# Ectomycorrhizal fungal diversity associated with endemic Tristaniopsis spp. (Myrtaceae) in ultramafic and volcano-sedimentary soils in New Caledonia

Muhammad Waseem, Marc Ducousso, Yves Prin, Odile Domergue, Laure

Hannibal, Clarisse Majorel, Philippe Jourand, Antoine Galiana

► To cite this version:

Muhammad Waseem, Marc Ducousso, Yves Prin, Odile Domergue, Laure Hannibal, et al.. Ectomy-corrhizal fungal diversity associated with endemic Tristaniopsis spp. (Myrtaceae) in ultramafic and volcano-sedimentary soils in New Caledonia. Mycorrhiza, 2017, 27 (4), pp.407-413. 10.1007/s00572-017-0761-4 . ird-04927889

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SHORT NOTE

# Ectomycorrhizal fungal diversity associated with endemic *Tristaniopsis* spp. (Myrtaceae) in ultramafic and volcano-sedimentary soils in New Caledonia

Muhammad Waseem<fnref n="1" />,<fnref n="2" /> · Marc Ducousso<fnref n="1" /> · Yves Prin<fnref n="1" /> · Odile Domergue<fnref n="3" /> · Laure Hannibal<fnref n="4" /> · Clarisse Majorel<fnref n="4" /> · Philippe Jourand<fnref n="4" /> · Antoine Galiana<fnref n="1" />

Received: 31 August 2016 / Accepted: 4 January 2017

© Springer-Verlag Berlin Heidelberg 2017

**Abstract** New Caledonian serpentine (ultramafic) soils contain high levels of toxic heavy metals, in particular nickel, (up to 20 g kg⁻<fnref n="1" />) and are deficient in essential elements like carbon, nitrogen and phosphorus while having a high magnesium/ calcium ratio. Although previous studies showed that ectomycorrhizal symbioses could play an important role in the adaptation of the endemic plants to ultramafic soils (FEMS Microbiol Ecol 72:238–49, 2010), none of them have compared the diversity of microbial communities from ultramafic vs non- ultramafic soils in New Caledonia. We explored the impact of edaphic characteristics on the diversity of ectomycorrhizal (ECM) fungi associated with different endemic species of *Tristaniopsis* (Myrtaceae) growing under contrasting soil condi- tions in the natural ecosystems of New Caledonia. ECM root tips were thus sampled from two different ultramafic sites (Koniambo massif and Desmazures forest) vs two volcano-

sedimentary ones (Arama and Mont Ninndo). The molecular characterization of the ECM fungi through partial sequencing of the ITS rRNA gene revealed the presence of different dom- inant fungal genera including, both soil types combined, *Cortinarius* (36.1%), *Pisolithus* (18.5%), *Russula* (13.4%), *Heliotales* (8.2%) and *Boletellus* (7.2%). A high diversity of ECM taxa associated with *Tristaniopsis* species was found in both ultramafic and volcano-sedimentary soils but no significant differences in ECM genera distribution were observed between both soil types. No link could be established between the phy- logenetic clustering of ECM taxa and their soil type origin, thus suggesting a possible functional—rather than taxonomical—ad- aptation of ECM fungal communities to ultramafic soils.

**Keywords** Serpentine soils · *Cortinarius* · *Pisolithus* · *Russula* · Nickel tolerance · Fungal diversity

**Electronic supplementary material** The online version of this article (doi:10.1007/s00572-017-0761-4) contains supplementary material, which is available to authorized users.

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Antoine Galiana
antoine.galiana@cirad.fr

Introduction

The tropical archipelago of New Caledonia is located in the South West Pacific, about 1200 km east from Australia. Because of its contrasted geological setting, the main island of New Caledonia known as “La Grande Terre” (Supplemental Fig. S1) presents a large variety of soils, that can be grouped into two major categories: clayey and chertz soils developed over volcano-sedimentary rocks (basalts and siliceous sediments) that cover about 2/3 of the main island and metalliferous soils developed over ultramafic rocks (peridotites and serpentinites) that cover about 1/3 of the main island (Fritsch 2012). Compared to volcano sedimentary bedrocks, the soils developed on ultramafic bedrocks are rich in mineral deposits of chromium, cobalt, iron, manganese, and nickel and are intensively exploited by the mining industry (Perrier et al. 2006). Low available concentrations of major

<footnote n="1">¹ CIRAD, UMR113 CIRAD/INRA/IRD/SupAgro/UM, Laboratoire des Symbioses Tropicales et Méditerranéennes, Campus International de Baillarguet, TA A-82/J, F-34398 Cedex 5 Montpellier, France</footnote>

<footnote n="2">² Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan</footnote>

<footnote n="3">³ INRA, UMR113 CIRAD/INRA/IRD/SupAgro/UM, Laboratoire des Symbioses Tropicales et Méditerranéennes, Campus International de Baillarguet, TA A-82/J, F-34398 Cedex 5 Montpellier, France</footnote>

<footnote n="4">⁴ IRD, UMR113 CIRAD/INRA/IRD/SupAgro/UM, Laboratoire des Symbioses Tropicales et Méditerranéennes, F-98848 Noumea Cedex, New Caledonia</footnote>
plant nutrients (N, P, K); unbalanced high Mg/Ca ratio; and high levels of phytotoxic heavy metals, in particular Ni, are also regarded as typical characteristics of ultramafic soils (Brooks 1987). In addition, low organic matter contents and poor water retention are often associated to ultramafic soils resulting in an environment with depressed nutrient and water levels (Brooks 1987). This diversity of soils, combined with the tropical climatic conditions and the geographical isolation of the archipelago, led to a high terrestrial biodiversity on the island, i.e. 3371 vascular plant species among which 80% are endemic (Jaffré et al. 1987; Morat et al. 2012), and enabled to classify New Caledonia as one of the highest hotspots of bio-diversity in the world (Myers et al. 2000).

Previous studies performed in New Caledonia have described the diversity of plants (Jaffré et al. 1987) as well as those of their associated fungi (Perrier et al. 2006, Jourand et al. 2010a) and bacterial communities (Navarro et al. 1999; Héry et al. 2003; Chaintreuil et al. 2007; Bordez et al. 2016). Some of these studies have suggested that ectomycorrhizal symbioses could play an important role in the adaptation of the endemic plants to ultramafic soils. However, none of these studies have compared the diversity of microbial communities from ultramafic vs non-ultramafic soils in New Caledonia. A few studies performed in other regions have indicated that ECM communities from serpentine soils had similar levels of diversity as those from non-serpentine environments (Urban et al. 2008; Moser et al. 2009; Branco and Ree 2010). The tolerance of ectomycorrhizal fungi to high levels of toxic metals in soil in particular might be essential for the establishment of plants sensitive to metal-rich environments (Krznaric et al. 2009; Jourand et al. 2010b). The exploration of ECM fungi and their associated microbial flora in naturally metalliferous soils may reveal useful organisms for bioremediation. Such investigation could also lead to the identification of potential model organisms for population and functional studies on the adaptation of microorganisms to serpentine soils, more particularly to high concentrations of Ni, in New Caledonian as in other ultramafic soils. In our study, we selected four species of the ectomycorrhizal *Tristaniopsis* genus belonging to the Myrtaceae family, which ectomycorrhizal status has been confirmed in previous studies (Perrier et al. 2006). In addition to their ecological role and occurrence as a dominant species in protected areas of New Caledonia, these *Tristaniopsis* spp. were chosen considering that they are the only endemic species of the same genus that have the ability to grow on both metalliferous (ultramafic) and non-metalliferous (volcano-sedimentary) soils. Among the four *Tristaniopsis* tree species selected, *Tristaniopsis ninndoensis* is known to be distributed on volcano-sedimentary soils exclusively, *Tristaniopsis glauca* and *Tristaniopsis guillainii* on ultramafic soils

only, while the ubiquitous *Tristaniopsis calobuxus* species is able to colonize both soil types (L'Huillier et al. 2010).

In this work, our objectives were (1) to characterize the ECM fungal diversity associated with *Tristaniopsis* spp. from both ultramafic and volcano-sedimentary soils in New Caledonia by internal transcribed spacer (ITS) sequencing and (2) to test the hypothesis that ECM fungal communities would vary between both soil types with the occurrence of possible indicator species and specific adaptations.

## Materials and methods

### Site description and soil characterization

The study sites were located on the two major soil types of the “Grande Terre” island, i.e. ultramafic and volcano-sedimentary (non-ultramafic) soils. A full description of the ultramafic sites including climate, geology and vegetation structure was already reported by Perrier et al. (2006). The two ultramafic studied sites, that are mainly composed of ferralsol (Jourand et al. 2010a), include (i) Koniambo massif (21° 00′ 25″ S, 164° 49′ 53″ E; alt 450–890 m) located in the Northern Province and (ii) Forêt Desmazures (22° 12′ 10″ S, 166° 37′ 29″ E; alt 340 m) found in the South of the “Grande Terre” island (Supplemental Table S1). The two volcano-sedimentary sites mainly represented by ferric acrisol (Gillespie and Jaffré 2003) were Mont Ninndo (20° 16′ 39″ S, 164° 11′ 35″ E; alt 350 m) and Col d’Arama (20° 16′ 59″ S, 164° 11.48″ E; alt 240 m), both situated in the extreme North of the “Grande Terre” island (Supplemental Table S1). As described in details by Jourand et al. (2010a), the soils of the ultramafic and volcano-sedimentary sites highly differed in their physico-chemical properties. Both soils from Koniambo massif and Desmazures forest derived from ultramafic bedrocks and showed typical properties of serpentine soils, such as low Ca to Mg ratio, high level of Mg and low contents of major nutrient elements such as N, P and K. On the other hand, Fe and Ni contents were dramatically higher on serpentine as compared to volcano-sedimentary soils, in particular Ni and diethylene triamine penta acetic acid (DTPA)-Ni (available Ni) that were respectively 83 and 190 times higher in ultramafic soils than in volcano-sedimentary soils.

### Site selection and sampling

The ECM root samples were collected from *Tristaniopsis* stands naturally growing on ultramafic and volcano-sedimentary soils. Only stands dominated by *Tristaniopsis* spp. were selected for the root collections. Four *Tristaniopsis* species were sampled: *T. calobuxus* Brongn. & Gris, *T. glauca* Brongn. & Gris, *T. guillainii* Vieill. ex Brongn. & Gris and *T. ninndoensis* J.W. Dawson (Supplemental Table S1). The
ubiquitous species *T. calobuxus*, growing on both ultramafic and volcano-sedimentary soils, was sampled in three sites of the Koniambo massif (TG-15, Camp des Chinois and Sommet), in Desmazures forest and in Col d'Arama. *T. glauca* and *T. guillainii*, that are exclusive to ultramafic soils, were found in Desmazures forest and Koniambo massif sites, respectively. The endemic *T. ninndoensis* was exclusively found in the volcano-sedimentary Mont Ninndo site. After removing gently the upper layers of moss and litter at the base of trees, 20 to 100 g of ECM roots was collected per tree with their adhering soil. In total, three plots per site were systematically selected for the sampling of ECM roots tips and three trees, separated from one another by 10–15 m, were sampled in each plot, so that nine trees per site were selected. The ECM root tips were isolated from the field-collected root samples before a morphotyping step in laboratory where one representative per differentiated morphotype was selected from each root sample. Ectomycorrhizal tips were observed under a binocular microscope, carefully separated from roots, before being rapidly processed for subsequent molecular analyses.

## ITS sequencing and phylogenetic analysis

Total genomic fungal DNA was extracted and purified from ECM root tips using the DNAeasy Plant Qiagen Kit (Qiagen, Courtabœuf, France). Approximately 650-base pair (bp) fragment of nuclear ribosomal ITS rDNA containing the 5.8S region was amplified with specific primers ITS1-f (5'-CTTG GTCATTTAGAGGAAGTAA-3') (Gardes and Bruns 1993) and ITS4 (5'TCCTCCGCTTATTGATATGC-3') (White et al. 1990). PCR reactions were prepared in a total volume of 25 µL containing aliquots of 1 µL of genomic DNA at 50 ng mL⁻¹, 1 mM of each primer, 1.5 U of Taq DNA polymerase (Promega, Charbonnières, France), Promega Taq polymerase buffer, 2 mM MgCl₂ and 0.2 mM of each dNTP. PCR was performed with a DNA thermocycler Mastercyclers Eppendorf system (Eppendorf, Hamburg, Germany) programmed as follows: one cycle for 3 min at 95 °C followed by 40 cycles at 95 °C for 30 s, 53 °C for 1 min, 2 min at 72 °C and a final extension for 10 min at 72 °C. PCR products were separated by electrophoresis in 1% w/v agarose gel in Tris/acetic acid/EDTA (TAE) buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3) with ethidium bromide at 10 mg mL⁻¹ in the running buffer (Moyersoen et al. 2003). Amplified ITS products were purified using the Qiagen Gel extraction Kit (Qiagen). DNA sequencing was performed by Macrogen, using ABI chemistry and an ABI3730 capillary electrophoresis-based genetic analyzer (Macrogen, Seoul, Korea). Each set of sequences was considered as a different fungal taxon and the consensus sequence was saved and compared with the NCBI database using the BlastN algorithm (Altschul et al. 1997). All sequences matched fungal genera; however, only the ones confidently

assigned to an ECM genus (with at least the first five BLAST hits belonging to one genus) were retained for analyses. ITS sequences (including ITS1-f, 5.8S rRNA gene and ITS4) from this study were then aligned together with reference ITS sequences using ClustalX (Thompson et al. 1997) and corrected manually using Genedoc (Nicholas et al. 1997). The phylogenetic tree based on ITS sequences was constructed with MEGA v5.05 (Tamura et al. 2011) computer program through neighbour-joining method. The tree was bootstrapped with 100 replications of each sequence to evaluate the reliability of tree topology, and the GTR + G model was used to calculate the genetic distances. An uncultured Ascomycota (GenBank accession no. AY970289) was used as an outgroup of the phylogenetic tree. The nucleotide sequences of the ITS region amplified with ITS1-f and ITS4 primers were deposited in GenBank nucleotide database under a set of accession numbers as follows: JN847446 to JN847504 and JN872841 to JN872862 (Supplemental Table S2).

## Statistical analyses

The relative abundance of each ECM taxon (as characterized by ITS sequence) was estimated based on the number of successfully sequenced ECM root tips obtained from ultramafic and volcano-sedimentary soils. To compare species accumulation and richness estimates between soil types, rarefaction curves (Colwell et al. 2004) including 95% confidence intervals curves were computed using the EstimateS program (v. 8.2; Colwell 2009) for all sample sets. This corrects for differences in sampling size and allows virtual reduction of the size of all samples to that of the smallest one. The Shannon diversity index and the Chao2 richness estimator were also calculated using the EstimateS software to estimate the fungal diversity sampled from ultramafic and volcano-sedimentary soils. We used the $\chi^2$ independence test at $p = 0.95$ applied to the number of occurrences of each ECM taxon in each soil to check whether the distribution of the various ECM taxa was influenced by soil type.

## Results

### Ectomycorrhizal fungal diversity

From the four *Tristaniopsis* species investigated, 76 root samples were collected from 20 trees in *T. calobuxus* and nine trees per species in *T. guillainii*, *T. glauca* and *T. ninndoensis*. From these samples, 150 ECM root tips were isolated following a morphotyping step where one representative per differentiated morphotype was selected from each root sample, which correspond to about two morphotypes per root sample on average. Finally, 97 ECM root tips were successfully amplified and sequenced, of which 76% originated from ultramafic soils vs 24%
from volcano-sedimentary soils (Supplemental Table S3). All sequences obtained were searched in the Genbank database in order to assign them a major ECM group, when possible. An ITS-based phylogenetic tree was constructed from the sequences of the most representative ECM taxa as presented in Fig. 1. The phylogenetic analysis revealed that the uncultured fungal isolates obtained from the different soil and plant species origins were affiliated to a variety of ECM lineages, more particularly to *Cortinarius*, *Pisolithus*, *Scleroderma* and, to a lesser extent, to *Boletellus*, *Lactarius* and *Russula*. Only *Cortinarius*, *Pisolithus* and *Russula* lineages included isolates from both soil origins (*Russula* sequence from volcano-sedimentary soil not shown in Fig. 1).

## Composition of the ECM community according to soil type

The overall proportion of the different ECM genera in both soils and in each *Tristaniopsis* species investigated is reported in Fig. 2. Our study revealed a high diversity of ECM fungi, with almost similar fungal richness and proportions of ECM genera in ultramafic and volcano-sedimentary soils. Seventy-four distinct ECM taxa (i.e. diverging sequences as described in Supplemental Table S2) belonging to 10 ECM genera were identified from the ultramafic soils vs 23 taxa grouped into eight genera from volcano-sedimentary soils. As shown in Fig. 2, in ultramafic soils, the ECM *Cortinarius* genus appeared to be the most abundant taxon (39%), followed by *Pisolithus* (19%) and *Russula* (11%), all *Tristaniopsis* species combined. Seven other genera were also present but in lower proportions (<10%): *Boletellus*, *Helotiales*, *Scleroderma*, *Tomentella*, *Lactarius*, *Thelephora* and *Tricholoma*. Similar proportions of these fungal ECM taxa were found in volcano-sedimentary soils, even though *Russula* appeared to be more abundant than *Pisolithus* in volcano-sedimentary soils, unlike the ultramafic soils (Fig. 2). Nevertheless, as attested by the χ2 independence test applied to the number of occurrences of each ECM taxon in each soil type, all *Tristaniopsis* species combined, the different ECM genera did not tend to associate with a particular soil type (observed χ2 = 6.32 for *df* = 9 at 5% level). When considering all ECM taxa and all *Tristaniopsis* species combined, the ultramafic soils had a larger diversity than volcano-sedimentary soils as attested by the Shannon index (2.70 vs 2.42, respectively), although the latter were less intensively sampled. Despite the unbalanced sampling intensity between both soils, as almost three times more sequenced ECM root tips originated from ultramafic soils, species accumulation curves obtained from the ultramafic and volcano-sedimentary soils exhibited similar shapes, without signs of saturation (Supplemental Fig. S2) suggesting that diversity did not greatly vary between the two soil types, as illustrated in Fig. 2, and that an increased sampling effort would have resulted in the detection of other

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**Fig. 1** Phylogenetic tree of Basidiomycota from ultramafic and volcano-sedimentary soils in New Caledonia based on their ITS rRNA gene sequences. The maximum likelihood tree was obtained using MEGA v5.05 (Tamura et al. 2011) based on the GTR + G model. Bootstrap probabilities were estimated by 100 resampling. *Bar* inferred nucleotide substitutions per nucleotide. GenBank accession numbers of the reference sequences are given in parentheses. WME sequences in *red characters* were obtained from volcano-sedimentary soils and those in *black characters* from ultramafic soils. The tree was rooted using an Ascomycota sequence as outgroup

ECM taxa in both soil types. Among the different estimators of species richness calculated, Chao2 estimates a total of 16 taxa for ultramafic soils, with a 95% confidence interval ranging from 12 to 52. In the case of volcano-sedimentary soils, Chao2 estimates a total of 14 taxa with a 95% confidence interval ranging from 9 to 50.

**Composition of the ECM community according to host species**

As illustrated in Fig. 2, although a few differences were observed in ECM diversity of the four *Tristaniopsis* host species, they displayed similar fungal taxa compositions overall. Three of the most represented ECM genera among 10, i.e. *Cortinarius*, *Pisolithus* and *Russula*, occurred in the four host plant species and in both soil types. However, the comparison between the proportions of each ECM genus in the four host species suggests that some ECM genera were preferably associated with certain *Tristaniopsis* species. In absolute terms, *Cortinarius* appeared to be more frequently associated with *T. calobuxus* than with *T. glauca* or *T. guilainii* in Desmazures

and Koniambo sites, respectively. A similar trend was also found for *Pisolithus* associated with *T. calobuxus*. However, the respective proportions of the two latter ECM genera were not significantly different between *T. calobuxus* and the other *Tristaniopsis* species (according to the χ² test at 5% level). On the opposite, *Thelephora* and *Tricholoma* appeared to be exclusively associated with *T. calobuxus*, though in some rare cases. However, the χ² independence test applied to the number of occurrences of each ECM taxon in each host plant species did not show any ECM specificity towards a particular host species in ultramafic and in volcano-sedimentary soils (χ² = 31.06 for df = 45 at 5% level).

**Discussion**

If most studies reported highly diverse ECM fungal communities from a variety of environments and geographic locations (Leyval et al. 1997; Joner et al. 2006; Perrier et al. 2006; Smith and Read 2008; Branco and Ree 2010; Diedhiou et al. 2010; Jourand et al. 2010a), extreme and constrained environments are generally considered to host a lower diversity (Rothschild and Mancinelli 2001). Our study revealed that the ECM fungal diversity observed in *Tristaniopsis* spp. was independent to soil type. We reported equally complex and diverse fungal communities in both ultramafic and volcano-sedimentary soils, even though we were not able to fully describe the ECM fungal communities across the different sites and soil types. The presence of all major and representative fungal taxa including *Cortinarius*, *Pisolithus*, *Russula* and *Thelephora*, as well as other genera within the

<figure><img src="image_6.png" /><figcaption>**Fig. 2** Number of ECM root tips in the different ECM taxa collected from the ultramafic (UM) and volcano-sedimentary (VS) sites and *Tristanopsis* species</figcaption></figure>
Ascomycota and Basidiomycota in both soil types suggests that these ECM taxa may have widespread ability to cope with such extreme edaphic conditions. To our knowledge, this is the first detailed study describing ECM communities associated to *Tristaniopsis* or to any other plant genus/species in both ultra-mafic and volcano-sedimentary soils and more generally in serpentine vs non-serpentine soils for this multispecific and widespread genus. Previous research on other plant-microbes associations in ultramafic soils have suggested that these soils act not only as an extreme environment but also as a selective force on several groups of organisms (Rothschild and Mancinelli 2001; Perrier et al. 2006; Amir et al. 2007; Jourand et al. 2010a). However, in accordance with our observations, more specific studies indicated that ECM communities from serpentine soils had similar levels of diversity as those from non-serpentine environments (Urban et al. 2008; Moser et al. 2009; Branco and Ree 2010; Bordez et al. 2016). For instance, Branco and Ree (2010) who compared ECM fungal communities associated to oak tree from adjacent serpentine and non-serpentine soils in northeastern Portugal through ITS sequencing revealed the presence of closely related ECM fungal taxa in both soil types. These authors characterized a large diversity of taxa belonging to Agaricales, Boletales, Russulales and Thelephorales along with several genera within Ascomycota, across serpentine and non-serpentine soils, showing that serpentine soils were able to support fungal communities as rich and complex than those of non-serpentine ones (Bordez et al. 2016). Moser et al. (2009) reported similar results while comparing ECM fungal communities associated to *Quercus garryana* roots on serpentine and non-serpentine soils in southwestern Oregon (USA). They found abundant and almost similar ECM fungal communities on both soil types, which included *Cenococcum geophilum*, *Genea harknessii*, *Inocybe* sp., *Sebacina* sp., *Tomentella* sp. and *Tuber candidum* as the most dominant taxa. Our present study corroborated these findings, suggesting that ultramafic soils might not be a strong evolutionary agent for the selection of particular ECM fungal taxa. Other studies carried out on arbuscular mycorrhizal fungi also reported similar community patterns and high specific richness in both serpentine soils and non-serpentine soils, suggesting an important and widespread phenotypic plasticity across the fungal kingdom (Schechter and Bruns 2008; Fitzsimons and Miller 2010; Daghino et al. 2012). In addition, the most abundant ECM genera found to be associated with *Tristaniopsis* in ultramafic soils were similar to those associated with other host plant species in diverse studies performed on serpentine soils (Moser et al. 2009; Branco 2010; Jourand et al. 2010a). For example, Branco and Ree (2010) recently reported the dominance of *Boletellus*, *Cortinarius*, *Russula* and *Tomentella* on serpentine soils while studying the ECM fungal communities associated to holm oak tree (*Quercus ilex* L.) in Portugal. Likewise, the occurrence of *Pisolithus* (Jourand et al. 2010a) and *Lactarius* (Moser et al. 2009) was reported on ultramafic soils in other parts of the world. Despite the wide

phylogenetic distribution of taxa found within ECM fungal communities, no particular taxon could be assigned to a given soil type. While phylogenetic clustering is driven by habitat filtering when functional traits are conserved (Cavander-Bares et al. 2004; Jourand et al. 2010a), we did not find such phylogenetic clustering in ECM from serpentine soils, so that the possibility of habitat filtering associated with trait conservatism may be ruled out. Therefore, more information is needed for a thorough understanding of ECM fungal evolutionary patterns on serpentine soils. In addition, although ultramafic soils are generally deficient in essential plant nutrients, the New Caledonian ones studied here contain higher levels of nitrogen and phosphorus than volcanosedimentary soils. Moreover, the host plants, that provide carbohydrates to ECM fungi through the symbiotic association, could also act as protecting niches for the survival and the development of ECM fungi, thus mitigating the adverse edaphic constraints found in the extreme environments (Gehring et al. 1998). This increased availability of nutritive elements in New Caledonian ultramafic soils might explain the high diversity observed as well as the apparent absence of fungal specialization to ultramafic conditions, as already observed in other serpentine ecosystems (Branco and Ree 2010). More research is needed to understand the adaptation strategies used by ectomycorrhizal fungi for coping with edaphic stress in relation to high levels of heavy metals.

**Acknowledgements** The Agence Nationale de la Recherche, ANR 07 BIODIV ULTRABIO, financially supported this research work. We are also thankful to the Higher Education Commission of Pakistan for supporting this work through a PhD fellowship.

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