Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant
Gilbertiodendron dewevrei rain forest in Cameroon
Helvyne C. Micha€
*, Adrien Taudi
, Nogaye Niang
, Cheikh Ndiaye
, Mathieu Sauve
ee Onguene Awana
e De Kesel
, Seynabou S
, Abdala G. Di
, Violette Sarda
, Omar Sadio
*,and Amadou M. B^
Laboratoire Commun de Microbiologie, IRD/UCAD/ISRA, BP 1386 Dakar, S
Institut de Syst
e (ISYEB –UMR 7205 –CNRS MNHN, UPMC, EPHE), Mus
eum national d’Histoire naturelle,
es, 57 rue Cuvier, CP50, 75005 Paris, France
UMR 5175, CEFE –CNRS –Universit
e de Montpellier –Universit
e Paul Val
ery Montpellier –EPHE, Montpellier, France
Department of Plant Taxonomy and Nature Conservation, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
Laboratoire des Symbioses Tropicales et M
eennes, UMR113- INRA/AGRO-M/CIRAD/IRD/UM2-TA10/J, Campus International de
Baillarguet, 34398 Montpellier Cedex 5, France
Laboratoire de Biologie et Physiologie V
e des Sciences Exactes et Naturelles, Universit
e des Antilles, BP 592, 97159
a-Pitre, Guadeloupe, France
Soil, Water and Atmosphere Department, Institute of Agriculture Research for Development, BP. 2123 Yaound
Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Botanic Garden Meise, Nieuwelaan 38, BE-1860 Meise, Belgium
IRD, UMR 195 LEMAR (UBO/CNRS/IRD/Ifremer), BP 1386, CP 18524 Dakar, S
Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests
and are often invoked as a potential contributor to monodominance. We identiﬁed ectomycorrhizal fungi in a monodominant Gilbertio-
dendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermedi-
ate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic
N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps)
than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations sug-
gest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the ﬁrst report of the Tuber-Hel-
vella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal
communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational
taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on
root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no
evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the
G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.
Abstract in French is available with online material.
N; Caesalpinioideae; common ectomycorrhizal network; ectomycorrhiza; Fabaceae subfamily; internal transcribed spacer; sporocarps.
MANY TREES FORM ECTOMYCORRHIZAL SYMBIOSES WITH DIVERSE
BASIDIOMYCOTA AND ASCOMYCOTA, which play a key role in many
tropical forests (B^aet al. 2012), affecting tree growth and nutrient
absorption as well as protection against pathogens. These associa-
tions also produce sporocarps of ectomycorrhizal (EM) fungi,
many of which are of economic interest (Yun & Hall 2004). Most
known EM tree species are found in temperate and boreal
regions (Smith & Read 2008, Van Der Heijden et al. 2015).
Despite recently increasing interest (B^aet al. 2012), tropical EM
associations are poorly understood, particularly in monodominant
Monodominant forests are large stands in which a single tree
species comprises more than 60 percent of canopy-level trees
(Torti & Coley 1999, McGuire 2007). Factors that create and
maintain monodominant forests within a matrix of otherwise
high-diversity tropical rain forests remain unexplained (Peh et al.
Received 22 February 2016; revision accepted 25 July 2016.
*These authors equally contributed to this work.
Corresponding author; e-mail: email@example.com
ª2016 The Association for Tropical Biology and Conservation 1
BIOTROPICA 0(0): 1–12 2016 10.1111/btp.12415
2011). The observation that monodominant forests typically con-
tain ectomycorrhizal trees has led to the hypothesis that EM
fungi, supported by adult trees, could facilitate the establishment
of seedlings growing under limited light, thereby competitively
excluding non-EM seedlings (Torti & Coley 1999, McGuire
2007). Enhanced establishment and survival of EM seedlings
near mature EM trees have been reported in monodominant or
mixed stands (Newbery et al. 2000, Onguene & Kuyper 2002,
McGuire 2007). In some cases, seedlings are integrated into an
EM fungal network already supported by mature trees (Selosse
et al. 2006, McGuire 2007, Corrales et al. 2016a). Recently, a
Panamanian monodominant species (Oreomunnea mexicana, Juglan-
daceae) was shown to have potential for such common mycor-
rhizal networks (CMNs) (Corrales et al. 2016a). Although some
aspects remain controversial, CMNs are known to drive nutrient
transfers (including carbon) between plants in some conditions
(Selosse et al. 2006, Teste et al. 2010, Klein et al. 2016). Plants
receiving carbon from CMNs are found in temperate and some
tropical ecosystems, and the compounds received from EM fun-
gal partners usually increase their natural
C abundance (Selosse
& Roy 2009). These plants also show high N content and
abundance, indicating modiﬁcations of their nitrogen nutrition,
although the mechanism for this remains poorly understood
(Selosse & Roy 2009, Selosse et al. 2016). Candidates for nutrient
transfer can thus be detected by isotopic analyses. However, iso-
topic analyses of seedlings in a mixed Guinean EM forest where
adults and seedlings shared most EM fungi failed to detect
resource transfer to seedlings (Diedhiou et al. 2010). To the best
of our knowledge, resource transfer has not yet been tested in
monodominant tropical rain forests.
The Fabaceae timber tree Gilbertiodendron dewevrei (De Wild.)
J. Leonard of the subfamily Caesalpinioideae (hereafter caesalpin-
ioids) provides a case of almost monospeciﬁc forest stands
(>90% of the canopy trees) on >1000–5000 ha patches in the
Congo basin (Letouzey 1985, Hart 1995, Peh et al. 2011), where
this species dominates in all subcanopy age categories (Hart
1990, 1995). These stands harbor diverse EM sporocarps (e.g.
Buyck 1993, 1994, 1997, Verbeken & Walleyn 2010). Fassi (1960)
described ectomycorrhizae (ECMs) on G. dewevrei, but the distri-
bution of EM fungal communities on roots of the different age
categories of G. dewevrei remains unknown.
Although EM fungi have been identiﬁed on G. ogoouense and
Gilbertiodendron sp. roots from mixed Cameroon forests (Tedersoo
et al. 2011), our study is the ﬁrst to investigate aboveground and
belowground EM fungal communities of G. dewevrei and their
potential role on seedlings. Gilbertiodendron dewevrei maintains high
densities of seedlings, suggesting that established trees may inocu-
late seedlings, and perhaps even provide carbon resources
through CMNs (Onguene & Kuyper 2002, McGuire 2007). To
identify the potential for CMNs between adult trees and seed-
lings, we determined the EM fungal communities of different age
categories by performing ECM barcoding and sampling sporo-
carps to increase the data set and to obtain speciﬁc references
for barcodes. We investigated the following: (1) if trees and seed-
lings share EM fungal taxa; (2) if the composition of the EM
fungal communities varies among different age categories; and (3)
if EM fungi provide carbohydrates and nitrogen to G. dewevrei
STUDY AREA.—We conducted this project in Southeast Cameroon
during the rainy seasons of 2009–2012 (Fig. S1). We located our
study area within an 8 ha forest near Nkondon I. Annual rainfall
averaged 1512 mm and fell mainly from August to November
near our study area (Meyibot I: Djuikouo et al. 2010). The area is
densely forested with G. dewevrei, which comprises 81.1 percent
of the basal area and densely regenerates (Djuikouo et al. 2010).
We randomly located three representative sites (0.5-ha per
site) within the 8 ha forest: S1 (2°49002.400N, 13°56054.800E), S2
(2°49030.600N, 13°56040.200 E), and S3 (2°49053.200N, 13°56018.200
E). Sites S1 and S2 were 970 m apart, while sites S2 and S3 were
977 m apart. Soil properties are analyzed in supporting informa-
tion (Table S3). A single individual of EM Uapaca sp. (at S1) and
several individuals of Carapa procera (a non-EM Meliaceae, Bereau
et al. 1997, Wang & Qiu 2006), Irvingia gabonensis (a non-EM
Irvingiaceae, Onguene & Kuyper 2001), and Pentaclethra macro-
phylla (a non-EM Fabaceae, Henkel et al. 2002) co-occurred with
G. dewevrei. We divided each site into ﬁve rectangles of
100 m 910 m in which we identiﬁed G. dewevrei individuals
from three main age categories: seedlings, intermediate trees, and
large trees (Table S1).
SAMPLING STRATEGY AND MORPHOTYPING OF ECMS.—At each site,
we sampled ECMs from six large trees, six intermediate trees,
and 48 seedlings during the rainy season of 2010. Sampled trees
were at least 8 m apart. For each large or intermediate tree, eight
soil cores were taken at 1–2 m from the base of the tree trunk
by inserting a 15 cm diameter soil corer to a depth of 30 cm (ap-
proximately 300 g of fresh soil remained attached to the roots) at
sites locally devoid of seedlings. We randomly chose seedlings (6–
15 leaves, and height <1 m) and fully harvested them by digging
with a spade (20 cm width and 28 cm depth) to recover approxi-
mately 300 g of fresh soil. All cores were stored in a cooler for
<5 days before being processed. Thus, we evaluated 144 cores
containing the roots of large trees, 144 cores containing these of
intermediate trees, and 144 root systems of seedlings.
We gently washed all root systems over a 250-lm sieve
under running tap water, dispersed them in a dish of water, and
examined them at 109magniﬁcation under a dissecting micro-
scope for EM root tips. EM root tips were classiﬁed into mor-
photypes according to Thoen and B^a (1989). We counted the
numbers of ECMs and noncolonized root tips to determine the
percentage of EM colonization. In total, we morphotyped 49,888
We maximized the detection of EM fungal diversity at the
barcoding step by choosing representatives of different morpho-
types: we subsampled for molecular analyses 15 EM root tips
from large and intermediate trees and two EM root tips per seed-
ling (i.e., 96 EM root tips per site) by maximizing the number of
ella Ebenye et al.
morphotypes represented. These 828 samples were stored in
CTAB buffer according to Sene et al. (2015).
DNA EXTRACTION AND SEQUENCING.—We extracted sporocarp
(Table S2) and ECM DNA using an Extract-N-Amp
PCR Kit (Sigma-Aldrich, St. Louis, U.S.A.). We performed PCR
ampliﬁcation and sequencing of the internal transcribed spacers
and 5.8S region (ITS) of the ribosomal DNA with primers ITS1-
F and ITS4, or otherwise ITS1-F and ITS4B as in Sene et al.
(2015). We edited sequences using CodonCode Aligner v.4.1.1
(LI-COR, Inc., MA). We partitioned sequences (accession num-
bers KR819005 to KR819138) into operational taxonomic units
(OTUs) by grouping sequences with more than 97 percent simi-
larity level. In the following section, ‘species’describe fungal taxa
identiﬁed from sporocarps, and ‘OTUs’describe taxa identiﬁed
by barcoding. OTUs were identiﬁed at the species level when a
sequence presented more than 97 percent full-length similarity to
(i) sequences derived from sporocarps in this study or (ii) well-
identiﬁed sequences from the GenBank database using the
BLAST-N algorithm. We identiﬁed OTUs at the genus or family
level based on BLAST-N results. Taxa identiﬁed from sporocarps
and root tips were assigned to the phylogenetic lineages of EM
fungi deﬁned by Tedersoo et al. (2010a). We considered Helo-
tiales to be EM fungi, although some debate exists in this
respect, and some species may not be EM; Sordariales and Euro-
tiales are likely not EM taxa but were taken into account since
our knowledge of EM taxa remains limited in African tropical
NANALYSES.—We investigated the natural
N abundance in seedlings to assess, by comparison with older
autotrophic plants or EM fungi, whether they showed any iso-
topic enrichment that may reﬂect the gain of compounds from
CMNs. At each site in 2010, we harvested ﬁve leaves from large
trees, intermediate trees, and seedlings of G. dewevrei,aswellas
large trees of Pentaclethra macrophylla, an arbuscular mycorrhizal
(AM) tree, as a control for autotrophic biomass. We also col-
lected G. dewevrei seeds to determine the isotopic abundance of
reserves that contribute to early seedling growth, as well as n=3
replicates taken from the pileus of sporocarps of ﬁve fungal spe-
cies (Clavulina sp.2, Lactiﬂuus longipes,Russula brunneoderma,Russula
sp.1, and Scleroderma sp., Table S4) to estimate the isotopic abun-
dance of the fungal biomass. All leaves were from similar light
conditions and distance to soil and therefore had comparable
photosynthetic conditions and CO
sources. Isotopic abundances
were measured as in Tedersoo et al. (2007) and expressed in d
N values in parts per thousand relative to international
standards (respectively, V-PDB and atmospheric N
STATISTICAL ANALYSES.—We performed one-way ANOVA with
XLSTAT2010 software (Addinsoft SARL, Paris) to compare (i)
soil properties at the three sites and (ii) stable isotope abundances
between ectomycorrhizal trees, arbuscular mycorrhizal trees,
seeds, and EM sporocarps. Data were normally distributed. Sig-
niﬁcant differences between pairs were revealed by a Newman-
Keuls test, which is more sensitive to differences than a post-hoc
Tukey test (Abdi & Williams 2010). Species-accumulation curves,
estimates of species richness (ﬁrst-order jackknife), and diversity
indices (Fisher’s alpha, Shannon index) were inferred using Esti-
mateS software version 9.1.0 (Colwell 2013). We assessed the
effects of site, age category, and their interaction on the EM fun-
gal community composition via perMANOVA (permutational
multivariate analysis of variance, Anderson 2001) analysis, using
the function Adonis from the R package Vegan 2.2-1 (Oksanen
et al. 2015). Due to high inter-individual variance, we merged
individual fungal communities by site and age. We, therefore, per-
formed NMDS on the community matrix with OTUs as rows
and nine communities (three sites *three ages). For each signiﬁ-
cant factor detected by perMANOVA (site and tree category), we
computed pairwise perMANOVA (Anderson 2001) on balanced
subsamples (e.g., to test for the difference in EM fungal composi-
tion between sites S1 and S2, we sampled the maximum number
of individuals at each site while keeping the number of samples
per site equal). The relative abundance of EM fungal taxa was
calculated as the ratio of the number of ECMs or sporocarps of
a given taxon over the total number of ECMs or sporocarps per
plot and site. Relative frequency of EM fungal lineages was calcu-
lated as the ratio of the number of occurrences of a given lineage
over the total occurrence of lineages per plot and per site. The a
type I error was set at 5 percent.
SPOROCARP IDENTIFICATION.—The 1030 sampled sporocarps
(Tables 1 and S2) fell into 11 EM fungal lineages proposed by
Tedersoo et al. (2010a), including 10 from Basidiomycota and 1
from Ascomycota (Fig. 1): /russula-lactarius-lactiﬂuus contributed
the most species (21), followed by /boletus (5), /amanita (5), /
cantharellus (5), /clavulina (4), /ramaria-gautieria (2), /cortinarius
(1), /pisolithus-scleroderma (1), /thelephora-tomentella (1), /tri-
choloma (1), and /tuber-helvella (1). Depending on the site, 5–9
lineages were fruiting during our surveys. The most fruiting spe-
cies were found in /russula-lactarius-lactiﬂuus, followed by /bole-
tus (Fig. S2). Morphological and molecular identiﬁcation
(GenBank accessions in Table S4) showed 59 species dominated
by Russula concolora Buyck, R. diffusa Buyck, Russula sp.16, Russula
sp.15, Boletaceae 4, Russula sp.5, Lactiﬂuus sesemotani (Beeli) Buyck,
and Russula sp.2, each contributing more than 3 percent of the
total abundance of sporocarps (Fig. 2). At the species level, the
communities revealed nonuniform composition for the three
sites, with 14–34 EM fungal species recorded per site (Fig. 2).
The number of EM fungal species that occurred in two sites ran-
ged from 6 (S1 and S2) to 12 (S1 and S3). The 55 EM fungal
species collected in 2010 (when above- and belowground sam-
pling can be compared) were similar in number to the other sam-
pling years (52, 51, and 50 species in 2009, 2011, and 2012,
respectively; Table S2). Of the 55 species collected in 2010, /rus-
sula-lactarius-lactiﬂuus (25 species) was most prevalent, followed
by /boletus (12), /amanita (4), /cantharellus (4), /clavulina (4), /
ramaria-gautieria (2), /cortinarius (1), /pisolithus-scleroderma (1),
Ectomycorrhizal fungi of G. dewevrei 3
TABLE 1. Number of EM root tips, sporocar ps and OTUs, percentage of mycorrhizal colonization, species richness, and diversity indices from G. dewevrei seedlings (S), intermediate trees (T), and large trees (LT) in a monodominant
forest in Cameroon.
Site 1 Site 2 Site 3 All sites
S T LT S T LT S T LT S T LT
Total No. of
EM root tips
8604 6607 9420 4681 3919 3789 5803 3455 3610 19,088 13,981 16,819
92.9% a 83.4% b 87.6% c 91.3% a 85.3% b 94.2% ab 94.5% a 88.1% b 85.5% b 92.9% a 85.0% b 88.5% b
No. of EM root
101 93 101 91 92 86 81 98 85 273 283 272
No. of sequences 78 81 72 72 67 61 74 59 65 224 207 198
No.ofOTUs3345 382442 332941 35656863
29.2 2.4 38.5 2.6 33.5 4.4 21.7 2.9 38.7 4.2 32.5 2.5 25.5 3.1 41.0 5.0 33.8 1.7 62.0 3.8 67.3 2.5 63.0 2.2
43.2 3.2 60.0 3.7 54.8 3.6 32.8 3.0 63.9 3.8 46.7 3.3 40.0 3.3 69.5 3.8 46.9 3.2 85.9 4.6 84.1 4.0 76.9 3.6
3.1 0.1 3.6 0.0 3.3 0.1 2.6 0.1 3.5 0.0 3.4 0.0 2.9 0.1 3.6 0.0 3.4 0.0 3.7 0.0 4.0 0.0 4.0 0.0
alpha per age
23.0 5.1 48.6 12.3 32.7 7.7 12.5 2.6 48.9 12.4 29.7 6.8 17.1 3.7 59.6 15.9 33.2 7.8 31.9 3.6 36.0 4.1 30.9 3.5
alpha per site
35.01 5.49 33.45 5.55 31.90 5.35 22.20 3.03
148 74 172 394
29 13 33 55
12.7% 9.2% 19.0% 65.3%
44.8% 46.2% 33.3% 27.3%
At each site, percentages followed by different alphabets differ signiﬁcantly (P<0.05).
Number of OTUs rareﬁed at n=59 sequences for each site and n=198 sequences for all sites, respectively.
Collected in 2010 (year where above- and belowground samplings can be compared).
Percentage of OTUs occurring on seedlings, intermediate trees, and large trees at each site.
Percentage of OTUs found belowground corresponding to species forming sporocarps at each site.
ella Ebenye et al.
/thelephora-tomentella (1), and tricholoma (1), and (Table S2).
Species-accumulation curves reached an asymptote (Figs. S3 and
S4A), indicating that the sporulating EM fungal diversity was well
recovered at all sites.
MOLECULAR IDENTIFICATION OF EM FUNGI ON ROOT TIPS.—Over
all sites, 49,888 EM root tips were observed from seedlings,
intermediate trees, and large trees. The percentage of EM roots
ranged from 83.4 to 94 percent, depending on age category
(Table 1). Of the 828 EM root tips subsampled for DNA analy-
sis, 629 (76%) were successfully sequenced and revealed 75 dis-
tinct OTUs (Table 1) from 11 different EM fungal lineages
(Fig. S5). The 75 OTUs included 68 Basidiomycota (90.7% of
the total abundance of OTUs) and 7 Ascomycota (9.3%, Fig. 3).
Among basidiomycetes, /russula-lactarius-lactiﬂuus contributed
the most (40% of all OTUs), followed by /thelephora-tomentella
(23%), /sebacina (12%), /clavulina (8%), /amanita (2.5%), /bole-
tus (2.5%), and /cantharellus (2.5%). Among ascomycetes, the
most highly represented lineages were /marcelleina-peziza gerardii
(4%), /sordariales (2.5%), /elaphomyces (1.5%), and /helotiales
(1.5%). The abundance of OTUs on root tips varied among sites
(Fig. S6): among OTUs representing >5 percent of the total num-
ber of tips, Thelephoraceae 2 dominated S1 (7.3%), Russula sp.5,
Thelephoraceae 1 and Thelephoraceae 2 dominated S2 (10.5%,
10.5%, and 6.5%, respectively), and Russula sp.16, Russulaceae
11, Russula sp.13, and Sebacinaceae 2 dominated S3 (8%, 6.5%,
5%, and 5%, respectively) (Figs. S6 and 3). Overall, 15 OTUs
(20%) matched species found as sporocarps: nine species from
/russula-lactarius-lactiﬂuus, three from /clavulina, one from /
amanita, /boletus, and /thelephora-tomentella. The similarity
between belowground and aboveground EM fungal taxa at each
site ranged from 33.3 to 46.2 percent (Table 1). When consider-
ing OTUs from all sites and all samples, the species-accumulation
curves reached an asymptote, suggesting that the EM fungal
community was exhaustively sampled at this scale (Fig. S4A). We
detected a total of 119 EM fungal taxa from EM root tips and
sporocarps (Table S4).
ECTOMYCORRHIZAL DISTRIBUTION AMONG STUDIED SITES.—The
number of OTUs detected on root tips was 71, 65, and 63 at S1,
S2, and S3, respectively (Table 1; Fig. S6). Species-accumulation
curves indicate that the EM fungal community was not exhaus-
tively sampled at each site and that there were no signiﬁcant dif-
ferences in the EM fungal species richness among sites
(Fig. S7A). This contrasts with the accumulation curves for
sporocarps, which suggest that although S1 and S3 had similar
diversity, S2 had less than half diversity (Fig. S7B). Although the
three sites shared 69.3 percent (52/75) of OTUs on roots, com-
position differed markedly among sites (df =628, F=2.99,
P=0.001) based on perMANOVA (Table 2). The NMDS
FIGURE 1. Some EM sporocarps harvested under the three age categories of Gilbertiodendron dewevrei: (A) Cantharellus congolensis Beeli, (B) Cantharellus rufopunctatus
var. rufopunctatus (Beeli) Heinem., (C) Clavulina sp.1, (D) Gomphaceae sp.1, (E) Lactiﬂuus pelliculatus (Beeli) Buyck, (F) Lactarius melanogalus Heim., (G) Lactiﬂuus long-
ipes (Verbeken) Verbeken, (H) Lactiﬂuus sesemotani (Beeli) Buyck, (I) Pulveroboletus aberrans Heinem. & Goss.-Font., (J) Russula concolora Buyck, (K) Russula diffusa Buy-
ck, (L) Russula sp.4, (M) Russula subﬁstulosa var. apsila Buyck, (N) Thelephoraceae sp., (O) Tylopilus sp.3, (P) Xerocomus virescens.
Ectomycorrhizal fungi of G. dewevrei 5
ordination (stress =0.09) showed an incomplete separation of
communities based on site (Fig. 4B).
ECTOMYCORRHIZAL COMPOSITION BETWEEN LARGE TREES,
INTERMEDIATE TREES,AND SEEDLINGS.—The percentage of EM
root tips was signiﬁcantly higher for seedlings than for other age
categories at all sites (except for large trees at S2, Table 1).
Ectomycorrhizal fungal species-accumulation curves per age cate-
gory did not reach an asymptote, suggesting that the EM fungal
community was not exhaustively sampled in the three age cate-
gories of trees at each site (Fig. S7A), or even when pooling all
sites (Fig. S4B). In all, we detected 65, 68, and 63 OTUs on
seedlings, intermediate trees, and large trees, respectively
(Table 1). The three age categories shared 49 OTUs, representing
FIGURE 2. Abundance distribution of EM species found among sporocarps collected over 4 years at each site.
FIGURE 3. Abundance distribution of OTUs on EM root tips of G. dewevrei seedlings, intermediate trees, and large trees at all sites.
ella Ebenye et al.
65.3 percent of the diversity found on roots (Table 1; Fig. S8),
and all fungal lineages detected were shared except/helotiales,
which occurred only on seedlings and intermediate trees at all
sites (Fig. S5). S3 had the highest proportion of OTUs shared
between the three tree age categories (19%), followed by S1
(12.7%) and S2 (9.2%, Table 1). The most abundant OTUs,
Thelephoraceae sp.2, Russula sp.5, Russula sp.16, and Russulaceae
sp.11, were associated with the three age categories at all sites
(Fig. 3). Our analyses of diversity (Table 1) revealed homoge-
neous patterns, with no difference among tree categories for
Fisher’s alpha (ANOVA: df =55, F=0.97, P=0.385; Table 1)
in accordance with the species-accumulation curves (Fig. S4B).
However, there was a signiﬁcant difference for Shannon diversity
index (ANOVA: df =55, F=4.002, P=0.024) among tree cat-
egories (intermediate tree: 1.83 0.33; large tree: 1.69 0.38;
seedlings: 1.40 0.67). Using pairwise t-test with P-value adjust-
ment, the only signiﬁcant difference in diversity was found for
seedlings against intermediate tree (t-test: t=2.529, P=0.022).
There is a weak but signiﬁcant effect of the tree age categories
(perMANOVA: df =628, F=2.16, P<0.001) in the EM fun-
gal community composition (Table 2) reﬂected in NMDS ordina-
tions (Fig. 4A). Pairwise perMANOVA detected signiﬁcant
differences between seedlings and large trees (df =407,
F=2.28, P<0.001), as well as between seedlings and interme-
diate trees (df =407, F=2.41, P<0.001), but not between
large trees and intermediate trees (df =407, F=1.40, P=0.06,
N Natural Abundances.—At all three sites, the foliage
of G. dewevrei and the AM species P. macrophylla was strongly
C (Figs. 5A–C) compared with EM sporocarps: the
foliage and seeds of G. dewevrei were, on average, depleted by 9.3
per mille and 7.9 per mille, respectively, for d
C and by 8.5 per
mille and 9.6 per mille, respectively, for d
increased with age for the G. dewevrei category at two sites (S1
and S2, Figs. 5A–C), whereas the
C abundance of foliage of
the AM P. macrophylla was not different from that of large G. dew-
evrei trees (Figs. 5A–C). Foliage of G. dewevrei did not differ sig-
niﬁcantly as a function of age category in
N abundance and C/
N (which did not differ between sites, data not shown)
(Figs. 5D–E and S10) but had signiﬁcantly higher values than
those of P. macrophylla. Seeds had higher C/N and similar
abundance compared with foliage for G. dewevrei. EM sporocarps
TABLE 2. Nonparametric perMANOVA on Bray-Curtis distance to test the effects of
site and age category of G. dewevrei on the distribution of OTUs.
SS MS FR
(A) Effect of site and tree category
Site 2.883 1.44152 2.9904 0.00938 0.001
Age category 2.089 1.04468 2.1672 0.00680 0.001
Site 9age category 3.968 0.99206 2.0580 0.01291 0.001
(B) Pairwise comparison
S1 vs. S2 2.080 1.03988 2.1315 0.00993 0.001
S1 vs. S3 1.437 1.43740 2.9514 0.00688 0.001
S2 vs. S3 1.022 1.02154 2.0895 0.00488 0.001
(C) Pairwise comparison
Seedlings vs. large trees 1.111 1.11090 2.2852 0.0056 0.001
Large trees vs.
0.688 0.68799 1.4008 0.00344 0.063
1.320 1.32043 2.4109 0.00663 0.001
SS, sums of squares; MS, mean squares.
FIGURE 4. Nonmetric multidimensional scaling ordination (NMDS) of EM fungal community (stress =0.09). The two ﬁgures represent the effect of age cate-
gory of G. dewevrei (A) and site (B) on EM fungal community composition in seedlings (S), intermediate trees (T), and large trees (LT) at sites 1, 2, and 3. Colored
points represent EM fungal taxa by family. Black squares represent communities.
Ectomycorrhizal fungi of G. dewevrei 7
N abundance and lower C/N than all plants (except
P. macrophylla for C/N, Figs. 5D–E and S10).
At all three sites of the studied monodominant G. dewevrei forest,
we detected fewer EM fungal taxa from sporocarps (59 species
from 1030 sporocarps) than from EM root tips (75 OTUs from
828 barcoded EM root tips). EM fungal diversity on root tips
differed signiﬁcantly depending on tree age category and site.
However, a core community was shared among the three investi-
gated tree age categories (colonizing 65.3% of the barcoded
ECMs) and among the three sites (69.3%). Shared fungi can
form CMNs that link together G. dewevrei plants of different age
categories growing in close vicinity, but we found no evidence
for nutrient transfer from trees to seedlings based on the seed-
lings’natural isotopic abundances.
LIMITED EM FUNGAL LINEAGE DIVERSITY BUT LARGE SPECIFIC
DIVERSITY.—The 119 EM fungal taxa identiﬁed here belong to
only 16 EM phylogenetic lineages sensu Tedersoo et al. (2010a).
This high species diversity from a limited number of EM fungal
lineages is consistent with other tropical rain forests, and it com-
plies with the view that tropical forests have a lower diversity of
EM phylogenetic lineages than temperate forests (which usually
display >20 lineages: Tedersoo & Nara 2010). For monodomi-
nant tropical EM forests, Smith et al. (2013) documented 11 EM
fungal lineages in a Guyana forest dominated by the dipterocarp
Pakaraimaea dipterocarpaceae, and Corrales et al. (2016a) found 13
EM lineages on Oreomunnea mexicana in Panama. For mixed tropi-
cal EM forests, Smith et al. (2011) found 17 lineages on three
caesalpinioid species from Guyana; Tedersoo et al. (2011) found
18 lineages on 11 caesalpinioids and one Phyllanthaceae in
Cameroon; Peay et al. (2010) identiﬁed 12 EM fungal lineages
under dipterocarps in a Malaysian rain forest; and Diedhiou et al.
(2010) identiﬁed seven EM fungal lineages on four caesalpinioids
and one Phyllanthaceae in a Guinean rain forest.
While comparison between studies is difﬁcult because of dif-
ferences in sampling methodologies and failure to saturate spe-
cies-accumulation curves, values of Fisher’s alpha, a diversity
FIGURE 5. Mean values of d
C(&) at site 1 (A), site 2 (B), and site 3 (C), and of d
N(D,&) and C/N (E) for the three sites pooled, since they did not dif-
fer signiﬁcantly for any age category. Glt, Git, and Gs: G. dewevrei large trees, intermediate trees, and seedlings, respectively; Pm: AM P. macrophylla large trees; S:
EM sporocarps. Se: seeds of G. dewevrei. Signiﬁcant differences revealed by Newman-Keuls tests are indicated by different letters (P<0.05).
ella Ebenye et al.
index that corrects for sampling intensity (Table 1), fall in the
range of values reported for tropical forests (Corrales et al.
2016a): based on this index, the diversity is lower than in mon-
odominant Oreomunnea mexicana (Fisher’s alpha =89.5) but higher
than in Coccoloba uvifera forests (Fisher’s alpha =3.67 for seedlings
and 3.32 for adult trees; Sene et al. 2015) or under Pakaraimaea
dipterocarpaceae (Fisher’s alpha =19.8; Smith et al. 2013); yet they
are in the range of mixed EM tropical forests, which is equally
large (Fisher’s alpha =4–183; Corrales et al. 2016a). Thus, the
lower host tree diversity does not necessarily translate into lower
fungal diversity, and ranges of diversity are large in both mixed
and monodominant forests. This ﬁnding contrasts with the view
that EM diversity correlates with that of tree species (e.g., Dickie
2007, Ishida et al. 2007, Tedersoo et al. 2008) and suggests that
additional factors drive fungal diversity Richard et al. For exam-
ple, host density (Table S1) inﬂuences EM fungal richness (Teder-
soo et al. 2014), perhaps because a greater availability of roots
provides more resources. Similarly, a recent origin of the EM
symbiosis and/or stress conditions may reduce the fungal diver-
sity (Sene et al. 2015). Finally, the lack of difference in richness of
EM fungal taxa between the mixed and monodominant forests
may, in part, be due to the dominance of generalist EM fungi
(Onguene & Kuyper 2002, Richard et al.Diedhiou et al. 2010,
Henry et al. 2015).
Although our sampling is globally saturated, many OTUs
detected belowground were not found aboveground and vice
versa. Sixty OTUs in all were found on roots only, while 44
(58.9% of sporocarps) were obtained from sporocarps exclusively.
Only 15 OTUs (28.7% of roots tips and 41.1% of sporocarps)
were detected from both approaches. Surveys of sporocarps may
have overlooked inconspicuous sporocarps of the /thelephora-
tomentella and /sebacina lineages (Horton & Bruns 2001), as
well as hypogeous species if any were present. More unexpect-
edly, species well represented by sporocarps, such as those in the
taxa /ramaria-gautieria, /tuber-helvella, /tricholoma, and /piso-
lithus-scleroderma, were not found on root tips, although they
are known to form ECMs (B^aet al. 2012): they may have a lower
investment in EM formation than the other lineages, although we
cannot exclude that their ECMs were deeper than the sampled
soil. Similarly, belowground EM fungal species were sometimes
absent aboveground, e.g. the lineages /sebacina, /marcelleina-
peziza gerardii, /elaphomyces, and /sordariales. Some /russula-
lactarius-lactiﬂuus and /thelephora-tomentella species that were
dominant on root systems were absent from the sporocarp sur-
vey, while the /boletus, /cantharellus, and /clavulina species
dominant on sporocarps were less abundant on root systems.
Similarly, the few species of ascomycetes fungi found from lin-
eages /elaphomyces (1), /sordariales (2), and /helotiales (1) were
present on root only. Interestingly, the lineages /elaphomyces cur-
rently emerge as an African clade requiring further research
(Buyck et al. 2016). These discrepancies further conﬁrm that
although the /russula-lactarius-lactiﬂuus and /thelephora-tomen-
tella lineages clearly dominated this G. dewevrei monodominant
forest, epigeous sporocarps are not perfect indicators of below-
ground richness (see also Gardes & Bruns 1996, Baptista et al.
2015). Moreover, sporocarps revealed >29lower species diversity
in S2 compared with S1 or S3, while OTU diversities on roots
did not differ, the discrepancies have also been reported in other
studies (e.g., Baptista et al. 2015). A better view of the EM fungal
diversity is achieved by combining sporocarp and ECM surveys,
as concluded for other temperate and tropical ecosystems
(Richard et al. 2005, Diedhiou et al. 2010, Sene et al. 2015).
The few species of ascomycetes detected may be due to
their speciﬁc ecological requirements more than a methodological
issue (see Tedersoo & Smith 2013). Previous studies in tropical
ecosystems also found low diversity of EM ascomycetes (Peay
et al. 2010, Smith et al. 2011, Tedersoo et al. 2011, Henry et al.
2015, 2016). Among the 16 discovered EM fungal lineages, /tu-
ber-helvella (one OTU, GenBank accession number KR819045)
had not previously been reported from an African tropical forest,
to the best of our knowledge (see Tedersoo et al. 2007, 2010a,
2011, Jairus et al. 2011, B^aet al. 2012). In addition to the Holar-
tic genus Tuber (Bonito et al. 2010), the /tuber-helvella lineage
includes taxa distributed in the austral regions (Tedersoo & Nara
2010). Members of this lineage are probably poorly represented
in the Paleotropic forests. There was also a noticeable absence of
common Holarctic and Austral EM fungal lineages such as /
cenococcum and /laccaria, as well as some panglobal EM fungal
lineages (e.g., /entoloma, /hebeloma-alnicola, and /hysterangium).
SIMILAR STRUCTURE BUT DIFFERENT COMPOSITIONS OF EM FUNGAL
COMMUNITIES AMONG THE AGE CATEGORIES.—The composition of
EM fungal communities differed as a function of the G. dewevrei
age category,with communities on seedlings differing from those
on older trees (Table 2). Soil properties were unlikely to drive
between-sites differences (Supporting information, Table S3).
These results partly differ from those obtained by Corrales
et al. (2016a) for Oreomunnea mexicana, the only other example of
monodominant forests investigated for age effect in which the
EM fungal communities of the three age categories did not differ.
Moreover, Corrales et al. (2016b) did not ﬁnd evidence for CMN
in O. mexicana based on mesh exclosure experiments and isotopes
analyses. Variation in community composition among sites is con-
sistent with high spatial turnover of tropical EM fungal commu-
nities (even at the kilometer scale: Smith et al. 2011), a patchiness
also known from temperate ecosystems (Richard et al. 2005).
Even so, 19 of the 49 OTUs that occurred in more than
one age category were most abundant on root tips for all three
age categories (38.7% of all sequences). The 30 remaining OTUs
were rare (28.1%). This suggests that the most abundant partners
of nearby conspeciﬁc adult trees may be a source of inoculum
for seedlings and potentially for CMN, although physical links are
not directly proven here. Similar observations were made in mul-
ti-aged stands of tropical rain forests. For instance, adults and
seedlings of monodominant Coccoloba uvifera forests share three
EM fungal taxa representing 80 percent of the EM colonization
(Sene et al. 2015). In mixed forests of Madagascar, 88 percent of
ECMs from adults are formed by EM fungal taxa also found on
seedlings, but they encompass less than a half of the taxonomic
diversity (Henry et al. 2015). In a mixed tropical rain forest in
Ectomycorrhizal fungi of G. dewevrei 9
Guinea, EM fungi shared by adults and seedlings represent 79
percent of the EM colonization (Diedhiou et al. 2010). It is difﬁ-
cult to determine whether this simply represents a sampling bias
(with common species more likely to reveal a link to different
age categories) or whether a multi-age strategy is linked to com-
monness in EM fungal species. The EM fungal taxa richness
(based on jackknife 1 and Fisher’s alpha) was not signiﬁcantly dif-
ferent for the three tree categories (Table 1), consistently with
observations on Oreomunnea mexicana (Corrales et al. 2016a).
NOEVIDENCE FOR NUTRIENT TRANSFER FROM TREES TO
SEEDLINGS.—Plants from temperate regions that rely on CMNs
are enriched in
N, and total N (Selosse & Roy 2009,
Selosse et al. 2016), reﬂecting values from the biomass of EM
fungi. The values observed here for EM fungi were concordant
with this pattern, but we detected no
N and total N deviation
in understory seedlings versus adult plants (Fig. 5D). For
seedlings were even slightly depleted compared with adult plants,
which is the opposite of what could be predicted based on car-
bon transfer. Since the main factors likely to inﬂuence
abundance were controlled (distance to soil to avoid different
levels of input of CO
from soil respiration; light level to avoid
different rates of photosynthesis), this trend was rather unex-
pected. We consider two possible explanations. First, the result
may be affected by carbon from the seed reserves, which are
C (Cernusak et al. 2009): this carbon source may
have been used to build cell wall polymers earlier in plant devel-
opment. A more sensitive analysis of recently synthesized sol-
uble circulating sugars may reveal some isotopic differences
(Hynson et al. 2012). Second, the carbon issuing from photosyn-
thesis is likely depleted in
C for two reasons. Seedlings are
generally more shaded than adult plants, so that systemically cir-
culating carbon may be more
C depleted (a lower photosyn-
thetic rate entails a better fractionation against
et al. 1989). Moreover, smaller trees are closer to the soil and
experience a less elevated
abundance than higher trees
due to the surrounding soil and plant respiration, combined
with poor ventilation in the understory (L€uttge 2008). The same
reasons could apply for the lower
C abundance in intermedi-
ate trees compared with large trees, because they also occupy a
lower position in the canopy.
These results and values are consistent with those reported
by Diedhiou et al. (2010), who found no isotopic evidence for
carbon transfer between seedlings and adults of several EM trees
species, including EM caesalpinioid legumes, in mixed rain forest
from a South Guinea forest. We do not fully reject the possibility
of carbon transfer that does not follow the same physiological
pathway as that reported in other CMNs formed by EM fungi,
and thus has different isotopic particularities and fractionations.
Moreover, the tendency of seedlings to have a lower
dance, as discussed above, may even offset any marginal contri-
bution of fungal C to their biomass. On one hand, we failed to
ﬁnd evidence that a nursery effect of CMNs by way of C transfer
could contribute to monodominance; on the other hand, the fact
that seedlings could connect to CMNs that are likely pre-
established at the expense of older trees may be relevant in terms
of carbon budget. This may indeed explain the observed high
EM rate of the seedlings, which is higher than for trees
Our study highlights the high speciﬁc diversity, but poor lin-
eage diversity, of EM fungal communities associated with roots
of G. dewevrei in monodominant stands.Although EM fungal
communities varied between growth stages of G. dewevrei, the
three age categories had partially overlapping EM fungal commu-
nities, and potentially formed CMNs between adults and seed-
lings. Nevertheless, there was no evidence of carbon transfer
from adults to seedlings. CMNs and their impact on the nutri-
tion, growth, and ﬁtness of regenerating seedlings should be fur-
ther investigated experimentally in G. dewevrei monodominant
This research was funded by the Projet Pilote Regional (PPR)
FTH-AC (IRD Cameroon) to A.M. B^a. Micha€ella Ebenye Hel-
vyne Christelle received a grant from the Institut de Recherche
pour le Developpement (IRD). The molecular analysis was partly
supported by the grant ‘Diversite des champignons mycorhiziens
des plantes’(DivMyc) from the network Bibliotheque Du Vivant
(INRA, CNRS & MNHN) to M.-A. Selosse. We warmly
acknowledge David Marsh for English corrections, and the editor
and three anonymous referees for improving previous versions of
Additional Supporting Information may be found online in the
supporting information tab for this article:
TABLE S1. Mean number of stems of G. dewevrei seedlings,
intermediate, and large trees (as deﬁned in main text) in each
TABLE S2. Number of EM fungal species per EM fungal lin-
eage from sporocarps in each year and all sites.
TABLE S3. Soil physical and chemical properties of the three
studied sites (depth 0–30 cm, n=3, measurement per site).
TABLE S4. Ectomycorrhizal fungal taxa recovered from
sporocarps and root tips of the three age categories of G. dewevrei
in the three sites.
FIGURE S1. Location of studied sites in southeast Cameroon.
FIGURE S2. Distribution of EM fungal lineages from sporo-
carps at each site over 4 years.
FIGURE S3. Species-accumulation curves of sporocarps col-
lected at each year and at all sites.
FIGURE S4. Species-accumulation curves with ECMs and
sporocarp sampling effort at all sites (A), and on EM root tips of
the three age categories of G. dewevrei at all sites (B) in 2010.
FIGURE S5. Distribution of EM fungal lineages on root tips
at each site (A) and on roots tips of seedlings, intermediate trees,
and large trees at all sites (B).
ella Ebenye et al.
FIGURE S6. Distribution of OTUs on EM root tips of seed-
lings, intermediate trees, and large trees of G. dewevrei at site 1
(A), site 2 (B), and site 3 (C).
FIGURE S7. Species-accumulation curves of OTUs on EM
root tips of seedlings, intermediate trees, and large trees (A) and
sporocarps (B) at each site in 2010.
FIGURE S8. Venn diagram showing the number of OTUs
shared by seedlings, intermediate trees, and large trees of G. dew-
FIGURE S9. Rarefaction curves of the percentage of OTUs
shared by the three age categories (seedlings, intermediate, and
FIGURE S10. Mean values of d
N(&) and C/N at site S1
(A, D), S2 (B, E), and S3 (C, F).
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