Loïc Yung
email: loic.yung@univ-lorraine.fr

Coralie Bertheau

Flavien Tafforeau

Cyril Zappelini

Benoit Valot

François Maillard

Marc-André Selosse

Chloé Viotti

Philippe Binet

Geneviève Chiapusio

Michel Chalot

Partial overlap of fungal communities associated with nettle and poplar roots when co-occurring at a trace metal contaminated site

Keywords: Ectomycorrhizal fungi, Environmental metabarcoding, Fungal network, Metal-enriched sediments, Phytomanagement, Root-associated mycobiome, Urtica dioica L

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Phytomanagement is a remediation technique based on plant species to restore and revalorize polluted sites while producing biomass [START_REF] Cundy | Brownfields to green fields: Realising wider benefits from practical contaminant phytomanagement strategies[END_REF][START_REF] Evangelou | Phytomanagement: Phytoremediation and the Production of Biomass for Economic Revenue on Contaminated Land[END_REF].

Fast-growing trees such as poplars (Populus sp.) allow the production of a high amount of valuable plant biomass for renewable energy [START_REF] Kidd | Agronomic Practices for Improving Gentle Remediation of Trace Element-Contaminated Soils[END_REF][START_REF] Pandey | Energy crops in sustainable phytoremediation[END_REF]. At the same time, trees limit the dispersal and risks of soil contaminants, while promoting the spontaneous biodiversity [START_REF] Parraga-Aguado | Usefulness of pioneer vegetation for the phytomanagement of metal(loid)s enriched tailings: Grasses vs. shrubs vs. trees[END_REF]. Recent approaches on the optimisation of phytomanagement systems pointed out the importance of assemblages between crops and spontaneous species [START_REF] Parraga-Aguado | The importance of edaphic niches and pioneer plant species succession for the phytomanagement of mine tailings[END_REF] to ensure a more efficient and gentle remediation [START_REF] Boisson | Potential of copper-tolerant grasses to implement phytostabilisation strategies on polluted soils in South D. R. Congo: Poaceae candidates for phytostabilisation[END_REF]. The spontaneous vegetation biodiversity is generally restricted by the soil physico-chemical properties, limiting the panel of species able to develop and compete with the tolerant species [START_REF] Macnair | Heavy metal tolerance in plants: A model evolutionary system[END_REF]. Much less is known on how this plant biodiversity is shaping its root microbiota in this context.

The stinging nettle (Urtica dioica L.) colonizes a vast diversity of nitrophilic environments and particularly anthropogenic areas [START_REF] Balabanova | Bioavailability and bioaccumulation characterization of essential and heavy metals contents in R. acetosa, S. oleracea and U. dioica from copper polluted and referent areas[END_REF][START_REF] Shams | Aspects of phytoremediation for chromium contaminated sites using common plants Urtica dioica, Brassica napus and Zea mays[END_REF][START_REF] Viktorova | Native Phytoremediation Potential of Urtica dioica for Removal of PCBs and Heavy Metals Can Be Improved by Genetic Manipulations Using Constitutive CaMV 35S Promoter[END_REF]. It has thus been frequently observed as a companion species in a wide range of nitrogen-rich environments such as Salicaceous plantations at both natural [START_REF] Cronk | Salix transect of Europe: variation in ploidy and genome size in willow-associated common nettle, Urtica dioica L. sens. lat., from Greece to arctic Norway[END_REF] and anthropized sites [START_REF] Yung | Insect Life Traits Are Key Factors in Mercury Accumulation and Transfer within the Terrestrial Food Web[END_REF]. Nettle has recently raised growing interest in the domain of phytomanagement because it (i) constitutes a new resource of high-quality herbaceous fibres for the manufacture of biomaterials [START_REF] Bacci | Fiber yield and quality of fiber nettle (Urtica dioica L.) cultivated in Italy[END_REF][START_REF] Virgilio | The potential of stinging nettle (Urtica dioica L.) as a crop with multiple uses[END_REF][START_REF] Jeannin | Native stinging nettle (Urtica dioica L.) growing spontaneously under short rotation coppice for phytomanagement of trace element contaminated soils: Fibre yield, processability and quality[END_REF][START_REF] Jeannin | Are nettle fibers produced on metal-contaminated lands suitable for composite applications?[END_REF], (ii) promotes the biodiversity of local entomofauna [START_REF] James | Beneficial insects associated with stinging nettle, Urtica dioica Linnaeus, in central Washington State[END_REF][START_REF] Perrin | The role of the perennial stinging nettle, Urtica dioica, as a reservoir of beneficial natural enemies[END_REF][START_REF] Yung | Insect Life Traits Are Key Factors in Mercury Accumulation and Transfer within the Terrestrial Food Web[END_REF] and (iii) spontaneously grows in contaminated sites [START_REF] Murtic | Assessment of wild plants for phytoremediation of heavy metals in soils surrounding the thermal power station[END_REF][START_REF] Paukszto | Using stinging nettle (Urtica dioica L.) to assess the influence of long term emission upon pollution with metals of the Tatra National Park area (Poland)[END_REF][START_REF] Viktorova | Native Phytoremediation Potential of Urtica dioica for Removal of PCBs and Heavy Metals Can Be Improved by Genetic Manipulations Using Constitutive CaMV 35S Promoter[END_REF].

The success of phytomanagement depends on the capacity of plants to colonize harsh environments, which is partly related to their ability to interact with soil microorganisms, and especially to establish symbiotic relationships with some of them. Among these microorganisms, arbuscular mycorrhizal (AM) [START_REF] Miransari | Arbuscular Mycorrhizal Fungi and Heavy Metal Tolerance in Plants[END_REF], ectomycorrhizal (EM) [START_REF] Gil-Martínez | Ectomycorrhizal Fungal Communities and Their Functional Traits Mediate Plant-Soil Interactions in Trace Element Contaminated Soils[END_REF] fungi and Dark Septate Endophytes (DSE) [START_REF] Deng | Fungal endophytes and their interactions with plants in phytoremediation: A review[END_REF] are major drivers of plant growth and response to abiotic stress and phytopathogens [START_REF] Otero-Blanca | Phytoremediation and Fungi: An Underexplored Binomial[END_REF][START_REF] Selosse | Symbiotic microorganisms, a key for ecological success and protection of plants[END_REF]. The use of fungal inoculum has been described as a phytoremediation enhancer by improving uptake and accumulation of trace metals (TM) by plants in phytoextraction application or by limiting the translocation of TM from roots to shoots during phytostabilisation (Berthelot et al., 2016a;[START_REF] Khan | Role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation[END_REF][START_REF] Ma | Potential of plant beneficial bacteria and arbuscular mycorrhizal fungi in phytoremediation of metal-contaminated saline soils[END_REF]Pepper et al., 2015). While the success of field inoculation is rather unpredictable [START_REF] Gerhardt | Opinion: Taking phytoremediation from proven technology to accepted practice[END_REF][START_REF] Selosse | Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation[END_REF][START_REF] Selosse | Structure and Dynamics of Experimentally Introduced and Naturally Occurring Laccaria sp. Discrete Genotypes in a Douglas Fir Plantation[END_REF] due to various abiotic (e.g., sensitivity to TM) and biotic parameters (e.g., competition with the local microbiome), the risks of failure may be mitigated by considering indigenous strains, which may have developed a tolerance for TM [START_REF] Wubs | Soil inoculation steers restoration of terrestrial ecosystems[END_REF]. Characterisation of in-situ plant-associated microbiome appears as an essential preliminary step for identifying rhizospheric microorganisms that may actively participate to phytomanagement processes.

In the temperate climate zone, poplars are among the few cultivated trees that form tripartite symbiotic associations with EM and AM fungi [START_REF] Gehring | Environmental and genetic effects on the formation of ectomycorrhizal and arbuscular mycorrhizal associations in cottonwoods[END_REF][START_REF] Teste | Dual-mycorrhizal plants: their ecology and relevance[END_REF]. As symbiotic fungi play crucial roles in soil fertility, colonisation, plant nutrition, metal uptake, accumulation or restriction in plants [START_REF] Smith | Mycorrhizal Symbiosis[END_REF][START_REF] Van Der Heijden | Mycorrhizal ecology and evolution: the past, the present, and the future[END_REF], poplar microbiome have been well studied in phytomanagement contexts [START_REF] Durand | Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site[END_REF]Foulon et al., 2016b;[START_REF] Schmidt | Distinct Communities of Poplar Endophytes on an Unpolluted and a Risk Element-Polluted Site and Their Plant Growth-Promoting Potential In Vitro[END_REF][START_REF] Vitali | Environmental pollution effects on plant microbiota: the case study of poplar bacterial-fungal response to silver nanoparticles[END_REF][START_REF] Zappelini | Diversity and complexity of microbial communities from a chlor-alkali tailings dump[END_REF].

Recent studies highlighted the positive effects of EM and AM inoculation on poplar cultivars growing at a multi-contaminated site [START_REF] Ciadamidaro | Poplar biomass production at phytomanagement sites is significantly enhanced by mycorrhizal inoculation[END_REF][START_REF] Phanthavongsa | Effect of mycorrhizal inoculation on metal accumulation by poplar leaves at phytomanaged sites[END_REF].

However, mycobiome associated with nettle have been barely studied in natural or anthropogenic biota. A study combining in-situ and mesocosm approaches concluded that the nettle rhizosphere was devoid of any mycorrhizal structures and revealed that the AM fungus Glomus mosseae was not able to develop in the presence of nettle [START_REF] Vierheilig | Resistance ofUrtica dioica to mycorrhizal colonization: a possible involvement ofUrtica dioica agglutinin[END_REF]. The authors speculated that a rhizome protein called Urtica dioica agglutinin (UDA) with antifungal properties [START_REF] Broekaert | A chitin-binding lectin from stinging nettle rhizomes with antifungal properties[END_REF]) could be involved in AM avoidance. In addition, this inhibitory effect could even affect the mycobiome of neighbouring plants [START_REF] Fontenla | Negative influence of non-host plants on the colonization of Pisum sativum by the arbuscular mycorrhizal fungus Glomus mosseae[END_REF].

The present study aimed at characterizing, using Illumina Mi-seq sequencing, the mycobiome associated with nettle and poplar roots co-occurring in an agrosystem set up on a contaminated site, where two areas were clearly distinguished by their TM concentrations. We hypothesised that poplar and nettle would harbour distinct root-associated fungal communities: poplar mycobiome would be dominated by EM and AM fungi; at the opposite, nettle communities would be composed of endophytic fungi (i.e. fungi colonizing root tissues asymptomatically [START_REF] Wilson | Endophyte: The Evolution of a Term, and Clarification of Its Use and Definition[END_REF]) with a low fungal diversity as described above. We further hypothesised that the level of TM contamination would alter the root-associated mycobiomes by reducing root fungal diversity and by increasing the diversity and abundance of TM-tolerant fungi, as mentioned elsewhere [START_REF] Giller | Heavy metals and soil microbes[END_REF][START_REF] Zappelini | Diversity and complexity of microbial communities from a chlor-alkali tailings dump[END_REF].

Material and methods

Site description

The study site located at Fresnes-sur-Escaut (France, 50°25'47.9"N 3°35'07.8"E) is a nettle-poplar agrosystem on a dredged sediment disposal site, contaminated with TM, that operated between 1978 and[START_REF] Bååth | Effects of heavy metals in soil on microbial processes and populations (a review)[END_REF] Approximately 200 000 m 3 of silt and sand from the dredging of l'Escaut canal were deposited, resulting in heterogeneous contamination with As, Cd, Cu, Ni, Pb and Zn within the surface horizons (0-50 cm). The experimental site is divided into two areas differing by the level of TM: the low trace-metal (LTM) and the high trace-metal (HTM) areas. The HTM area has mean soil concentrations of Zn (2231 mg/kg), Pb (425 mg/kg), Cu (108 mg/kg) and Cd (63 mg/kg), on average 20-, 9-, 4-and 100-fold higher than the LTM area, respectively [START_REF] Phanthavongsa | Effect of mycorrhizal inoculation on metal accumulation by poplar leaves at phytomanaged sites[END_REF]. A phytomanagement field trial was implemented in 2011 on this site (covering both areas) with plots of poplar Skado (P. trichocarpa × P. maximowiczii) or I214 (P. deltoides x P. nigra) as a short-rotation coppice (SRC; 2200 stems/ha). A detailed description of the experimental design is provided in [START_REF] Ciadamidaro | Poplar biomass production at phytomanagement sites is significantly enhanced by mycorrhizal inoculation[END_REF] and [START_REF] Phanthavongsa | Effect of mycorrhizal inoculation on metal accumulation by poplar leaves at phytomanaged sites[END_REF]. Five years after planting, the spontaneous herbaceous layer evolved in the same way as that of another similar experiment set up in eastern France, with a dominance of the stinging nettle under poplars with a coverage rate depending on the poplar cultivar [START_REF] Yung | Insect Life Traits Are Key Factors in Mercury Accumulation and Transfer within the Terrestrial Food Web[END_REF]. In order to get rid of this variable and appreciate the contaminant effect on the microbiomes associated with nettle and poplar roots, we selected only one plot for each poplar cultivar (i.e. Skado or I214) for both the HTM and LTM areas, each of 504 m² (28m x 18m), i.e. 4 plots in all. These plots are of sufficient size to allow for replicate collection.

Sample collection and preparation

The sampling was conducted in November 2016, consisting of root samples from the upper 20-cm layer of soil from 9 random poplars and 9 adjacent nettles for each of the four studied plots. Comparisons between soil and poplar root mycobiomes were done and available in our previous studies [START_REF] Durand | Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site[END_REF]Foulon et al., 2016bFoulon et al., , 2016a)). A total of three pseudo-replicates of thin roots were sampled from each tree and mixed to obtain one composite per poplar individual, whereas the entire root system of nettles was collected. For each plant, the root samples were carefully sorted so as not to be mixed, the soil was carefully removed in three successive baths in sterilized distilled-water, and the smallest roots were selected and separated from larger roots by sampling them with a scalpel. The samples were freeze-dried and stored at -20 °C before molecular analysis. Thus, we considered both the endophytic and epiphytic fungi from the rhizoplane.

Molecular method

The environmental DNA from the roots was extracted with the PowerSoil DNA isolation Kit following the manufacturer's instructions (MoBio Laboratories, Inc., Carlsbad, CA, USA). The PCR was performed using the specific fungal primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') [START_REF] Gardes | ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts[END_REF][START_REF] White | 38 -Amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics[END_REF]. We selected these primers to allow for comparison with our previous dataset obtained on a similar plantation, at a different site [START_REF] Durand | Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site[END_REF]. These primers has also been found as one of the most competent in uncovering the fungal diversity of plant root [START_REF] Bainard | Increasing the frequency of pulses in crop rotations reduces soil fungal diversity and increases the proportion of fungal pathotrophs in a semiarid agroecosystem[END_REF][START_REF] Huang | Plant pathological condition is associated with fungal community succession triggered by root exudates in the plant-soil system[END_REF]. PCR was performed in a reaction volume of 35 µL using 0.5 µM forward and reverse primer each, 200 µM dNTPs, 1 µL of Phire hot start II DNA polymerase (1 U/µl), 26.25 µL of 5X Phire reaction buffer (Thermo Fisher Scientific, Inc. USA), 10 ng template DNA and sterilized water to reach a final volume of 35 µl. The PCR conditions were 98 °C for 30 sec, followed by 25 cycles of 98°C for 5 sec, 57.2 °C for 5 sec and 72 °C for 15 sec, followed by a final extension step at 72 °C for 1 min. These primers targeted a short portion of the fungal ITS region, resulting in amplicons of small size (~300 bp), appropriated for Illumina sequencing. The DNA quality and quantity were assessed by agarose gel electrophoresis using the ImageLab software (Bio-Rad, USA) and with a Qubit TM dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA) using the Qubit 4 fluorometer. The partial fungal ITS region [START_REF] Wang | ITS1: a DNA barcode better than ITS2 in eukaryotes?[END_REF] of 6 and 7 of nine poplar and nettle samples (i.e. with the concentration of environmental DNA required) were sequenced with the Illumina MiSeq platform (Microsynth AG, Switzerland).

Bioinformatic analysis

The sequence data were processed using Mothur [START_REF] Schloss | Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities[END_REF]. First, all raw read pairs were joined at the overlapping region to create consensus sequences. Then several steps of filtering were performed, consisting in removing homopolymers, ambiguous sequences and sequences with an inappropriate length (shorter than 220 bp and greater than 400 bp). The 18S RNA region amplified by the ITS1F primer [START_REF] Toju | High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples[END_REF] was removed by in-silico PCR to target precisely the ITS1 region. After quality filtering, reads were then clustered into OTUs using the swarm tools [START_REF] Mahé | Swarm v2: highly-scalable and high-resolution amplicon clustering[END_REF] with a difference value of 1 and fastidious option enable. The OTU clustering method has been repeatedly shown to be powerful for recovering the richness and composition of the fungal community [START_REF] Bell | Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny[END_REF][START_REF] Durand | Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site[END_REF]Foulon et al., 2016b;[START_REF] Huang | Plant pathological condition is associated with fungal community succession triggered by root exudates in the plant-soil system[END_REF][START_REF] Nguyen | FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild[END_REF]. We removed singleton OTUs to avoid technical artefacts and overestimation of the number of species [START_REF] Tedersoo | Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages[END_REF]. The taxonomic assignment of OTUs was performed using the UNITE [START_REF] Kõljalg | Towards a unified paradigm for sequence-based identification of fungi[END_REF] database by a naïve Bayesian approach [START_REF] Wang | Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy[END_REF].The presence of plant sequences was verified using the BLAST tool of the GenBank database and removed. Each fungal OTU was further assigned to functional or morphological groups of fungi using FUNguild [START_REF] Nguyen | FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild[END_REF]. For each assignment, three confidence rankings (i.e., "possible": suspected, "probable": fairly certain, "highly probable": absolutely certain) referring to previously peer reviewed data were given. We only considered the functional and morphological assignation with at least a "probable" confidence ranking. Finally, the guilds related to a similar function were grouped to obtain a total of six main guilds, based on the literature.

Data analysis

Alpha diversity indices (OTU richness, Chao estimation, Shannon diversity index, inverse of the Simpson diversity index) were calculated using MOTHUR. Samples were rarefied at 33 000 sequences, corresponding to the smallest number of sequences detected in a sample. No sample was excluded. To verify that sequencing depth allowed covering most of the fungal diversity of our root samples, Good's coverage was calculated as C = [1 -(n / N)] × 100 (%), where "n" is the number of OTUs, and "N" is the number of sequences [START_REF] Good | The Population Frequencies of Species and the Estimation of Population Parameters[END_REF].

Statistical analyses were performed with R software v.3.6.1 (R Development Core Team, 2019). All the considered variables were tested for their homoscedasticity (Bartlett or Levene tests) and normal distribution (Shapiro-Wilk test) and compared using analysis of variance (ANOVA) followed by a Tukey HSD test. Where necessary, data were "log" transformed to improve normality. Data that did not fit a normal distribution after transformation were analysed with non-parametrical tests (Kruskal-Wallis test by ranks). First we calculated and drew rarefaction curves for each experimental condition of nettle and poplar, using "rarecurve" function available in the R Vegan package. The shape of the curve is a graphic display of the relative estimated species accumulation rates. All diversity and richness indices and the mean number of OTUs were then compared between all experimental conditions and plants through ANOVA followed by a Tukey HSD test.

To summarize differences in fungal OTU composition between the experimental conditions for both the I214 and Skado plots, we used two-dimensional non-metric Multi-Dimensional Scaling (NMDS) plots based on Bray-Curtis dissimilarity matrix (k = 2) using the metaMDS function of the R Vegan package. Permutational multivariate analyses of variance (PERMANOVA) based on Bray-Curtis dissimilarity were applied to determine the potential effects of the plant species (nettle vs poplar) and the level of contamination (LTM vs HTM) on fungal communities for both I214 and Skado plots. To assess the influence of the studied factors on the fungal classes, genera, EM fungal taxa and guilds, the experimental conditions were compared using a Kruskal-Wallis test, followed by a post-hoc test with a Holm correction of the p-value.

NMDS and PERMANOVA analyses were further performed on the EM fungal guilds.

For the whole fungal community and the six most abundant guilds taken individually, for nettle and poplar, we tested the correlation and the overlapping between the OTU composition using Mantel test (ade4 package) and by calculating the Morisita-Horn similaritity index (SpadeR package), respectively. The relative abundances of EM fungal OTUs were represented by a heatmap, from the pheatmap R package. Similarly, a hierarchical cluster analysis of the relative abundances of EM fungal OTUs from the various experimental conditions was represented by a dendrogram expressing the linkage between species and between the experimental conditions.

Results

Diversity of mycobiome associated with nettle and poplar roots

Illumina MiSeq sequencing of ITS1 amplicons obtained from 26 root samples of poplar and 25 root samples of nettle generated a total of 3,481,223 non-chimeric fungal sequences.

The initial total number of sequences obtained per sample ranged from 33,615 to 140,409. The sample with the lowest number of sequences was selected for subsampling, revealing a total number of 1833 non-singleton OTUs for 1,683,000 fungal reads, and constituting our final dataset. Rarefaction curves calculated for each experimental condition of nettle and poplar tended to reach an asymptotic profile, suggesting that the sequencing depth was sufficient to cover the fungal diversity of our root samples. Additionally, the measured Good's coverage values were greater than 99% for each condition and in every sample, suggesting a representative sampling. Rarefaction curves for nettles clearly exhibited divergent trends in LTM vs. HTM areas. For a same number of reads on the asymptote, the HTM plots reached a number of OTUs reduced by approximately 30%, as compared to the LTM plots (Figure S1).

Concerning poplar, the curve for the I214-HTM plot was clearly lower than those corresponding to the three other experimental conditions.

The total number of fungal OTUs was comparable for nettle (1218) and poplar (1321).

Among them, nettle and poplar shared 766 OTUs, representing 63% of the total number of OTUs for nettle and 58% for poplar. The mean number of fungal OTUs associated with the roots of nettle and poplar are detailed in Table 1 for each experimental condition. The highest mean number of OTUs was obtained for Skado roots from the HTM area, whereas the lowest was obtained for nettles within the same area, with a significant difference (P < 0.05). Only 7.1% and 7.5% of the total number of OTUs were shared between the four conditions for nettle and poplar, respectively. Additionally, the percentage of OTUs specific to each condition ranged between 8.5% and 19.9% for nettle and between 11.2% and 14.8% for poplar (Figure 1). For nettle, this number of specific OTUs was 2-fold higher in the LTM area (35.4%) compared to the HTM area (17.3%), whereas it was comparable for poplar (23 vs 26%).

Depending on the experimental condition, nettle shared between 47.3% and 63.0% of their OTUs with poplar, while poplar shared between 39.5% and 44.0% of their OTUs with nettle (Figure 1). While the obtained mean numbers of fungal OTUs were comparable between nettle and poplar, the estimated number of fungal OTUs (using the Chao model) showed a higher species richness in poplar roots, except for the I214-HTM condition (Table 1). The species richness of the HTM area was lower than the LTM area for nettle, while this tendency was only noticeable for I214 poplars, as suggested by the rarefaction curves (Figure S1). For nettle, we collected an average of 65% and 54% of the estimated number of OTUs (Chao estimation) for the LTM and HTM areas, respectively, whereas we obtained 69% for HTM versus 48% for LTM for poplar.

Overall, when considering the individual diversity estimators related to poplar or nettle, no apparent influence of the studied factors level of contamination was noticeable. However, NMDS analysis revealed a clear clustering of fungal communities according to the plant species (nettle vs poplar) and the level of contamination (LTM vs HTM) for the I214 and Skado plots (Figure 2). Thus, while the fungal diversity did not seem to be significantly influenced by the experimental condition (Table 1), the fungal composition of nettle and poplar appeared contrasted and affected by the level of TM contamination and the plant species (Figure 2).

NMDS and PERMANOVA indicated substantial differences in the composition of fungal communities between nettle and poplar (Figure 2), the plant species factor explaining 24% and 17% of the variance in the fungal community for I214 and Skado plots, respectively (P < 0.001;

Table S1). The level of TM had a significant effect on the fungal communities, both for nettle grown under I214 (28%) and Skado (37%) and for poplar, although to a lesser extent (26% for I214, and 16% for I214 Skado) (all P < 0.01; Figure 2, Table S1). PERMANOVA analysis for plant species ("species") and level of TM ("contamination") factors and their interaction, and conducted separately by species (contamination effect) and contamination (species effect) are provided under each NMDS, with the following legend: ***, P < 0.001; **, P < 0.01; * P < 0.05.

Taxonomic characterization of the mycobiome of nettle and poplar roots

The fungal communities associated with roots of plants growing at the sediment disposal site studied were dominated by the phylum Ascomycota in nettle roots (84.5% of total relative abundance) whereas Basidiomycota (43.2%) and Ascomycota (52.3%) were more equally represented in poplar roots (the Ascomycota/Basidiomycota ratio was significantly higher for nettle than for poplar; P < 0.05).

Among all samples, we detected 29 and 28 distinct fungal classes for nettle and poplar, respectively. Among them, the most abundant classes were Pezizomycetes, Agaricomycetes, Leotiomycetes, Dothideomycetes, Sordariomycetes, Mortierellomycetes, Eurotiomycetes and Olpidiomycetes (total relative abundance of each > 0.5%; Figure S2). For this study, Dothideomycetes and Leotiomycetes, as abundant classes, characterized the mycobiome of nettle roots. In contrast, poplar roots were characterized by Agaricomycetes, representing 20-66% of all sequences, depending on the experimental condition (Figure S2). Pezizomycetes were also well represented and similarly abundant in both plants.

For poplars, 79% of the fungal sequences were successfully assigned to a genus (73% for I214 vs 85% for Skado), whereas only 54% were successfully assigned for nettle (Figure 3).

The genus Kotlabaea was the most abundant genus associated in nettle roots (26.5% of the total number of fungi associated with nettle), followed by three unknown genera belonging to Helotiales (17.5%), Didymosphaeriaceae (8.7%) and Phaeosphaeriaceae (5.8%). The genera Tetracladium (5.7%) and Hymenoscyphus (2.5%) were the most abundant nettle-associated

Leotiomycetes and were also present in poplar roots with comparable relative abundances (Figure 3). For poplar, the most abundant genera were Inocybe (17.1% of the total number of fungi associated with poplar), Tuber (10.8%), Hebeloma (7.3%), Tomentella (6.9%) and Geopora (7.1%), the latter being almost absent in nettle roots (< 0.02%) (Figure 3). The genus Olpidium (Olpidiomycetes), accounting for 1.3% and 1.4% of the fungal sequences for nettle and poplar, respectively, was also well represented.

We detected significant differences in terms of fungal composition between the two poplar cultivars. Tomentella, Geopora, Hymenogaster, Hebeloma and Clavulina were more represented in roots of the Skado cultivar, while Helvella and Kotlabaea were mostly associated with I214 cultivar (P < 0.05), but each of these trends were only significant for one of the two considered areas (i.e., LTM or HTM; P < 0.05; Figure 3, Table S2). Differences were also noticed between mycobiomes of nettles grown in I214 plots (high density of nettle) and those grown in Skado plots (low density of nettle). Inocybe, Genabea, Helvellosebacina and Hebeloma were more abundant in roots of nettles grown in the Skado plot (P < 0.05), whereas

Olpidium and Calyptella were more abundant in the I214 plot (P < 0.05). These results were significant for only one of the two considered areas (Figure 3, Table S2). Pezizomycetes were significantly influenced by TM contamination : first, their relative abundance was significantly higher at the HTM area for nettles, both the I214 (10% for LTM vs. 32% for HTM; P < 0.05)

and Skado (7% vs. 68%; P < 0.05) plots. Second, the same trend was observed on poplar but only statistically supported for the Skado cultivar, (6% vs. 41%; P < 0.05; Figure S2). Among

Pezizomycetes, the genus Kotlabaea significantly increased in the HTM area (for I214 plots: 0.1% vs. 31.7%; for Skado plots: 2.5% vs. 65.1%; P < 0.05; Figure 3). Similarly, the genus Tuber was more represented in the poplar roots collected from the HTM area (for the I214 cultivar: 6.2 % vs. 17.6%; for the Skado cultivar 4.6% vs. 15.2%; P), however, the high between-sample variability did not allow to statistically support this. Agaricomycetes associated with both poplar and nettle were influenced by TM as their abundance was significantly reduced in the HTM areas (P < 0.05), except for nettle in the I214 plots (Figure S2). This reduction of Agaricomycetes in the HTM area was mainly due to the decrease of the Inocybe genus, the most abundant of all Agaricomycetes for nettle (34%) and poplar (40%) (Figure 3). When considering nettle and poplar together, the relative abundance of Inocybe species decreased from LTM-to HTM area, namely from 31.9 to 1.5% for I214 plots and from 24.1 to 8.1% for Skado plots (P < 0.05 in each case; Figure 3). A similar but less pronounced trend was also noticeable for the genus Tomentella within the I214 plots. S2.

Functional characterization of the mycobiome of nettle and poplar roots

Among the 1218 and 1321 fungal OTUs recorded in nettle and poplar roots (Figure 1), 70.4% and 83.1%, respectively, were successfully assigned to a functional guild with at least a "probable" confidence ranking using FUNguild [START_REF] Nguyen | FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild[END_REF]. This functional assignment revealed a majority of saprotrophic fungi (from 18 to 69% depending on the condition), followed by plant pathogens (6 -15%) and EM fungi (1 -30%) in the nettle roots whereas EM fungi (26 -75%), mainly represented by Tomentella, Geopora, Hymenogaster, Hebeloma and Clavulina genera, were the most abundant guild in poplar roots (Figure 4). This guild was significantly more abundant in roots of Skado poplar, compared to I214 poplar (63% vs 26% for HTM area, P < 0.05; Figure 4). EM fungi were abundant on nettle roots and, for the LTM area, even more on nettle grown with the Skado cultivar than on those grown with I214 (30% vs 2%; P < 0.05). Endophytes were poorly represented in the poplar roots in contrast to nettle (0.1% vs. 9.2% of all sequences; P < 0.05; Figure 4). Twelve OTUs assigned to AM fungi were found in nettle and poplar roots, accounting for less than 0.05% of all sequences (not shown). Plant pathogenic fungi were equally represented in poplar and nettle roots, without any influence of the studied factors (Figure 4).

Functional guild also revealed several differences between soil contamination levels.

Indeed, the relative abundance of nettle saprotrophs was higher in the HTM area (statistically supported only for the Skado plots; P < 0.05) (Figure 4). This result was driven by Kotlabaea, the most abundant genus within this guild (Figure 3): an unidentified species from this genus, accounting for 66% of all the saprotrophs associated with nettle, was indeed significantly more present in the HTM areas (P < 0.001). EM fungi associated with poplar (particularly Inocybe and Tuber) and nettle (particularly Inocybe) tended to decrease in the HTM area, which was statistically supported for I214 plot in the case of poplar (75% vs 26%, P < 0.05) and for Skado plot in the case of nettle (30% vs 6%; P < 0.05; Figure 4).

Overlap of poplar and nettle microbial communities

According to the Mantel test, nettle and poplar total fungal communities under I214 (P < 0.05) or Skado (P < 0.01) plots were significantly correlated (Figure 5), while the corresponding Morisita-Horn similarity index did not corroborate this result. However, similarity indexes and Mantel tests converged for nettle and poplar root-associated animal pathogenic fungi, EM fungi and saprotrophs that were significantly correlated (Mantel test, P < 0.05) and overlapped (Morisita-Horn index < 0.5; Figure 5). Despite the low relative abundance of EM fungi on nettle under I214 plots (Figure 4), nettle and poplar composition of EM fungi were strongly correlated (P < 0.001, Figure 5) for I214, while it was not the case for Skado plots. Interestingly, the saprotrophic guild of the two plant species were only correlated and overlapped at I214 plots, where the most abundant genera Kotlabaea was driving the trend.

Consequently, these results suggested that nettle and poplar roots shared large part of their fungal communities. Although there was a significant relationship between EM fungi associated with the two plants, the NMDS analysis showed that EM fungi of nettle and poplar clustered for each host, but not together, suggesting host differentiation (Figure 6.A). EM fungi were significantly more represented (59.6% vs 9.7%; P < 0.05) and diverse (330 vs 146 OTUs; P < 0.05) in poplar than in nettle roots (Figure 4 and 6.C). Moreover, the ratios between nettle and poplar EM richnesses were similar for each condition (ranging from 0.64 to 0.83). A Venn diagram showed a higher species richness of EM fungi associated with poplar and particularly with the Skado cultivar According to a hierarchical cluster analysis of the relative abundances of EM fungal OTUs , LTM poplars were mostly colonized by the EM Inocybe rimosa, Tomentella sp., Helvella elastica, Inocybe semifulva, Clavulina sp. and Hebeloma eburneum (Figure S3). The EM OTUs Geopora cervina and Tuber rapaeodorum were abundant in roots of poplar grown at the HTM area (Figure S3). Most of these taxa were poorly represented in the nettle roots, except for Inocybe rimosa, which was the most abundant EM fungus in both plants (Figure S3).

(
Indeed, this species accounted for 4% of nettle sequences (95% from the Skado plots) and 12.3% of poplar sequences (52% from the Skado plots). Additionally, this species was more abundant in roots of nettle grown with the Skado cultivar (P < 0.05), and when associated with poplar, it was sensitive to the soil contamination (P < 0.05) (Figure S3).

Discussion

EM fungi dominated the poplar mycobiome

In this study, the mycobiome associated with poplar roots were clearly dominated by several EM fungal species, such as the Agaricomycetes Inocybe rimosa, Tomentella sp., and Hebeloma spp., and the Pezizomycetes Tuber rapeodorum and Geopora cervina. We also detected the presence of AM fungi belonging to the Rhizophagus and Glomus genera, although with a rather low number of sequences (< 0.001%), partially due to the choice that was made to target the ITS1 region, which is not the most adapted region for the study of Glomeromycota [START_REF] Beeck | Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies[END_REF]. The recorded EM fungal species are known fungal symbionts of poplar [START_REF] Podila | Populus Rhizosphere and the Ectomycorrhizal Interactome[END_REF] that improve plant health by enhancing resistance to diverse stresses like drought, salinity, heavy metals and pathogens, etc. (van der [START_REF] Van Der Heijden | Mycorrhizal ecology and evolution: the past, the present, and the future[END_REF]. Particularly, they facilitate the adaptation of plants to TE stress, promoting host growth and phytomanagement of TM-contaminated soils [START_REF] Gil-Martínez | Ectomycorrhizal Fungal Communities and Their Functional Traits Mediate Plant-Soil Interactions in Trace Element Contaminated Soils[END_REF].

In a previous study on a Hg-enriched sediment disposal site, the same experimental design was set up in 2011. [START_REF] Durand | Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site[END_REF] showed that EM fungi, and particularly the Tuber and Geopora genera also dominated the roots of Skado poplar at this site. These taxa seemed to be largely abundant in TM contaminated sites [START_REF] Bonito | Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants[END_REF][START_REF] Guevara | New North American truffles (Tuber spp.) and their ectomycorrhizal associations[END_REF][START_REF] Lacercat-Didier | New mutualistic fungal endophytes isolated from poplar roots display high metal tolerance[END_REF]. Moreover, these EM fungi were not only related to the physico-chemical properties of these sites, but rather to the juvenil tree stage that promotes some Pezizales (Geopora spp., Tuber spp.) or Agaricales (Inocybe spp.) (Foulon et al., 2016b;[START_REF] Hrynkiewicz | The significance of rotation periods for mycorrhiza formation in Short Rotation Coppice[END_REF][START_REF] Lacercat-Didier | New mutualistic fungal endophytes isolated from poplar roots display high metal tolerance[END_REF]. Hebeloma or Inocybe species, which

were well represented at the studied site, are "early stage" fungi capable of colonizing roots of trees established in virgin or disturbed habitats [START_REF] Smith | Mycorrhizal Symbiosis[END_REF]. In our dataset, only 7%

of the variability were explained by the cultivar factor when performing a PERMANOVA on the poplar dataset (data not shown), indicating that the mycobiomes of Skado and I214 roots were comparable, consistently our previous results (Foulon et al., 2016a): in our previous work, the mycobiome of eight poplar cultivars grown at a TM-contaminated site, exhibited slight differences, especially between I214 and Skado roots. Together with the present study, the only discrepancy between the two communities concerned the Laccaria genus, only recorded under the I214 cultivar in small abundance. This similarity between the two studies might be linked to the similar TM composition of the two sites and the similar age of the poplar plantation (7years in Foulon et al., (2016a) vs 6-years in the present study).

Saprotroph and endophytic fungi dominated the nettle root mycobiome

The overall fungal composition of nettle roots differed from that of poplar, despite their co-occurrence. Most of the dominant species associated with nettles were only assigned to the family or order level, reflecting a lack of information concerning the nettle mycobiome.

Dominant species on nettle roots were mostly endophytic or pathogenic fungi, such as Tetracladium sp., Olpidium spp., or saprotroph belonging to the Helotiales and Pleosporales orders as previously observed for the non-mycorrhizal plant Arabis alpina [START_REF] Almario | Rootassociated fungal microbiota of nonmycorrhizal Arabis alpina and its contribution to plant phosphorus nutrition[END_REF]. The most abundant OTU was assigned to the Kotlabaea genus, accounting for more than 26% of all sequences. This species belongs to the Pyronemataceae, one of the largest family of Pezizales traditionally considered saprotrophic, although the trophic strategies of most species remain undocumented [START_REF] Perry | A phylogenetic overview of the family Pyronemataceae (Ascomycota, Pezizales)[END_REF]. Indeed, an increasing number of them are being identified as EM [START_REF] Tedersoo | Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages[END_REF]. We classified Kotlabaea spp. as saprotrophic based on [START_REF] Tedersoo | Fungal biogeography. Global diversity and geography of soil fungi[END_REF], but uncertainties remained. Overall, knowledge about taxonomy and functional role of these root-associated fungi of nettle needs to be enriched by fungal isolation and characterisation experiments, which has not yet been performed to our knowledge.

The degree of contamination differently affected the mycobiomes of poplar and nettle

According to a previous pedogeochemical characterization of the soils from the two areas, Zn and Cd concentrations were the most contrasted parameters between the two areas [START_REF] Phanthavongsa | Effect of mycorrhizal inoculation on metal accumulation by poplar leaves at phytomanaged sites[END_REF]. Other soil parameters were rather comparable, suggesting that the level of TM is one of the main factor influencing the soil fungal communities.

A slight reduction of fungal diversity was noticed for plants grown at the most contaminated area (HTM), which is commonly observed [START_REF] Bååth | Effects of heavy metals in soil on microbial processes and populations (a review)[END_REF][START_REF] Giller | Heavy metals and soil microbes[END_REF].

Indeed, while certain fungal microorganisms can tolerate large concentrations of non-essential heavy metals (e.g. Al, As, Cd, Hg, Pb), plant mycobiome is generally affected [START_REF] Gadd | Metals, minerals and microbes: geomicrobiology and bioremediation[END_REF].

Other studies showed that TM tended to reduce the amount of soil microbial biomass as well as the species richness, changing microbial structure in favor of tolerant taxa and modifying the soil functioning [START_REF] Giller | Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review[END_REF][START_REF] Giller | Heavy metals and soil microbes[END_REF].

Some mycorrhizal fungi isolated from contaminated soils develop adaptations to tolerate metals [START_REF] Colpaert | How metal-tolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution[END_REF][START_REF] Gonçalves | Evidence of adaptive tolerance to nickel in isolates of Cenococcum geophilum from serpentine soils[END_REF], which make them particularly suitable for phytomanagement applications. Indeed, plants inoculated with metal-tolerant EM fungi [START_REF] Adriaensen | Copper-adapted Suillus luteus, a symbiotic solution for pines colonizing Cu mine spoils[END_REF][START_REF] Redon | Differential effects of AM fungal isolates on Medicago truncatula growth and metal uptake in a multimetallic (Cd, Zn, Pb) contaminated agricultural soil[END_REF] or DSE [START_REF] Likar | Isolates of dark septate endophytes reduce metal uptake and improve physiology of Salix caprea L[END_REF] showed enhanced tolerance when compared to plants inoculated with non-tolerant strains. In our observations, Agaricomycetes, and particularly the Inocybe and Tomentella genera, when associated with nettle or poplar, were the most impacted by contamination. Although significantly reduced, these two EM fungi were still well represented at the heavily contaminated area on poplar roots. Additionally, the Inocybe genus has already been isolated [START_REF] Repáč | Inoculation of Picea abies (L.) Karst. seedlings with vegetative inocula of ectomycorrhizal fungi Suillus bovinus (L.: Fr.) O. Kuntze and Inocybe lacera (Fr.) Kumm[END_REF], and successfully tested as an inoculum strain on coniferous trees [START_REF] Flores-Rentería | Ectomycorrhizal pre-inoculation of Pinus hartwegii and Abies religiosa is replaced by native fungi in a temperate forest of central Mexico[END_REF], which supports its interest in bioremediation applications. Their usefulness and functional importance in nettle plantation deserves further analyses (see below).

In our study, members of the Pezizomycetes seemed to be the most tolerant fungi as the Kotlabaea genus associated with nettle and the Tuber genus, associated with poplars, were more represented in the area with the highest concentration of Cd, Zn, Cu and Pb. This increase of Perizomycetes related to soil contamination has already been detected in hydrocarboncontaminated soils [START_REF] Bell | Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny[END_REF][START_REF] Tardif | The Willow Microbiome Is Influenced by Soil Petroleum-Hydrocarbon Concentration with Plant Compartment-Specific Effects[END_REF]. Indeed, an untested possibility is that hydrocarbons are also present at our site because canals dredged sediments generally contain various contaminants [START_REF] Besser | Assessment of Sediment Quality in Dredged and Undredged Areas of the Trenton Channel of the Detroit River, Michigan USA, using the Sediment Quality Triad[END_REF][START_REF] King | Changing contaminant mobility in a dredged canal sediment during a three-year phytoremediation trial[END_REF].

A significant fraction of EM fungi were shared by poplar and nettle roots

Despite the noticeable differences mentioned above in terms of fungal composition between nettle and poplar, our results suggested that their respective mycobiome could be influenced by the other when co-occurring. More specifically, fungal taxa associated with abundant plants seemed more likely to colonize neighbouring plants than if the later were alone.

Indeed, taxa characteristics of the nettle mycobiome (e.g. Helvella, Kotlabaea) where more represented in the I214 plot where nettle covered up to 60% of the herbaceous layer, compared to the Skado plot where nettle and other plants were rare. Similarly, an influence of Skado poplar on the mycobiome of neighbouring nettles was possibly detected, since a significant part (9.7%) of EM OTUs, which were characteristic of poplars mycobiomes, contributed to the mycobiome of nettle roots, which might be surprising as this plant is a recognised nonmycorrhizal plant [START_REF] Vierheilig | Resistance ofUrtica dioica to mycorrhizal colonization: a possible involvement ofUrtica dioica agglutinin[END_REF]. Nettle roots were carefully washed and sorted, making contaminations by the adjacent soil or poplar roots unlikely. Yet their presence on the rhizoplane or as true endophytes (i.e. with root penetration) deserves further investigations.

Indded, the ability of some EM fungi to colonize AM (and more generally non-EM) plants has already been described for several clades based on molecular detection and direct observations, mainly for Sebacinaceae [START_REF] Selosse | Do Sebacinales commonly associate with plant roots as endophytes?[END_REF][START_REF] Weiß | Sebacinales Everywhere: Previously Overlooked Ubiquitous Fungal Endophytes[END_REF] and Tuber spp. [START_REF] Gryndler | Tuber aestivum association with non-host roots[END_REF][START_REF] Schneider-Maunoury | Is Tuber melanosporum colonizing the roots of herbaceous, non-ectomycorrhizal plants?[END_REF], 2020). In addition, some suspicion exists, based on molecular detection only, for Thelephoraceae and Inocybaceae [START_REF] Schneider-Maunoury | Is Tuber melanosporum colonizing the roots of herbaceous, non-ectomycorrhizal plants?[END_REF], 2020), Pyronemataceae [START_REF] Hansen | A phylogeny of the highly diverse cupfungus family Pyronemataceae (Pezizomycetes, Ascomycota) clarifies relationships and evolution of selected life history traits[END_REF] and Helotiales [START_REF] Wang | Toward a phylogenetic classification of the leotiomycetes based on rDNA data[END_REF]. Such a dual, EM and endophytic interaction has been viewed as a persistence of the evolutionary past of EM fungi, if they evolved from endophytic species: in the so-called 'waiting room hypothesis', endophytism is considered as a niche from which the tighter and more elaborate mycorrhizal symbiosis can evolve [START_REF] Schneider-Maunoury | Is Tuber melanosporum colonizing the roots of herbaceous, non-ectomycorrhizal plants?[END_REF][START_REF] Selosse | Do Sebacinales commonly associate with plant roots as endophytes?[END_REF][START_REF] Van Der Heijden | Mycorrhizal ecology and evolution: the past, the present, and the future[END_REF]. It was recently commentated that many fungi have more complex niches (e.g., dual niches) than what is commonly agreed, which could be explained by such evolutionary trajectories [START_REF] Selosse | Time to re-think fungal ecology? Fungal ecological niches are often prejudged[END_REF][START_REF] Thoen | In vitro evidence of root colonization suggests ecological versatility in the genus Mycena[END_REF].

Recently, [START_REF] Taschen | Insight into the truffle brûlé: tripartite interactions between the black truffle (Tuber melanosporum), holm oak (Quercus ilex) and arbuscular mycorrhizal plants[END_REF] demonstrated that Tuber melanosporum growth gains benefit from the non-EM plants it colonizes, while it has a negative effect on them.

Additionally, the EM trees colonized by this fungus as ectomycorrhizal partners gain, at least in terms of nitrogen content, from the interaction between truffles and non-EM plants. The possibility of an overlooked interaction between EM-and AM-or non-mycorrhizal plants growing close to host trees is further supported by the fact that connection and the exchange of carbon between plant sharing the same belowground ectomycorrhizal network were demonstrated for trees [START_REF] Klein | Belowground carbon trade among tall trees in a temperate forest[END_REF][START_REF] Selosse | Mycorrhizal networks: des liaisons dangereuses?[END_REF], even when one of the plant was herbaceous [START_REF] Selosse | Do Sebacinales commonly associate with plant roots as endophytes?[END_REF]. The functional implications for plants of an extensive sharing of non-mycorrhizal fungi remained unclear and highly dependant on the plant-fungus association. In case they are beneficial, and this happens even for non-mycorrhizal endophytic fungi such as dark septate endophytes in non-mycorrhizal plants [START_REF] Liu | Dark septate endophytes colonizing the roots of 'non-mycorrhizal' plants in a mine tailing pond and in a relatively undisturbed environment, Southwest China[END_REF][START_REF] Yung | Dark septate endophytes isolated from non-hyperaccumulator plants can increase phytoextraction of Cd and Zn by the hyperaccumulator Noccaea caerulescens[END_REF], there may be mutual reinforcement of the two plant species by way of coordinated responses to stresses, nutritional sharing, etc. Yet, the benefit can be asymmetric, or even relevant for one plant species only. Moreover, an intriguing possibility is pathogen spillover [START_REF] Mordecai | Pathogen impacts on plant communities: unifying theory, concepts, and empirical work[END_REF], i.e. one plant promotes the spread of pathogens that harm competitors more strongly than itself. In this context, further investigations are now necessary to conclude about the exact nature and functionality of this poplar-nettle EM sharing and its consequence for this agroforestry system.

Inocybe rimosa, the most abundant EM fungal species found on nettle roots, accounting for approximately 4% of the total fungal sequences, is a major candidate for such studies. The presence of I. rimosa is reported in TM-polluted soils [START_REF] Huang | Ectomycorrhizal fungal communities associated with Masson pine (Pinus massoniana Lamb.) in Pb-Zn mine sites of central south China[END_REF][START_REF] Krpata | Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution[END_REF] and, although its ability to colonize roots of non-EM plant has not been previously investigated,

Inocybaceae were already detected in some AM plants [START_REF] Schneider-Maunoury | Is Tuber melanosporum colonizing the roots of herbaceous, non-ectomycorrhizal plants?[END_REF]. Any facilitation mechanism through EM fungi could contribute to the dominance of nettles within Salicaceous SRC in natural [START_REF] Cronk | Salix transect of Europe: variation in ploidy and genome size in willow-associated common nettle, Urtica dioica L. sens. lat., from Greece to arctic Norway[END_REF] and contaminated sites [START_REF] Yung | Insect Life Traits Are Key Factors in Mercury Accumulation and Transfer within the Terrestrial Food Web[END_REF]. As a conclusion, we taxonomically and functionnaly characterized the fungal microbiomes of a spontaneous species (i.e., Urtica dioica L.) co-occuring with cultivated crops (i.e., Populus spp.) at a phytomanaged area contaminated with TM. Our results suggested that nettle and poplar had distinct root-associated fungal communities, albeit sharing many common ectomycorrhizal fungi and particularly the genus Inocybe, which is surprising given the nonmycorrhizal status of nettle. These results suggest to reconsider the ecological niches of fungi.

Figure 1 :

 1 Figure 1: Venn diagrams showing the overlap of the fungal communities according to the poplar cultivar plot (I214 and Skado) and the level of TM in soil (LTM and HTM), for nettle (left) and poplar (right). These diagrams present the number of total (above the ellipses), specific (within non-intersected ellipses) and shared OTUs (intersected ellipses). The five couples of circles provide the total number of OTUs for nettle (left) and poplar (right) and the shared number (intersection) for the whole dataset (center) and for each experimental conditions (bottom).

Figure 2 :

 2 Figure 2: Non-parametric multidimensional scaling (NMDS) plot of fungal communities associated with nettles and poplars grown at LTM and HTM areas, for I214 (left) and Skado (right) plots, using the Bray Curtis dissimilarity measure. Small and large circles represent the individual samples and the centroids of the different temperatures, respectively. Results from a

Figure 3 :

 3 Figure 3: Relative abundance of the 20 most represented fungal genera (based on the taxonomic assignment of OTUs), belonging to 7 classes, according to the level of TM in soil (LTM and HTM) for nettle (orange bubbles) and poplar (blue bubbles) grown in the I214 or Skado plots. The classes with a relative abundance <0.5% have been gathered in the "others" group. For each genus, the related results from Kruskal-Wallis pairwise or Tukey HSD tests are provided in the TableS2.3.3. Functional characterization of the mycobiome of nettle and poplar roots

Figure 4 :

 4 Figure 4: Relative abundance of the four most abundant fungal guilds associated with nettles and poplars grown at the LTM or HTM area, for I214 (left) and Skado (right) plots. Data are the means ± SE (n = 6 or 7). For each class, bars designated with the same letter(s) are not significantly different (Kruskal-Wallis, P < 0.05).

Figure 5 :

 5 Figure 5: Correlations (Mantel test) and similarity (Morisita-Horn index) between the OTUs composition of nettle and poplar, for the whole fungal community ("Total Fungi") and the six most abundant guilds depending on the poplar cultivar plot. The size of the circles is proportional to the P-value for the Mantel test and to the index value for Morisita-Horn. Two communities with a Morisita-Horn index ≥0.5 are considered similar. P-value of the Mantel tests are represented with the following legend***, P < 0.001; **, P < 0.01; * P < 0.05.

  Figure 6.B), confirming poplar as the main host for EM fungi. As observed above for poplar, nettle EM fungi were more represented in terms of richness (115 vs 65 OTUs) and abundance (27.1 vs 1.7% of the total number of sequences) under the Skado plots as compared to the I214 plots. Among the 138 OTUs associated with nettle, 102 OTUs were shared between nettle and poplar (Figure 6.B). Only 29 of the 115 EM OTUs (representing 1.6% of the total abundance of EM fungi) under the Skado plots and 8 of the 65 EM OTUs (11.7% of the total abundance) under the I214 plots were specific to nettle (Figure 6.B). These results suggested that most of the EM fungi associated with nettle were related to these hosted by poplar.

Figure 6 :

 6 Figure 6: (A) Non-metric multidimensional scaling (NMDS) analysis of the EM fungal guild structure associated to poplar or nettle roots, (B) a Venn diagram based on EM OTUs and (C) the EM OTU richness, depending on the plant species (poplar or nettle), the poplar cultivar plot and the level of contamination (LTM and HTM).

  However, further research about the mycobiome of nettle roots and particularly in the case of co-cropping at phytomanagement sites, especially to assess morphological (e.g. by fluorescent in-situ hybridization; Schneider-Maunoury et al., 2020) and functional evidence of interactions with EM fungi, is now awaited. I. rimosa is present in the CBS Filamentous fungi and Yeast Collection as CBS 210.55. It will be used in further experiments, for poplar x nettle experiments to test its growth between the two plants and assess its positive or negative or no influence on nettle.

Table 1 :

 1 Results of the Illumina MiSeq sequencing of ITS1 amplicons, including richness and diversity indices of the mycobiomes associated to I214 and Skado poplar roots, or to nettle roots growing in the I214 or Skado plots, at low trace-metal (LTM) and the high trace-metal (HTM) areas. Mean values and standard deviations (mean ± SD) are provided for each experimental condition.The "total number of sequences" corresponds to datasets before subsampling at 33,000 sequences, while the other parameters are calculated after subsampling. Values designated with the same letters are not significantly different (Kruskal-Wallis test, P < 0.05).

							NETTLE											POPLAR
				LTM					HTM					LTM						HTM
			I214	Skado		I214	Skado		I214	Skado	I214	Skado
	Number of samples			6			6			6			7			6			7			6			7
	Mean total
	number of	52,264	73,778	68,434	70,185	69,479	63,116	55,243	61,067
	sequences
	Mean OTUs observed	175 ab (± 56)	168 ab (± 69)	122 b (± 23)	109 b (± 52)	178 ab (± 19)	196 a (± 30)	156 ab (± 38)	204 a (± 43)
	Chao estimation	248 c (± 56)	280 bc (± 61)	221 c (± 25)	210 c (± 64)	362 ab (± 31)	410 a (± 74)	232 c (± 33)	358 ab (± 70)
	Shannon index	2.3 ab (± 0.8)	2.7 a (± 1.0)	2.2 ab (± 0.6)	1.4 b (± 1.0)	2.4 ab (± 0.5)	2.7 a (± 0.5)	2.9 a (± 0.7)	2.9 a (± 0.5)
	Inverse of
	Simpson diversity	6.0	a	(± 5.4)	8.9	a	(± 6.1)	5.8	a	(± 3.4)	3.1	a	(± 2.8)	5.7	a	(± 2.3)	7.7	a	(± 5.3)	10.1	a	(± 5.7)	8.9	a	(± 6.1)
	index

Acknowledgments

The authors gratefully acknowledge the funding provided We thank VNF (Voies Navigables de France) and Valérie Bert (INERIS) for providing us with access to the experimental site at Fresnes-sur-Escaut.

Funding sources

This work was supported by ADEME, France, under grant No. 1772C0018, PHYTOFIBER Project, by ANR, under grant n°2010-INTB-1703-03 BIOFILTREE (ANR Programme Blanc International) and the Fondation de France and ADEME for their financial support to LY under a PhD grant.