Forest Ecology and Management 358 (2015) 202–211
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Forest Ecology and Management
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Asteropeia mcphersonii, a potential mycorrhizal facilitator for ecological restoration in Madagascar wet tropical rainforests ⇑ Charline Henry a, , Jeanne-Françoise Raivoarisoa b, Angélo Razafimamonjy b, Heriniaina Ramanankierana c, Paul Andrianaivomahefa b, Marc-André Selosse d, Marc Ducousso e a AgroParisTech, Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD/INRA/CIRAD/Montpellier SupAgro/Université Montpellier, TA10J, 34398 Montpellier Cedex 5, France b Ambatovy, Immeuble Tranofitaratra, 7ème étage, Rue Ravoninahitriniarivo, Ankorondrano, Antananarivo 101, Madagascar c Centre National de Recherches sur l’Environnement, Laboratoire de Microbiologie de l’environnement, Antananarivo, Madagascar d Institut de Systématique, Évolution, Biodiversité, ISYEB, UMR 7205, CNRS, MNHN, UPMC, EPHE, Muséum national d’Histoire naturelle, Sorbonne Universités, 57 rue Cuvier, CP50, 75005 Paris, France e CIRAD, Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD/INRA/CIRAD/Montpellier SupAgro/Université Montpellier, TA10C, 34398 Montpellier Cedex 5, France article info abstract
Article history: Ecological restoration in severely disturbed environments can fail because of lack of knowledge of the Received 21 April 2015 functioning of the original ecosystem. Nevertheless, facilitating establishment between plant species Received in revised form 10 September can help accelerate ecological succession, especially in stressful environments. Mycorrhizal symbiosis 2015 plays a key role in plant growth, particularly in harsh environments, and could also play a role in facilita- Accepted 11 September 2015 tion between plants, as mycorrhizal fungi can form a mycelial network that simultaneously interacts with the root systems of several plant species. In a high-elevation Malagasy tropical rainforest on acidic and iron-rich soil surrounding an active mining site, four genera of ectomycorrhizal plants are locally abun- Keywords: dant: Leptolaena, Sarcolaena, Uapaca and Asteropeia. A floristic survey showed that only Asteropeia seed- Ecological restoration Ectomycorrhizal community lings can grow on bare soil. Molecular analysis of ectomycorrhizal fungi ITS rDNA enabled us to Nurse plant describe ectomycorrhizal communities and their distribution among these four plant genera. Asteropeia Russulaceae, Boletales, Cortinariaceae and Thelephoraceae are abundant in these forests. There is exten- Madagascar sive sharing between ectomycorrhizal communities associated with Asteropeia mcphersonii and other ecto- mycorrhizal plants. There are also many mycorrhizal fungi species which are common to ectomycorrhizal communities of seedlings and adult trees. This abundance of generalist fungi allows us to envisage the use of A. mcphersonii in the ecological restoration of the mine site. Planting ectomycorrhizal fungi in the bare soil at the beginning of ecological restoration could allow them to grow, thereby establishing a source of inoculum to colonize other ectomycorrhizal plants and consequently facilitate their establishment. Ó 2015 Published by Elsevier B.V.
1. Introduction 1994; Callaway and Walker, 1997), suggests that interactions shift from mainly negative (i.e. competition) to mainly positive (i.e. Ecological restoration in environments disturbed by human facilitation) with increasing stress. This hypothesis has received activities, especially those where the substrate is bare and a suc- much observational support (e.g. Callaway et al., 2002) and may cession has to be restarted, is particularly challenging (Wong, apply in particular under conditions of extreme stress (He and 2003), and is often limited by insufficient knowledge of the pre- Bertness, 2014). disturbance ecosystem. In particular, the original community A particular form of facilitation which is useful in plant commu- may contain mechanisms that allow facilitation, i.e. the positive nity restoration is referred to as the ‘‘nurse effect” when an adult influence of one species on another that may be instrumental in plant (the nurse plant) promotes the establishment of seedlings restoration. Even though this assumption is under debate (e.g. (the target plants; Niering et al., 1963). It may be decisive for plant Bakker et al., 2013), stressful environments are likely to promote establishment in primary or secondary successions, especially in facilitation between plants. The so-called stress gradient hypothe- harsh environments (Walker and del Moral, 2003), and sometimes sis, which was proposed 20 years ago (Bertness and Callaway, involves microbial mediation (Duponnois et al., 2011). Mycorrhizal symbiosis, which concerns 80% of land plant species (van der
⇑ Corresponding author. Heijden et al., 2015; Wang and Qiu, 2006), is a mutualistic associ- E-mail address: [email protected] (C. Henry). ation between a fungus and the roots of a plant, which often http://dx.doi.org/10.1016/j.foreco.2015.09.017 0378-1127/Ó 2015 Published by Elsevier B.V. C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211 203 depends on fungal colonization for its survival. This relationship is forest on sandy soil in southeast Madagascar; Tedersoo et al., crucial for both partners, since the plant supplies carbon to the soil 2011). Yet many mining projects require forest restoration on the fungus and, as a reward, receives nutrients and water from the fun- bare, hostile soils which remain after exploitation. Here we report gus through a symbiotic organ, the mycorrhiza (Smith and Read, a study conducted in the altitudinal tropical rain forests growing 2008). Mycorrhizal symbiosis plays a key role in plant nutrition on acidic and iron-rich soils at the foot of the Ambatovy mining and also in plant defence against soil biotic and abiotic aggression project, which will require restoration in the near future. In the (Selosse et al., 2004). For example, some ectomycorrhizal fungi mature forest, four species which are locally abundant as adults give the host plant resistance to heavy metals (Jourand et al., are ectomycorrhizal: Asteropeia mcphersonii (Asteropeiaceae; 2010), and, the absence of fungal partners in stressful environ- Ducousso et al., 2008), Leptolaena sp. and Sarcolaena sp. (Sarcolae- ments can thus seriously limit plant development (Cázares et al., naceae; Ducousso et al., 2004), and Uapaca densifolia (Phyllan- 2005; Feldhaar, 2011). taceae; Wang and Qiu, 2006). Seedlings of A. mcphersonii, One major reason why mycorrhizal symbiosis can help facilita- Leptolaena sp and U. densifolia are quite abundant, whereas tion is its network structure, by way of individual mycelia that col- Sarcolaena sp. seedlings are rare. We looked for species with a onize different plants, sometimes of different species (Selosse et al., potential nurse effect to help re-establish ectomycorrhizal tree 2006; Simard et al., 2012): one plant can thus provide fungal diversity by way of fungal partners. To locate an ideal nurse plant inoculum to another, in the form of already established and sup- to fulfil feature #1, a floristic survey was undertaken to identify ported mycelia. Such facilitation between plants where mycor- plants which spontaneously regenerate in the degraded environ- rhizas mediate a nurse effect has already been demonstrated in ment. Then, the ectomycorrhizal fungi spontaneously associated ectomycorrhizas (Horton et al., 1999; Richard et al., 2009), a kind with trees in different undisturbed sites were identified by molec- of mycorrhizal association common in trees and shrubs in most ular barcoding to check that the potential nurse species share fungi temperate and in some tropical regions (Smith and Read, 2008). with the seedlings (feature #2) and adults (feature #3a) of other This facilitation process implies that nurse and target plants are target species, or that the fungal communities in adults and seed- non-specific and share a large proportion of their ectomycorrhizal lings overlap (features #3b). We thus sought to determine if one of fungal partners. Indeed, this occurs in most cases (Smith and Read, the ectomycorrhizal trees combined all four features, which would 2008) and even host plants associating with a small number of facilitate the installation of other ectomycorrhizal genera in the ectomycorrhizal fungi do harbour generalist fungi, as exemplified framework of ecological restoration. by the genus Alnus (Bent et al., 2011; Bogar and Kennedy, 2013; Roy et al., 2013). Moreover, studies showing a preferential associ- 2. Materials and methods ation between certain fungi and certain plants (Ishida et al., 2007; Morris et al., 2008; Tedersoo et al., 2008, 2010) do not support 2.1. Study site strict specificity. Ectomycorrhizal fungal communities often dis- play fungal sharing between host species in temperate (e.g. Ambatovy mine extends over 1800 ha in Madagascar between Richard et al., 2005; Ryberg et al., 2009) and tropical environments latitudes 18°5201900S and 18°4904700S and between longitudes (Diédhiou et al., 2010; Smith et al., 2011, 2013). Still, the trend may 48°1902200E and 48°1700200E(Fig. 1), at an average altitude of vary among ecosystems, and in a wet Tasmanian sclerophyllous 1000 m above sea level. This is a mosaic of land locally disturbed forest, half the fungal species sampled more than once were only by human practice and primary forests in which 1759 plant species found on a single host species (Tedersoo et al., 2008). were identified, including 209 which are considered to be rare in Our research aims to advance our understanding of the ecology Madagascar (Dickinson and Berner, 2010). The climate is altitudi- of mycorrhizal communities associated with natural forests, so as nal wet tropical with stable temperatures of around 25 °C through- to improve forest management for sustainable ecological restora- out the year. According to the Dynatec Corporation (Canada), tion of disturbed tropical forests. Understanding the structure of annual rainfall is about 1700 mm. More rain falls between Decem- ectomycorrhizal fungal communities associated with a plant com- ber and February, when monthly rainfall exceeds 230 mm. The soil munity may suggest how to use ectomycorrhizal networks to accel- is an eroded oxisol rich in iron, with diverse concretions such as erate ecological restoration after degradation, and especially how to fragments of cuirass (Table 1). It is acidic (pH = 4) and has low min- prepare a fungal community which favors the establishment of tar- eral content, and the minerals present are poorly available. get species. Candidate nurse plants have three ideal features that Although highly diversified, the canopy at the study sites does support the formation of ectomycorrhizal networks: (#1) The nurse not exceed 15 m in height. The vast majority of tree species are plant is a pioneer plant, i.e. able to grow alone on degraded soils arbuscular mycorrhizal, but ectomycorrhizal tree species such as such as bare soils; (#2) It shares some of its ectomycorrhizal fungal A. mcphersonii and U. densifolia are locally dominant, making them partners with seedlings of ectomycorrhizal target species to be important in terms of the functioning in the ecosystem. The shrub reintroduced. Thus, the nurse plant pre-cultivates the fungi stratum is dominated by the non-ectomycorrhizal genera required for the establishment of ectomycorrhizal target species; Gaertnera, Macaranga, Schefflera, Homalium, Tina, Canthium and (#3a) At the adult stage, the nurse plant still shares some fungal Dicoryphe. In areas disturbed by mining activities, grasses like partners with adults of other species present in the original ecosys- Emila citrina (Asteraceae), Ageratum conyzoides (Asteraceae), Bidens tem. Thus, the nurse plant can promote mycorrhizal infection of pilosa (Asteraceae), Sida rhombifolia (Malvaceae) and Clidemia hirta ectomycorrhizal target species at all developmental stages. In cases (Asteraceae), shrubs like Trema orientalis (Cannabaceae), Solanum where more than one species exhibits these three features, the best mauritianum (Solanaceae), Harungana madagascarienis (Hyperi- one shares the most ectomycorrhizal partners with target species. caceae), Psiadia altissima (Asteraceae) and the invasive Lantana Alternatively, (#3b) seedlings and adult plants should share a large camara (verbanaceae) appear first. These species are non- proportion of their ectomycorrhizal communities to ensure the mycorrhizal or arbuscular mycorrhizal (AM). AM inoculum is not presence of fungal partners compatible with adults, even if mature a limiting factor for AM plant establishment because T. orientalis trees were previously absent from the restored forest. and L. camara, which are AM mycotrophic and abundant, may pro- Madagascar is a diversity hotspot (Myers et al., 2000) whose mote AM soil inoculum. rainforests exhibit high biodiversity and a high level of endemism The present study concerned the most abundant ectomycor- (Phillipson et al., 2006), but whose associated mycorrhizal commu- rhizal species in natural forests: U. densifolia, Leptolaena sp., A. nities have been little studied (with the exception of a littoral mcphersonii and Sarcolaena sp. 204 C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211
Fig. 1. Location of the study site (18°4904700Sto18°5201900S and 48°1700200Eto48°1902200E).
Table 1 Ectomycorrhizal plant host and main characteristics of the three investigated sites.
Site number 1 2 3 Date of sampling March 2013 March 2013 March 2014 GPS coordinates Latitude 18°50057.1200S18°51010.5400S18°51010.3700S Longitude 48°18047.1400E48°18057.4100E48°19002.0500E Ectomycorrhizal plant species present as adults Asteropeia mcphersonii xxx Uapaca densifolia xxx Leptolaena sp. xxx Sarcolaena sp. xx Ectomycorrhizal plant species present as seedlings Asteropeia mcphersonii xx Uapaca densifolia xx Leptolaena sp. xx Soil main characteristics Soil particles >2 mm 67% 67% 84% Organic matter 15% 15% 5% pH (eau) 3.7 4.03 4.09 Total Fe (g/kg)⁄ 504 553.88 446.25 Total nitrogen (‰) 3.49 3.35 1.05 Available phosphorus (mg/kg) (Olsen method) 4.4 4.8 2.6
⁄ ICP-AES chemical assay after total solubilization using a mixture of hydrofluoric, nitric and perchloric acids, with silica volatilization, according to NF-ISO 14869-1.
2.2. Characterization of the ability of ectomycorrhizal species to spread the circulation of mining vehicles at Ambatovy. Over 500 m along by seedlings the border zone of each of these pathways, inventory plots were established at each location where a group of seedlings (of any of The seed dispersal ability of ectomycorrhizal trees in the for- the five ectomycorrhizal genera) developed and covered an area est is low since most seedlings grow in the vicinity of an adult with a radius of more than 3 m. In each plot, we made five repli- mother tree. Consequently, seedlings are concentrated in small cate inventories on 0.4 m2 areas that (i) did not overlap (ii) were areas. We investigated which seedlings are spontaneously pre- randomly selected by throwing a circular hoop onto the group of sent and abundant in a degraded environment in order to iden- seedlings. A replicate of a similar study was conducted in the tify candidate nurse plants for use in ecological restoration adjacent forest on the side closest to the regenerating plot, at a (feature #1). To this end, we looked for regeneration in March distance of 10 m from the pathways: in the forest, we made five 2013 and 2014 on the 15 m wide pathways cleared to enable replicate inventories, within a circle with a radius of 3 m. In each C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211 205
0.4 m2 area, all individuals of each ectomycorrhizal genus were reduced to their only known ectomycorrhizal family, Thele- recorded. phoraceae. For all other OTUs, homologies in GenBank allowed affiliations at the family level. OTUs similar to sequences of fungi 2.3. Ectomycorrhizal sampling already found on ectomycorrhizal roots, but without clear taxo- nomic affiliation, were named ‘‘unknown 1” to ‘‘unknown 3”. We investigated the ectomycorrhizal community of adults and seedlings in order to determine the potential overlap between host species and developmental stages (features #2 and #3). In the 2.5. Data analyses established forest, we sampled three 100 m2 square plots (for char- acteristics of the plots, see Table 1). In each plot, 20 ectomycor- The R Vegan package was used for statistical analyses (Oksanen rhizas from at least three different pieces of root were collected et al., 2013; R Development Core Team, 2011). To evaluate the by root tracking (to be sure of the identity of the tree) in at least quality of the sampling effort, a rarefaction curve was calculated three adult individuals per ectomycorrhizal species present at the using the Specaccum function in the R environment. As adults site. For seedlings, five 4 m2 plots containing the most seedlings and seedlings were not harvested at the same sites, to avoid spatial of each species were chosen at each site. In each of these plots, aggregation bias, statistical analyses were performed separately for all ectomycorrhizas were taken from at least four individual seed- them. To test the effect of site, plot and host plant species on the lings per species studied. We were not able to find a site with suf- ectomycorrhizal community associated with an individual tree ficient Sarcolaena sp. seedlings to include this species in the study (seedlings and adults), a stratified multi-response permutation for seedlings. This sampling strategy allowed us to study: (i) fungal procedure (MRPP) was performed using the MRPP function in the sharing between adults of the four species in plots 1 and 3 (feature R environment, a non-parametric procedure to test the hypothesis #2), (ii) fungal sharing between seedlings of the three species stud- that there is no difference between groups (here, a group is com- ied in plots 2 and 3 (feature #3a) and (iii) fungal sharing between posed of all ectomycorrhizal OTUs associated with the same host adults and seedlings in plots 2 and 3 (feature #3b). Ectomycor- species). This procedure calculates a distance matrix between each rhizal root tips were cleaned in tap water, sorted and stored at sample (list of the fungal OTUs associated with one individual tree) 4 °C in a 0.5 mL aliquot tube filled with CTAB buffer (2% using the Chao index and then the average distance between sam- cetyltrimethylammonium bromide, 100 mM Tris–HCl (pH 8.0), ples within each group (delta). The procedure then performs ran- 1.4 M NaCl, 20 mM EDTA) until analysis. dom permutations (n = 999) of samples in each group only within the specified strata. The significance of the test is based 2.4. Molecular analyses on the proportion of deltas from permutations which are lower than the observed delta. The Chao index takes into account species Each root tip was cleaned with distilled water, dried and sub- which were not observed during sampling in a given sample (Chao jected to DNA extraction using a modified protocol with the et al., 2005). To account for the phylogenetic distance between REDExtract-N-Amp Tissue PCR Kit (Sigma–Aldrich, Saint Louis, mycorrhizal community components from each site, plot and host MO, USA), in which each root tip was first placed in only 50 lLof plant species, we used the Unifrac metric distance (Lozupone and extraction buffer and incubated in a thermal cycler set at 91 °C Knight, 2005) based on the similarity between OTUs. To obtain for 11 min. Then, 50 lL of dilution solution was added for storage the Unifrac matrix distance between each pair of individual adult at 4 °C for 24 h and then at 20 °C. The internal transcribed spacer trees, representative sequences of each OTU were aligned using (ITS) region of the rDNA was amplified by PCR of 1.5 lL of each ClustalX 1.81 software (Thompson et al., 1997). Some sequences extract using the primer pair ITS1F (Gardes and Bruns, 1993) and were removed to optimise the recovery length of the set of ITS4 (White et al., 1990) according to the protocol of the sequences used, and the alignment was then manually corrected REDExtract-N-Amp Plant PCR Kit (Sigma–Aldrich, Saint Louis, using GeneDoc software (Nicholas and Nicholas, 1997). A MO, USA) using a thermal cycler (Veriti 96 well, Applied Biosys- neighbour-joining phylogenetic tree was built with Mega 5.05 soft- tems). PCR products were visualized under UV light in 1% agarose ware (Tamura et al., 2011) to test the phylogeny with the bootstrap gel stained with 0.3 mg/L ethidium bromide. Samples which method (1000 bootstrap replications) and using the Kimura yielded multiple PCR products were subjected to a second PCR 2-parameter model. The resulting tree was used to make the using the basidiomycete-specific primer ITS4B (Gardes and abundance-weighted Unifrac distance matrix using FastUniFrac Bruns, 1993) and ITS1F, based on the assumption that root- tools available from unifrac.colorado.edu (Hamady et al., 2010). inhabiting endophytes or contaminants are often ascomycetes, The same procedure as that used for adult trees was used to obtain whereas mycorrhizal fungi are often basidiomycetes (e.g., the Unifrac distance matrix between each pair of individual seed- Tedersoo and Smith, 2013). Both strands were sequenced by lings. Because of individual effects on host tree mycorrhizal com- GenoScreen (Lille, France) using the BigDye Terminator v3.1 kit, munity composition, all sequences belonging to one host tree using each of the two PCR primers. Sequences were edited using species cannot be considered as a homogeneous data set. Conse- ChromasPro v1.7.5 and merged into operational taxonomic units quently, Unifrac jackknife clustering could not be used to test the (OTUs), a proxy for molecular species, based on 97% similarity effect of host species on the phylogenetic composition of mycor- using the CD-HIT suite web server. The taxonomic affiliation of rhizal communities. To test this effect, an MRPP was performed these OTUs was tentatively obtained by a BLASTN search against using the Unifrac distance matrix. The putative grouping of host the National Center for Biotechnology Information (NCBI) data- tree species into clusters was visualized after principal coordinate base. One representative sequence of each OTU was deposited in analysis (PCoA) using the Unifrac distance (Lozupone and Knight, GenBank (Accession Numbers KP754013 to KP754128). When the 2005). To check the ability of A. mcphersonii to share part of its BlastN match was below 90% and did not allow assignment of ectomycorrhizal community with other host trees, sharing the family, a conservative taxonomic assignment OTU was con- between A. mcphersonii ectomycorrhizal communities and other ducted at the order level for Boletales and Agaricales (except, ectomycorrhizal hosts was represented by a bar diagram according respectively, for Sclerodermataceae and Tricholomataceae + to the development of the tree: seedling or adult, and according to Cortinariaceae, for which >90% homologies in GenBank allowed the study site. Similarly, the ability of seedlings and adults of all affiliations at the family level), as well as Sebacinales, Hymeno- ectomycorrhizal hosts to share part of their community at the chaetales, Sordariales and Polyporales. Thelephorales were two sites was represented by Venn diagrams, which were 206 C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211
Table 2 belong to a putative ectomycorrhizal fungal taxon (n = 34). The Abundance of ectomycorrhizal seedlings of the four tree genera in the three open absence of a plateau in accumulation curves (Fig. 2) indicates that areas investigated (A, B and C) and in the adjacent forest areas investigated in 2013 our sampling effort was not sufficient to evidence all OTUs present for spontaneous regeneration. Data from five 0.4 m2 inventories on each area. at each site, so that less frequent species are likely missing. Open areas Corresponding forest Singletons (OTUs found only once in our samples) represented areas 44% of all OTUs, amounting to less than 9% of the total root tip 0 0 0 A BCAB C abundance. Russulaceae and Boletales were the most diverse and Asteropeia mcphersonii 5 (65)a 5 (19) 5 (63) 3 (7) 5 (16) 5 (10) abundant groups at the three sites (Fig. 3). Depending on the site, Uapaca densifolia 0 (0) 0 (0) 0 (0) 2 (2) 4 (13) 4 (5) these two lineages represented between 60% and 90% of total Leptolaena sp. 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (2) abundance and between 41% and 81% of all OTUs. Cortinariaceae Sarcolaena sp. 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) and Thelephoraceae were also abundant (Fig. 3). a Number of 0.4 m2 inventories containing a least one seedling (with number of seedlings found in the sum of all areas from the site in brackets). 3.3. A community depending on spatial effects
An MRPP using Chao indices indicated an effect of site on the mycorrhizal community associated with seedlings (observed delta: 0.8967; expected delta: 0.943; P = 0.001) and with adults (observed delta: 0.8816; expected delta: 0.9228; P = 0.001). An MRPP stratified by site also indicated a plot effect on seedlings col- lected from different plots, (observed delta: 0.7254; expected delta: 0.943; P = 0.001). Phylogenetic trees required to obtain the Unifrac distance matrix were successfully obtained for adult trees on 49 OTUs (out of 85) representing 274 ECM sequences (out of 356) and for seedlings on 38 OTUs (out of 51) representing 160 ECM sequences (out of 194). An MRPP performed using Unifrac dis- tances or the Chao indice produced similar results. A site effect was observed in adult trees (observed delta: 7.751; expected delta: 9.723; P = 0.041) and a plot effect in seedlings (observed delta: 3.482; expected delta: 4.618; P = 0.029). Thus, the mycorrhizal community is strongly affected by spatial effects and statistical analysis testing for the effect of the host species on the mycorrhizal community must be stratified by site for adults and by plot for seedlings.
3.4. A community largely shared between host species Fig. 2. OTU accumulation curve calculated by cumulating all 4 study sites and all 4 host species as adults or seedlings, depending on the number of molecularly A total of 66% of the OTUs obtained from more than one root tip characterized ectomycorrhizas. The order of ectomycorrhizas on the x-axis was drawn at random 1000 times. The curve represents the average of 1000 random were found on more than one host plant species, and represented samples. The envelope around the curve represents the 95% confidence interval of 75% of the total abundance of ectomycorrhizas (83% when single- the actual value. tons were excluded). The 17 most common OTUs were all found on more than one host plant species, and nine of them were found on the four host species. An MRPP using Chao indices (stratified by produced using the function venn.diagram in the VennDia- site for adults and by plot for seedlings) did not show that the ecto- gram package using the R environment (Hanbo, 2012). mycorrhizal communities associated with one individual varied according to its species, among adult trees (observed delta: 3. Results 0.9242; expected delta: 0.9228; P = 0.361) or among seedlings (observed delta: 0.935; expected delta: 0.943; P = 0.514). Similarly, 3.1. Asteropeia mcphersonii can spread by seedlings stratified MRPP using the Unifrac distance did not suggest that the phylogenetic composition of mycorrhizal communities was Along the four transects investigated, we identified only three affected by host tree species for adults (observed delta: 8.537; open areas (A, B and C; Table 2) on which ectomycorrhizal tree expected delta: 9.723; P = 0.145) or for seedlings (observed delta: seedlings regenerated in 2013, and no new area in 2014. While 3.934; expected delta: 4.618; P = 0.124). Therefore, no species dis- seedlings of A. mcphersonii, U. densifolia and Leptolaena sp. were played better sharing of its fungal partners, as confirmed by PCoA identified in at least some 0.4 m2 inventories of the adjacent forest (Fig. 4), which did not group mycorrhizal communities associated areas, only A. mcphersonii was found in 0.4 m2 inventories in open with individual trees or seedlings by host species. areas. We found a total of 147 A. mcphersonii seedlings across open There was thus strong sharing of ectomycorrhizal communities areas A, B and C, but no seedlings of Uapaca, Leptolaena or Sarco- between A. mcphersonii, the candidate nurse plant, and other target laena (Table 2). Because of its ability to grow on bare soil, A. species (Fig. 5). Depending on the species and on the site, adults of mcphersonii was thus judged to a candidate nurse plant. the target species shared 25–33% of OTUs (representing 35–58% of ectomycorrhizas) with A. mcphersonii adults. The trend was even 3.2. A diversified community dominated by Russulaceae and Boletales more pronounced in seedlings of the target species, which shared 35–55% of OTUs (representing 64–79% of ectomycorrhizas) with Among a total of 1857 ectomycorrhizal root tips, 634 provided a A. mcphersonii seedlings (unfortunately, we were not able to docu- tractable DNA sequence, corresponding to 150 OTUs at a 97% ment this for Sarcolaena sp.). For a sampling effort of over 200 ecto- homology threshold. Among these, 35 OTUs (48 sequences) were mycorrhizas (Fig. 6), the percentage decrease in OTUs specific to not used for the analysis described below because they did not one single tree species was weak and tended to a value of ca. C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211 207
Fig. 3. Taxonomic diversity of ectomycorrhizal communities at sites 1, 2 and 3 expressed as taxon number (OTU, left column) or abundance among ectomycorrhizal root tips (myc, right column). ‘‘n”, number of root tips sampled by site.
Fig. 4. PCoA representation of distances between mycorrhizal communities associated with each individual in seedlings (left diagram) and in adults (right diagram). Black circles: A. mcphersonii; gray circles: U. densifolia; empty circles: Leptolaena sp.; crosses: Sarcolaena sp.
65% of the whole OTU diversity for our range of sampling effort. At ectomycorrhizas from adults were formed by fungi also found on sites 2 and 3, designed to compare ectomycorrhizal communities seedlings. At site 3, 87% of ectomycorrhizas found on seedlings between seedlings and adults (Table 2), taking into account only were also found on adult trees (Fig. 7). For the whole set of host mycorrhizas found on Leptolaena sp., U. densifolia and A. mcpher- species, there was therefore strong sharing of ectomycorrhizal sonii, which were all present as adults and seedlings, the propor- communities between seedlings and adults. tion of OTUs shared by at least two host species was 33% (67% as ectomycorrhizas) for seedlings and 27% (55% as mycorrhizas) for adults. 4. Discussion
4.1. A high specific richness in spite of extreme soil conditions 3.5. A community largely shared between developmental stages We are aware of only one similar molecular analysis of an ecto- At sites 2 and 3, the difference in the success rate of molecular mycorrhizal community in Madagascar, in a southern lowland characterization showed unbalanced sampling between seedlings rainforest on sandy neutral soil (Tedersoo et al., 2011), where 46 and adults. At site 2, 101 ectomycorrhizas from adult trees and OTUs were identified on Asteropeia sp. In our study, we observed 154 from seedlings (+52%) were successfully characterized. At site 116 OTUs on four host species from 587 successfully sequenced 3, 211 ectomycorrhizas from seedlings and 304 from adult trees root tips, confirming that ectomycorrhizal fungal communities (+44%) were successfully characterized. At site 2, 88% of can be highly diversified in this tropical ecosystem. A similarly 208 C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211
Fig. 5. Fungal sharing between target species on the one hand (Uapaca, Leptolaena,orSarcolaena) and Asteropeia on the other hand, at the different sites, in seedlings (A) and in adults (B). Sharing is expressed as the percentage of shared OTUs (OTU) and as the percentage of ectomycorrhizas belonging to shared OTUs (myc).
high number of OTUs was associated with (i) three legume trees in a tropical forest in Guyana (118 OTUs for 1020 mycorrhizal root tips; Smith et al., 2011); (ii) more than 40 ectomycorrhizal tree species in a Malaysian dipterocarp rainforest (146 OTUs for 589 mycorrhizal root tips; Peay et al., 2010); (iii) three tree species in a Tasmanian dry sclerophyllous forest (123 OTUs; Tedersoo et al., 2008); (iv) two sympatric Californian Quercus species (140 OTUs for 3072 mycorrhizal root tips; Morris et al., 2008); and (v) Mediterranean Quercus ilex and Arbutus unedo (140 OTUs for 558 mycorrhizal root tips; Richard et al., 2005). However, we found many more OTUs than Tedersoo et al. (2010), who found only 38 OTUs in an Amazon rainforest despite a large sampling effort (60 root samples; 15 15 cm and down to 10 cm in depth) and strong host diversity. Interestingly, in the Amazon forest, ectomycorrhizal trees were grouped in small discontinuous patches located far away from each other, thus limiting the spread of fungi which propagate by mycelial growth (Peay et al., 2007). In the forest we studied, ectomycorrhizal trees are abundant and scattered throughout the forest, allowing fungi to grow irrespective of their exploration strategy or mode of dispersal. As is often the case in ectomycorrhizal species-rich assemblages (Morris et al., 2009; Smith et al., 2011; Tedersoo et al., 2008), the accumulation curve Fig. 6. Proportion of OTUs collected on a single host species as a function of the (Fig. 2) does not reach a plateau, suggesting that additional inven- sampling effort, calculated by cumulating all 4 study sites and all 4 host species as tory effort would reveal new, but rare, OTUs. adults or seedlings. The order of appearance of ectomycorrhizas on the x-axis was Our observations were made in the context of extreme soil con- drawn at random 1000 times. Each circle represents a random draw and the red ditions (Table 1). The pH was similar at the three study sites ( 4), curve represents the average of 1000 random re-samplings. (For interpretation of the references to color in this figure legend, the reader is referred to the web version and, as a consequence, it was not possible to establish any positive of this article.) or negative correlation between species richness and pH. However, C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211 209
Fig. 7. Venn diagrams comparing fungal communities between seedlings and adults, expressed as taxon number (OTU, left column) or abundance among ectomycorrhizal root tips (ectomycorrhizas, right column), at site 2 (top) and site 3 (bottom). Each diagram pools the four species studied, and the size of the circles is proportional to the total number of OTUs or ectomycorrhizal root tips.
this study showed that highly acidic, iron-rich and nutrient-poor lineage, almost absent from our data, is one of the most abundant soil can support a rich ectomycorrhizal community. This result is (Smith et al., 2011, 2013; Tedersoo et al., 2010). in agreement with the results of other studies which showed that serpentine soils, which are also nutrient-poor and rich in heavy metals, support diversified ectomycorrhizal fungal communities 4.3. Preponderance of generalist fungi (Branco and Ree, 2010; Moser et al., 2008). The impact of pH on ectomycorrhizal community species richness is not well under- Our study showed that there was no effect of host plant species stood. In plantations of Eucalyptus with soil pH ranging from 4.1 on the mycorrhizal community associated with an individual tree. to 6.6, soil acidity was positively correlated with species richness We can thus be certain that most OTUs in primary forests are able as estimated from fruit body surveys (Lu et al., 1999). However, to colonize many if not all host plants, and can thus be considered no change in the species richness of ectomycorrhizal communities as generalists. In this study, we showed that the proportion of gen- on root tips was recorded in acidic hardwood forests when the pH eralist OTUs increases very slowly with sampling effort: 34% of of the soil was experimentally increased from 4.4 to 5.9 (Kluber generalist OTUs for 200 ectomycorrhizas sampled, to 37% for 587 et al., 2012). Yet, at our three study sites, pH was below 4.1, and ectomycorrhizas sampled (Fig. 6). This means that when increasing in these conditions inorganic phosphorus binds to Al3+ ions and the sampling effort, the emergence of new OTUs (which are neces- is consequently largely unavailable for plants. Although available sarily initially present on only one tree genus) is nearly compen- phosphorus at low concentrations affects the composition of ecto- sated for by the discovery of rare OTUs on other tree species mycorrhizal communities (Morris et al., 2008), it does not appear (these OTUs thus no longer look specific). The abundance of rare to reduce their richness (Kluber et al., 2012), and our study con- OTUs (Fig. 2) therefore means that a tremendous sampling effort firms this result. Moreover, the soils we studied resulted from would be necessary to provide direct evidence of the sharing of the progressive erosion of an old substrate, which gave time for ectomycorrhizal communities between tree species. Such a sam- adaptive evolution to occur. One major conclusion of our work is pling effort can only be achieved using next-generation DNA thus that very low pH and available phosphorus in soils allow high sequencing. Thus, in the present study, the preponderance of gen- species richness of ectomycorrhizal fungi. eralist fungi is shown by the absence of an effect of host plant spe- cies on the mycorrhizal community. Yet shared OTUs were also the most frequently sampled because a fungus associated with many genera of host plants has a greater chance of being harvested. 4.2. An ectomycorrhizal fungal community typical for African tropical But we can also hypothesize that generalist fungi are actually more rainforests common because it gives them a competitive advantage (Selosse et al., 2006), because of increased resilience to the loss of one host The ectomycorrhizal fungal communities in our study were species, e.g. after disturbances such as cyclones, which are partic- dominated by Russulales, Boletales and, to a lesser extent, by ularly common in this region. Fungi able to colonize different host Thelephoraceae and Cortinariaceae. This is in full accordance with species have an increased likelihood of finding a host tree after a published observations in African tropical rainforests (Diédhiou disturbance, at the beginning of the next succession. Beyond adap- et al., 2010; Tedersoo et al., 2007, 2011) but differs from what tive reasons, it is likely that the molecular modality of plant–fungal has been described in rainforests in Guyana, where the Clavulina interactions (which is poorly understood in ectomycorrhizas) 210 C. Henry et al. / Forest Ecology and Management 358 (2015) 202–211 targets the universal mechanisms present in most plants and fungi hypothesis, facilitation can offset competition in the harsh condi- that evolved ectomycorrhizal ability and thus entails tions plants will face during mine site ecological restoration. non-specificity as a default state in lineages commutating to an ectomycorrhizal niche. 5. Conclusion Similar results have been recorded in most multi- ectomycorrhizal host tropical ecosystems, where ectomycorrhizal We show that one of the four plant species studied, fungi associated with more than one host represent the majority A. mcphersonii, can grow in bare soils and open areas (feature of mycorrhizal abundance (Diédhiou et al., 2010; Smith et al., #1). A. mcphersonii thus fulfilled the four requirements defined in 2011). But contrary to Ishida et al. (2007) and Tedersoo et al. the introduction. A. mcphersonii seedlings share a large proportion (2010), who showed strong host preference for some fungi, we of their ectomycorrhizal partners with seedlings of the other ecto- did not detect any influence of the host species on ectomycorrhizal mycorrhizal target species (feature #2). Its introduction in early community composition. Moreover, our study indicates that in this ecological restoration could pre-cultivate ectomycorrhizal fungi, tropical forest, generalist fungi dominate on both seedlings and which will then be able to colonize target ectomycorrhizal species adult trees. As Diédhiou et al. (2010) observed at sites where seed- to be reintroduced at the site. As A. mcphersonii adults also share a lings and adults co-occurred, the proportion of generalist OTUs large proportion of their ectomycorrhizal partners with other adult (OTUs present on at least two species at the same development ectomycorrhizal species (feature #3a), the process of facilitation stage) was higher on seedlings than on adults. However, the indi- between species would continue throughout plant succession. vidual effect in the sample has to be disentangled from the species Finally, as the same ectomycorrhizal fungi colonise seedlings and effect to avoid confusing preferential association with one or more adults (feature #3b), fungi which colonise the four seedling species individuals and preferential association with a species. In other at the beginning of restoration would thereafter be able to colonise words, the proportion of generalists can be compared between adult trees during forest ageing and thereby counterbalance the seedlings and adults only if the individual effect is the same in each initial lack of adult host trees in the restored forest. This forest case. When the ratio of the average number of mycorrhizas sam- ecology research enables us to develop jointly with Ambatovy pled per individual to the number of individuals sampled is high, new tools for forest management, in the framework of ecological the individual effect is greater. Here, this ratio was higher for adult restoration. trees than for seedlings because the number of ectomycorrhizas on seedlings was much lower, making the difference in specificity Acknowledgements between seedlings and adults difficult to compare (an apparent increase in the proportion of shared OTUs with age may simply The authors wish to thank the project ANR-12-ADAP-0017 reflect a reduction of the individual effect). ADASPIR, the Doctoral School SIBAGHE and AgroParisTech for In greenhouse experiments, it has been demonstrated that funding C.H’s travel expenses. They thank members of the ‘‘Envi- when two host plants grow close to each other, specific fungi can ronment” team of the Ambatovy mine for their kind and efficient colonize both species if they have already massively colonized technical help, for providing logistics and accommodation during the usual host (Bogar and Kennedy, 2013; Massicotte et al., each field trip. They thank David Marsh and Daphne Goodfellow 1994). Thus, in the slightly disturbed forests under study, the for providing language help. Finally, they thank Fabien Buissart abundance of and proximity between host tree species may favor for help in preparation of Fig. 3. M.-A. S. was funded by internal the strong sharing we observed within ectomycorrhizal communi- grants from the Institut de Systématique, Évolution, Biodiversité ties in both adults and seedlings. (UMR7205). Finally, in our study, seedlings and adults shared a large part of their ectomycorrhizal community (feature #3b). 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