# Ectomycorrhizal Communities Associated with the Legume Acacia spirorbis Growing on Contrasted Edaphic Constraints in New Caledonia

Anne Houles, Bryan Vincent, Magali David, Marc Ducousso, Antoine Galiana, Farid Juillot, Laure Hannibal, Fabian Carriconde, Emmanuel Fritsch, Philippe Jourand

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Anne Houles, Bryan Vincent, Magali David, Marc Ducousso, Antoine Galiana, et al.. Ectomycorrhizal Communities Associated with the Legume Acacia spirorbis Growing on Contrasted Edaphic Constraints in New Caledonia. Environmental Microbiology, 2018, 76 (4), 10.1007/s00248-018-1193-1. ird-04927886

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# Ectomycorrhizal Communities Associated with the Legume *Acacia spirorbis* Growing on Contrasted Edaphic Constraints in New Caledonia

Anne Houles<fnref n="1" />,<fnref n="2" /> · Bryan Vincent<fnref n="3" /> · Magali David<fnref n="4" /> · Marc Ducousso<fnref n="1" /> · Antoine Galiana<fnref n="1" /> · Farid Juillot<fnref n="4" /> · Laure Hannibal<fnref n="3" /> · Fabian Carriconde<fnref n="5" /> · Emmanuel Fritsch<fnref n="4" /> · Philippe Jourand<fnref n="3" />

Received: 29 June 2017 / Accepted: 12 April 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018

## Abstract

This study aims to characterize the ectomycorrhizal (ECM) communities associated with *Acacia spirorbis*, a legume tree widely spread in New Caledonia that spontaneously grows on contrasted edaphic constraints, i.e. calcareous, ferralitic and volcano-sedimentary soils. Soil geochemical parameters and diversity of ECM communities were assessed in 12 sites representative of the three mains categories of soils. The ectomycorrhizal status of *Acacia spirorbis* was confirmed in all studied soils, with a fungal community dominated at 92% by Basidiomycota, mostly represented by/tomentella-thelephora (27.6%), /boletus (15.8%), /sebacina (10.5%), /russula-lactarius (10.5%) and /pisolithus-scleroderma (7.9%) lineages. The diversity and the proportion of the ECM lineages were similar for the ferralitic and volcano-sedimentary soils but significantly different for the calcareous soils. These differences in the distribution of the ECM communities were statistically correlated with pH, Ca, P and Al in the calcareous soils and with Co in the ferralitic soils. Altogether, these data suggest a high capacity of *A. spirorbis* to form ECM symbioses with a large spectrum of fungi regardless the soil categories with contrasted edaphic parameters.

**Keywords** *Acacia spirorbis* · Ectomycorrhizas · New Caledonia · Fungal diversity · Edaphic constraint

## Introduction

New Caledonia is a tropical archipelago located in the south-west Pacific, about 1200 km east from Australia. Because of a contrasted geological history, New Caledonia presents a wide variety of soils that can be grouped into three major categories: clayey and chert soils developed upon volcano-

sedimentary rocks (basalts, schists and chert-type siliceous sediments) that cover about two thirds of the main island; ferralitic soils developed upon ultramafic rocks (peridotites and serpentinites) representing about one third of the main island and more or less humic and aluminous soils developed upon coral reef formations that are mainly found in the Loyalty islands [1]. This diversity of soils, combined with the tropical climatic conditions and the geographical isolation of the archipelago, led to the high terrestrial biodiversity of the island, i.e. 3371 vascular plant species among which 80% are endemic [2]. Among the vascular plants identified in New Caledonia, Labillardière [3] reported *Acacia spirorbis*, which belongs to the Fabaceae family and the Mimosoideae subfamily. This legume named *Acacia spirorbis* subsp. *spirorbis*, abbreviated into *A. spirorbis* along the following text, is now considered as a species of the phyllodinous Australian acacias clade and is geographically located only in the New Caledonian archipelago [4]. *A. spirorbis* is able to colonize and grow on a large variety of soils (i.e. calcareous in the Loyalty islands; volcano-sedimentary and ferralitic soils on the main island that show a large range of concentrations of major plant nutrients (Ca, N, P and K) and high levels of potentially toxic heavy metals (Co, Cr, Mn and Ni) as well

**Electronic supplementary material** The online version of this article (https://doi.org/10.1007/s00248-018-1193-1) contains supplementary material, which is available to authorized users.

<figure><img src="image_3.png" /></figure>

Marc Ducousso
marc.ducousso@cirad.fr

<footnote n="1">¹ CIRAD, UMR082 LSTM, TA A-82/J, Campus International de Baillarguet, 34398 Cedex 5 Montpellier, France</footnote>

<footnote n="2">² Koniambo Nickel SAS, Vavouto, BP 679, 98860 Koné, New Caledonia</footnote>

<footnote n="3">³ IRD, UMR040 LSTM, 98848 Nouméa Cedex, New Caledonia</footnote>

<footnote n="4">⁴ IRD, UMR206 IMPMC, 98848 Nouméa Cedex, New Caledonia</footnote>

<footnote n="5">⁵ Institut Agronomique néo-Calédonien (IAC), Axe 2, 98800 Nouméa, New Caledonia</footnote>
as pH values between 4 and 8). This plant is naturally wide-
spread in New Caledonia, with a large distribution along the
archipelago up to an altitude of 400 m, and it was used as a
tool in ecological restoration strategies for ferralitic outcrops
after nickel mining [5]. This legume species has the capacity
to develop a triple root symbiosis with the nitrogen fixing
bacteria *Bradyrhizobium* sp. [6] and with both arbuscular
(AM) and ectomycorrhizal (ECM) fungi [7, 8]. In a previous
study, it has been shown that *A. spirorbis* was able to form
ECM symbiosis with the fungus *Pisolithus albus* in soils de-
veloped upon volcano-sedimentary or ultramafic rocks [9]. It
has also been recently demonstrated that the ECM fungus
*P. albus* plays a key role in adaptation of *A. spirorbis* to
ferralitic soils by enhancing the uptake of major nutritive ele-
ments such as Ca, K and P and reducing the polymetallic
toxicity related to the large amounts of cobalt, chromium,
manganese and nickel naturally present in most New
Caledonian ferralitic soils [8]. Such adaptations of
*A. spirorbis* related to its ability to develop symbiotic associ-
ation with a specific ECM fungus raises the question of the
actual diversity of the ECM fungal community associated
with this plant species, in relation with the various edaphic
conditions encountered by this legume. Very few studies have
reported comparisons between ECM fungal communities of a
same tree species facing different edaphic constraints, mainly
because the distribution of most ECM tree species is limited
by soil constraints. For example, a study focused on *Quercus*
spp. that is able to grow on both ferralitic and non-ferralitic
soils reported similar structures of ECM communities associ-
ated with this plant in both soil types [10]. Similarly, the com-
parison of fungal ECM communities from plants growing on
both serpentine and non-serpentine soils concluded that the
serpentinic edaphic constraints did not limit but might even
promote the ECM diversity [11, 12]. However, it is often
assumed that links exist between (i) the distribution of tropical
tree diversity, (ii) the distribution of tropical ectomycorrhizal
diversity and (iii) the local-scale edaphic variations [13]. More
recently, Roy et al. [14] found that the richness of the ECM
community associated with *Alnus* spp. was significantly influ-
enced by soil parameters (mineral vs organic), with about 26%
of richness variation that could be explained by the soil pa-
rameters in combination with the host plant of origin. This
latter result is in agreement with the conclusion of the recent
review article by Tedersoo et al. [15], indicating that environ-
mental traits such as climatic and edaphic conditions strongly
structure the fungal biodiversity at the global scale. In New
Caledonia, the contrasted edaphic conditions might also struc-
ture the diversity of the ECM fungal communities encoun-
tered by the legume *A. spirorbis* that is able to colonize a large
variety of soils. The aims of the present study were (i) to
characterize the general ECM status of *A. spirorbis* on the
different soil types on which this plant species grows, (ii) to
analyse the structure of the ECM fungal communities able to

develop symbiotic associations with this legume species in
situ, and (iii) to compare the structures of ECM communities
of the three soil categories. The hypothesis is that *A. spirorbis*
has a strong capacity to develop ECM with a large spectrum of
fungi regardless of the very contrasting edaphic parameters. In
order to perform this study, the diversity of the ECM fungal
community associated with *A. spirorbis* naturally growing on
the three major types of soils in New Caledonia, i.e. soils de-
veloped upon calcareous, ultramafic and volcano-sedimentary
rocks, was assessed. For that purpose, ECM root tips of
*A. spirorbis* were sampled in several sites representative of the
three major soil types for identification and molecular charac-
terization of symbiotic partners by ITS sequencing. The
resulting ECM fungal diversity associated with *A. spirorbis*
was established and then analysed according to the soil type.

**Materials and Methods**

**Site Description, Soil Sampling and Chemical Analyses**

The sites for sampling were chosen as they all presented spon-
taneous gregarious populations of *A. spirorbis*. The geograph-
ical position of the sites where the soil samples and ECM roots
from *A. spirorbis* were collected are shown on the map in
Fig. 1, and the detailed location of the study sites and soil
descriptions are reported in Table 1 [17]. These soils were
classified into three major categories as recently reported in
the pedological survey of New Caledonia by Fritsch [1]. Four
soils were classified into the calcareous (CA) soil category:
two Gibbsic Ferralsols, Humic (WNK and MUC), one
Gibbsic Ferralsols, Magnesic (WAN) and one Rendzic
Leptosol, Humic (EAS). Four soils were classified into the
ferralitic (FE) soil category: one Geric Plinthosol (TIE), one
Magnesic Ferralsol (PAND-1), one Abruptic Ferralsol
(PAND-2) and one Haplic Ferralsol (PIND). Four soils were
classified into the volcano-sedimentary (VS) soil category:
one Leptic Cambisol (POUM MB), one Albic Acrisol
(POUM SI), one Haplic Vertisol (VERT), and one Haplic
Cambisol (SERP). For each of these 12 soils, three samples
of 1 L each representing the 0–30-cm horizon were collected.
This depth was considered as representative of the bulk soil
horizon that is mainly explored by the *A. spirorbis* roots. The
concentration of elements (Si, Mg, Ca, Na, K, P, Fe, Mn, Al,
Ni, Cr, Co, P) in the soil samples was determined at the ISO
9002 certified Laboratoire des Moyens Analytiques (LAMA)
of the Centre IRD in Noumea (New Caledonia) by ICP-AES
after alkaline fusion [18]. The organic carbon (Corg) and ni-
trogen (Norg) contents were determined following the
Walkley-Black and Kjeldahl methods, respectively, as previ-
ously described in [19]. The full geochemical characteristics
of the 12 soils sampled are reported in ESM Table 1A–C and
data are summarized in Table 2. The geochemical data of each
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Map of the New Caledonian archipelago including the three major soil categories (i.e. calcareous, ferralitic and volcano-sedimentary soils) and showing the location of the sites where ectomycorrhizal fungi were sampled from roots of *Acacia spirorbis*</figcaption></figure>

site were analysed by a principal component analysis (PCA) in order to test the pooling of the sites according to their geochemical properties. All statistical analyses were carried out using the *R* software (https://www.r-project.org/) as described below.

## Sampling of Ectomycorrhizal Root Tips

In each selected site, ectomycorrhizal root tips were collected from roots of *A. spirorbis* specimens in dense populations, aged from 2 to 15 years, and dispersed across an area of about 1 ha. Ectomycorrhizas were randomly collected on five trees per site, with a minimal distance of 10 m between each specimen. For each tree, ectomycorrhizas were collected using the root tracking method that consists in following the roots from

the largest trunk-connected roots toward the finest ones where ectomycorrhizas preferentially occur [19]. Numbers of ECM collected on each site are reported in Table 3. Immediately after sampling, the ectomycorrhizal root tips were placed in sealed vials and directly transferred into liquid nitrogen. Back to the laboratory, the samples were stored at – 20 °C before DNA extraction for sequencing.

## Molecular Methods

Total DNA was extracted from individual ectomycorrhizal root tips using the DNeasy Plant Mini Kit (QIAGEN, Courtabœuf, France) following the protocol described by Jourand et al. [9]. A fragment of ribosomal DNA of approximately 650 bp of the Internal Transcribed Spacer regions (ITS) was amplified using
<table><caption>**Table 1** Summary data of sampled sites where ectomycorrhizas of *A. spirorbis* were collected: description, references as reported in Fig. 1, location with geographical coordinates and soil description and WRB classification as reported by Fritsch [1]</caption><thead><tr><th>Soil geochemical category</th><th>Site name</th><th>Location and coordinates</th><th>Soil description and WRB classification</th></tr></thead><tbody><tr><td rowspan="6">Calcareous</td><td>EAS</td><td>Atoll's ledge, Pointe de Easo (Lifou)<br>S 20° 47' 12.23"; E 167° 07' 21.72" (alt 25 m)</td><td>Very thin dark-brown humic soil<br>Rendzic leptosol (humic)</td></tr><tr><td>MUC</td><td>Atoll's central plateau, Mucaweng (Lifou)<br>S 20° 44' 57.54"; E 167° 09' 09.34" (alt 40 m)</td><td>Thin dark yellow-brown soil<br>Gibbsic ferralsol (humic)</td></tr><tr><td>WAN</td><td>Atoll's central plateau, Wanaham (Lifou)<br>S 20° 44' 43.56"; E 167° 13' 17.88" (alt 31 m)</td><td>Thin yellow-brown soil<br>Gibbsic ferralsol (magnesic)</td></tr><tr><td>WNK</td><td>Atoll's central plateau, Wanek (Lifou).<br>S 20° 44' 49.33"; E 167° 05' 00.86" (alt 41 m)</td><td>Red-brown soil with calcareous rocks<br>Gibbsic ferralsol (humic)</td></tr><tr><td>TIE</td><td>Colluvium at the bottom of the Tiebaghi ultramafic regolith<br>S 20° 28' 19.20"; E 164° 15' 35.16" (alt 69 m)</td><td>Red pisolithic soil<br>Geric plinthosol</td></tr><tr><td>PIND</td><td>Pindaï peninsula, East Nepoui<br>S 21° 19' 55.45" E164° 58' 19.24" (alt 65 m)</td><td>Red-brown soil<br>Haplic ferralsol</td></tr><tr><td rowspan="2">Ferralitic</td><td>PAND-1</td><td>Bottom of the Koniambo ultramafic regolith<br>S 21° 01' 35.84"; E 164° 46' 58.68" (alt 38 m)</td><td>Brown soil<br>Magnesic ferralsol</td></tr><tr><td>PAND-2</td><td>Colluvium of pisolithic material (Pandanus valley)<br>S 21° 01' 58.80"; E 164° 46' 40.97" (alt 31 m)</td><td>Brown clayey soil with colluvium<br>pisolithic cover<br>Abruptic ferralsol</td></tr><tr><td rowspan="5">Volcano-sedimentary</td><td>POUM MB</td><td>Golone peninsula, Poum Malabou<br>S 20° 17' 32.56"; E 164° 05' 22.60" (alt 48 m)</td><td>Brown gravel-rich soil<br>Leptic cambisol</td></tr><tr><td>POUM SI</td><td>Podzols beach (Poum)<br>S 20° 13' 38.50"; E 164° 04' 44.52" (alt 14 m)</td><td>Silicic bleached soil<br>Geric acrisol (Albic)</td></tr><tr><td>VERT</td><td>Black soils plain on basalts, Pouembout<br>S 21° 07' 59.59"; E 164° 54' 55.64" (alt 16 m)</td><td>Black yey soil<br>Haplic vertisol</td></tr><tr><td>SERP</td><td>Serpentinic basement of the Tia plateau ultramafic regolith<br>S 21° 10' 27.74"; E 164° 52' 40.47" (alt 75 m)</td><td>Thin silty grey-brown soil (can be gravel-rich)<br>Haplic cambisol</td></tr></tbody></table>

alt altitude, WRB world reference base for soil resources [16]

primers ITS1F: 5'-TTTCCGTAGGTGAACCT-3' [16] and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' [18]. The PCR reaction used for this amplification was prepared in a total volume of 25 µL containing a 1 µL aliquot of genomic DNA diluted to 1/20; 1 µM of each primer; 1.5 units of Taq DNA polymerase (Promega, Charbonnières, France); 5 µL of 5X Promega Taq polymerase buffer; 2 mM of MgCl₂ and 200 µM of dNTP Promega. Amplification was performed with a Mastercycler® thermocycler Eppendorf (Eppendorf, Hamburg, Germany) programmed as follows: 1 cycle of 3 min at 94 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 55 °C, 1 min at 72 °C and a final extension of 10 min at 72 °C. PCR products were checked by electrophoresis on a 1% agarose gel (Sigma) in 0.5% TBE buffer with 2 µL ethidium bromide (BET, 10 µg/mL for 100 mL gel). A size marker 1000 pb (Promega) was used to estimate the weight of the different fragments. The DNA bands were then visualized by fluorescence under UV light and photographed. Samples with simple and well-defined bands were then sequenced with primers ITS4 and ITS1F by the Macrogen Company (Macrogen, Seoul, Korea) using a genetic analysis based on capillary electrophoresis ABI3730.

## Data and Statistical Analyses

The molecular data were analysed as follow: the ITS1F and ITS4 sequences were analysed and assembled into contigs with the Sequencher® software (Genes code Corporation,

Ann Harbor, Michigan, USA). The algorithm ITSx [20, 21] was used to remove the parts of 18S and 28S regions and keep the ITS highly variable sub-regions and analyses were carried out using PlutoF Unite [22]. The total DNA sequences were assigned to operational taxonomic units (OTU) using a 97% sequence similarity cutoff homology of the ITS sequences, as reported in Smith et al. [23] by using the software CD-Hit Est® [21]. The fungal phylogenetic lineages were assigned using the MassBLASTer algorithm and then identified in reference to the PlutoF Unite fungal database [22] following the fungal nomenclature reported by Tedersoo et al. [24].

Then, to investigate the richness and the diversity of ECM communities associated with *A. spirorbis*, we standardized the samples using rarefaction curves and extrapolation methods as previously reported in the Chao et al. method [25]. Rarefaction provides a method of comparison between different communities, whereby each community is “rarefied” back to an equal number of sampled specimens. Extrapolation methods are useful to validate any convergence between the observed and estimated diversity [25]. The level of diversity for each site was evaluated by calculation of the observed Shannon *H'* and Simpson *D* indexes based on the sample coverage of each soil category as reported in ESM Fig. 1 and Table 4 [27]. In addition, the estimated and asymptotic richness index ⁰D, which counts species equally without regarding their relative abundance, Shannon diversity index ¹D, which counts species in proportion to their abundance, and the
<table><caption>**Table 2** Geochemical characteristics of the bulk soils. All data are means ± stdv of element content measured in soil samples gathering data of soil sites belonging to a same soil category. For pH and each soil element, a non-parametric test of Kruskal-Wallis was applied to</caption><thead><tr><th rowspan="2">Geochemical characteristics</th><th rowspan="2"></th><th colspan="3">Soil type according to the geochemical soil analyses</th><th rowspan="2">Kruskall wallis<br>Statistic test<br>p value</th></tr><tr><th>Calcareous</th><th>Ferralitic</th><th>Volcano-sedimentary</th></tr></thead><tbody><tr><td>pH</td><td></td><td>7.6 ± 0.8 a</td><td>6.1 ± 0.5 b</td><td>5.7 ± 1.1 b</td><td>&lt; 0.001</td></tr><tr><td rowspan="10">Total element soil<br>contents (mg/kg)</td><td>C</td><td>7420 ± 3521 a</td><td>2322 ± 2004 b</td><td>2858 ± 2627 b</td><td>0.007362</td></tr><tr><td>N</td><td>5500 ± 4845 a</td><td>999 ± 780 b</td><td>1006 ± 923 b</td><td>0.003292</td></tr><tr><td>P</td><td>6471 ± 2662 a</td><td>81 ± 59 b</td><td>168 ± 180 b</td><td>&lt; 0.001</td></tr><tr><td>K</td><td>267 ± 124</td><td>336 ± 469</td><td>2807 ± 3222</td><td>0.459</td></tr><tr><td>Ca</td><td>78,585 ± 81,909 a</td><td>1443 ± 1968 b</td><td>13,035 ± 20,923 b</td><td>&lt; 0.001</td></tr><tr><td>Mg</td><td>23,234 ± 34,731</td><td>29,087 ± 41,762</td><td>21,199 ± 20,005</td><td>0.8723</td></tr><tr><td>Na</td><td>585 ± 475 b</td><td>352 ± 194 b</td><td>4017 ± 4825 a</td><td>0.02781</td></tr><tr><td>Al</td><td>141,574 ± 75,691 a</td><td>29,079 ± 19,879 b</td><td>49,952 ± 37,457 b</td><td>0.004413</td></tr><tr><td>Fe</td><td>109,749 ± 58,963 b</td><td>317,453 ± 137,596 a</td><td>46,231 ± 35,582 c</td><td>&lt; 0.001</td></tr><tr><td>Co</td><td>43 ± 26 b</td><td>608 ± 295 a</td><td>45 ± 39 b</td><td>&lt; 0.001</td></tr><tr><td>Cr</td><td>570 ± 233 b</td><td>20,833 ± 9919 a</td><td>569 ± 581 b</td><td>&lt; 0.001</td></tr><tr><td>Mn</td><td>3103 ± 1768 a</td><td>7714 ± 6942 a</td><td>668 ± 516 b</td><td>&lt; 0.001</td></tr><tr><td>Ni</td><td>44 ± 20 b</td><td>5653 ± 2783 a</td><td>524 ± 772 b</td><td>&lt; 0.001</td></tr><tr><td>Si</td><td>13,420 ± 10,698 c</td><td>100,194 ± 57,151 b</td><td>301,606 ± 97,483 a</td><td>&lt; 0.001</td></tr></tbody></table>

detect differences between soil types of which *p* value is reported. When Kruskal-Wallis *p* value test was < 0.05, a pairwise Wilcoxon test was used to assess the different levels of significance that letters a, b or c indicate at *p* < 0.05

Simpson diversity index $^2$D, which discounts all but the dominant species in the assemblage, were calculated using the *R* package of iNEXT [26]. To test the sampling method, the observed and estimated richness sample completeness curves were constructed using the iNEXT online software (http://chao.stat.nthu.edu.tw/wordpress/software_download/; [25]).

A chi-square independence test and dissimilarity Jaccard indices was applied to compare the distribution of ECM lineages between soil types. Correlations between the diversity of ECM lineages distribution among soils and their edaphic

parameters were calculated ranking the geochemical characteristics of the soils into a Euclidian matrix. The distribution of OTUs per site reflecting the diversity of ECM communities was ranking into a Bray-Curtis dissimilarity matrix using abundance data [28]. A non-parametric Mantel test was then performed in order to check potential correlations between the Bray-Curtis and the Euclidian matrices. The combination of both data (i) the ECM lineages and (ii) the edaphic parameters, all according to sites sampling and type of soils, is represented with a non-metric multidimensional scaling (NMDS) analysis.

<table><caption>**Table 3** Summary of ECM data collected on roots of *A. spirorbis* in the 12 studied sites: number of ECM collected, ITS DNA sequences obtained with their accession number as deposited in DDJB bank, the</caption><thead><tr><th rowspan="2">Soil geochemical<br>category</th><th rowspan="2">Site name</th><th rowspan="2">Total number of<br>ECM root tips<br>sampled</th><th rowspan="2">Number of<br>DNA ITS<br>sequences<br>obtained</th><th rowspan="2">ITS DNA sequence<br>accession<br>number in DDJB<br>bank</th><th rowspan="2">Number of<br>OTU at 97%<br>represented</th><th colspan="2">Diversity indexes</th></tr><tr><th colspan="2">Observed</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th>Shannon<br>H'</th><th>Simpson<br>D</th></tr></thead><tbody><tr><td rowspan="5">Calcareous</td><td>EAS</td><td>49</td><td>25</td><td>LC122057 to LC122081</td><td>11</td><td>2.20</td><td>0.09</td></tr><tr><td>MUC</td><td>18</td><td>5</td><td>LC122082 to LC122086</td><td>4</td><td>1.33</td><td>0.10</td></tr><tr><td>WAN</td><td>43</td><td>19</td><td>LC122241 to LC122259</td><td>8</td><td>1.84</td><td>0.15</td></tr><tr><td>WNK</td><td>18</td><td>5</td><td>LC122260 to LC122264</td><td>4</td><td>1.33</td><td>0.10</td></tr><tr><td>TIE</td><td>62</td><td>12</td><td>LC122200 to LC122211</td><td>8</td><td>2.27</td><td>0.06</td></tr><tr><td rowspan="5">Ferralitic</td><td>PIND</td><td>33</td><td>16</td><td>LC122116 to LC122131</td><td>11</td><td>2.45</td><td>0.11</td></tr><tr><td>PAND-1</td><td>21</td><td>13</td><td>LC122087 to LC122099</td><td>7</td><td>2.35</td><td>0.03</td></tr><tr><td>PAND-2</td><td>48</td><td>16</td><td>LC122100 to LC122115</td><td>13</td><td>1.77</td><td>0.17</td></tr><tr><td>POUM MB</td><td>77</td><td>41</td><td>LC122132 to LC122172</td><td>18</td><td>1.35</td><td>0.24</td></tr><tr><td>POUM SI</td><td>46</td><td>12</td><td>LC122173 to LC122184</td><td>5</td><td>2.15</td><td>0.11</td></tr><tr><td rowspan="4">Volcano-sedimentary</td><td>VERT</td><td>55</td><td>29</td><td>LC122212 to LC122240</td><td>11</td><td>1.92</td><td>0.11</td></tr><tr><td>SERP</td><td>38</td><td>15</td><td>LC122185 to LC122199</td><td>8</td><td>1.94</td><td>0.09</td></tr><tr><td>Global<br>sampling</td><td>488</td><td>208</td><td></td><td>108</td><td>2.15</td><td>0.11</td></tr></tbody></table>

corresponding number of OTU generated and the observed Shannon *H'* and Simpson *D* diversity indexes per site

*ITS* internal transcribed spacer, *OTU* operational taxonomic unit, *DDJB* DNA Data Bank of Japan
Permanova tests were carried out with the ADONIS function included in the Vegan R package to test the influence of the edaphic parameters on the ECM distribution. As the statistical analyses based on the Bray-Curtis matrices generated by the dataset of OTU with and without singletons were very similar, only non-singleton OTUs were used in the NMDS analyses because the dataset may be influenced by low sampling that can yield misleading inferences about composition. All statistical analyses were carried out using the packages Vegan 2.0-6, RVAideMemoire 0.9-54 and Inext R, all in R 2.15.1 software [29].

# Results

## Soil Properties

The geochemical characteristics per soil category (i.e. calcareous, ferralitic and volcano-sedimentary soils) are summarized in Table 2 and the whole data per site are reported in ESM Tables 1A–C. For pH and most of the element soil contents, the non-parametric test of Kruskal-Wallis showed significant differences between soil types ($p < 0.05$) as reported in Table 2. The results of a principal component analysis (PCA) of the soil geochemical characteristics (including total element contents and pH) are shown in Fig. 2. These results confirmed that the selected sites could be distributed into three main groups according to the soil geochemical characteristics: sites EAS, MUC, WAN and WNK can be grouped as calcareous soils (CA soils); sites PAND-1, PAND-2, PIND and TIE can be grouped as ferralitic soils (FE soils) and sites POUM SI, POUM MB, SERP and VERT can be grouped as volcanosedimentary soils (VS soils). The results of the statistical analyses (Table 2) indicated that CA soils were mainly characterized by significantly high levels of major plant nutrients (i.e. N and P) and also a high Ca/Mg ratio (3:1), while FE and VS soils had low contents in C, N and P and presented a strongly unbalanced Ca/Mg ratio (1:1.6 in VS soils to 1:20 in Fe soils). VS-soils were characterized by significantly high contents of Na and Si. Regarding potentially toxic elements, CA-soils were characterized by significantly high contents of Al, while VS soils presented quite low levels of Fe and Mn. Finally, FE soils differed significantly from the two other categories of soils, with very high Fe content and also high contents in potentially toxic heavy metals: Co, Cr, Mn and Ni.

## Diversity and Richness of ECM Fungi Communities Associated with A. spirorbis

All the ITS DNA sequences obtained in this study were deposited in the DNA Data Bank of Japan (DDJB) from the accession numbers LC122057 to LC122264. Summary data concerning ITS sequences distribution in the studied sites are

presented in Table 3. Full data presenting DDJB sequence accession numbers, OTU and Blast results with both genus and lineage identifications are available in ESM Table 2. A total of 488 mycorrhizal root tips were collected from 60 A. spirorbis trees distributed throughout 12 selected sites and three soil categories (Table 3). Successful ITS sequences were obtained from 208 ECM root tips and grouped into 76 different identified OTUs (including 41 singletons) corresponding to fungal lineages identified by Blast (ESM Table 2). Our results indicated that the majority of ECM taxa found in association with A. spirorbis belonged to the Basidiomycota division (92% of the total number of OTUs). The relative abundance of the 208 sequences according to the 76 identified OTUs and their distribution into 15 lineages, as presented in Fig. 3, showed that the most representative Basidiomycota ECM taxa found belonged to the /tomentella-thelephora (27.6%), /boletus (15.8%), /sebacina (10.5%), /russulalactarius (10.5%) and /pisolithus-scleroderma (7.9%) lineages. The rarefaction curves in ESM Fig. 1 build with observed and estimated sample coverage validates the quality of our sampling. For calcareous and volcano-sedimentary sites, the sample coverage reach approximately 90 and 80% of sample coverage observed, and for the ferralitic sites, approximately 55%. The estimated sample coverage indicates that by doubling this sampling, the sample coverage for calcareous and ferralitic sites change by less than 5% and for ferralitic sites the sample coverage reaches 60%.

## Relationship between ECM Communities and Edaphic Conditions of Soil Categories

The distribution of OTUs into lineages according to the three soil categories yielded contrasted features (Fig. 4). In CA soils, three Basidiomycota ECM lineages reached 96.2% of the total number of OTUs identified, namely /sebacina (41%), /tomentella-thelephora (37%) and /inocybe (18%). In FE soils, 11 Basidiomycota ECM lineages amounted to 92.4% of the OTUs, the most represented ones being /botelus (25%), /pisolithus-scleroderma (17.9%), /russula-lactarius (12.8%), /tomentella-thelephora (10.2%) and /cortinarius (7.7%). In VS soils, eight Basidiomycota ECM lineages reached 88.1% of the OTUs, mostly represented by /tomentella-thelephora (31%), /botelus (16.7%), /pisolithus-scleroderma (14.3%) and /russula-lactarius (14.3%). The distribution of the OTUs relative abundance according to both fungal lineages and soil categories is reported in ESM Fig. 2. The chi square independence statistical test confirmed that the lineages distribution found in CA soils was significantly different from those found in FE soils (chi square = 74.3, df = 28, $p$ value = 4.5e-06 < 0.05) and in VS soils (chi square = 47.2, df = 13, $p$ value = 9.0e-06 < 0.05). However, no significant differences were observed in the lineages distribution found in FE and VS soils (chi square = 11.2, df = 12, $p$ value =
<table><caption>**Table 4** Data summary of samples (sequences and OTUs), sample coverage, observed and asymptotic estimated indexes of diversity of the OTUs distribution according to the soil category based on “iNext” algorithm R Package as reported by Hsieh et al. [26]</caption><thead><tr><th rowspan="3">Soil category</th><th rowspan="3"></th><th colspan="3">Samples</th><th colspan="6">Diversity indexes</th></tr><tr><th colspan="2">Sequence number</th><th rowspan="2">Sample* coverage %</th><th colspan="2">Richness <sup>0</sup>D</th><th colspan="2">Shannon (<sup>1</sup>D)</th><th colspan="2">Simpson (<sup>2</sup>D)</th></tr><tr><th>CA</th><th>FE</th><th>Observed**</th><th>Estimated</th><th>Observed</th><th>Estimated</th><th>Observed</th><th>Estimated</th></tr></thead><tbody><tr><td></td><td>CA</td><td>54</td><td>27</td><td>90</td><td>18</td><td>22.1 ± 4.8</td><td>2.71</td><td>2.92 ± 0.12</td><td>0.923</td><td>0.941 ± 0.012</td></tr><tr><td></td><td>FE</td><td>57</td><td>39</td><td>55</td><td>33</td><td>225.6 ± 155</td><td>3.04</td><td>4.21 ± 0.45</td><td>0.917</td><td>0.934 ± 0.024</td></tr><tr><td></td><td>VS</td><td>97</td><td>42</td><td>80</td><td>36</td><td>90.6 ± 37</td><td>3.14</td><td>3.57 ± 0.19</td><td>0.937</td><td>0.946 ± 0.011</td></tr></tbody></table>

CA calcareous, FE ferralitic, VS volcano-sedimentary

*Sample coverage was estimated according to Chao et al. method [27] and as reported in ESM Fig. 1

**Un-repeated OTUs

0.5139 > 0.05). These results are reinforced by the Jaccard indices of dissimilarity indicating CA soil lineages distribution is more different of the other soils types, with, respectively, values 0.918 and 0.810 for CA-soils compared to FE and VS soils, and 0.579 for FE soils compared to VS soils. To evaluate the diversity of ECM fungi colonizing *A. spirorbis*, the observed and estimated richness of Shannon and Simpson indices were calculated (Table 4). The results showed that the observed species richness <sup>0</sup>D differed from the asymptotic estimated richness for both FE and VS soils. The observed and estimated Shannon <sup>1</sup>D converged to similar values for the ECM distribution found in CA and VS soils (averaged variation of 5% between observed and estimated indexes), while a gap of 27% difference could be noticed between observed and estimated indexes in FE soils. Finally, the Simpson <sup>2</sup>D for the ECM distribution found in the three categories of

soils showed close values between observed and estimated indexes with only 3.7% differences in mean. A Mantel test between the Bray-Curtis matrix of the ECM diversity and the Euclidian matrix of the soil properties (i.e. pH, Corg/Norg ratio, total element contents as reported in ESM Table 1A-C) showed a significant correlation between these two parameters (at *p* < 0.01) as reported in ESM Table 3. These positive results allowed to perform a Permanova analysis showing multiple significant correlations (*p* < 0.05) between the edaphic parameters as pH, the elements content in soils (i.e. Ca, P, Al and Co) and the ECM OTUs distribution (ESM Table 4). In addition, no significant differences could be observed when considering OTU data with or without singletons (ESM Table 4). Consequently, only non-singleton OTUs were used in a non-metric multidimensional scaling (NMDS) analysis in order to avoid misleading inferences about composition that

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Principal component analysis of the pH, the C/N ratio and the total element contents in the studied soils. Confidence ellipses (95%, R package) group sites into soils categories, i.e. CA = calcareous soils; FE = ferralitic soils and VS = volcano-sedimentary soils</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Distribution of the OTUs relative abundance (%) according to fungal lineages [24]</figcaption></figure>

could have erased from influence on the dataset by low sampling. The NMDS results showed that the three soil categories harboured different fungal communities although those of ferralitic and volcano-sedimentary soil types shared the most abundant lineages, in particular /tomentella-thelephora, and at a lesser extent /boletus /pisolithus-scleroderma and /russula-lactarius (Fig. 5). By contrast, the calcareous soils predominantly harboured the /sebacina and /inocybe lineages as well as /tomentella-thelephora which was the most ubiquitous and abundant ECM lineage found in the three soil types (Fig. 5). In addition, as reported in ESM Table 4, the Permanova results indicated that the variation of the ECM diversity in the study sites was significantly explained by major edaphic parameters as pH (31.4%), soil contents of P (45.5%), Ca (28.4), Al (29.5%) and Co (26.1%).

## Discussion

The originality of this study is to bring (i) new data about ectomycorrhizal diversity of a legume from tropical southern hemisphere and (ii) to focus on one endemic *Acacia* that colonized a tropical island [4] and which is able to grow on a large and contrasted variety of soils.

## Ectomycorrhizal Status and Global Diversity of the ECM Community Associated with A. spirorbis

Australian phyllodinous acacias have been described for their ability to form symbiotic associations with ECM fungi [30]. The present study confirms the ECM status of *A. spirorbis* that belongs to the phyllodinous acacias [4] and it reports the existence of 76 fungal OTUs grouped into 15 ectomycorrhizal lineages. Among the total of OTUs, 92% belong to the Basidiomycota. The dominant ECM lineages of

Basidiomycota found were /tomentella-thelephora, /boletus, /sebacina, /russula-lactarius and /pisolithus-scleroderma. Compared to other previous studies about the diversity of ECM fungi associated with native or introduced plant species [23, 31, 32], the results of the present study indicate that the diversity of ECM fungi associated with *A. spirorbis* in New Caledonia is similar to the native range of ECM from other plant species. Indeed Smith et al. [31] listed 161 ectomycorrhizal species associated with native *Quercus douglasii* in California. Ducoussou et al. [32] reported 38 ectomycorrhizal species associated with the introduced *Eucalyptus robusta* in Madagascar. The recent study by Smith et al. [23] on the ectomycorrhizal community of *Pakaraimaea dipterocarpacea* and *Dicymbe jenmanii* in a neotropical forest of Guyana listed 52 OTUs distributed into 11 ectomycorrhizal groups based on 255 sequences analysed. Considering the lack of data about the diversity of ECM communities associated with acacias, this study brings then original data about *A. spirorbis* to form symbiotic associations particularly with a large spectrum of ECM fungi on different edaphic constraints.

## Diversity of the Ectomycorrhizal Communities as a Function of the Soil Category

Regarding the ECM diversity and the lineages richness associated with *A. spirorbis*, our results indicate that Basidiomycota are dominant whatever the type of soil. Concerning the diversity of the ECM communities associated with *A. spirorbis* as a function of the soil category our data showed that there is no significant difference between the ECM communities from ferralitic (FE) and volcano-sedimentary (VS) soils. The main common lineages for these both type of soil are /botelus, /tomentella-thelephora, /pisolithus-scleroderma and /russula-lactarius. These results
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Distribution of the lineages according the three categories of soils: CA = calcareous; FE = ferralitic; VS = volcano-sedimentary</figcaption></figure>

are in agreement with previous results reported by Moser et al. [10] that showed no significant difference between the ECM communities associated with *Quercus* spp. grown on serpentinic (ferralitic) and non-serpentinic soils. Moreover, our results (i.e. 12 lineages in FE soils compared to 10 lineages in VS soils) confirm that, despite their large contents in potentially toxic metals, low contents in nutrients (P, K) and strongly unbalanced Ca/Mg ratio, FE soils do not limit the diversity of the ectomycorrhizal community associated with *A. spirorbis*. Such a conclusion is in agreement with the results already reported for serpentinic (ferralitic) soils by Branco [11] and Branco and Ree [12]. These results are in agreement with those from previous studies focused on the ectomycorrhizal status and ECM fungal species richness associated with other dominant ECM plants in New Caledonia

such as *Melaleuca* spp., *Nothofagus* spp., *Tristaniopsis* spp. and *Sannantha* spp. [8, 9, 33, 34]. Moreover, when *A. spirorbis* grow in association with *Melaleuca* spp. or *Tristaniopsis* spp., it shares the same ECM lineages such as /tomentella-thelephora, /russula-lactarius and /pisolithus-scleroderma [8, 9, 33, 34]. Regarding the presence of /cortinarius lineage only found in FE soils, this result is in agreement with the recurring occurrence of *Cortinariaceae* as ectomycorrhizal fungi reported for plants such as *Tristaniopsis* spp. and *Nothofagus* spp. only growing on soils developed upon ultramafic rocks that are equivalent to our FE soils [33, 34]. On the opposite, the diversity of ECM communities associated with *A. spirorbis* on CA soils is very low and significantly different from those found on FE and VS soils; this low diversity may be linked to high soil fertility (e.g. [35, 36]). Notwithstanding, it is important to consider that CA soils were sampled in one of the Loyalty Islands, Lifou that emerged during the Miocene, while the soils of the main island developed on bedrocks aged of 35 MA [1]. So, the low ECM fungal diversity associated with *A. spirorbis* on CA soils may result as a weak fungal colonisation of these soils due to the island isolation. This would be in agreement with the island biogeographical theory regarding dynamics of fungi species: the fungal island colonisation is mainly driven by spore dispersion but plant host colonisation is limited by distance and so impacts the ECM richness [37]. On such isolated CA soils, the results showed also the dominance of Basidiomycota as observed in UF and VS soils but only four lineages were found in association with *A. spirorbis* dominated by /thelephora-tomentella and /sebacina lineages. The presence of /thelephora-tomentella lineages in these CA soils is in agreement with the results of previous studies that reported the occurrence of *Tomentella* species as ECM symbionts for plants growing on coral reef island [38]. Moreover and not surprisingly the /thelephora-tomentella lineage is dominant: such lineage is known to host ubiquitous taxa (e.g. [39]) that are common and dominant ECM fungi, colonizing a large spectrum of diverse environments, especially in primary successional habitats [40, 41]. Altogether, our results about the diversity of the ECM communities associated with *A. spirorbis* in the three studied soil categories indicated that this legume has the capacity to develop symbiotic associations with a large spectrum of ECM fungi regardless the contrasted edaphic parameters.

## Ectomycorrhizal Diversity in Relation with Edaphic Parameters

The diversity of the ECM communities associated with *A. spirorbis* showed significant correlations with some major edaphic parameters for each of the three soil categories (Fig. 4). First, the distribution of the ECM diversity between sites is correlated with soil pH, the highest diversity being
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Two-dimensional NMDS ordination of the OTU distribution (without singletons) as fungal lineage patterns according to the sampling sites and the geochemical soil parameters including the pH, the C/N ratio and the total element contents. Blue squares represent OTUs grouped into lineages according Tedersoo et al. [24], numbers are lineage code. Red stars represent the 12 sites (Table 1) with a specific position in the graph according to their fungal lineages. Edaphic parameters are positioned with arrow according to the Permanova calculation, and asterisks indicate the level of significant</figcaption></figure>

found in the most acidic soils (i.e. pH = 5.7 ± 1.1 for VS soils and pH = 6.1 ± 0.5 for FE soils compared to pH = 7.6 ± 0.8 for CA soils). This result is in agreement with previous results of a larger-scale study on the description of forest fungal diversity, which showed a negative correlation between the fungal richness and the number of OTUs for most fungal taxonomic groups and the increase in soil pH [42]. Second, in our study, the diversity of the ECM communities was found to negatively correlate with the Ca and P contents of the selected soils, with a larger diversity of the ECM communities in the soils with the lowest Ca and P contents (i.e. VS and FE soils). Such a negative correlation between the P contents of the soil and the ECM diversity might be explained by the fact that the limitation of phosphorous in soils is known to promote the growth of ECM fungi since a major role of ECM fungi is to provide P to their host plants [43, 44]. Similarly, the negative correlation between the Ca contents of the soil and the ECM diversity might be explained by the fact that a low Ca content in soil is known to enhance the occurrence and diversity of ECM fungi [45, 46], mechanisms supporting these enhancement are still unknown. Altogether, our results are in agreement with the recent hypothesis that major edaphic parameters such as Ca, P and pH play a key role in structuring the soil fungal diversity at the global scale [15]. Moreover, our results

relationship with the lineage distribution (*p* < 0.05; **p** < 0.01). Confidence ellipses (95%, R package) group lineages according soil category i.e. calcareous (CA), ferralitic (FE) and volcano-sedimentary (VS). Lineages codes: 1 /tomentella-thelephora; 2 /boletus; 3 /pisolithus-scleroderma; 4 /unspecified Boletaceae; 5 /russula-lactarius; 6 /cortinarius; 7 /inocybe; 8 /amanita; 9 /tricholoma; 10 /unspecified Agaricales; 11 /sebacina; 12 / clavulina; 13 /peziza; 14 /unspecified Ascomycota; 15 /dothideomycetes incertae sedis

also show some significant correlations between the diversity of the ECM communities and the contents in potentially toxic elements in the soils, as already reported by De Beeck et al. [47]. Indeed, in FE soils, the diversity of the ECM community is positively correlated with the Co content, while in CA soil, it is negatively correlated with the Al content. Such correlations between (i) edaphic parameters and (ii) the ECM communities associated with *A. spirorbis* suggest that ECM fungi might play a significant role in the adaptation of the plant to the various soils as already demonstrated when the ECM fungus *P. albus* was inoculated on *A. spirorbis* grown on an artificial substrate similar to Fe soils [8].

**Acknowledgments** This work was conducted as part of an industrial agreement through the French program of PhD training research CIFRE with grant number N° 2013/1434 involving the industrial company Koniambo Nickel SAS. In addition, this work was supported by the French National Agency for Research (ANR) financial support through the BIOADAPT Program 2012 [grant number ANR-12-ADAP-0017 ADASPIR].

## Compliance with Ethical Standards

**Conflict of Interest** The authors declare that they have no conflict of interest.
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