f u n g a l e c o l o g y 6 ( 2 0 1 3 ) 2 5 6 e2 6 8
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/funeco
Evolution of nutritional modes of Ceratobasidiaceae
(Cantharellales, Basidiomycota) as revealed from publicly
available ITS sequences
Vilmar VELDREa, Kessy ABARENKOVa, Mohammad BAHRAMa, Florent MARTOSc,1,
a
~
Marc-Andre SELOSSEb, Heidi TAMMa, Urmas KOLJALG
, Leho TEDERSOOa,d,*
a
Institute of Ecology and Earth Sciences, University of Tartu, 40 Lai, 51005 Tartu, Estonia
Centre d’Ecologie Fonctionnelle et Evolutive, CNRS, UMR 5175, 1919 Route de Mende, 34293 Montpellier cedex 5, France
c
Universite de La Reunion, Peuplements Vegetaux et Bioagresseurs en Milieu Tropical (UMR C53), Equipe Dynamiques ecologiques au sein des
ecosystemes naturels, 15 Avenue Rene Cassin, BP 7151, 97715 Saint-Denis cedex 9, France
d
Natural History Museum of Tartu University, 46 Vanemuise, 51046 Tartu, Estonia
b
article info
abstract
Article history:
Fungi from the Ceratobasidiaceae family have important ecological roles as pathogens,
Received 1 September 2012
saprotrophs, non-mycorrhizal endophytes, orchid mycorrhizal and ectomycorrhizal sym-
Revision received 5 March 2013
bionts, but little is known about the distribution and evolution of these nutritional modes.
Accepted 6 March 2013
All public ITS sequences of Ceratobasidiaceae were downloaded from databases, annotated
Available online 24 April 2013
with ecological and taxonomic metadata, and tested for the non-random phylogenetic
Corresponding editor:
€ vard Kauserud
Ha
distribution of nutritional modes. Phylogenetic analysis revealed six main clades within
Ceratobasidiaceae and a poor correlation between molecular phylogeny and morphologicalecytological characters traditionally used for taxonomy. Sequences derived from soil
Keywords:
(representing putative saprotrophs) and orchid mycorrhiza clustered together, but
Ceratobasidium
remained distinct from pathogens. All nutritional modes were phylogenetically conserved
Ectomycorrhiza
in the Ceratobasidiaceae based on at least one index. Our analyses suggest that in general,
Evolutionary ecology
autotrophic orchids form root symbiosis with available Ceratobasidiaceae isolates in soil.
Orchid mycorrhiza
Ectomycorrhiza-forming capability has evolved twice within the Ceratobasidiaceae and it
Phylogeny
had a strong influence on the evolution of mycoheterotrophy and host specificity in certain
Rhizoctonia
orchid taxa.
Saprotrophepathogen continuum
ª 2013 Elsevier Ltd and The British Mycological Society. All rights reserved.
Thanatephorus
Introduction
Fungi play a fundamental role in nutrient and carbon cycling
in terrestrial ecosystems and sediments. They act as
decomposers of dead organic matter, provide mineral nutrition to plants as mycorrhizal symbionts, or devastate plant
populations as phytopathogens. Despite their major ecological and economic importance, the taxonomic and
* Corresponding author. Institute of Ecology and Earth Sciences, University of Tartu, 40 Lai, 51005 Tartu, Estonia. Tel./fax: þ372 7376222.
E-mail address: [email protected] (L. Tedersoo).
1
Current address: School of Life Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville, Pietermaritzburg 3209, South
Africa.
1754-5048/$ e see front matter ª 2013 Elsevier Ltd and The British Mycological Society. All rights reserved.
http://dx.doi.org/10.1016/j.funeco.2013.03.004
Evolution of nutritional modes in Ceratobasidiaceae
ecological complexity of many important taxa remains poorly
understood because of the ephemeral habit, and the lack of
fruit bodies in most fungal groups.
The family Ceratobasidiaceae (Cantharellales, Basidiomycota) consists of the two closely related sexual genera, Ceratobasidium and Thanatephorus along with their Rhizoctonia
asexual forms. They form one of such ‘cryptic’ fungal groups
that play important ecological roles as crop pathogens, orchid
mycorrhizal symbionts, saprotrophs and endophytes
(Parmeter 1970; Sneh et al. 1996; Roberts 1999). These fungi
spend most of their life cycle in the morphologically simple
asexual (i.e. ‘anamorphic’) stage during which they can only
be observed macroscopically as irregular sclerotia or, for
phytopathogens, as necrotic lesions in the tissues of a tremendous range of host plants. Even when the sexual (i.e.
‘teleomorphic’) stage occurs, it represents an inconspicuous,
fragile, web-like layer of generative hyphae covering living
leaves and stems of hosts, or plant debris (Roberts 1999). Difficulties with the induction of fruiting in culture and the lack
of morphological characters to distinguish among biological
species have considerably complicated taxonomic and ecological research on these fungi.
To provide a practical method for identification, plant
pathologists implemented a tractable test based on number of
nuclei per cell and anastomosis compatibility in co-culture
instead of morphology-based binomial taxonomy (Carling
1996; Sharon et al. 2008). Traditionally, strains of binucleate
Rhizoctonia (BNR; binucleate strains are occasionally placed in
the anamorphic genus Ceratorhiza) and multinucleate Rhizoctonia (MNR) are considered anamorphs of Ceratobasidium
and Thanatephorus, respectively, although a few species of
Thanatephorus are binucleate (Roberts 1999). Both the MNR and
BNR are separated into anastomosis groups (AG), including 13
MNR AGs (AG-1 to AG-13) and 16 BNR AGs (AG-A to AG-S,
excluding AG-J, AG-M and AG-N; Sharon et al. 2008), some of
which are further divided into anastomosis subgroups based
on more detailed analysis of anastomosis compatibility,
morphology, pathogenicity and other criteria. All multinucleate AGs correspond to the teleomorphic species T.
cucumeris (anamorph Rhizoctonia solani). The binucleate AG-A,
AG-B(o), AG-D, AG-P and AG-Q correspond to Ceratobasidium
cornigerum, whereas AG-Ba and AG-Bb correspond to
Ceratobasidium setariae. However, teleomorphs of the remaining binucleate AGs remain unknown (Roberts 1999).
Teleomorphs of Ceratobasidiaceae are characterized by
aseptate basidia (a derived, apomorphic feature), but also selfreplicating spores and large indeterminate sterigmata
(ancestral features; Roberts 1999). Numerous minor genera
have been described within the Ceratobasidiaceae, but later
synonymized with either Ceratobasidium (characterized by a
single layer of basidia arising from horizontally branching
hyphae of <10 mm in diameter) or Thanatephorus (characterized by multiple layers of basidia arising from vertically
branching hyphae of >10 mm in diameter; Roberts 1999).
Roberts (1999) included the genera Waitea and Scotomyces in
the Ceratobasidiaceae, but molecular phylogenetic analyses
place both taxa outside Ceratobasidiaceae (Larsson 2007; K-H.
Larsson, pers. comm. 02.08.2012). We, therefore, consider the
Ceratobasidiaceae family to consist of the two genera Ceratobasidium and Thanatephorus only. The Ceratobasidiaceae is
257
currently considered a peripheral member of the order Cantharellales, although its phylogenetic relations to other
members of this group are not well resolved (Moncalvo et al.
2006; Hibbett et al. 2007).
Most attention to the members of the Ceratobasidiaceae
has been sparked by their role as widespread soil-borne crop
pathogens. Their necrotrophic capability is remarkably nonspecific and affects a multitude of plant taxa. There is a continuum between the necrotrophic parasitism and saprotrophy
in the Ceratobasidiaceae. Most strains possess some saprotrophic capability, but aggressive pathogens are poor saprotrophic competitors and depend on nutrients acquired from
living plant tissue (Papavizas 1970). By contrast, strains that
have lost their ability to infect and cause serious damage to
living organs, may function as commensal or even mutualistic
endophytes (i.e. fungi that grow diffusely in tissues, without
causing any visible symptoms; Sen et al. 1999) and increase
their hosts’ resistance to pathogenic strains (Sneh 1998).
However, little is known about the frequency and role of
Ceratobasidiaceae endophytes (Sen et al. 1999).
As a further major interaction, the Ceratobasidiaceae
includes a large number of taxa that form orchid mycorrhiza
(OrM). Orchids have an unusual relationship with their
mycorrhizal fungi compared to other mycorrhizal plants: they
receive all nutrients, including carbon, from their fungi during
their heterotrophic germination (Smith & Read 2008;
Dearnaley et al. 2012). At the adult stage, most orchids develop
photosynthetic capability, but clearly continue to benefit from
their mycobionts by receiving mineral nutrients. Some species associated with Ceratobasidium allow a net carbon flow
from adult orchids to the fungus (Cameron et al. 2006, 2008),
while in some other species, adult orchids still obtain carbon
from fungi (Selosse & Roy 2009; Yagame et al. 2012). Thus, the
balance between costs and benefits for fungi remains poorly
understood in OrM (Dearnaley et al. 2012), and we treat here
the association between autotrophic orchids and their
mycorrhizal fungi as symbiotic along the mutualismeparasitism continuum (Egger & Hibbett 2004). As a side finding in the
research on OrM, it was discovered that some Ceratobasidiaceae isolates also form ectomycorrhiza (EcM) (Warcup 1991;
Yagame et al. 2008, 2012; Bougoure et al. 2009), and indeed
some Ceratobasidiaceae have been sporadically reported in
community analyses of EcM fungi (e.g. Rosling et al. 2003).
However, the phylogenetic and biogeographic distribution of
these relatively uncommon EcM groups remains poorly
understood (Tedersoo et al. 2010).
Development of molecular methods has led to rapid
unravelling of the systematics and ecology of microbes in the
past few decades. Barcoding with the Internal Transcribed
Spacer (ITS) region of the ribosomal DNA locus has become a
standard means of identification in fungi (Schoch et al. 2012).
ITS sequences offer suitable resolution for identification of the
Ceratobasidiaceae strains and provide an invaluable tool for
differentiating AGs and their subtypes both from cultured
strains, soil and plant tissue (Johanson et al. 1998; Sharon et al.
2006). Phylogenetic analyses suggest that most AGs are
monophyletic, but still correspond to complexes of molecular
and ecological species (Gonzales et al. 2001, 2006; Sharon et al.
2006, 2008). AG subtypes usually have different ecological
niches and their within-group ITS sequence similarity is over
258
97e98 %; AG subtypes could be, therefore, considered as
molecular operational taxonomic units (MOTUs; Vilgalys &
Cubeta 1994; Gonzales et al. 2001; Sharon et al. 2006). This
level of ITS sequence similarity roughly matches the barcoding gap in most groups of Basidiomycota and Ascomycota
(Schoch et al. 2012). The International Nucleotide Sequence
Databases (INSD) have accumulated over 3 000 ITS sequences
of the Ceratobasidiaceae that provide a rich reference material
for molecular identification and inferring evolutionary
hypotheses.
Here, we utilize the wealth of accumulated ITS sequence
data to address the evolution of ecological strategies, biogeographic patterns and host specificity in the Ceratobasidiaceae.
In particular, we hypothesized that both the pathogenic and
EcM isolates are phylogenetically clustered but distinct from
other modes of nutrition, assuming that these biotrophic
strategies require specific genetic adaptations. We also tested
a null hypothesis that both the OrM and soil-derived saprotrophic isolates are evenly distributed across the Ceratobasidiaceae family and we expected that orchids preferentially
associate with non-pathogenic isolates. Based on the results
of several case studies that focused on specific host taxa or
geographic regions (e.g. Kuninaga et al. 1997; Waterman et al.
2011), we predicted that several Ceratobasidiaceae MOTUs
display strong patterns in biogeography and host specificity.
Materials and methods
Data assembly
All available full-length ITS sequences belonging to the Ceratobasidiaceae were retrieved from INSD in Jun. 2010. A direct
search by taxonomic assignment to the family Ceratobasidiaceae or the genera Ceratobasidium, Thanatephorus, Rhizoctonia or Ceratorhiza in GenBank (3 167 sequences), search for
matching unidentified sequences (299 additional sequences)
using the web tool emerencia (Nilsson et al. 2005) and the 1 000
best BLAST matches for highly deviating sequences AJ633124
(49 matching sequences) and AB000014 (one matching
sequence) retrieved a total of 3 516 INSD entries. In addition,
84 sequences were included from the UNITE database
(Abarenkov et al. 2010a). All sequences were aligned with
MAFFT (Katoh & Toh 2008) and inspected by use of the program Seaview (Gouy et al. 2010) to identify short and lowquality ITS sequences. Non-Ceratobasidiaceae taxa, other
genes, reverse complementary, chimeric and low-quality
sequences were detected as outlined in Tedersoo et al.
(2011). Sequences of Ceratobasidiaceae were often mislabelled or misidentified in INSD. Of 3 188 sequences named as
Ceratobasidiaceae, 51 ITS sequences (1.6 %) belonged to other
families within Cantharellales or to other orders of Agaricomycetes, especially Polyporales (incl. 28 sequences of Bjerkandera adusta; Table S1). Conversely, 10 ITS sequences
belonging to the Ceratobasidiaceae were wrongly assigned to
other groups of fungi or other eukaryote kingdoms in INSD. In
addition, 249 Ceratobasidiaceae sequences were assigned to
the kingdom Fungi or phylum Basidiomycota without further
specification. Visual inspection and the programs Reverse
Complementary Checker (Nilsson et al. 2010b) and Chimera
V. Veldre et al.
Checker (Nilsson et al. 2010a) revealed 19 reverse complementary and ten chimeric sequences. The reverse complementary sequences were re-orientated and kept in the
analyses. All chimeric sequences, 361 sequences representing
other taxonomic groups or genes, 26 highly divergent
sequences that potentially belong to Ceratobasidiaceae and
946 partial (>100 bp missing) or low-quality sequences were
excluded. Sequences with low read quality or of chimeric
nature were tagged accordingly in the INSD copy of the UNITE
database as implemented in the PlutoF workbench
(Abarenkov et al. 2010b). The remaining 2 257 nearly fulllength ITS sequences formed the ‘full conservative’ dataset
that is central in the subsequent analyses. Because AG-D-II
and AG-H were only represented by notoriously low-quality
or incomplete sequences, a ‘liberal’ dataset including an
additional 285 ITS sequences was also analyzed. In addition,
all 44 nuclear rDNA Large Subunit (LSU) sequences (at least
900 bp) of the Ceratobasidiaceae were retrieved from INSD and
UNITE. These were supplemented with a reference dataset of
95 sequences covering the Cantharellales and most other
orders of the Agaricomycotina to determine a suitable outgroup for the ITS-based analysis.
Metadata on isolation source, interacting taxon, locality
and AG were compiled for all full-length ITS sequences from
INSD and associated publications (Table S1). Isolation source
and substrate formed a basis for the statistical metadata
analysis. The main sources of isolation included diseased
tissue of crop plants (implying pathogenic interaction), roots
of orchids (OrM interaction) and soil (see below), followed by
ectomycorrhizal root tips (EcM interaction) and healthy nonmycorrhizal root or symptomless above-ground tissue of
wild plants (endophytic interaction; Sen et al. 1999). Some
non-mycorrhizal isolates from crop plants were found to be
non-pathogenic or even beneficial in experimental studies,
and were therefore treated as endophytes. Soil-derived
sequences were separated into entries obtained from soils
of: (1) crop fields by plant pathologists in studies targeting
pathogens; or (2) natural or semi-natural ecosystems
addressing soil fungal communities. The first category was
included among pathogens, while the latter group was considered suggestive of saprotrophy, although we anticipate
that pathogenic and EcM strains may also be present in the
soil of natural habitats. All annotated metadata are publicly
available in the UNITE database and through a link-out function in the European Nucleotide Archive (ENA).
Phylogenetic analyses
The full-length ITS sequences ranged from 580 to 680
nucleotides. The final alignments subjected to phylogeny
€ ytynoja &
reconstruction were created with PRANKSTER (Lo
Goldman 2005) using default parameters, and corrected
manually. Two segments of ITS1 (ca. 15e30 and ca. 20e60 bp,
varying among sequences) and one segment of ITS2 (ca.
10e20 bp) were omitted from phylogenetic analyses, because
these were highly variable and could not be reliably aligned
across the whole dataset. The length of individual sequences
was thereby reduced to 540e580 nucleotides. The final full
alignment spanned 1 178 sites, of which 571 were variable and
444 were informative. For Maximum Likelihood (ML) and
Evolution of nutritional modes in Ceratobasidiaceae
Bayesian phylogeny reconstruction, RAxML 7.2.8 (Stamatakis
2006) and MrBayes 3.1 (Huelsenbeck & Ronquist 2001) were
respectively used. Only RAxML was able to handle the full
dataset with reasonable speed. In all RAxML analyses,
GTR þ G þ I evolutionary model and 1 000 fast bootstrap replicates were used. To use both methods for comparison to
confirm validity of tree topology and to reduce tree size for
convenient display, the dataset was collapsed into 288 entries
by clustering the sequences with BLASTclust (Biegert et al.
2006) based on 99 % sequence similarity and 90 % coverage
criteria (99 % threshold was used instead of 97 %, to keep all
anastomosis subgroups in separate clusters). The longest
sequence of each cluster served as a representative sequence.
Phylograms were constructed from this ‘collapsed conservative’ dataset with both RAxML and MrBayes (burnin ¼ 2 000; evolutionary model GTR þ G þ I as revealed from
Modeltest; Posada & Crandall 1998). All Bayesian analyses in
this study were run for 10 million generations sampled every
1 000 generations. Burn-in value was determined according to
at which generation the log likelihood scores reached stationary level. To include all ecological metadata in a single
tree, a phylogram was constructed directly from the ‘full
conservative’ dataset with RAxML. Patristic distances (pairwise total path length between two terminals) were exported
from this full dataset tree using PDAP package of Mesquite
2.75 (Midford et al. 2003; Maddison & Maddison 2009), and
subjected to statistical analyses regarding the nutritional
mode of sequenced isolates (see below). Trees were visualized
together with metadata using the online tool iTOL (Letunic &
Bork 2007; http://itol.embl.de/), and are publicly available as
shared projects of the user ‘CeratobasidiumThanatephorus’
(case sensitive). For improved readability, clade names of the
Ceratobasidiaceae are labelled based on the phylogenetic
distribution of known AGs preceded by a slash (Moncalvo et al.
2002).
The LSU sequences were aligned with PRANKSTER using
default parameters and minimal manual correction. The LSU
alignment spanned 1 622 sites, of which 706 were variable and
517 were informative. LSU phylograms were generated both
with RAxML and MrBayes (GTR þ G þ I evolutionary model as
revealed from Modeltest) to assess the monophyly of the
Ceratobasidiaceae and to obtain an outgroup for rooting ITS
phylograms. All alignments are available in TreeBase (accession TB2:S13952).
To remove pseudoreplicates from the analyses of distribution of nutritional modes, we excluded redundant
sequences from each study by keeping the longest representative sequence from each MOTU (barcoding threshold of
97 % sequence similarity and 90 % of coverage) per study,
resulting in 508 representative sequences. The Net Relatedness Index (NRI, standardized mean of distances between all
pairs of sequences from compared categories) and the Nearest
Taxon Index (NTI, standardized mean of distances between
pairs of the nearest neighbours) were calculated based on
patristic phylogenetic differences of an ultrametric tree to test
clustering and overdispersion of nutritional modes on larger
and smaller phylogenetic scales, respectively (Webb 2000).
Sampling species as random draws from the phylogeny
without replacement served as a null model and statistical
significance was calculated based on 999 permutations as
259
implemented in Phylocom (Webb et al. 2008). To address
phylogenetic distinctness of nutritional modes, we calculated
the pairwise Nearest Taxon distances (NTD) for all these ecological guilds. Differences between observed and randomized
NTD that exceeded 2 SD of the randomized dataset were
considered statistically significant. In addition, pairwise Unifrac distances were calculated in the Fast Unifrac server
(http://bmf2.colorado.edu/unifrac/) by use of 999 permutations. Unifrac distance metric measures the phylogenetic
distance among communities by calculating the proportional
length of the tree branches that lead to descendants from each
single community but not from both communities, and tests
whether this distance is different from a random draw from
the communities (Hamady et al. 2010). All these distance
metrics were calculated with and without the pathogen-rich/
mainly-MNR clade to assess the robustness of results. Significance values of the Unifrac pairwise distances were subjected to reduction of false discovery rate by use of
BenjaminieHochberg FDR correction.
Based on the ML phylogram of the ‘full conservative’
dataset, we generated ancestral state reconstructions for
major clades and well-supported subgroups as implemented
in the Ape package of R (Paradis et al. 2004). We particularly
focused on the evolution of EcM habit within the Ceratobasidiaceae, because EcM sequences were mainly concentrated in
two subclades. All nutritional modes were allowed to change
to any other state at different probabilities, because no a priori
model exists.
Biogeographic patterns and host specificity of the most
common MOTUs and major clades were assessed based on
both the full and non-redundant datasets. Because of highly
biased sequence availability in different countries and hosts,
we sought the patterns of endemicity on a continental scale
and host specificity at the plant tribe (OrM) and family
(pathogens) levels.
Results
Taxonomic and phylogenetic patterns
The Bayesian (ASDSF ¼ 0.011) and ML analyses revealed similar tree topology in the LSU-based phylogenetic reconstruction. The LSU-based phylogeny confirmed monophyly of
the Ceratobasidiaceae within Cantharellales (Fig 1). The
Tulasnellaceae clade was inferred as a sister group to the
Ceratobasidiaceae, but with no statistical support. Within the
Ceratobasidiaceae, Thanatephorus (syn. Uthatobasidium) fusisporus formed a basal branch that was supported in both the
Bayesian (PP ¼ 1.0) and ML (BS ¼ 95) analyses. Only the /BD
clade was not represented by LSU sequences, but it was nested
among four other clades that were only partly supported
(PP ¼ 0.70; BP ¼ 75) as a sister group to the /fusisporus clade
based on the ITS analysis (Fig 2). Therefore, we rooted the ITS
phylogram at the /fusisporus clade. The topology of the LSU
phylogram was congruent with the ITS phylogram. The
/mainly-MNR clade was monophyletic and nested within the
BNR clades in both the LSU- and ITS-based analyses.
In the ‘collapsed conservative’ ITS dataset, phylograms
revealed in both the ML and Bayesian analyses (ASDSF ¼ 0.019)
260
V. Veldre et al.
class TREMELLOMYCETES
class DACRYMYCETES
Auriculariales
Cantharellales
core group
(Cantharellaceae,
Hydnaceae,
Clavulinaceae)
Botryobasidiaceae
Tulasnellaceae
Cantharellales
/fusisporus
/AK
/GLO
/CHI
Sebacinales
Thelephorales
class AGARICOMYCETES
/mainlyMNR
Ceratobasidiaceae
AY463475 Tremella mesenterica
AY586693 Paullicorticium ansatum
96
100
AY463403 Dacrymyces sp
AY701526 Calocera cornea
EU118672 Stypella papillata
96
AY586653 Elmerina holophaea
94
AF506492 Auricularia mesenterica
AF506493 Exidia glandulosa
AY586654 Exidiopsis calcea
AF506476 Sistotrema sernanderi
100
AY756071 Minimedusa pubescens
AM259216 Sistotrema confluens
97
AF347095 Hydnum repandum
81
77
AJ606042 Sistotrema alboluteum
AF506473 Sistotrema brinkmannii
EU118616 Clavulina cinerea
DQ089013 Botryobasidium botryosum
100
EU118607 Botryobasidium subcoronatum
EU118629 Haplotrichum curtisii
AY586657 Haplotrichum conspersum
AY585831 Gloeotulasnella cystidiophora
100
AB369929 Epulorhiza sp
100
84
AB369928 Epulorhiza sp
AB369933 Epulorhiza sp
95 UDB013030 Thanatephorus fusisporus
AF518664 Thanatephorus fusisporus
DQ369859 Thanatephorus cucumeris AG-3
Thanatephorus cucumeris AG-3
78 AF354064
AF354078 Thanatephorus cucumeris AG-5
AF354066 Thanatephorus cucumeris AG-8
AF354070 Thanatephorus cucumeris AG-2-BI
DQ097887 Thanatephorus cucumeris AG-2-2
AF354069 Thanatephorus cucumeris AG-8
DQ097888 Thanatephorus cucumeris AG-8
AF354119 Thanatephorus cucumeris AG-8
AF354068 Thanatephorus cucumeris AG-8
AF518655 Thanatephorus cucumeris
DQ917658 Thanatephorus cucumeris
AF354072 Thanatephorus cucumeris AG-4-HGII
100
AF354077 Thanatephorus cucumeris AG-4-HGIII
AF354073 Thanatephorus cucumeris AG-4-HGII
AF354074 Thanatephorus cucumeris AG-4-HGII
AF354076 Thanatephorus cucumeris AG-4-HGIII
AF354075 Thanatephorus cucumeris AG-4-HGIII
AF354081 Ceratobasidium sp CAG-4
AF354096 Thanatephorus cucumeris AG-6
AF354058 Thanatephorus cucumeris AG-1-IC
AF354060 Thanatephorus cucumeris AG-1-IA
AF354059 Thanatephorus cucumeris AG1-IB
AF354084 Ceratobasidium sp CAG-7
AF354083 Ceratobasidium sp CAG-6
100
AF354080 Ceratobasidium sp CAG-3
AF354061 Thanatephorus cucumeris AG-6
AF354062 Thanatephorus cucumeris AG-6
AF354111 Thanatephorus cucumeris AG-10
AF354071 Thanatephorus cucumeris AG-10
AF354079 Thanatephorus cucumeris AG-11
AF354065 Thanatephorus cucumeris AG-9
AF354063 Thanatephorus cucumeris AG-2-1
AF354092 Ceratobasidium sp AG-A
AF354091 Ceratobasidium sp AG-K
83 DQ097889 Ceratobasidium sp AG-G
AF354093 Ceratobasidium sp AG-L
AF354094 Ceratobasidium sp AG-O
78 FJ207506 Ceratobasidiaceae sp
92 DQ520098 Ceratobasidium sp FO38200
AY293171 Ceratobasidium sp GEL5602
AY634127 Ceratobasidiaceae sp
100
AY505557 Piriformospora indica
100
EU625999 Sebacina vermifera
100
DQ520103 Craterocolla cerasi
AY745701 Tremellodendron sp
78
AY586711 Sarcodon imbricatus
99
AY586658 Hydnellum gracilipes
AY586635 Bankera fuligineoalba
EU118674 Tomentellopsis bresadoliana
AY586636 Boletopsis grisea
AY586726 Vuilleminia macrospora
93
AY586652 Dendrothele maculata
96
EU118639 Laetisaria fuciformis
AY463401 Corticium roseum
AY885164 Waitea circinata
100
EU118636 Jaapia argillacea
EU118637 Jaapia ochroleuca
AY586679 Hypochniciellum subillaqueatum
AY586629 Anomoporia bombycina
EU118610 Ceraceomyces borealis
AY586628 Amylocorticium subincarnatum
AY586639 Byssocorticium pulchrum
96
AY586632 Athelia decipiens
AY463480 Tylospora asterophora
AY586662 Hygrophorus olivaceoalbus
AY586685 Clitocybe nebularis
EU118620 Cristinia helvetica
AY586680 Mallocybe agardhii
AY586681 Inocybe fibrosa
EU118622 Cystidiodontia laminifera
AY586646 Clavaria fumosa
EU118673 Tapinella atrotomentosa
93
AY586715 Suillus luteus
AY586645 Chroogomphus rutilus
AY586723 Tylopilus felleus
AY586659 Hygrophoropsis aurantiaca
EU118643 Leucogyrophana mollusca
97
EU118657 Phlebia unica
82
EU118653 Phanerochaete sordida
EU118665 Scopuloides hydnoides
EU118668 Steccherinum fimbriatum
AF506432 Gloeocystidiellum aspellum
100
AF506413 Lactarius volemus
AF506462 Russula nauseosa
AF506489 Wrightoporia lenta
AF506396 Albatrellus ovinus
100
AY586714 Subulicystidium sp
AY586720 Trechispora nivea
EU118621
Cyphellostereum laeve
95
EU118631 Hyphodontia alutaria
90
71
AY586665 Hymenochaete rubiginosa
AY586722 Tubulicrinis subulatus
EU118628 Gomphus clavatus
AF336259
Hysterangium stoloniferum
70
AY885165 Phallus hadriani
100
AJ406479 Anthurus archeri
88
AF139943 Aseroe arachnoidea
EU118641 Lentaria dendroidea
AY586682 Kavinia himantia
AF139975 Sphaerobolus stellatus
88
100
0.1
AF336251 Geastrum rufescens
AF287859 Geastrum saccatum
Corticiales
Jaapiales
Amylocorticiales
Atheliales
Agaricales
Boletales
Polyporales
Russulales
Trechisporales
Hymenochaetales
Gomphales
Hysterangiales
Phallales
Gomphales
Geastrales
Fig 1 e Large subunit maximum likelihood phylogram demonstrating the phylogenetic placement of Ceratobasidiaceae
among Agaricomycotina. Tremella mesenterica serves as outgroup. Values above branches indicate bootstrap support ‡70 %,
while thick branches indicate high posterior probabilities (PP ‡ 0.95) as revealed from a parallel Bayesian analysis. Bar
indicates 0.1 substitutions per site.
Evolution of nutritional modes in Ceratobasidiaceae
75
1
3
2
2
1
2
2
1
1
1
3
3
1
1
3
4
7
1
5
1
7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1 1
2 2
1 1
5 15
1 23
1
1
1
1
2
1
5
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
1
2
5
8
1
7
1
3
1
3
1
1
1
1
1
3
1
5
3
1
3
3
1
6
4
8
1
1
1
1
1
1
1
3
3
3 26
2 3
2
1
5
1
4
1
2
5
1
3
2
2
1
1
1
1
1
2
1
2
1 1
7 11 16
1
1
1 1 1
1
2
1
4
1 1
1 9
9 13
AG-C
1
AG-C
1
2
1
1
3
1
1
1
2
1
1
3
4
1
1
1
1
5
8
EcM2
(/ceratobasidium2;
Tedersoo et al., 2010)
AG-D-III
1
1
1
1
AG-Ba
AG-B(o)
2 13
1 1
4 57
1 5
1 2
1 1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
6
1
1
1
1
1
1
1 1
3 13
3 3
1 1
1 8
1 3
AG-Bb
1
1
1
1
1
1
1
1
1
1
1
2
1
1
2
1
3
1
1
3
9
1
1
1
1
1
1
1
1
9
AG-G
1
1
1
1
AG-L
AG-O
1 16
3
4
2
2
1
2
58
1
1
1
1
AG-D-I
1
1
1
1
1
1
1
1
1
2
1
1
AG-I
1
2 10
1 1
1 1
1 1
7
1
7
1
1
1
2
1
1
AG-I
1
1
1
2 36
1 1
1 2
1 8
1
1
3
/fusisporus
1
1
/CHI
1
1
Sapr Endoph EcM
1
8
1
1
/BD
75
Pathog OrM
1 1
1 1
1 1
1 1
2 5
1 1
1 1
1 1
1 1
1 1
1 10
1 2
2 2
1 1
1 1
4 24
1 1
1 1
1 1
1 1
1 1
1 2
2 7
1 2
1 1
1 1
1 1
1
1
2
3
1
1
1
1
1
3
9
1
1
1
1
1
1
3
9
2
EcM1
(/ceratobasidium1;
Tedersoo et al., 2010)
AG-A
1
1
1
1
1
1
1
1
1
1
/GLO
Unkn
AF472302
AF472303
AF504008
DQ028824
AF503973
86
DQ093780
FJ809766
100
EU218895
86
GQ223450
AY634129
100
UDB008679
UDB008680
AJ242890
AJ242896
DQ061931
DQ672286
DQ028808
89
93
100 EF090498
EF090492
DQ028800
DQ028797
DQ028798
DQ028799
DQ028818
99
DQ028817
AJ242882
100
EU730859
84 96
DQ356407
DQ356409
70
AJ419929
AJ716305
89
AM901722
100
93
GU985226
AB290020
AM901962
72
85
100
UDB008709
UDB008720
97
UDB008678
EU645602
AJ419928
EF536968
AJ242901
DQ421054
UDB008719
94 AB290021
GU446633
AM697947
AM697948
UDB008710
DQ520098
AY634126
UDB008661
100
AM231372
FJ552883
AJ633124
100
88
100 DQ273373
FJ660483
98
FN298243
DQ493565
FJ807984
EU645652
79
GU083181
91
AJ419931
EF100192
FJ237067
DQ093652
FM866376
98
UDB008670
GQ175312
100
GQ175305
FJ688125
DQ028791
DQ028796
FJ231393
100
AB198709
AB198714
DQ278930
98 AB198699
AB198702
87
AB198703
AY618223
80
100AB449170 UDB008676
UDB008674
89
AJ318421
GQ221863
96
FJ440188
FJ440200
74
92 DQ102430AB286930
AJ427400
AF503964
DQ084013
DQ307249
AF503998
96
AF472288
AF503970
98
FJ788720
EU516900
EU516785
EU810056
DQ028823
82
FJ788669
82
DQ028822
AJ427403
FJ788668
FJ788674
UDB008717
FJ788673
78
EF433959
EU516903
DQ672300
77
EU090897
AY634163
97 100
DQ672269
99
DQ672278
85
AY970109
DQ182419
73
FJ613066
FM866369
94
DQ084001
AJ318442
100
AJ242875
EU195338
AM040889
UDB008725
AJ242898
DQ223780
AB286936
FJ362331
97
79
AM697940
AF472285
100
AB432931
UDB004860
GQ175295
100
GQ175292
GQ175304
76 AB432932
AB432928
UDB004466
FR731600
AB303048
FR731598
FR731599
FJ788681
94
100
AF200518
AF200514
FJ763573
100
FJ763577
FJ763578
AY927354
EU591775
93 AJ242903
75
DQ102426
72
DQ102425
DQ279055
DQ672314
77
DQ672313
73
DQ672315
EF429313
AY927338
100 AJ242887
DQ672328
94
/mainly-MNR clade (continued)
0.05
100
/AK
A
261
AG-K
AG-A
Fig 2 e ITS maximum likelihood phylogram of the reduced conservative dataset, ancestral states of selected nodes and frequency
of occurrence in different nutritional modes. The phylogram was rooted at the /fusisporus clade. Values above branches indicate
bootstrap support ‡70 %, while thick branches indicate high posterior probabilities (PP ‡ 0.95) as revealed from a parallel Bayesian
analysis. Bar indicates 0.05 substitutions per site. Circular diagrams below branches indicate the probability of nutritional modes
being ancestral: red, pathogenic (pathog); black, saprotrophic (sapr); blue, orchid mycorrhizal (OrM); yellow, ectomycorrhizal (EcM);
purple, endophytic (endoph); white, unknown (unkn). The table of counts of nutritional modes is based on the full conservative
dataset e the first sub-column excluding and second sub-column including redundant sequences (99 % similarity within a study).
262
V. Veldre et al.
Unkn
100
94
77
EU810045
EU810026
AJ000202
AB275641
AF153806
AF354099
97
DQ301760
DQ279022
GU937740
AJ301901
71 DQ885780
DQ279017
DQ278936
84
DQ279019
AF354084
88
AJ318435
GU937739
AF153784
GU937738
DQ672268
FJ788709
AF354101
79
AF354102
AF153783
AY433813
AY634121
94
DQ672312
DQ672311
FJ746906
81
AY586167
DQ093646
AY684922
90
AM901911
AF478452
FJ435099
77
73
AB019025
64
U57740
100
AF354114
EU591766
EU730848
100
EU730840
EU730809
AF153802
DQ356413
FJ788716
FJ851083
87
AM697936
AM697938
AM697934
100
AF354110
98
FJ492108
AB054876
AB054875
U57885
EU591761
U57729
98
96
U57722
U57727
AB000030
U57744
EU244841
U57880
U57882
81
U57883
U57881
FJ553367
DQ913036
FJ480916
AF308623
99
EU591791
U57734
DQ278980
96
100
FJ492149
FJ492143
100
FJ435097
EU244844
EF017212
83
DQ421056
DQ421057
EU480292
86
FJ788707
96
FJ788711
FJ746909
92
DQ356408
DQ421055
DQ021449
U19954
92
DQ102447
EU375545
FJ492106
HM117643
97
100
UDB008690
UDB008714
94
EU218892
EU135906
UDB008721
FJ788721
97 FJ492085
AJ242888
AF222795
AF354081
DQ408294
DQ102441
AY684921
DQ102434
82
DQ279014
DQ102433
100 EF197798
89
EF197799
AJ868444
89
99
AF067641
AB000025
EU591762
GU937735
GU937736
AB286941
90
DQ278931
AJ301899
DQ173058
AY665171
73
AB479196
FJ440209
GU570159
FJ667265
AF354060
3
2
1
1
1
2
1
1
1
2
1
1
1
1
1
Pathog OrM
1 9
1 21
1 1
9
1 3
3 4 7
1 1 3
1 1
1
1
3 3 3
1
1
1
1 1
1 2
2
1 1 1 3
1
1 2 2
2 3 2 4
2 1 2
1 2 3
1 1 1 1 2
1 1
1 6
3 5
10 23
1 1
3
2
1
5
2
1
2
1
3
1
2 7
1 1
8 18
9 30
1 1
1 1
1 2
1 2
2 3
2 8
1 6
2 2
4 10
1
2 4
1 1
1 1
1 1 1
12 23 15
1
1
1 1 1
1
1
1 1
1
1
1
1
1 1 1
1
1 1 1
33 13312
2
1 1
1
1
2 3 4
1 1
11 11314
1 1 2
1
1
AG-13
AG-12
AG-7
AG-R
1
1
AG-S
1
1
1
1
1
1
1
1
1
3
AG-6
AG-Fb
AG-6
1
1
AG-7
AG-E
AG-5
AG-2-3
AG-11
3
1
1
1
AG-11
AG-8
4
2
2
2
AG-2-BI
1
89
1
1
1
1
1
1
1
1
1
4
2
36
83
5
1
1
6
76
6
3 7
3 4 2 4
1 1 4 22
1 2
10 20 18 74
1 1
1 1
1
9 28 13
1 1 1
1
1
1
1 1
1
1 1
4
2
1
1 6 2
11 16 6
1 1 1
7 16 3
3 3 2
1 1
2 2
1 1
1 1
1
1 1 1
1 1 1
6 54 21
1 1 1
1
1
Sapr Endoph EcM
1
90
1
1
1
5
1
8
16
1
7
21
2
6
3
AG-2-1
1
1
/mainly-MNR
B
AG-2-2
AG-9
1
1
5
1
1 1
1 1
2 2
2 12
1
1
1
1
2
1
1
1
AG-3
AG-10
AG-4-HGIII
AG-4-HGI
1
1
5
1
2
1
1
4
AG-4-HGII
AG-Fa
1
1
AG-1-ID
AG-1-IF
AG-1-IB
AG-1-IC
AG-P
1
1
1
195
2
1
1
1
AG-1-IA +
AG-1-IE
Fig 2 e (continued).
produced comparable results that differed only in statistical
support (Fig 2). Phylograms of the ‘full conservative’ dataset
displayed slight differences in tree topology and weaker
branch support, perhaps due to a greater amount of noise
owing to accumulation of erroneous base calls in larger
datasets.
All ITS-based phylogenetic analyses resulted in similar tree
topology and revealed six major clades (Fig 2). All MNR AGs
were clustered in one well-supported clade (/mainly-MNR)
together with some BNR AGs (AG-E, AG-F, AG-P, AG-R, AG-S).
The remaining BNR sequences were divided between other
clades, four of which included known AGs (/AK, /BD, /CHI
and /GLO). The /fusisporus clade was not associated with any
described AGs, but contained a sequence of the binucleate
teleomorph T. fusisporus (DQ398957). The /mainly-MNR, /AK
and /CHI clades were supported in the Bayesian analysis
(PP 95 %), whereas the /mainly-MNR, /AK, /CHI and /fusisporus clades received bootstrap support over 70 % in the ML
analysis. Thus, the monophyly of the /BD and /GLO clades is
poorly supported despite their consistent formation in all
different analyses and datasets.
We found no clear distinction between the teleomorph
genera Thanatephorus and Ceratobasidium (Fig S1). While most
Thanatephorus fruit-bodies (predominantly T. cucumeris) were
placed in the /mainly-MNR clade, fruit-bodies with Thanatephorus morphology also appeared in the binucleate /GLO
Evolution of nutritional modes in Ceratobasidiaceae
263
(T. theobromae) and /fusisporus clades (T. fusisporus). The
majority of Ceratobasidium fruit bodies (including most C. cornigerum specimens) belonged to the BNR clades, but four
sequences labelled as C. cornigerum were scattered in the
/mainly-MNR clade (among AG-E, AG-P, AG-R and AG-4-HGI).
Most of the AGs were monophyletic, including AG-1, AG-4,
AG-B and AG-D that in turn contained monophyletic subgroups
(BS > 70; Fig 2, Fig S2). By contrast, the AG-Fa formed a distinct
monophyletic group (BS ¼ 82) near the base of the /mainly-MNR
clade, whereas AG-Fb constituted a subgroup of the multinucleate AG-6. AG-2 was polyphyletic as a whole, but each of its
subgroups was monophyletic. Although AG-C, AG-I and AG-7
have not been separated into subgroups, members of these
AGs were distributed in seemingly unrelated subclades within
the /CHI (AG-C, AG-I) and /mainly-MNR (AG-7) clades. AG-S and
AG-Q were represented by only one and two sequences,
respectively, and therefore the monophyly of these AGs cannot
be assessed. A large proportion of the /mainly-MNR clade was
covered with known AGs (Fig 2B), while the majority of terminal taxa of the BNR clades lacked anastomosis grouping
information (Fig 2A).
Ecological patterns
Based on metadata, putative pathogens, OrM symbionts,
saprotrophs and EcM symbionts were represented by 1 129
(50.0 %), 401 (17.8 %), 69 (3.1 %) and 73 (3.2 %) sequences,
respectively (Table 1). The putative ecology of 597 (26.5 %)
sequences remained unknown due to the lack of metadata
about the isolation source. In total, 82.9 % of sequences of
putative pathogens were affiliated to the /mainly-MNR clade.
Only 14.3 % of sequences representing other ecological strategies belonged to this clade. Sequences of putative EcM fungi
clustered either in the /CHI or in the /GLO clade. A group of
Australian EcM isolates sampled by Bougoure et al. (2009) was
exceptional as each of these strains comprised two ITS copies,
one clustering with other EcM sequences in the /GLO clade
and another clustering with non-EcM sequences in the /BD
clade. Phylogenetic analyses and ancestral state reconstructions suggested that EcM habit has evolved twice in the
Ceratobasidiaceae (P < 0.01), but revealed no ancestral mode
of nutrition with confidence, except that EcM habit is unlikely
to be ancestral (Fig 2A).
Based on sequence clustering at 97 % similarity cut-off, the
‘full conservative’ dataset was divided into 157 MOTUs of
which 53 (33.8 %) were represented by a single sequence and
23 (14.6 %) were represented by only two sequences. Damaged
plant tissue, orchid roots, soil, EcM root tips, and healthy
shoot tissue or non-mycorrhizal root tissue were the dominant isolation source in 52 (33.1 %), 47 (29.9 %), 14 (8.9 %), 14
(8.9 %) and 7 (4.5 %) MOTUs, respectively (Table 1). The main
isolation source of 13 (8.2 %) MOTUs remains unknown,
whereas sequences of ten MOTUs (6.4 %) were obtained from
multiple sources in equal abundance (usually a combination
of pathogenic, OrM and/or soil-derived sequences). The ‘liberal’ dataset was divided into 252 MOTUs, including an additional 84 singletons, eight doubletons, and three taxa with
more than two sequences.
The six major clades of the Ceratobasidiaceae differed in
the available taxonomic information and relative sampling
depth. The /mainly-MNR clade comprised 68.4 % of sequences,
but only 38.9 % of MOTUs, indicating relative oversampling of
harmful pathogens by plant pathologists. Anastomosis
grouping information was available for 64.0 % of MOTUs in the
/mainly-MNR clade, but only for 16.4 % of species in the BNR
clades taken together. No AGs were assigned to members of
the /fusisporus clade.
All nutritional modes except endophytes (0.1 < P < 0.5)
were significantly phylogenetically clustered compared to the
null model based on the NRI index and inclusion or exclusion
of the /mainly-MNR clade (P < 0.05 in all cases; Table 2). Based
on NTI index, all nutritional modes except pathogens (P > 0.5)
were significantly phylogenetically clustered. In all analyses,
OrM fungi displayed the strongest phylogenetic clustering
among all modes of nutrition. The NTD pairwise distance
metric revealed several phylogenetic dissimilarities among
the nutritional modes (Table 2). OrM sequences differed from
pathogens (NTD ¼ 20.63; P < 0.001) and EcM fungi
(NTD ¼ 3.21; 0.01 < P < 0.05), but not from endophytes and
saprotrophs (0.1 < P < 0.5). EcM fungi were significantly different from pathogens (NTD ¼ 19.15; P < 0.001) and saprotrophs (NTD ¼ 3.36; 0.01 < P < 0.05), whereas endophytes
differed significantly only from pathogens (NTD ¼ 5.59;
0.001 < P < 0.01). Saprotrophs were phylogenetically clearly
distinct from pathogens (NTD ¼ 8.18; 0.001 < P < 0.01). The
Unifrac pairwise distance metric corroborated these findings
of phylogenetic differentiation (i.e. separation of communities; Table 2).
Biogeographic patterns and host specificity could be
addressed in sufficient detail only in the seven most common
MOTUs (found in more than five studies; representing AG-1IA, AG-2-1, AG-2-2, AG-4-HGI, AG-4-HGII, AG-5, AG-A) due to
Table 1 e Distribution of sequences and MOTUs (97 % similarity threshold; in parentheses) representing different
nutritional modes among six major clades of Ceratobasidiaceae. For MOTUs, only the most common nutritional mode is
scored
EcM
/mainly-MNR
/AK
/BD
/CHI
/GLO
/fusisporus
Total
0 (0)
0 (0)
13 (1)
15 (7)
45 (6)
0 (0)
73 (14)
Endophyte
3
4
2
7
14
0
30
(1)
(0)
(2)
(3)
(1)
(0)
(7)
OrM
47
1
151
90
46
24
359
(11)
(0)
(13)
(9)
(8)
(6)
(47)
Pathogen
Saprotroph
936 (34)
86 (4)
34 (6)
21 (4)
52 (4)
0 (0)
1 129 (52)
26 (3)
4 (0)
5 (3)
25 (3)
8 (4)
1 (1)
69 (14)
Unknown
531
18
24
13
9
2
597
(9)
(0)
(1)
(3)
(0)
(0)
(13)
Multiple equal
- (3)
- (0)
- (0)
- (5)
- (2)
- (0)
- (10)
Total
1 543
113
229
171
174
27
2 257
(61)
(4)
(26)
(34)
(25)
(7)
(157)
264
V. Veldre et al.
Table 2 e Phylogenetic clustering and pairwise phylogenetic distances of nutritional modes. Unifrac pairwise distances
indicate phylogenetic dissimilarity and these are given to the left from the diagonal; nearest taxon (NT) pairwise distances
are given to the right from the diagonal. For net Relatedness index (NRI), nearest taxon index (NTI) and NT, positive values
denote phylogenetic clustering and negative values indicate overdispersion (or avoidance, NT). Significant values are
highlighted in bold
na
EcM
Endophyte
OrM
Pathogen
Saprotroph
Unknown
18
19
82
216
35
138
NRI
3.46
1.94
7.44
1.58
3.57
1.58
NTI
5.34
0.83
6.87
2.64
2.67
3.17
Pairwise Unifrac\NTD (comdistNT) phylogenetic distances
EcM
Endophyte
OrM
Pathogen
Saprotroph
Unknown
e
0.74
0.83
0.93
0.83
0.92
1.59
e
0.76
0.84
0.76
0.84
L3.21
0.82
e
0.84
0.74
0.85
L19.15
L5.59
L20.63
e
0.83
0.61
L3.36
1.24
0.47
L8.18
e
0.81
L20.45
L7.05
L18.43
L11.04
L9.62
e
a The number of non-redundant sequences (one sequence per MOTUs per study).
limited sample size. All these MOTUs were distributed in
several continents and most of these displayed no evidence
for host specificity, infecting a wide range of plant families
(Fig S1). Only AG-3 showed some host specificity, infecting
predominantly species of the Solanaceae family, particularly
potato (Solanum tuberosum). Anastomosis groups that were
relatively widely distributed in the phylogenetic tree (AG-C,
AG-I, AG-2, AG-7) all had wide geographic distribution in the
Northern hemisphere, with sympatric occurrence of MOTUs.
In OrM, there was no evidence for specificity between orchid
tribes and MOTUs or major clades of the Ceratobasidiaceae
(Fig S1). Because most orchid species were each represented
by only a single study and geographic origin, we cannot
address OrM specificity at orchid species level. At higher
taxonomic level, most of the six major clades had cosmopolitan distribution and none of them were restricted to a single
continent or biome (Fig S1).
Discussion
Phylogenetic implications
Phylogenetic analyses revealed that the Ceratobasidiaceae is
monophyletic within the Cantharellales and the /mainly-MNR
clade is nested within BNR groups. Within Ceratobasidiaceae,
the /fusisporus clade was inferred as a basal branch given the
position of T. fusisporus isolates in the LSU phylogram.
Therefore, we rooted the ITS phylogram at the /fusisporus
clade, but conservatively anticipate the unlikely possibility
that the true rooting point lies within this clade. Use of
protein-encoding genes will be necessary for further in-depth
phylogenetic reconstruction of the Ceratobasidiaceae
(Gonzales et al. 2006).
We found strong phylogenetic evidence for nonmonophyly in both the teleomorph genera and the nuclear
count-based system of anamorphs. Species of the two teleomorph genera Ceratobasidium and Thanatephorus were scattered across several major clades, indicating that both taxa are
polyphyletic and the morphological characters (nuclear state,
septation of basidia, shape of sterigmata) used for the generic
distinction are unconserved. Indeed, differences between the
genera Ceratobasidium and Thanatephorus are subtle and
intermediate forms exist (Roberts 1999), so that the characteristic features may reverse in Ceratobasidiaceae evolution.
Our data support that the simplest basidioma shape characteristic for Ceratobasidium (Roberts 1999) is likely ancestral and
at least evolved several times into the more complex Thanatephorus basidioma shape. Although all MNR strains were
concentrated in the /mainly-MNR clade, a few BNR AGs (AG-E,
AG-F, AG-P, AG-R, AG-S) were deeply nested within this clade
(Gonzales et al. 2006; Sharon et al. 2008). The topology found
suggests that the multinucleate organization is a derived
feature that arose multiple times in the Ceratobasidiaceae
evolution, and/or with many reversions to the binucleate
state. While there seems to have been one main shift from
binucleate to multinucleate organization, at least one more
change is evident based on the sister relationship between
AG-1 and AG-P. Although most AGs were monophyletic, large
AGs usually consisted of multiple MOTUs that were not
closely related (as low as 81 % ITS sequence similarity between
different subgroups of the same anastomosis group; Gonzales
et al. 2001; Sharon et al. 2006, 2008). The polyphyly of teleomorph genera and some AGs as well as multiple shifts in the
number of nuclei imply that both the morphology-based and
anastomosis behaviour-based taxonomies lack species-level
resolution in the Ceratobasidiaceae. The limited number of
known morphological characters available for fungi, especially for taxa with no or resupinate fruit-bodies, often results
in lower taxonomic resolution when using morphology, as
compared to molecular features (Taylor et al. 2006).
Evolution of nutritional modes
Although represented in several major clades, both the
pathogenic, OrM, EcM, endophytic and putatively saprotrophic isolates displayed phylogenetically clustered distribution within the Ceratobasidiaceae. This indicates that
these ecological roles tend to be evolutionarily conserved.
Sequences derived from OrM and soil formed a phylogenetically coherent group, and the fact that they originated from
studies with different aims and sampling protocols emphasizes this result. Assuming that soil-derived isolates mostly
represent saprotrophs, it seems that orchids preferentially
Evolution of nutritional modes in Ceratobasidiaceae
establish symbiosis with the less aggressive side of the
pathogen-saprotroph continuum in the Ceratobasidiaceae.
One could conclude that association with pathogenic strains
could result in death of the orchids before they germinate or
reach maturity and become available for sampling (Taylor
et al. 2003). However, some orchids germinate as successfully with isolates from strongly pathogenic AGs as with those
from non-pathogenic or weakly pathogenic AGs in vitro
(Masuhara et al. 1993; Masuhara & Katsuya 1994; Pope & Carter
2001). Alternatively, pathogenic isolates could be simply less
available for orchids in soil, because pathogens tend to be
more limited to tissues and rhizosphere of infected plants. We
tested the latter hypothesis by separately considering only the
MOTUs found from soil. There was no significant difference in
the association of OrM isolates with non-pathogenic or
pathogenic MOTUs (chi-square test: n ¼ 29; P ¼ 0.775), which
lends no support for an avoidance strategy. Therefore, our
analyses suggest that in general, orchids associate with
available Ceratobasidiaceae strains in soil irrespective of their
pathogenicity.
Sequences of plant pathogens were phylogenetically distinct from sequences of all other nutritional modes. Phylogenetic differences between soil-derived sequences and
pathogens may represent a trade-off between abilities to
compete for nutrients in debris dispersed in soil and to attack
living plant tissue. To our knowledge, phylogenetic distinctness of saprotrophic and parasitic guilds has not been
explicitly tested at the genus or family level in other taxonomic complexes of fungi, but this could be a common phenomenon in basidiomycetes as suggested by published
phylograms (e.g. Cryptococcus: Findley et al. 2009). Within
Ceratobasidiaceae, pathogenic groups were potentially
derived from putative soil saprotrophs in several instances
(Sharon et al. 2006). One could argue that soil-derived isolates
are non-pathogenic to most plants in natural conditions
where they evolved, but may become aggressive against fertilized crop plants or when introduced to exotic habitats
(Wingfield et al. 2001; Desprez-Lousteau et al. 2007). This is
certainly an uncommon phenomenon in the Ceratobasidiaceae family, because pathogenic isolates are relatively wellsampled.
Bougoure et al. (2009) reported that several cultured strains
contained two alleles (cf. ‘cistrons’) that shared only 80 %
ITS sequence similarity. While one of the alleles belonged to
the /ceratobasidium1 EcM lineage in the /GLO clade, the other
allele was nested in the distantly related /BD clade, within
other OrM and soil-derived isolates. This may represent a case
of hybridization or gene transfer between an EcM fungus and a
distantly related non-mycorrhizal fungus (Bougoure et al.
2009). Some anastomosis groups (AG-1, AG-2, AG-B) encompass lineages with only 81e83 % similarity (Sharon et al. 2006,
2008), which may allow the coexistence of distantly related
nuclei and thus create a possibility for recombination events
(Xie et al. 2008). Such hybridization within sympatric,
anastomosis-compatible groups may result in shifts in host
range and give rise to novel harmful pathogens (Brasier 2000;
Desprez-Lousteau et al. 2007).
Sequences isolated from EcM root tips fell consistently into
two well-supported lineages in the BNR /GLO and /CHI clades.
Ancestral state reconstructions support these findings
265
indicating that EcM lifestyle has secondarily arisen twice in
the Ceratobasidiaceae. Tedersoo et al. (2010) referred to these
EcM fungal lineages as /ceratobasidium1 and /ceratobasidium2, respectively, but provided no phylogenetic support for
this hypothesis. Present data indicates that the /ceratobasidium1 lineage includes both EcM and OrM strains from SW
Japan, Thailand, Australia, Malaysia, Zambia and Madagascar,
suggesting a subtropical and tropical distribution. The Japanese (Yagame et al. 2008) and Australian (Bougoure et al. 2009)
OrM strains readily form EcM in experimental conditions and
participate in tripartite interactions with mycoheterotrophic
(non-photosynthetic) orchids in nature. The evolution of EcM
habit in this lineage may have facilitated the development of
mycoheterotrophy in several orchid taxa (Chamaegastrodia
sikokiana and Rhizanthella gardneri), because mycoheterotrophic orchids usually associate with EcM fungi in temperate
habitats. It is believed that EcM fungi provide a more stable
and reliable carbon source compared to saprotrophic fungi
(Dearnaley et al. 2012). The /ceratobasidium2 lineage represents a collection of sequences from EcM root tips in several
community analyses in the boreal and temperate forests of
Europe, North America and Japan, suggesting a circumboreal
distribution. Recently, Yagame et al. (2012) showed that Platanthera minor, a partially mycoheterotrophic (¼ mixotrophic)
green orchid, associates with both the /ceratobasidium1 (cf.
types I1 and I2) and /ceratobasidium2 EcM lineages (I4). Both
EcM lineages lack a sister group with well-defined nutritional
mode, and the ancestral state reconstruction was unable to
resolve the ancestral mode of nutrition for them.
Biodiversity and biogeography
Sequence clustering reveals that both the /mainly-MNR and
especially BNR clades of the Ceratobasidiaceae comprise high
cryptic diversity of taxa that lack sequenced representative
cultures and fruit bodies. Culturing and anastomosis tests of
pathogenic isolates have been performed for decades by
phytopathologists, but soil-derived and orchid rootassociated strains have usually escaped these trials,
although they are often isolated. Moreover, recent in situ
molecular identification studies from roots and soil have
provided an unprecedented wealth of information about the
diversity of BNR clades, suggesting that many Ceratobasidiaceae taxa have never been obtained into pure culture. Both the
‘full conservative’ and ‘liberal’ datasets revealed a large
number of MOTUs represented by only one or two sequences,
indicating that part of the uncultured richness is yet to be
captured by molecular techniques. Because sequence analysis
of soil and root material is performed mostly in boreal and
temperate regions, tropical soils probably contribute to a large
proportion of the undetected richness and understanding of
biogeographic patterns. For example, the ‘full conservative’
dataset included 11 Ceratobasidiaceae MOTUs from 15 orchid
union Island (Martos et al. 2012). The present
species in Re
evaluation of biogeography and host specificity was restricted
to the most common MOTUs of pathogens and OrM fungi and
to the six major clades. While the major clades have cosmopolitan distribution, the common species lack both host specificity and endemism that may be at least partly ascribed to
anthropogenic dispersal for crop pathogens. We cannot rule
266
out the possibility that host specificity occurs at the strain
level as shown for the FusariumeGibberella complex (Ma et al.
2010). In OrM fungi, the lack of specificity for plant groups is
consistent with the facultative nature of this symbiosis for
fungi, as revealed by the lack of phylogenetic fidelity on the
fungal side (Martos et al. 2012).
The use of only ITS sequences for inferring biogeographic
and ecological questions has, however, a few limitations.
First, tracing the origin and specificity of pathogens is best
approached by use of population genetics techniques, because
the ITS region has insufficient resolution at the population
level. Second, ITS-based phylogenetic trees often exhibit low
phylogenetic resolution because of abundant insertions and
deletions that are difficult to handle by alignment and phylogenetic programs. Another source of error is the paucity of
metadata. Both phylogenetic uncertainty and missing metadata render the results of evolutionary ecology studies less
statistically supported due to greater noise to signal ratio and
reduced sample size, respectively. However, such noise is
unlikely to bias the qualitative patterns when it is distributed
randomly along the phylogram.
Conclusions
Our global analysis of public ITS sequences of the Ceratobasidiaceae family sheds light onto phylogenetic relations and
distribution of ecological strategies within this large, ecologically and economically important fungal family. All major
nutritional modes such as saprotrophy, pathogenic, OrM, EcM
and endophytic interactions were phylogenetically conserved.
Although pathogens have arisen multiple times independently (Gonzales et al. 2001, 2006), they are phylogenetically
distinct from most other functional guilds. Orchid root symbionts are phylogenetically overlapping with putative saprotrophs from soil samples, suggesting that saprotrophic strains
from natural soils are more easily accessible for orchids. EcM
lifestyle has evolved separately in two major clades of Ceratobasidiaceae. Probably through improved carbon nutrition, at
least one of these events may have triggered loss of photosynthesis in certain orchid taxa associating with these clades
of Ceratobasidiaceae. Our study emphasizes that the Ceratobasidiaceae is both functionally and taxonomically highly
diverse, and that the classical morphological, cytological and
anastomosis investigations have limited power of resolution
at both the species and genus levels. Further sampling in
natural habitats, especially in tropical sites, will probably
enable us to recover many novel species and to address
biogeographic patterns within the Ceratobasidiaceae.
Acknowledgements
This work was initially led by V. Veldre, but was finalized by
co-authors following the accidental death of the first author.
We thank A. Stamatakis and A. Aberer for advice regarding
phylogenetic analyses. The bulk of this project was funded
from Estonian Science Foundation grants PUT171, 8235 and
9286, and FIBIR. M.-A. Selosse is funded by the Agence
V. Veldre et al.
Nationale de la Recherche (ANR program SYSTRUF), and
gion Re
union.
F. Martos by the Re
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.funeco.2013.03.004.
references
Abarenkov K, Nilsson RH, Larsson KH, Alexander IJ, Eberhardt U,
Erland S, Høiland K, Kjøller R, Larsson E, Pennanen T, Sen R,
Taylor AFS, Tedersoo L, Ursing B, Vr
alstad T, Liimatainen K,
~ ljalg U, 2010a. The UNITE database for
Peintner U, Ko
molecular identification of fungi e recent updates and future
perspectives. New Phytologist 186: 281e285.
Abarenkov K, Tedersoo L, Nilsson RH, Vellak K, Saar I, Veldre V,
Parmasto E, Prous M, Aan A, Ots M, Kurina O, Ostonen I,
~ geva J, Halapuu S, Po
~ ldmaa K, Toots M, Truu J, Larsson K-H,
Jo
~ ljalg U, 2010b. PlutoF e a web based workbench for
Ko
ecological and taxonomic research, with an online
implementation for fungal ITS sequences. Evolutionary
Bioinformatics 6: 189.
€ ding J, Lupas A, 2006. The MPI
Biegert A, Mayer C, Remmert M, So
Toolkit for protein sequence analysis. Nucleic Acids Research 34:
W335eW339.
Bougoure J, Ludwig M, Brundrett M, Grierson P, 2009. Identity and
specificity of the fungi forming mycorrhizas with the rare
mycoheterotrophic orchid Rhizanthella gardneri. Mycological
Research 113: 1097e1106.
Brasier C, 2000. The rise of the hybrid fungi. Nature 405: 134e135.
Cameron DD, Johnson I, Read DJ, Leake JR, 2008. Giving and
receiving: measuring the carbon cost of mycorrhizas in the
green orchid, Goodyera repens. New Phytologist 180:
176e184.
Cameron DD, Leake JR, Read DJ, 2006. Mutualistic mycorrhiza in
orchids: evidence from plantefungus carbon and nitrogen
transfers in the green-leaved terrestrial orchid Goodyera
repens. New Phytologist 171: 405e416.
Carling D, 1996. Grouping in Rhizoctonia solani by hyphal
anastomosis reaction. In: Sneh B, Jabaji-Hare S, Neate S,
Dijst G (eds), Rhizoctonia Species: taxonomy, molecular biology,
ecology, pathology and disease control. Kluwer Academic
Publishers, Dordrecht, The Netherlands, pp. 37e47.
Dearnaley JDW, Martos F, Selosse M-A, 2012. Orchid mycorrhizas:
molecular ecology, physiology, evolution and conservation
aspects. In: Hock BE (ed), Fungal Associations, 2nd edn. The
Mycota IX Springer, Berlin, Germany.
Desprez-Lousteau M-L, Robin C, Buee M, Courtecuisse R,
Garbaye J, Suffert F, Sache I, Rizzo DM, 2007. The fungal
dimension of biological invasions. Trends in Ecology and
Evolution 22: 472e480.
Egger KN, Hibbett DS, 2004. The evolutionary implications of
exploitation in mycorrhizas. Canadian Journal of Botany 82:
1110e1121.
Findley K, Rodriguez-Carres M, Metin B, Kroiss J, Fonseca A,
Vilgalys R, Heitman J, 2009. Phylogeny and phenotypic
characterization of pathogenic Cryptococcus species and closely
related saprobic taxa in the Tremellales. Eukaryotic Cell 8: 353.
Gonzales D, Carling DE, Kuninaga S, Vilgalys R, Cubeta MA, 2001.
Ribosomal DNA systematics of Ceratobasidium and
Thanatephorus with Rhizoctonia anamorphs. Mycologia 95:
1138e1150.
Evolution of nutritional modes in Ceratobasidiaceae
Gonzales D, Cubeta MA, Vilgalys R, 2006. Phylogenetic utility of
indels within ribosomal DNA and beta-tubulin sequences
from fungi in the Rhizoctonia solani species complex. Molecular
Phylogenetics and Evolution 40: 459e470.
Gouy M, Guindon S, Gascuel O, 2010. SeaView version 4: a
multiplatform graphical user interface for sequence
alignment and phylogenetic tree building. Molecular Biology
and Evolution 27: 221e224.
Hamady M, Lozupone C, Knight R, 2010. Fast UniFrac: facilitating
high-throughput phylogenetic analyses of microbial
communities including analysis of pyrosequencing and
PhyloChip data. The ISME Journal 4: 17e27.
Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF,
€ cking R, 2007. A
Eriksson OE, Huhndorf S, James T, Kirk PM, Lu
higher-level phylogenetic classification of the Fungi.
Mycological Research 111: 509e547.
Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference
of phylogenetic trees. Bioinformatics 17: 754e755.
Johanson A, Turner HCC, McKay GJ, Brown AE, 1998. A PCR-based
method to distinguish fungi of the rice sheath-blight complex,
Rhizoctonia solani, R. oryzae and R. oryzae-sativae. FEMS
Microbiological Letters 162: 289e294.
Katoh K, Toh H, 2008. Recent developments in the MAFFT
multiple sequence alignment program. Briefings in
Bioinformatics 9: 286e298.
Kuninaga S, Natsuaki T, Takeuchi T, Yokosawa R, 1997. Sequence
variation of the rDNA ITS regions within and between
anastomosis groups in Rhizoctonia solani. Current Genetics 32:
237e243.
Larsson K-H, 2007. Re-thinking the classification of corticioid
fungi. Mycological Research 111: 1040e1063.
Letunic I, Bork P, 2007. Interactive Tree Of Life (iTOL): an online
tool for phylogenetic tree display and annotation.
Bioinformatics 23: 127e128.
€ ytynoja A, Goldman N, 2005. An algorithm for progressive
Lo
multiple alignment of sequences with insertions. Proceedings of
the National Academy of Sciences, USA 102: 10557e10562.
Ma L-J, van der Does HC, Borkovich KA, Coleman JJ, Daboussi MJ,
Di Pietro A, Dufresne M, Freitag M, Grabherr M, Henrissat B,
2010. Comparative genomics reveals mobile pathogenicity
chromosomes in Fusarium. Nature 464: 367e373.
Maddison WP, Maddison DR, 2009. Mesquite: a modular system for
evolutionary analysis. Version 2.6. URL: http://mesquiteproject.org.
Masuhara G, Katsuya K, 1994. In situ and in vitro specificity
between Rhizoctonia spp. and Spiranthes sinensis (Persoon)
Ames, var. amoena (M. Bieberstein) Hara (Orchidaceae). New
Phytologist 127: 711e718.
Masuhara G, Katsuya K, Yamaguchi K, 1993. Potential for
symbiosis of Rhizoctonia solani and binucleate Rhizoctonia with
seeds of Spiranthes sinensis var. amoena in vitro. Mycological
Research 97: 746e752.
Midford P, Garland T, Maddison W, 2003. PDAP Package of Mesquite.
URL: http://mesquiteproject.org/pdap_mesquite.
Martos F, Munoz F, Pailler T, Kottke I, Gonneau C, Selosse M-A,
2012. The role of epiphytism in architecture and evolutionary
constraint within mycorrhizal networks of tropical orchids.
Molecular Ecology 21: 5098e5109.
Moncalvo J-M, Nilsson RH, Koster B, Dunham SM, Bernauer T,
Matheny PB, Porter TM, Margaritescu S, Weiss M, Garnica S, 2006.
The cantharelloid clade: dealing with incongruent gene trees and
phylogenetic reconstruction methods. Mycologia 98: 937.
Moncalvo J-M, Vilgalys R, Redhead SA, 2002. One hundred and
seventeen clades of euagarics. Molecular Phylogenetics and
Evolution 23: 357e400.
Nilsson RH, Kristiansson E, Ryberg M, Larsson KH, 2005.
Approaching the taxonomic affiliation of unidentified
sequences in public databases e an example from the
mycorrhizal fungi. BMC Bioinformatics 6: 178.
267
Nilsson RH, Abarenkov K, Veldre V, Nylinder S, de Wit P,
Brosche S, Alfredsson JF, Ryberg M, Kristiansson E, 2010a. An
open source chimera checker for the fungal ITS region.
Molecular Ecology Resources 10: 1076e1081.
Nilsson RH, Veldre V, Wang Z, Eckart M, Branco S, Hartmann M,
Quince C, Godhe A, Bertrand Y, Alfredsson JF, 2010b. A note on
the incidence of reverse complementary fungal ITS sequences
in the public sequence databases and a software tool for their
detection and reorientation. Mycoscience 52: 278e282.
Papavizas GC, 1970. Colonization and growth of Rhizoctonia
solani in soil. In: Parmeter JR (ed), Rhizoctonia solani, Biology
and Pathology. University of California Press, Berkeley, CA,
USA, pp. 108e122.
Paradis E, Claude J, Strimmer K, 2004. APE: analyses of phylogenetics
and evolution in R language. Bioinformatics 20: 289e290.
Parmeter JR (ed), 1970. Rhizoctonia solani, Biology and Pathology.
University of California Press, Berkeley, CA, USA.
Pope EJ, Carter DA, 2001. Phylogenetic placement and host
specificity of mycorrhizal isolates belonging to AG-6 and AG-12
in the Rhizoctonia solani species complex. Mycologia 93: 712e719.
Posada D, Crandall KA, 1998. MODELTEST: testing the model of
DNA substitution. Bioinformatics 14: 817e818.
Roberts P, 1999. Rhizoctonia-forming Fungi. A Taxonomic Guide.
Royal Botanic Gardens, Kew, London, UK.
Rosling A, Landeweert R, Lindahl BD, Larsson K-H, Kuyper TW,
Taylor AFS, Finlay RD, 2003. Vertical distribution of
ectomycorrhizal fungal taxa in a podzol soil profile. New
Phytologist 159: 775e783.
Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL,
Levesque CA, Chen WFungal Barcoding Consortium, 2012.
Nuclear ribosomal internal transcribed spacer (ITS) region as a
universal DNA barcode marker for Fungi. Proceedings of the
National Academy of Sciences, USA. http://dx.doi.org/10.1073/
pnas.1117018109.
Selosse M-A, Roy M, 2009. Green plants that feed on fungi: facts and
questions about mixotrophy. Trends in Plant Science 14: 64e70.
Sen R, Hietala AM, Zelmer CD, 1999. Common anastomosis and
internal transcribed spacer RFLP groupings in binucleate
Rhizoctonia isolates representing root endophytes of Pinus
sylvestris, Ceratorhiza spp. from orchid mycorrhizas and a
phytopathogenic anastomosis group. New Phytologist 144:
331e341.
Sharon M, Kuninaga S, Hyakumachi M, Sneh B, 2006. The
advancing identification and classification of Rhizoctonia spp.
using molecular and biotechnological methods compared with
the classical anastomosis grouping. Mycoscience 47: 299e316.
Sharon M, Kuninaga S, Hyakumachi M, Naito S, Sneh B, 2008.
Classification of Rhizoctonia spp. using rDNA-ITS sequence
analysis supports the genetic basis of the classical
anastomosis grouping. Mycoscience 49: 93e114.
Smith SE, Read DJ, 2008. Mycorrhizal Symbiosis, 3rd edn. Academic
Press, London, UK.
Sneh B, Jabaji-Hare S, Neate SM, Dijst G (eds), 1996. Rhizoctonia
Species: taxonomy, molecular biology, ecology, pathology and
disease control. Kluwer Academic Publishers, Dordrecht, The
Netherlands.
Sneh B, 1998. Use of non-pathogenic or hypovirulent fungal
strains to protect plants against closely related fungal
pathogens. Biotechnology Advances 16: 1e32.
Stamatakis A, 2006. RAxML-VI-HPC: maximum likelihood-based
phylogenetic analyses with thousands of taxa and mixed
models. Bioinformatics 22: 2688e2690.
Taylor DL, Bruns TD, Szaro TM, Hodges SA, 2003. Divergence in
mycorrhizal specialization within Hexalectris spicata
(Orchidaceae), a nonphotosynthetic desert orchid. American
Journal of Botany 90: 1168e1179.
Taylor JW, Turner E, Townsend JP, Dettman JR, Jacobson D, 2006.
Eukaryotic microbes, species recognition and the geographic
268
limits of species: examples from the kingdom Fungi. Philosophical
Transactions of the Royal Society, Series B 361: 1947e1963.
Tedersoo L, May TW, Smith ME, 2010. Ectomycorrhizal lifestyle in
fungi: global diversity, distribution, and evolution of
phylogenetic lineages. Mycorrhiza 20: 217e263.
€ ßler A, Grelet G-A,
Tedersoo L, Abarenkov K, Nilsson RH, Schu
Kohout P, Oja J, Bonito GM, Veldre V, Jairus T, Ryberg M,
~ ljalg U, 2011. Tidying up international
Larsson K-H, Ko
nucleotide sequence databases: ecological, geographical and
sequence quality annotation of ITS sequences of mycorrhizal
fungi. PLoS One 6. e24940.
Vilgalys R, Cubeta MA, 1994. Molecular systematics and
population biology of Rhizoctonia. Annual Review of
Phytopathology 32: 135e155.
Warcup JH, 1991. The Rhizoctonia endophytes of Rhizanthella
(Orchidaceae). Mycological Research 95: 656e659.
Waterman RJ, Bidartondo MI, Stofberg J, Combs JK, Gebauer G,
Savolainen V, Barraclough TG, Pauw A, 2011. The effects of
above- and belowground mutualisms on orchid speciation
and coexistence. American Naturalist 177: E54eE68.
V. Veldre et al.
Webb CO, 2000. Exploring the phylogenetic structure of ecological
communities: an example for rain forest trees. American
Naturalist 156: 145e155.
Webb CO, Ackerly DD, Kembel SW, 2008. Phylocom: software for the
analysis of community phylogenetic structure and trait evolution.
Version 4.0.1. Available from URL: http://www.phylodiversity.
net/phylocom/.
Wingfield MJ, Slippers B, Roux J, Wingfield BD, 2001. Worldwide
movement of exotic forest fungi, especially in the tropics and
the Southern Hemisphere. Bioscience 51: 134e140.
Xie J, Yanping Y, Jiang D, Li G, Huang J, Li B, Hsiang T, Peng Y,
2008. Intergeneric transfer of ribosomal genes between two
fungi. BMC Evolutionary Biology 8: 87.
Yagame T, Orihara T, Selosse MA, Yamato M, Iwase K, 2012.
Mixotrophy of Platanthera minor, an orchid associated with
ectomycorrhiza-forming Ceratobasidiaceae fungi. New
Phytologist 193: 178e187.
Yagame T, Yamato M, Suzuki A, Iwase K, 2008. Ceratobasidiaceae
mycorrhizal fungi isolated from nonphotosynthetic orchid
Chamaegastrodia sikokiana. Mycorrhiza 18: 97e101.