Significant diversity and potential problems associated with inferring population
structure within the Cenococcum geophilum species complex
Greg W. Douhan1
Karyn L. Huryn
LeAnn I. Douhan
Department of Plant Pathology and Microbiology,
University of California, Riverside, California 92521
widely distributed and most recognized ectomycor-
rhizal fungus with a host range of more than 200 tree
species from 40 genera of both angiosperms and
gymnosperms. We conducted a phylogenetic analysis
on a large collection of isolates (n 5 74) from North
America and Europe based on glyceraldehyde 3-
phosphate dehydrogenase (gpd). A subset of isolates
(n 5 22) also was analyzed with the more conservative
LSU-rDNA locus. Significant nucleotide diversity was
detected (, 20%) in the gpd region and the LSU-
rDNA analysis supported that the C. geophilum
isolates studied were monophyletic but distinct from
two isolates, Am5-1 and N2-10, which previously were
used in population genetic studies of this species.
These results suggest that Am5-1 and N2-10 are likely
two undescribed species or even genera. Our results
suggest that C. geophilum sensu lato is a species
complex and support previous molecular, physiolog-
ical and morphological studies that have shown
significant diversity in C. geophilum. This study also
revealed that caution is advised when conducting
population genetic studies in C. geophilum due to the
possibility of pooling unrelated isolates. This poten-
tial problem also has implications for other fungal
taxa because cryptic species routinely have been
found in recent years based on molecular data.
Cenococcum geophilum is perhaps the most
Cenococcum geophilum is one of the most commonly
encountered soil fungi and is found in diverse
habitats throughout northern temperate regions
(Trappe 1964). C. geophilum is thought to play
a significant role in many ecosystems because it forms
ectomycorrhizal (EM) associations with a diverse array
of gymnosperm and angiosperm hosts (LoBuglio
1999). This mutualism allows the uptake of mineral
nutrients, organic nutrients and water for the host,
and in exchange the fungi receive photosynthates
(Smith and Read 1997). C. geophilum is one of the few
mycorrhizal species that routinely is identified based
on the morphology of the colonized roots where it
produces a dark black mantle with emanating stellate
hyphae (Trappe 1964). This fungus also is isolated
easily and cultured in vitro directly from sclerotia
found in the soil. Given the wide host range and
distribution, ease of experimental manipulation and
potential ecological importance, a considerable
amount of research has been conducted with this
species. Most studies have found considerable cultural
and physiological variation among isolates of C.
geophilum sensu lato collected from the same envi-
ronment as well as from diverse geographic regions
(e.g. LoBuglio 1999).
C. geophilum is also one of the many fungal species
in which the production of sexual or asexual spores is
not known to occur. However Ferna ´ndez-Toira ´n and
A´gueda (2007) have claimed to have found cleis-
tothecia of C. geophilum based on similar morphology
between the cleistothecia and sclerotia. No single
ascospore cultures or molecular methods were used
to confirm the identification so this finding remains
to be substantiated. The only known means of
reproduction for C. geophilum are the production of
mitotically derived sclerotia that can serve as dispersal
and survival structures. However recent population
genetic analyses have revealed considerable genotypic
diversity within and among populations of this fungus
(Jany et al 2002, LoBuglio and Taylor 2002, Panac-
cione et al 2001, Wu et al 2005). Based on restriction
fragment length polymorphism (RFLP) analysis of
the entire rDNA region, it has been suggested that C.
geophilum is either a heterogeneous species or is
a species complex (LoBuglio et al 1991). For example
LoBuglio et al (1991) detected 32 unique genotypes
out of 71 isolates collected from broad host and
geographic ranges. However some of this variation
was attributed to a Group-I intron (CgSSU intron)
found within the 39 end of the small subunit (SSU) of
rDNA (LoBuglio 1999). A phylogenetic analysis on
the same isolates was conducted with the ITS-rDNA
region. Shinohara et al (1999) found up to 4%
sequence divergence among the isolates and con-
cluded that C. geophilum was in fact a ‘‘single
taxonomic entity, possibly a single species’’ that was
extremely adaptable and widespread.
Accepted for publication 30 July 2007.
1Corresponding author. Fax: 951-827-4132; E-mail: gdouhan@
Mycologia, 99(6), 2007, pp. 812–819.
# 2007 by The Mycological Society of America, Lawrence, KS 66044-8897
We recently detected phylogenetically distinct
lineages or cryptic species of C. geophilum at the
spatial scale of a single soil sample in an oak-
woodland of California based on the analyses of
a glyceraldehyde 3-phosphate dehydrogenase (gpd)
gene, ITS-rDNA, a group I intron located in the 39
end of the SSU-rDNA and a portion of the mitochon-
drial SSU-rDNA (Douhan and Rizzo 2005). Moreover
C. geophilum isolates from Oregon, Alaska and Mary-
land also clustered within the California lineages,
suggesting this ‘‘species complex’’ has a wide geo-
graphic distribution. These results help explain the
large amount of physiological, phenotypic and genetic
differences reported among isolates of C. geophilum
from similar as well as diverse geographic regions
(LoBuglio 1999). However the ecological role that
these cryptic species play remains to be determined.
The objectives of this study were to broaden our
views of C. geophilum diversity by examining widely
distributed isolates from North America and Europe.
We chose to use the gpd locus because we have found
that it is highly variable and easy to PCR amplify. This
locus also shows significant congruence with other
loci that we have tested (Douhan and Rizzo 2005,
Douhan unpubl). We hypothesized that the gpd locus
would reveal even more cryptic diversity than we
previously have found from isolates mostly collected
from a single environment (Douhan and Rizzo 2005).
MATERIALS AND METHODS
Fungal isolates.—Both dried hyphal material and cultures
of C. geophilum were used. Information regarding the C.
geophilum isolates is provided (TABLE I). For cultured
material freshly transferred hyphal tips were transferred to
potato-dextrose agar plates and incubated 1–3 mo to allow
enough growth for DNA extraction. The hyphae were
scraped off the agar plates, freeze-dried, ground and the
DNA was isolated with a slightly modified phenol:chloro-
form extraction procedure of Lee and Taylor (1990). DNA
TABLE I. Collection information for the Cenococcum geophilum isolates used in this study
IsolateLocation and potential hostReference or source
2-11-1, 1-1-4, 3-10-3, 2-6-1,3-18-1, 1-19-1, 3-8-2, 2-3-1,
2-9-1, 1-1-7, 2-13-2, 1-7-11, 1-16-2, 1-7-8, 1-14-7,
1-7-1, 2-10-3, 3-9-2, 2-14-4, 1-5-4, 2-4-1, 3-11-1,
Btree1, I-2, I-3
N3-4 5 03-4-II, N2-10, S2-10
CGURNA 51.15, CGTAR 51.04, CGSCA 51.07,
CGTSCHA 51.08, CGWEG51.09, CGHOR 51.14,
CGPIL 51.27, CGCHEY51.36, CGLESPAC 51.52,
CGKLAU 51.53, CGBOD 51.01
MC149 5 A149
747M, 217M, 175M, 537M, 619M, 428R
Am2-1, Am4-1, Cre1-1, Cre2-1, H1-3, H4-1, H5-1,
Browns Valley, California; Quercus
Douhan and Rizzo 2005
Oregon; Quercus garryana
Maryland; Quercus sp.
Wickersham Dome, Alasja; Betula nana
Holland; Picea abies
Panaccione et al 2001
LoBuglio et al 1991
Switzerland; Picea abies
Alabama; Quercus velutina
Asturia, Spain; Betula pendula
Mary’s Peak, Oregon; Pseudotsuga menziesii
Blue River, Oregon; Pseudotsuga menziesii
Chugach, Alaska; Tsuga mertensiana
Newcomb, New York; Picea rubens
Newcomb, New York; Pinus strobus
Georgia; loblolly pine
Washington; Pseudotsuga menziesii
Newcomb, New York; Unknown
Spain; Quercus ilex
France; Epicea sp.
France; Fagus sylvatica
France; Fagus sylvatica
Susana Gonc ¸alves
Jany et al 2002
aThis isolate was sent to the senior author from Danniel Panaccione as 03-4-II but was found to be the same isolate as N3-4
from Panaccione et al 2001 (Danniel Panaccione, pers com).
DOUHAN ET AL: CENOCOCCUM GEOPHILUM
from the dried material provided by K. LoBuglio (Harvard
University Herbaria, USA) was extracted in a similar
PCR, recombination tests and phylogeny reconstruction.—PCR
was set up with 20 mL reaction mixtures containing 2 mL of
a 1:10–1:25 dilution of template DNA, 13 PCR buffer
(Invitrogen, Carlsbad, California), 2.5 mM MgCl2, 0.2 mM
each dNTP (Invitrogen), 7.5 mM of each primer and 0.5 U
of Taq polymerase (Invitrogen). For the gpd region primers
gpd 1 and gpd 2 were used as described in Berbee et al
(1999). Thermo-cycling conditions consisted of an initial
hold at 94 C for 3 min, followed by 25 cycles of 94 C (30 s),
60 C (30 s) and 72 C (1 min), and a final hold of 72 C for
8 min. For LSU-rDNA we used primers ITS1F (Gardes and
Bruns 1993) and LR3 (Hopple and Vilgalys 1994). Thermo-
cycling conditions were the same as for gpd except the
annealing temperature was lowered to 55 C. All amplifica-
tions were performed in a MyCycler (Bio-Rad Laboratories
Inc., Hercules, California). A subset of isolates was chosen
to sequence the LSU-rDNA region which represented the
diversity of the gpd phylogeny (see RESULTS). This additional
analysis also was done because gpd sequences from two of
the isolates were considerably divergent from the reset of
the sequences and because of the significant amount of
variation found in gpd. We wanted to test whether our C.
geophilum isolates represented a monophyletic group using
the more conserved LSU-rDNA locus.
For each reaction 2.5 mL was separated on a 1.5% agarose
gel, stained with SYBR Green I nucleic acid stain and viewed
under UV light. PCR products were cleaned with ExoSap-IT
(USB, Cleveland, Ohio) following the manufacturer’s
instructions. The gpd region was sequenced in both
directions whereas the LSU-rDNA was sequenced only in
one direction with LR3 using Big DyeH Terminator v3.1
chemistry (Applied Biosystems, Foster City, California).
Sequencing was performed at the Core Instrumentation
Facility (CIF) of the University of California at Riverside’s
Institute of Integrative Genome Biology. The sequences
were edited with Sequencher (version 4.6, Gene Codes
Corp., Ann Arbor, Michigan), aligned with Clustal X
(version 1.81) (Thompson et al 1997) and visually edited
in MacClade version 4 (Maddison and Maddison 2001). For
LSU-rDNA care was taken to use only sequences that had
strong chromatograms because only a single read was done.
Six analytical methods were used to test for recombina-
tion within the gpd region before phylogenetic analysis with
RDP (Recombination Detection Program, beta version 2.6:
htpp://darwin.uvigo.es). The specific recombination tests
that were used included RDP (Martin and Rybicki 2000),
GENECOV (Padidam et al 1999), Bootscanning (Salminene
et al 1995), MaxChi (Maynard Smith 1992), Chimaera
(Posada and Crandall 2001) and SiScan (Gibbs et al 1997).
Default settings in RDP were used for each test and a 5 0.05
was used to test for significance.
We previously identified a 42–44 bp indel from some of
our C. geophilum isolates from lineage II (Douhan and Rizzo
2005). The inclusion of worldwide samples increased the
size of the indel to 42–48 bp. This region was deleted from
isolates that had the ‘‘extra’’ bases as well as approximately
50 bp adjacent to the indel because the alignment was
ambiguous. Moreover this was also a region identified as
possibly recombinant (see RESULTS). Maximum parsimony
(MP) analysis was conducted with the heuristic search
procedure with 1000 random-addition sequence replicates
and tree-bisection-reconnection branch swapping were
conducted with PAUP* version 4.0 beta 10 (Swofford
2002). Confidence in tree topology was examined with
bootstrap with 10000 replicates using the ‘‘fast’’ stepwise
addition procedure. The LSU-rDNA tree was rooted with
Am5-1, whereas the gpd was midway rooted because the
divergent sequences found in isolates Am5-1 and N2-10
could not be aligned unambiguously (see RESULTS) to root
the tree and no other sequences in GenBank were related
closely enough to make a reliable alignment.
Testing for recombination in the gpd locus.—The gpd
locus was tested for recombination to ensure that it
was a proper locus to infer phylogenetic relationships
among the C. geophilum isolates in this study, because
preliminary analysis found substantial nucleotide
variation in this region. The gpd region for isolates
Am5-1 and N2-10 were not included in the analysis
because they were significantly divergent from the rest
of the gpd sequences and could not be aligned
unambiguously. Out of the six tests used to detect
recombination only the SiScan method detected any
potential recombination in gpd sequences. However
this method might be sensitive to evolutionary rate
variation along the length of an alignment and prone
to reporting false positives (Worobey et al 2002).
Nevertheless we removed the region where SiScan
revealed potential recombination, which was adjacent
to the indel. Moreover this putative recombinant
region also was difficult to align, which justified
deleting it from the final alignment. Analyses run on
the dataset after removal of this problematic region
showed no evidence of recombination. This was the
dataset that was used to estimate the phylogeny of our
isolates based on gpd.
Phylogenetic analyses.—MP analysis of the gpd region
(362 bp) for 74 isolates produced a tree that was
highly diverse with 290 constant sites, 19 uninforma-
tive sites and 53 informative sites (FIG. 1). Two isolates
were sequenced twice from independent cultures (I-3
5 I-3A and N3-4 5 03-4-II). All new sequences have
EU306912–EU306956). The gpd sequences for the
isolates from Douhan and Rizzo (2005) have been
deposited in GenBank.
Many of the terminal nodes had high bootstrap
support and in general many isolates clustered with
other isolates from the same general geographic
region (FIG. 1). However these geographic clusters
were dispersed across the tree and no bootstrap
support was found for the backbone of the phylogeny.
Isolates that clustered between distant regions were
found only for two clades. Isolates from Oregon
(A145) and Alaska (A175) clustered with an isolate
from Switzerland (CGURNA 51.15) but was not
supported by any bootstrap support. Another isolate
from Oregon (A166) clustered with a different isolate
from Switzerland (CGTAR 51.04) but was supported
only by a bootstrap value of 66%.
hydrogenase gene. Bootstrap support of 50% and above are indicated above nodes based on 10000 replicates.
Maximum parsimony analysis of Cenococcum geophilum isolates based on glyceraldehyde 3-phosphate de-
DOUHAN ET AL: CENOCOCCUM GEOPHILUM
MP analysis of the LSU-rDNA region (590 bp) for
a subset of the isolates (n 5 22) produced a well
resolved tree with 476 constant sites, 84 uninformative
sites and 30 informative sites (FIG. 2). Isolates Am5-1
and N2-10 with the divergent gpd sequences clustered
apart from the rest of the isolates with a bootstrap
support of 99%, and no additional support was found
for subclusters among the rest of the isolates. This
supports the hypothesis that all isolates except Am5-1
and N2-10 represent a monophyletic lineage.
above are indicated above nodes based on 10000 replicates.
Maximum parsimony analysis of Cenococcum geophilum isolates based on LSU-rDNA. Bootstrap support of 50% and
We found a significant amount of diversity in the gpd
phylogeny of North American and European isolates
of C. geophilum. However a high level of bootstrap
support was not found for the majority of the
backbone of the phylogeny and thus phylogeographic
inference could not be made. Two isolates in previous
studies of C. geophilum population genetics, Am5-1
and N2-5, were found to have gpd sequences that were
significantly divergent compared to the alignment of
the other sequences. We sequenced the more
conserved LSU-rDNA region for a subset of isolates
that represented the diversity of the gpd phylogeny.
This analysis supports monophyly for isolates, except
Am5-1 and N2-5. These results along with results from
Douhan and Rizzo (2005) suggest a phylogenetic
basis for the extensive phenotypic and physiological
differences that have been found for isolates of C.
geophilum sensu lato. These results also demonstrate
the potential problems with inferring population
genetic inferences due to the pooling of unrelated
isolates at scales of centimeters to hundreds of
Isolate Am5-1 was isolated and identified as C.
geophilum based on colony morphology from a beech
forest in France (Jany et al 2002). However randomly
amplified polymorphic DNA (RAPD) analysis placed
it on a long branch by itself away from many of the
other isolates. Our LSU-rDNA analysis suggests that
this taxon does not belong to the C. geophilum species
complex at all. BLAST of this isolate for the LSU
matched 99% to Melinomyces bicolor (AY394885),
a fungus that produces black ectomycorrhizal with
hardwoods and pines (Mitchell and Gibson 2006).
However the colony morphology on PDA is dark gray
whereas C. geophilum produces dark brown to black
colonies. Thus the identification of this isolate
remains unknown because likely there is insufficient
resolution in LSU for species identification. In
contrast a BLAST of this isolate for the gpd region
identified a sequence that closely matches (94%)
another fungus, Helicoma irregulare (DQ128090). The
sequences are different but a comparison to the
alignment among these two sequences and the others
clearly demonstrates their similarity (data not shown).
Descriptions of Helicoma species have similar mor-
phological characters as C. geophilum, such as dark
melanized hyphae and subterranean growth charac-
teristics in culture (Goos 1986). Therefore it is
possible that Am5-1 might have been derived from
a Helicoma ancestor and lost its ability to make spores.
However this is purely speculation and additional
studies are needed to test this hypothesis.
BLAST with the N2-10 LSU did not have any
significant hits (,88%) and the gpd sequence was
informative only in the broad sense in that a portion
of sequence aligned with some Loculoascomycetes.
Thus the identification of this isolate is also not
possible. Of interest, the closest LSU hits were from
Helicoma related species and Tsui and Berbee (2006)
found that the closest relative of one species, H. isiola,
was C. geophilum based on analysis of SSU and LSU
sequences. N2-10 was used in a population genetic
structure study based on amplified fragment length
polymorphism (AFLP) in which significant genotypic
diversity was found (Panaccione et al 2001). However,
on inspection of some of the figures from Panaccione
et al (2001), N2-10 has a unique RFLP-ITS pattern
compared to the rest of the isolates, is on a long
branch by itself in a phenogram based on AFLP data
and has an ITS fragment that is a different size than
the rest of the isolates that did not posses the Group I
intron in the 39 end of the small subunit of rDNA.
These findings along with our current results dem-
onstrate the potential problems associated with
pooling isolates that might not be related realistically
when inferring population structure. This also was
supported by a multigene analysis of 10 loci in which
the acceptance or rejection of random mating based
on gametic disequilibrium analyses was highly de-
pended on species concept in C. geophilum (Douhan
et al 2007).
For putative asexual fungi that lack spores and
spore-bearing structures, traditional species concepts
based on morphology might not be adequate to
properly identify a taxon to species. For the fungi the
ITS region, including partial LSU-rDNA, ITS-1, 5.8s,
ITS-2 and partial SSU-rDNA, has been the marker of
choice for differentiating fungi at the species level
and a substantial public database has grown, namely
GenBank (Bruns and Shefferson 2004, Bidartondo
and Gardes 2005, O’Brien et al 2005). However there
are examples of closely related fungi, such as the
Phialocephala fortinii complex, where ITS phylogenies
alone do not resolve species adequately (Gru ¨nig et al
2004). There are also examples of distinct species
based on biological and ecological data where ITS
sequences are identical or nearly identical among
species such as in the mushroom genus Armillaria
(Anderson and Stasovski 1992) and between the
ascomycete species Ceratocystis polonica and C.
laricicola (Harrington and Rizzo 1999).
For C. geophilum Shinohara et al (1999) published
a previous ITS phylogeny of many of the same isolates
and found only approximately 4% variability com-
pared to almost 20% in gpd region in this study, and
they suggested that C. geophilum was a cohesive
species because intraspecific diversity in other fungi
also has been reported (e.g. Shinohara et al 1999).
DOUHAN ET AL: CENOCOCCUM GEOPHILUM
However divergent lineages of C. geophilum were
found that occupied the same soil core (Douhan and
Rizzo 2005). If these organisms were functioning as
a cohesive ‘‘biological’’ species we would not expect
so much divergence at this scale because they
potentially could interact with one another. This
highlights the utility of using fine scale and macro-
scale sampling when trying to understand species
barriers within fungi, especially those in which any
type of cytoplasmic exchange of genetic material is
not known to occur. A similar pattern in bolete
parasites (Hypomyces spp.) also has been observed
(Douhan and Rizzo 2003). Within California isolates
from divergent AFLP clades, which also correspond to
ITS types, can be found at local scales but the same
AFLP types also could be found separated by more
than 600 km (Douhan and Rizzo 2003). If interbreed-
ing were occurring we would expect more homoge-
nized banding patterns from cohesive species.
Species concepts and the type of analysis are
important when inferring how a biological organism
reproduces and spreads. For the fungi and especially
for putative asexual species that lack significant
morphological differences, multigene genealogies
have become a popular approach. Taylor et al
(2000) advocated using the analyses of multiple genes
as a criterion to identify species within the fungi,
which they term the genealogical concordance
phylogenetic species recognition (GCPSR). They
suggest using multiple genes to determine the
transition from concordance to conflict among taxa,
which can be used to determine species boundaries
and potential recombination within a phylogenetic
species. Phylogenetic species for many morphospecies
within various fungal genera have been identified
with this approach with some examples including
Fusarium (Skovgaard et al 2002), Stachybotrys (Cruse
et al 2002), Coccidioides and some of its close relatives
(Koufopanou et al 2001). However this approach can
be vulnerable to sampling bias. For example we
previously analyzed four loci in a local population of
C. geophilum (Douhan and Rizzo 2005). Incongru-
ence in the datasets was apparent only when isolates
from outside the sampling location were included in
the analysis. Therefore we ask whether the local
population is not recombining and whether the
history of recombination is evident only in this
lineage due to past events. Moreover inclusion of
many more isolates from broader geographic regions
revealed much more diversity than found previously
(Douhan and Rizzo 2005) and also blurred the
phylogenetic relationships among C. geophilum iso-
lates. Douhan and Rizzo (2005) found three well
supported lineages of C. geophilum, whereas in the
present study no support could be found for deep
phylogenetic relationships and primarily only termi-
nal nodes had any support.
C. geophilum sensu lato clearly is widespread geo-
graphically and ecologically successful, which is
amazing given its inability to produce any type of
spore for dispersal. However recognizing C. geophilum
as an actual species complex helps to explain the
apparent success of this ubiquitous mycorrhizal
fungus. A detailed understanding of this species
complex awaits further study. Multigene genealogy
studies of C. geophilum populations sampled through-
out its known range likely will be needed to un-
derstand this species complex. Detailed biological
studies then may reveal associated phenotypic differ-
ences (morphological, physiological) among phylo-
genetic species within C. geophilum that might lead to
a better understanding of the ecology of mycorrhizal
Financial support of the Agricultural Experiment Station,
University of California at Riverside, is gratefully acknowl-
edged. We thank Jim Trappe, Danniel Panaccione, Francis
Martin and Susana Concalves for C. geophilum cultures,
Darlene Southworth for sclerotia samples, Kathy LoBuglio
for freeze dried mycelium, and Randolph Currah and
anonymous reviewers for helpful comments and sugges-
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