycotahave increased from c. 150 species in 1993 (Walker & Trappe,1993) to the current 244 species (Sch€ußler, 2013). When envi-ronmental sequences are considered, estimates range from 348small subunit (SSU) ribosomal RNA (rRNA) gene-based virtualtaxa (VTs) ( €Opik et al., 2013) to over 1600 internal transcribedspacer (ITS)-based species hypotheses (SHs) (K~oljalg et al.,2013). Evidently, even the taxonomic richness of this phylum isstill being documented, and therefore knowledge of the biologyof this group of fungi is far from complete.

A large proportion of Glomeromycota detected in ecologicalsurveys belong to ‘uncultivated’ sequence types (van der Heijdenet al., 2008). While it is clear that these fungi belong to theGlomeromycota, there are no related sequences available in thepublic databases that originate from cultured specimens, and it ishighly probable that even total sequencing of culture collectionsand herbaria would not close this gap (cf. Hibbett et al., 2011). Isthis discrepancy because these ‘undescribed taxa’ represent iso-lates that cannot be easily cultured? Or have we simply failed tocollect data from many ecosystems, thus ignoring species charac-teristic of nonmodel habitats? Relatively few habitat types havetraditionally dominated the AM fungal research and literature – arecent search revealed that 11 of the 24 most cited experimentalAM field studies sampled from old-field (post-agricultural)research sites (Web of Science; search terms: arbusc* mycorr*diversity; December 2013). If understudied habitats represent areservoir of ‘undescribed taxa’, then current AM fungal diversityestimates might be decidedly low.

The glasshouse bias

Getting an AM fungus into culture can be challenging. To cir-cumvent culturing, researchers have used spore communities insoil to study AM fungal natural diversity. However, this approachtends to underestimate true levels of diversity (Clapp et al., 1995;Hempel et al., 2007) because AM fungi differ in the amount andrate of sporulation (Carvalho et al., 2001; Oehl et al., 2004,2009; Escudero & Mendoza, 2005). In fact, sporulation of sometaxa has never been observed under experimental conditions(Merryweather & Fitter, 1995; Clapp et al., 2002; Husbandet al., 2002; Rosendahl & Stukenbrock, 2004).

As an alternative to studying spore communities in situ, manyresearchers rely on ‘trap cultures’ whereby model host plants aregrown with soil from an environmental sample under controlledconditions in order to trigger sporulation and provide morematerial for spore-based AM fungal identification. It is generallyaccepted, however, that these trap cultures are a poor

approximation of natural AM fungal communities (S�ykorov�aet al., 2007; Hazard et al., 2013). The act of culturing is akin tosevere disturbance; soil mixing fragments the soil mycelia anddestroys connections to the host plant. Such conditions shouldfavour the growth of disturbance-tolerant fungi, such as thosethat are more resilient to mycelial damage, invest more heavily inintra-radical structures, and can regenerate from root fragments(Chagnon et al., 2013). Disturbances of this kind are common inhuman-impacted ecosystems, although they occur to a lesserextent in natural systems.

In addition to disturbance, the prevailing practice of using asingle host, which is usually a disturbance-tolerant cultivated orgeneralist plant species, may favour the growth of more generalistfungi over those with specific host requirements. Although trap-culturing of AM fungi is an invaluable method for obtaining cul-tures for experimentation, it is highly biased towards a limitedfunctional group of AM fungi – the disturbance-tolerant general-ist species. Ultimately, because we depend so heavily on culturedAM fungi to understand Glomeromycotan biology via compara-tive studies and controlled experiments, our knowledge may beconfined to the disturbance-tolerant (ruderal) and generalistfungi. Thus, knowledge about other, nonmodel, disturbance-intolerant, specialist AM fungal species from nonmodel hosts andhabitats may be systematically lacking.

Given that human-impacted ecosystems (characterized byrecurrent heavy disturbance) probably contain more ruderal fungicompared with natural ecosystems (typically with lower distur-bance intensity and frequency), we expect that DNA-based AMfungal community surveys of human-impacted systems shouldyield a higher proportion of species that have been cultured, com-pared with community analyses of undisturbed, natural systems.Further, host growth strategy may be an important predictor ofthe proportion of undescribed AM fungal taxa in natural systems.That is, we expect agricultural cultivated plants (disturbance tol-erant) to host more AM fungal species that have been cultured ascompared with wild plants. To test these hypotheses, we per-formed a meta-analysis of AM fungal community studies andasked whether disturbed systems had a higher proportion ofdescribed (cultured) AM fungal taxa than natural, undisturbedsystems. We also explored the proportion of cultured AM fungaltaxa found associated with plant hosts of different growth strate-gies.

Materials and Methods

Data collection

Publications included in this meta-analysis were selected fromthe 249 publications represented in the MaarjAM database ( €Opiket al., 2010) and collected on 6 August 2013. MaarjAM is acurated, online, open-access AM fungal-specific sequence andmeta-data database that places SSU rRNA gene sequences intogroups called virtual taxa (VTs); the database also includes othermarkers such as the ITS and large subunit (LSU) rRNA gene, butthese are not subjected to VT delimitation. VTs are delimitedphylogenetically on the basis of bootstrap support of clades and

New Phytologist (2014) 204: 171–179 � 2014 The Authors

New Phytologist� 2014 New Phytologist Trustwww.newphytologist.com

Research

NewPhytologist172

sequence similarity ≥ 97% ( €Opik et al., 2010, 2014). The VTconcept implemented in this database provides an opportunity toexamine natural AM fungal communities at approximately spe-cies-level taxonomy, using a consistent and stable taxon assign-ment approach ( €Opik et al., 2010, 2014). The MaarjAMdatabase incorporates representative sequences of AM fungalsequence groups for every host plant species at every site (wherepossible) in the International Nucleotide Sequence Databases(INSD) (http://www.insdc.org/). The MaarjAM database alsoincorporates data for sequenced cultured morphospecies fromtaxonomic publications. In this manner, sequences from bothcultures and environmental samples are subjected to uniform VTdelimitation ( €Opik et al., 2014). This results in some VTs con-taining several morphospecies as a result of the relatively low vari-ation of the central fragment of the SSU, in particular inDiversisporales (€Opik et al., 2010, 2013). Currently, SSU rRNAgene sequences are available for 84 of 244 morphospecies and areincorporated in MaarjAM in 60 VTs; an additional 288 VTscontain no sequences from morphospecies ( €Opik et al., 2014).

Our approach used data already present in the MaarjAM data-base to assess differences in the proportion of cultured:unculturedVTs across different habitats and host identities. In this analysis,a VT is considered cultured if it contains at least one sequencefrom a cultured morphospecies. In this way, the occurrence ofthe cultured species can be recorded on the basis of DNAsequences, even in the absence of cultures from respective habitatsand hosts.

Publications were included in the meta-analysis if they met thefollowing criteria.

(1) Experimental set-up DNA sequences must have beenextracted from naturally occurring field sites. No sequences wereobtained from glasshouse or trap culture experiments. Only con-trol treatment sequence data were used when experimental condi-tions were tested in a study. For example, DNA extractions frominoculation or soil enrichment experiments were excluded inorder to represent only unmanipulated AM fungal communities.In experimental designs with a temporal component, multiplesampling events were pooled into a single representative commu-nity. As in other meta-analyses, multiple ecologically distinct sitesmay have been sampled within an experiment. To meet our crite-ria, an experimental location was treated as an independent sitewhen the location differed in vegetation type and abiotic environ-mental conditions.

(2) Sequences Publications targeting only the SSU rRNA genewere included in the analysis. The SSU rRNA gene is a relativelyconserved region of DNA that contains sufficient intraspecificvariation and phylogenetic signal to allow delimitation of AMfungal sequence groups approximately at the species level, orslightly higher ( €Opik et al., 2014). For this analysis, onlysequences covering the central fragment of the gene were consid-ered, because this marker region is most commonly used in AMfungus community surveys. Publications with sequences not cov-ering this region or not targeting all Glomeromycota, such asthose obtained using general fungal primers, AM fungal primers

with lower level taxon specificity, or primers targeting the ITSregion or LSU rRNA gene, were excluded. Accessions withoutrepresentative DNA sequences available and no INSD accessionnumber (referred to in MaarjAM as ‘dummy’ accessions) wereexcluded from the analysis in order to maintain consistent com-parisons between publications.

We considered AM fungi identified from soil and/or plantroot samples when considering communities from different habi-tats, but only AM fungi identified from plant roots when consid-ering communities from different plant hosts. Finally, weconsidered only data sets generated using Sanger sequencing,because of the low number of next-generation sequencing studiesavailable.

Once we had identified appropriate data sets, accessions con-taining sequences and related meta-data were extracted from theMaarjAM database. The sequences were used with their VT iden-tities as present in MaarjAM.

(3) Categorical variables Publications must have included eco-logical, environmental, and taxonomic meta-data to accuratelydetermine the categorical explanatory variables. The categoricalvariables for this meta-analysis were as follows.Cultured versus uncultured AM fungi. In this study, twogroups of AM fungal virtual taxa were distinguished: those thatincluded sequence(s) from morphologically identified speciesmaintained in a culture collection were considered ‘cultured’VTs; those composed entirely of environmental sequences, withno sequence from a cultured AM fungus, were considered ‘uncul-tured’ VTs.Habitats. The habitat classification scheme was modified fromthat used in the MaarjAM database meta-data (described in €Opiket al., 2010) in order to increase the statistical power of the analy-sis. Four categories of habitat were used: human impacted (agri-cultural and industrial), forest, grassland/savannah, and desert/shrubland.Cultivated versus noncultivated plant species. We considereddomesticated plants bred for food production as cultivated hostsand all other plant species noncultivated host plant species.Plant functional group. The following six categories were used:C3 grasses, C4 grasses, nitrogen (N)-fixing plants, woody plants,myco-heterotrophs and forbs (other than N-fixing plants andmyco-heterotrophs).

Meta-analysis

Two sets of independent variables (moderators) were used inthe meta-analysis as follows: habitat (with two levels: human-impacted and natural habitats, or four levels: human-impactedhabitats, forest, grassland/savannah and desert/shrubland), andhost plant species (with two levels: cultivated and noncultivatedplants; or for host plant functional groups with six levels: C3

grass, C4 grass, N-fixing plant, woody plant, myco-heterotrophand forb). The data matrices were compiled as occurrences ofVTs (presence/absence): (1) at each site, as defined in theexperimental set-up; and (2) associated with each host plantspecies. Individual VTs were categorized either as cultured

� 2014 The Authors

New Phytologist� 2014 New Phytologist TrustNew Phytologist (2014) 204: 171–179

www.newphytologist.com

NewPhytologist Research 173

or uncultured AM fungal taxa, as described in the categoricalvariable definition of cultured versus uncultured AM fungi.The total count of cultured and uncultured VTs was calcu-lated for each replicate (site or host species at a site) within amoderator.

For each study, we calculated data points for each site and alsofor each host species as follows:

Xcount of cultured VT

� �=�X

count uncultured VT

þX

count cultured VT�

¼ Proportion of cultured VT per replicate Eqn 1

This calculation methodology was used to create the datapoints for three data sets: (1) all data, (2) only Glomerales, and(3) only Diversisporales. AM fungi in the orders Paraglomeralesand Archaeosporales were not included as sub-data sets becauseof the small number of records in the MaarjAM database result-ing from infrequent detection in original case studies. Replicationlevels were variable among these data sets because GlomeralesVTs tend to dominate over Diversisporales in the original casestudies. Only replicates (sites or hosts) with at least one culturedor uncultured VT were analysed for the Glomerales or Diversi-sporales.

To adjust for differing sampling effort between published stud-ies, we weighted each value with a modified nonparametric weight-ing scheme, calculated as follows: weighting factor = (N9N)/(N +N), where N equals the number of replicate samples taken ateach site or for each host plant. This nonparametric weightingscheme has been used widely in the ecological literature (Hoeksema& Forde, 2008; Veresoglou et al., 2012).

Habitat effect

To determine the effect of habitat type on the proportion of cul-tured AM fungal taxa per site, first the cultured AM fungal VTproportion associated with human-impacted habitats was com-pared with the cultured AM fungal VT proportion in naturalhabitats. The parameter value in human-impacted habitats wasused as the intercept with which the coefficient for natural habi-tats was compared. Similarly, we determined differences amongthe three natural habitat types compared to the human-impactedhabitat intercept.

Host effect

To test whether host type affected the proportion of cul-tured AM fungal taxa per plant species, first the culturedAM fungal VT proportion associated with cultivated plantspecies was compared with that associated with noncultivatedplants. The parameter value for cultivated plant speciesserved as the intercept against which other categories werecompared. Similarly, we determined differences among thesix plant functional groups compared to the cultivated plantspecies intercept.

Statistical analysis

Statistical analyses were performed using R v3.0.2 (R Core Team,2013). All meta-analysis procedures were calculated with the‘metafor’ package (Viechtbauer, 2010) using a Knapp and Har-tung adjustment for mixed effects models to test for coefficientsignificance in all analyses (Knapp & Hartung, 2003). In thisapproach, one moderator level is assigned to be the model inter-cept and all other coefficients are evaluated against this to deter-mine the significance of differences.

Results

In this meta-analysis, data from 70 publications were used toinvestigate the effect of disturbance and host identity on the pro-portion of cultured AM fungal VTs across habitats and host plantspecies. The habitat data set incorporates a total of 162 sites from70 publications and the plant host data set incorporates 279 plantspecies from 62 publications (Supporting Information TablesS1–S2). There was no estimated residual heterogeneity in any ofthe analyses; therefore, these values are not reported.

Habitat effect

The average percentage of cultured Glomeromycota VTs per sitewas lower in natural than in human-impacted habitats(P = 0.008) (Fig. 1a). This was also true when only Glomeralesand Diversisporales were examined (Glomerales: P = 0.001; thesame trend in Diversisporales: P = 0.062) (Fig. 1b,c).

The average percentage of cultured Glomeromycota andGlomerales VTs per site was lower in forests and grasslands/savannahs, but not in deserts/shrublands, when compared withhuman-impacted habitats (Glomeromycota: grassland/savannah,P = 0.010; forest, P = 0.002; Glomerales: grassland/savannah,P = 0.001; forest, P = 0.001) (Fig. 2a,b). In the case of Diversi-sporales, only forest habitats (P = 0.021) had a lower averagepercentage of cultured VTs per site compared with the human-impacted habitats (Fig. 2c).

Host effect

The average percentage of cultured Glomeromycota VTs perplant species was lower in the case of noncultivated than culti-vated plant species (P = 0.002) (Fig. 3a). This was also truewhen only Glomerales was examined (P = 0.001)(Fig. 3b), butnot in the case of Diversisporales (P = 0.380) (Fig. 3c).

The average percentage of cultured Glomeromycota VTs perplant species was lower in the case of forbs (P = 0.004), N-fixingplants (P = 0.017), woody plants (P = 0.001), and myco-hetero-trophs (P = 0.001) as compared with cultivated plant species(Fig. 4a). C3 and C4 grasses were associated with a similar per-centage of cultured AM fungal taxa as cultivated plant hosts.Again, the same was true for the Glomerales (forbs, P = 0.001;N-fixing plants, P = 0.003; woody plants, P = 0.001; myco-het-erotrophs, P = 0.001) (Fig. 4b), but not for the Diversisporales(Fig. 4c).

New Phytologist (2014) 204: 171–179 � 2014 The Authors

New Phytologist� 2014 New Phytologist Trustwww.newphytologist.com

Research

NewPhytologist174

Discussion

Our understanding of Glomeromycotan biology has seen majorbreakthroughs in recent years: the publication of the first ge-nomes (Tisserant et al., 2013; Lin et al., 2014), taxonomicreorganization (Oehl et al., 2011; Redecker et al., 2013), re-evalu-ation of their (a)sexual nature (Lee et al., 2014; Riley et al.,2014), and a reassessment of fundamental symbiotic functioning(Kiers et al., 2011; Fellbaum et al., 2012). All this research relieson using cultured fungal specimens.

In this meta-analysis we demonstrate that cultured fungi con-stitute only a minor fraction of AM fungi in natural and human-impacted habitats. Even in human-impacted habitats theproportion of cultured taxa is less than half of all species foundwithin a site. These results highlight that our current understand-ing of Glomeromycota biology may be confined to a very smallgroup of species with specific growth strategies. AM fungi (andtheir functions) that do not exist within model hosts and habitatsmay play important roles in global ecosystems, but this is yet tobe explored.

Are there more easily cultured fungi in disturbed systems?

We found support for our hypothesis that disturbed systems har-bour more cultured AM fungi than natural habitats. Further-more, the proportion of cultured AM fungi among the total

number of species per site differed depending on habitat type. Inthis study, AM fungal communities described from forests andgrasslands contained fewer cultured AM fungi compared withhuman-impacted (disturbed) habitats.

We predicted that human-impacted systems would select forfungi that are more easily cultured. There is ample evidence thatdisturbance selects for phylogenetically conserved fungi (Johnsonet al., 1991; Helgason et al., 1998, 2007). These fungi typicallyexhibit traits that may be associated with disturbance tolerance,such as restricted soil mycelia (McGonigle & Miller, 1996; Hart& Reader, 2004) and germination from hyphal fragments(Klironomos & Hart, 2002).

To determine if there was a phylogenetic basis underlying thistrend, we analysed the data at the level of AM fungal order, asthere is evidence that AM fungi differ broadly in terms of life his-tory strategy between the Glomerales and the Diversisporales.Specifically, members of the Glomerales (Glomeraceae andClairoideoglomeraceae) are thought to exhibit more r-selectedtraits whereas members of the Diversisporales (Gigasporaceae,Acaulosporaceae, Pacisporaceae and Diversisporaceae) tendtowards more K-selected traits (such as large extraradical myce-lium, spore size and density and increased hyphal diameter; Hart& Reader, 2002; S�ykorov�a et al., 2007; Chagnon et al., 2013).These differences suggest that Diversisporales may contain ahigher proportion of uncultured fungi in undisturbed systems, asfungi in this group should be more abundant in undisturbed sys-tems, and less amenable to culturing. In fact, we observed the

016 5649 11349 113

Cul

ture

d ta

xa (%

)

****

Glomeromycota Glomerales Diversisporales

25

50

75

100

Impa

cted

Natural

.

Impa

cted

Natural

Impa

cted

Natural

(a) (b) (c)

Fig. 1 The proportion of cultured arbuscular mycorrhizal (AM) fungal taxaamong the total AM fungal taxa per site in human-impacted versus naturalsystems for all Glomeromycota (a), Glomerales (b), and Diversisporales (c).Symbols represent the weighted model coefficient percentage of culturedtaxa (cultured taxa/all taxa) per site� SE. The number of sites for eachhabitat category is shown in the figure, above respective moderator levels.Differences between moderator levels (human-impacted versus naturalsystems) were tested using a linear mixed-effect model. The statisticaldifferences comparing the natural habitat moderator level to the human-impacted moderator level intercept were tested. Significance: **, P ≤ 0.01;P ≤ 0.10.

49 25

**

***

38 50

**

49 24 38 50

**

16 17 21 18

*

0

Cul

ture

d ta

xa (%

)

Glomeromycota Glomerales Diversisporales

25

50

75

100

Impa

cted

Desert

-shrub

Forest

Grass-s

av

Impa

cted

Desert

-shrub

Forest

Grass-s

av

Impa

cted

Desert

-shrub

Forest

Grass-s

av

(a) (b) (c)

Fig. 2 The proportion of cultured arbuscular mycorrhizal (AM) fungal taxaamong all AM fungal taxa in different habitat types for all Glomeromycota(a), Glomerales only (b), and Diversisporales only (c). Symbols representthe weighted model coefficient percentage of cultured taxa (culturedtaxa/all taxa)� SE. The number of sites for each moderator level is shownin the figure, above respective moderator levels. The statistical differencesamong natural habitat moderator levels (desert/shrub, forests andgrassland/savannahs (grass/sav)) compared to human-impactedmoderator level intercept were tested using linear mixed-effect models.Significance: ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

� 2014 The Authors

New Phytologist� 2014 New Phytologist TrustNew Phytologist (2014) 204: 171–179

www.newphytologist.com

NewPhytologist Research 175

opposite pattern. Our results showed that the Glomerales con-tained significantly more uncultured taxa in forests and grass-lands/savannahs compared with human-impacted systems,whereas there was no difference in the proportion of unculturedtaxa among different types of sites for the Diversisporales.

There are several possible reasons for the observed differencebetween the Glomerales and Diversisporales. First, it could be

partially artifactual, that is, related to the lower species resolutionpower of the marker used, and the higher number of morphospe-cies grouped into a single VT in Diversisporales ( €Opik et al.,2010). There is evidence to suggest that SSU rDNA does notprovide good resolution to the species level in Diversisporalescompared with other Glomeromycotan groups (€Opik et al.,2013). This would result in fewer VTs and possibly a higher pro-portion of total VTs in this order being categorized as ‘cultured’.The discrepancy may also be attributable to the fact that moreDiversisporales morphospecies have been sequenced for the SSUrRNA gene (39%; 38 of 97 species) compared with Glomerales(only 23%; 25 of 109 species) (table 2 in €Opik et al., 2010).However, it is reasonable to assume that the most easily culti-vated Glomeromycota have been sequenced by now for the com-monly used markers. Thus, the proportion of sequenced speciesmay reflect the greater ease of cultivability of Diversisporales overGlomerales, meaning that the observed difference in the ratio ofcultured:uncultured taxa reflects the difference in the proportionof cultivable species between these groups. Furthermore, the cul-tivated species of Glomerales are represented in few clades of thatorder, while those in Diversisporales are more evenly distributedacross the order ( €Opik et al., 2010, 2013).

Taken together, the findings suggest that there might be func-tionally divergent groups present within Glomerales, the largestorder of Glomeromycota, which may be the reason why ourfunctional trait-based expectation was not supported by the data.

It is interesting that we detected no difference in the propor-tion of cultured fungi between human-impacted sites anddeserts/shrublands. The simplest explanation for this is that therehas been a considerable history of research in these environmentsand that these fungi are well represented in culture collectionssimply as a result of sampling intensity. But this trend may alsohave an ecological basis: because these systems are so resource-limited, there may be selection for a fungus to associate with thefirst host it encounters, that is, a more generalist host affiliation.This would result in fungi that are more easily grown in trap

8 8126 24526 253

*****

0

Cul

ture

d ta

xa (%

)

Glomeromycota Glomerales Diversisporales

25

50

75

100

Cultiva

r

Non-cu

ltivar

Cultiva

r

Non-cu

ltivar

Cultiva

r

Non-cu

ltivar

(a) (b) (c)

Fig. 3 The proportion of cultured arbuscular mycorrhizal (AM) fungal taxaamong all AM fungal taxa in cultivated and noncultivated host plantspecies for all Glomeromycota (a), Glomerales only (b), and Diversisporalesonly (c). Symbols represent the weighted model coefficient percentage ofcultured taxa (cultured taxa/all taxa)� SE. The number of host species ineach moderator level is shown in the figure, above respective moderatorlevels. Statistical differences between moderator levels (cultivar andnoncultivar) were tested using a linear mixed-effect model. The statisticalcomparison relates the moderator level to the cultivated plants intercept.Significance: ***, P ≤ 0.001; **, P ≤ 0.01.

***

*** *****

***

******

26 28 16 112 26 50 21

25Cul

ture

d ta

xa (%

)

Glomeromycota Glomerales Diversisporales

50

75

100

125

Cultiva

r

C 3 gr

ass

Forb

N-fixer

Woody

Myco-h

etero

0

–2526 27 16 107 24 50 21 8 11 8 46 6 6 4

C 4 gr

ass

C 3 gr

ass

C 4 gr

ass

C 3 gr

ass

C 4 gr

ass

Cultiva

rForb

N-fixer

Woody

Myco-h

etero

Cultiva

rForb

N-fixer

Woody

Myco-h

etero

(a) (b) (c)

Fig. 4 The proportion of cultured arbuscularmycorrhizal (AM) fungal taxa among all AMfungal taxa in different host types for allGlomeromycota (a), Glomerales only (b), andDiversisporales only (c). Symbols representthe weighted model coefficient percentageof cultured taxa (cultured taxa/all taxa)� SE.The number of host plant species in eachmoderator level is shown in the figure, aboverespective moderator levels. Statisticaldifferences among moderator levels (C3

grass, C4 grass,forb, nitrogen-fixing plant,woody plant and myco-heterotroph)compared to the cultivated host plantintercept were tested using a linearmixed-effect model. Significance:***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

New Phytologist (2014) 204: 171–179 � 2014 The Authors

New Phytologist� 2014 New Phytologist Trustwww.newphytologist.com

Research

NewPhytologist176

cultures, and thus better represented among cultured fungi. Wedo not yet understand, however, the role of stress in the fidelityof the host/AM fungus relationship.

Why are there more cultured fungi with certain plantgroups?

We found support for the hypothesis that host identity affectedthe proportion of uncultured AM fungal taxa in a given data set;that is, fungal communities growing with cultivated plants con-tained more cultured taxa compared with plants rarely used in trapcultures or glasshouse experiments (i.e. forbs, N-fixing plants,woody plants and myco-heterotrophs). There are several reasonswhy this might be true. First, it is unlikely that all fungi in an envi-ronmental sample will be able to form an association with a trapplant. While traditionally believed to be generalist and nonspecificin terms of host range, research from natural systems reveals a sce-nario where natural AM fungal communities preferentially associ-ate with a subset of available hosts (Vandenkoornhuyse et al.,2003; €Opik et al., 2009; Davison et al., 2011; Chagnon et al.,2012; Osanai et al., 2013). Thus, the fungi cultured on trap plantsrepresent only a fraction of the total community that is able to col-onize the trap plant. To compound this host effect even further,the plants used in trap cultures are most often those that grow wellunder glasshouse conditions (having a fast growth rate and beingdisturbance tolerant). When we recently surveyed the literature(search terms: arbusc* mycorr* diversity; Web of Science,November 2013), we found that of the 14 most cited AM fungalglasshouse studies, which include trap cultures or manipulativeexperiments, 10 used one of the following plant hosts: Trifolium,Sorghum, Allium, Prunella and Plantago (Bever et al., 1996; Streit-wolf-Engel et al., 1997; van der Heijden et al., 1998; Van Tuinenet al., 1998; Eom et al., 2000; Hodge et al., 2001; Klironomos,2003; Oehl et al., 2003, 2004; Munkvold et al., 2004).

This study indicates that forbs, N-fixers, woody plants andmyco-heterotrophs were associated with proportionately fewercultured AM fungal taxa. This may be because such hosts areunderrepresented in glasshouse and field studies. While there hasbeen some work with N-fixing hosts, these studies have typicallybeen carried out on agricultural hosts (i.e. Medicago andTrifolium). Woody AM hosts, despite their wide distribution inmany ecosystems, are grossly underrepresented, probably becauselong-lived perennials are hard to work with, but also becausemost of these species are tropical/subtropical, which are less rep-resented in the literature (€Opik et al., 2010, 2013; Kivlin et al.,2011).

It is worth noting that the Diversisporales did not show anydifference among plant hosts in terms of the proportion of cul-tured AM fungal taxa. That is, cultured and uncultured taxa fromthis group were equally represented across all plant functionalgroups. It may be that these fungi are less strict in their hostrequirements, which would make them more easily cultured, butthis has not been tested in the literature. This finding is surprisingbecause some members of this order are notoriously difficult toobtain, and keep, in culture, and particularly root organ culture(Klironomos & Hart, 2002).

Is our knowledge about the Glomeromycota biasedtowards cultured AM fungi?

Our results suggest that c. 60% of AM fungi that are currentlyknown from DNA sequences are uncultured. Natural, undis-turbed systems and wild plants are associated with more uncul-tured AM fungi than disturbed systems and cultivars. However,this value varies dramatically depending on the type of systemand the identity of the host. Given that the vast majority of cul-tured AM fungal isolates originate in cultivated plants andhuman-impacted sites, is our understanding of AM fungi limitedto these systems? Given that some systems and hosts are almostentirely undescribed by cultured AM fungi, our knowledge aboutnatural systems and wild plants may be more limited than previ-ously thought.

Are these fungi uncultured because they are unculturable orbecause they occur in sites or on hosts that have been overlooked?In this study, we suggest that culturability may be related to thelife history traits of the AM fungi. However, we cannot yet con-clude which traits are important determinants of culturability.The evidence on trait diversification in AM fungi is limited tobasic information about mycelial investment (Hart & Reader,2002; Johnson, 2010), spore size and propagule types(Klironomos & Hart, 2002), but more likely it is based on adiverse suite of traits, pertaining to dispersal, colonization andpersistence in an environment. More information on such traits,and their variability in different environmental contexts, isneeded to make better predictions about AM fungal communityassembly and functioning.

Acknowledgements

The authors wish to thank the Irving K. Barber School of Artsand Science URA for funding received by P.D.Z., and the Natu-ral Sciences and Engineering Research Council for fundingreceived by M.M.H. M.€O. receives funding from the EstonianResearch Council (grants 9050 and IUT20-28) and the Euro-pean Regional Development Fund (Centre of Excellence FIBIR).We are grateful to the three anonymous referees, S. Hart and theEditor for useful comments on the manuscript.

References

Bever JD, Morton JB, Antonovics J, Schultz PA. 1996.Host-dependent

sporulation and species diversity of arbuscular mycorrhizal fungi in a mown

grassland. Journal of Ecology 84: 71–82.Brundrett MC. 2009.Mycorrhizal associations and other means of nutrition of

vascular plants: understanding the global diversity of host plants by resolving

conflicting information and developing reliable means of diagnosis. Plant andSoil 320: 37–77.

Carvalho LM, Cac�ador I, Martins-Louc�~ao M. 2001. Temporal and spatial

variation of arbuscular mycorrhizas in salt marsh plants of the Tagus estuary

(Portugal).Mycorrhiza 11: 303–309.Chagnon P-L, Bradley RL, Klironomos JN. 2012. Using ecological network

theory to evaluate the causes and consequences of arbuscular mycorrhizal

community structure. New Phytologist 194: 307–312.Chagnon P-L, Bradley RL, Maherali H, Klironomos JN. 2013. A trait-based

framework to understand life history of mycorrhizal fungi. Trends in PlantScience 18: 484–491.

� 2014 The Authors

New Phytologist� 2014 New Phytologist TrustNew Phytologist (2014) 204: 171–179

www.newphytologist.com

NewPhytologist Research 177

Clapp JP, Helgason T, Daniell TJ, Peter J, Young W. 2002. Genetic studies of

the structure and diversity of arbuscular mycorrhizal fungal communities. In:

van Der Heijden MGA, Sanders IR, eds.Mycorrhizal ecology. Heidelberg,

Germany: Springer-Verlag, 201–224.Clapp JP, Young JPW, Merryweather JW, Fitter AH. 1995. Diversity of fungal

symbionts in arbuscular mycorrhizas from a natural community. NewPhytologist 130: 259–265.

Davison J, €Opik M, Daniell TJ, Moora M, Zobel M. 2011. Arbuscular

mycorrhizal fungal communities in plant roots are not random assemblages.

FEMS Microbiology Ecology 78: 103–115.Eom A-H, Hartnett DC, Wilson GWT. 2000.Host plant species effects on

arbuscular mycorrhizal fungal communities in tallgrass prairie. Oecologia 122:435–444.

Escudero V, Mendoza R. 2005. Seasonal variation of arbuscular mycorrhizal

fungi in temperate grasslands along a wide hydrologic gradient.Mycorrhiza 15:291–299.

Fellbaum CR, Gachomo EW, Beesetty Y, Choudhari S, Strahan GD, Pfeffer

PE, Kiers ET, B€ucking H. 2012. Carbon availability triggers fungal nitrogen

uptake and transport in arbuscular mycorrhizal symbiosis. Proceedings of theNational Academy of Sciences, USA 109: 2666–2671.

Hart MM, Reader RJ. 2002. Taxonomic basis for variation in the colonization

strategy of arbuscular mycorrhizal fungi. New Phytologist 153: 335–344.Hart MM, Reader RJ. 2004. Do arbuscular mycorrhizal fungi recover from soil

disturbance differently? Tropical Ecology 45: 97–112.Hazard C, Gosling P, van der Gast CJ, Mitchell DT, Doohan FM, Bending

GD. 2013. The role of local environment and geographical distance in

determining community composition of arbuscular mycorrhizal fungi at the

landscape scale. ISME Journal 7: 498–508.van der Heijden MGA, Bardgett RD, van Straalen NM. 2008. The unseen

majority: soil microbes as drivers of plant diversity and productivity in

terrestrial ecosystems. Ecology Letters 11: 296–310.van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel

R, Boller T, Wiemken A, Sanders IR. 1998.Mycorrhizal fungal diversity

determines plant biodiversity, ecosystem variability and productivity. Nature396: 69–72.

Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW. 1998. Ploughing

up the wood-wide web? Nature 394: 431.Helgason T, Merryweather JW, Young JPW, Fitter AH. 2007. Specificity and

resilience in the arbuscular mycorrhizal fungi of a natural woodland

community. Journal of Ecology 95: 623–630.Hempel S, Renker C, Buscot F. 2007. Differences in the species composition of

arbuscular mycorrhizal fungi in spore, root and soil communities in a grassland

ecosystem. Environmental Microbiology 9: 1930–1938.Hibbett DS, Ohman A, Glotzer D, Nuhn M, Kirk P, Nilsson RH. 2011.

Progress in molecular and morphological taxon discovery in Fungi and options

for formal classification of environmental sequences. Fungal Biology Reviews 25:38–47.

Hodge A, Campbell CD, Fitter AH. 2001. An arbuscular mycorrhizal fungus

accelerates decomposition and acquires nitrogen directly from organic material.

Nature 413: 297–299.Hoeksema JD, Forde SE. 2008. A meta-analysis of factors affecting local

adaptation between interacting species. American Naturalist 171: 275–290.Husband R, Herre EA, Turner SL, Gallery R, Young JPW. 2002.Molecular

diversity of arbuscular mycorrhizal fungi and patterns of host association over

time and space in a tropical forest.Molecular Ecology 11: 2669–2678.Johnson NC. 2010. Resource stoichiometry elucidates the structure and function

of arbuscular mycorrhizas across scales. New Phytologist 185: 631–647.Johnson NC, Zak DR, Tilman D, Pfleger FL. 1991. Dynamics of

vesicular-arbuscular mycorrhizae during old field succession. Oecologia 86:349–358.

Kiers ET, Duhamel M, Beesetty Y, Mensah JA, Franken O, Verbruggen E,

Fellbaum CR, Kowalchuk GA, Hart MM, Bago A et al. 2011. Reciprocalrewards stabilize cooperation in the mycorrhizal symbiosis. Science 333: 880–882.

Kirk PM, Cannon PF, Minter DW, Stalpers JA, eds. 2008. Ainsworth & Bisby’sdictionary of the fungi. Wallingford, UK: CABI Publishing.

Kivlin SN, Hawkes CV, Treseder KK. 2011. Global diversity and distribution of

arbuscular mycorrhizal fungi. Soil Biology and Biochemistry 43: 2294–2303.Klironomos JN. 2003. Variation in plant response to native and exotic arbuscular

mycorrhizal fungi. Ecology 84: 2292–2301.Klironomos JN, Hart MM. 2002. Colonization of roots by arbuscular

mycorrhizal fungi using different sources of inoculum.Mycorrhiza 12: 181–184.

Knapp G, Hartung J. 2003. Improved tests for a random effects meta-regression

with a single covariate. Statistics in Medicine 22: 2693–2710.K~oljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M,

Bates ST, Bruns TD, Bengtsson-Palme J, Callaghan TM et al. 2013. Towardsa unified paradigm for sequence-based identification of fungi.Molecular Ecology22: 5271–5277.

Lee SC, Sun S, Heitman J. 2014. Unseen sex in ancient virgin fungi. NewPhytologist 201: 3–5.

Lin K, Limpens E, Zhang Z, Ivanov S, Saunders DGO, Mu D, Pang E, Cao H,

Cha H, Lin T et al. 2014. Single nucleus genome sequencing reveals high

similarity among nuclei of an endomycorrhizal fungus. PLoS Genetics 10:e1004078.

McGonigle TP, Miller MH. 1996. Development of fungi below ground in

association with plants growing in disturbed and undisturbed soils. Soil Biologyand Biochemistry 28: 263–269.

Merryweather J, Fitter A. 1995. Phosphorus and carbon budgets: mycorrhizal

contribution in Hyacinthoides non-scripta (L.) Chouard ex Rothm. under

natural conditions. New Phytologist 129: 619–627.Moora M, Berger S, Davison J, €Opik M, Bommarco R, Bruelheide H, K€uhn I,

Kunin WE, Metsis M, Rortais A. 2011. Alien plants associate with widespread

generalist arbuscular mycorrhizal fungal taxa: evidence from a continental-scale

study using massively parallel 454 sequencing. Journal of Biogeography 38:1305–1317.

Munkvold L, Kjøller R, Vestberg M, Rosendahl S, Jakobsen I. 2004.High

functional diversity within species of arbuscular mycorrhizal fungi. NewPhytologist 164: 357–364.

Oehl F, Sieverding E, Ineichen K, M€ader P, Boller T, Wiemken A. 2003.

Impact of land use intensity on the species diversity of arbuscular mycorrhizal

fungi in agroecosystems of Central Europe. Applied and EnvironmentalMicrobiology 69: 2816–2824.

Oehl F, Sieverding E, Ineichen K, M€ader P, Wiemken A, Boller T. 2009.

Distinct sporulation dynamics of arbuscular mycorrhizal fungal communities

from different agroecosystems in long-term microcosms. Agriculture, Ecosystemsand Environment 134: 257–268.

Oehl F, Sieverding E, M€ader P, Dubois D, Ineichen K, Boller T, Wiemken A.

2004. Impact of long-term conventional and organic farming on the diversity

of arbuscular mycorrhizal fungi. Oecologia 138: 574–583.Oehl F, Sieverding E, Palenzuela J, Ineichen K, da Silva GA. 2011.

Advances in Glomeromycota taxonomy and classification. IMA Fungus 2:191–199.

€Opik M, Davison J, Moora M, Zobel M. 2014. DNA-based detection and

identification of Glomeromycota: the virtual taxonomy of environmental

sequences. Botany 92: 135–147.€Opik M, Metsis M, Daniell TJ, Zobel M, Moora M. 2009. Large-scale parallel

454 sequencing reveals host ecological group specificity of arbuscular

mycorrhizal fungi in a boreonemoral forest. New Phytologist 184: 424–437.€Opik M, Vanatoa A, Vanatoa E, Moora M, Davison J, Kalwij JM, Reier €U,

Zobel M. 2010. The online databaseMaarjAM reveals global and ecosystemic

distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota). NewPhytologist 188: 223–241.

€Opik M, Zobel M, Cantero JJ, Davison J, Facelli JM, Hiiesalu I, Jairus T,

Kalwij JM, Koorem K, Leal ME et al. 2013. Global sampling of plant roots

expands the described molecular diversity of arbuscular mycorrhizal fungi.

Mycorrhiza 23: 411–430.Osanai Y, Bougoure DS, Hayden HL, Hovenden MJ. 2013. Co-occurring grass

species differ in their associated microbial community composition in a

temperate native grassland. Plant and Soil 368: 419–431.Pyrozinski KA, Malloch DW. 1975. The origin of land plants: a matter of

mycotrophism. Biosystems 6: 153–164.

New Phytologist (2014) 204: 171–179 � 2014 The Authors

New Phytologist� 2014 New Phytologist Trustwww.newphytologist.com

Research

NewPhytologist178

R Core Team. 2013. R: a language and environment for statistical computing.Vienna, Austria: R Foundation for Statistical Computing. URL http://www.

R-project.org/

Redecker D, Kodner R, Graham LE. 2000. Glomalean fungi from the

Ordovician. Science 289: 1920–1921.Redecker D, Sch€ußler A, Stockinger H, St€urmer SL, Morton JB, Walker C.

2013. An evidence-based consensus for the classification of arbuscular

mycorrhizal fungi (Glomeromyota).Mycorrhiza 23: 515–531.Riley R, Charron P, Idnurm A, Farinelli L, Dalpe Y, Martin F, Corradi N.

2014. Extreme diversification of the mating type–high-mobility group

(MATA-HMG) gene family in a plant-associated arbuscular mycorrhizal

fungus. New Phytologist 201: 254–268.Rosendahl S, Stukenbrock EH. 2004. Community structure of arbuscular

mycorrhizal fungi in undisturbed vegetation revealed by analyses of LSU rDNA

sequences.Molecular Ecology 13: 3179–3186.Sch€ußler A. 2013. Glomeromycota: Species list. [WWW document] URL http://

schuessler.userweb.mwn.de/amphylo [accessed 12 December 2013].

Smith SE, Read DJ. 2008.Mycorrhizal symbiosis. Amsterdam, the Netherlands:

Academic Press, Elsevier.

Streitwolf-Engel R, Boller T, Wiemken A, Sanders IR. 1997. Clonal

growth traits of two Prunella species are determined by co-occurring

arbuscular mycorrhizal fungi from a calcareous grassland. Journal of Ecology 85:181–191.

Strullu-Derrien C, Kenrick P, Pressel S, Duckettt JG, Rioult JP, Strullu DG.

2014. Fungal associations in Horneophyto ligneri from the Rhynie Chert (c 407

million years old) closely resemble those in extant lower land plants; novel

insights into ancestral plant-fungal symbioses. New Phytologist. doi: 10.1111/nph.12805.

S�ykorov�a Z, Ineichen K, Wiemken A, Redecker D. 2007. The cultivation bias:

different communities of arbuscular mycorrhizal fungi detected in roots from

the field, from bait plants transplanted to the field, and from a greenhouse trap

experiment.Mycorrhiza 18: 1–14.Tisserant E, Malbreil M, Kuo A, Kohler A, Symeonidi A, Balestrini R, Charron

P, Duensing N, Frei dit Frey N, Gianinazzi-Pearson V et al. 2013. Genome of

an arbuscular mycorrhizal fungus provides insight into the oldest plant

symbiosis. Proceedings of the National Academy of Sciences, USA 110: 20117–20122.

Van Tuinen D, Jacquot E, Zhao B, Gollotte A, Gianinazzi-Pearson V. 1998.

Characterization of root colonization profiles by a microcosm community of

arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR.MolecularEcology 7: 879–887.

Vandenkoornhuyse P, Ridgway KP, Watson IJ, Fitter AH, Young JPW. 2003.

Co-existing grass species have distinctive arbuscular mycorrhizal communities.

Molecular Ecology 12: 3085–3095.Veresoglou SD, Menexes G, Rillig MC. 2012. Do arbuscular mycorrhizal fungi

affect the allometric partition of host plant biomass to shoots and roots? A

meta-analysis of studies from 1990 to 2010.Mycorrhiza 22: 227–235.Viechtbauer W. 2010. Conducting meta-analyses in R with the metafor package.

Journal of Statistical Software 36: 1–48.Walker C, Trappe JM. 1993. Names and epithets in the Glomales and

Endogonales.Mycological Research 97: 339–344.

Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Table S1 Data used in the habitat meta-analysis includingdetailed ecosystem and publication information

Table S2 Data used in the plant host meta-analysis includingdetailed plant, ecosystem, and publication information

Please note: Wiley Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.

www.newphytologist.com

www.newphytologist.com

np-centraloffice@lancaster.ac.uknp-usaoffice@lancaster.ac.uk

� 2014 The Authors

New Phytologist� 2014 New Phytologist TrustNew Phytologist (2014) 204: 171–179

www.newphytologist.com

NewPhytologist Research 179