f u n g a l b i o l o g y 1 1 5 ( 2 0 1 1 ) 5 6 9 e5 9 7
journal homepage: www.elsevier.com/locate/funbio
Review
Population genetics of ectomycorrhizal fungi: from current
knowledge to emerging directions
Greg W. DOUHANa,**,1, Lucie VINCENOTb,***,1, Herve GRYTAc,d, Marc-Andre SELOSSEb,*
a
Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA
Centre d’Ecologie Fonctionnelle et Evolutive, CNRS UMR 5175, Equipe Interactions Biotiques, 1919 Route de Mende,
F-34293 Montpellier cedex 5, France
c
Universite de Toulouse, UPS, UMR 5174 EDB (Laboratoire Evolution et Diversite Biologique), 118 route de Narbonne,
F-31062 Toulouse, France
d
CNRS, UMR 5174 EDB (Laboratoire Evolution et Diversite Biologique), F-31062 Toulouse, France
b
article info
abstract
Article history:
Ectomycorrhizal (EM) fungi are major microbial components of boreal, temperate and Med-
Received 15 November 2010
iterranean forests, as well as some tropical forest ecosystems. Nearly two decades of stud-
Received in revised form
ies have clarified many aspects of their population biology, based on several model species
6 March 2011
from diverse lineages of fungi where the EM symbiosis evolved, i.e. among Hymenomycetes
Accepted 12 March 2011
and, to a lesser extent, among Ascomycetes. In this review, we show how tools for individ-
Available online 21 March 2011
ual recognition have changed, shifting from the use of somatic incompatibility reactions to
Corresponding Editor:
dominant and non-specific markers (such as random amplified polymorphic DNA (RAPD)
Nicholas P. Money
and amplified fragment length polymorphism (AFLP)) and, more recently, to co-dominant
and specific markers (such as microsatellites and single nucleotide polymorphisms (SNPs)).
Keywords:
At the same time, the theoretical focus has also changed. In earlier studies, a major aim
Biogeography
was the description of genet size and popul/ation strategy. For example, we show how
Cryptic biological species
some studies supported or challenged the simple, classical model of colonization of new
Ecological strategies
forest stands by ruderal (R) species, propagating by spores and forming small genets, pro-
Fungal reproduction
gressively replaced in older forests by more competitive (C) species, propagating by myce-
Gene flow
lial growth and forming larger genets. By contrast, more recent studies give insights into
Isolation by distance
some genetic traits, such as partners’ assortment (allo- versus autogamy), genetic structure
Mating systems
of populations and gene flow that turn out to depend both on distance and on whether
Molecular markers
spores are animal- or wind-dispersed. We discuss the rising awareness that (i) many mor-
Population genetics
phospecies contain cryptic biological species (often sympatric) and (ii) trans- and inter-con-
Somatic incompatibility
tinental species may often contain several biological species isolated by distance. Finally,
we show the emergence of biogeographic approaches and call for some aspects to be
developed, such as fine-scale and long-term population monitoring, analyses of
* Corresponding author. Centre d’Ecologie Fonctionnelle et Evolutive, CNRS UMR 5175, Equipe Interactions Biotiques, 1919 Route de
Mende, F-34293 Montpellier cedex 5, France.
** Corresponding author. Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA. Fax: þ1 951
827 4132.
*** Corresponding author. Present address: IASMA-Fondazione Edmund Mach, Research and Innovation Center, via E. Mach 1, 38010 San
Michele all’Adige, Italy. Tel.: þ39 0461615507; Fax: þ39 0461650872.
E-mail addresses: [email protected], [email protected], [email protected]
1
G.W. Douhan & L. Vincenot equally contributed to this article and should be considered as associated first authors.
1878-6146/$ e see front matter ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.funbio.2011.03.005
570
G. W. Douhan et al.
subterranean populations of extra-radical mycelia, or more model species from the tropics,
as well as from the Ascomycetes (whose genetic idiosyncrasies are discussed). With the
rise of the ‘-omics’ sciences, analysis of population structure for non-neutral genes is expected to develop, and forest management and conservation biology will probably profit
from published and expected work.
ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
The mycorrhizal interaction between fungi and plant roots is
a common, worldwide symbiotic interaction (Smith & Read
2008; Tedersoo et al. 2010) that contributed to land colonization by terrestrial plants and to their posterior diversification
(Selosse & Le Tacon 1998). Although a limited fraction of the
land flora associates with ectomycorrhizal (EM) fungi, EM associations are dominant among the major forest trees in the
Mediterranean, temperate, boreal and some tropical regions,
from the families Pinaceae, Fagaceae, Nothofagaceae, Myrtaceae
and Dipterocarpaceae, making EM fungi ecologically relevant
(Smith & Read 2008). EM association involves an estimated
6000 (Brundrett 2002) to >20 000 fungal species (Rinaldi et al.
2008), and is thought to have evolved at least ten times
(Hibbett & Matheny 2009; Tedersoo et al. 2010) within the
Asco- and Hymenomycetes (a subclade of Basidiomycetes).
EM fungi have widely been recognised based on morphological characters, because some of them produce conspicuous
epigeous fruitbodies (Amanita, Boletus, Pisolithus, Russula,
Suillus.), but some are less obvious, producing hypogeous
(‘truffle-like’ fungi: Rhizopogon, Tuber.) or resupinate (Hydnellum, Thelephora.) fruitbodies or even no known fruitbodies,
such as the widespread Cenococcum geophilum. Beside their
major ecological roles in some forests, EM associations are
also economically important because they improve tree
growth (Le Tacon et al. 1992; Selosse et al. 1998) or are sources
of edible mushrooms: ca. 200 EM species are edible, such as
truffles in Europe and matsutake mushrooms in Asia, which
are both estimated to be worth over US$ 2 billion/y (Yun &
Hall 2004).
Although a number of reviews have focused on various aspects of EM community ecology (Dahlberg 2001; Horton &
Bruns 2001; Taylor 2002), to our knowledge no review in the
last 15 y has specifically been devoted to EM population genetics (Dahlberg & Stenlid 1995; except for one review in French,
Selosse 2001a), although Xu (2006) reviewed elements for molecular genetic analyses of fungal populations. Even though
scarce, in terms of frequency as compared with other organisms, publications on EM population genetics are accumulating linearly at a constant rate (Figs 1 and 2), signalling
a permanent interest in the EM research community. Many
studies have focused on some model EM taxa, such as Laccaria
spp., Hebeloma cylindrosporum, Tricholoma matsutake, Suillus
spp., Rhizopogon spp., C. geophilum, and Tuber ssp., but beside
these ‘model species’ investigations have also been accumulating for many other EM species from diverse ecosystems
(Tables 1 and S1). Here, we review how population genetics
studies have enhanced our knowledge about establishment,
maintenance and dynamics of EM populations, as well as
about the functioning of the EM symbiosis and biology of EM
fungi under natural conditions.
The complexity of EM communities
Compared with single-partner symbiotic systems, EM symbiosis in nature acts more like a network connecting a host plant
with several fungal species and individuals, and vice versa
(Selosse et al. 2006). Estimations of dozens of EM species sharing
the root system of the same host plant are commonly reported
(Dahlberg 2001; Horton & Bruns 2001; Richard et al. 2004; Hynes
et al. 2010). As a result, many fungal and host species co-exist
in a forest stand. Moreover, greenhouse and in vitro experiments
have also found large quantitative variation even among isolates of the same species for traits involved in mycorrhizal functioning, e.g. the capacity to form EM associations or to use soil
nutrients (e.g. di Battista et al. 1996; Guidot et al. 2005).
First attempts to understand the diversity and ecology of
communities of EM fungi were based on the distribution of epigeous fruitbodies and the below-ground distribution of colonised mycorrhizal root tips, as characterised by gross
morphology (e.g. Agerer 1991, 1995). However, the observed
root tip morphotypes often correlated poorly with described epigeous fungal fruitbodies (e.g. Nylund et al. 1995). More recently,
molecular tools such as restriction fragment length polymorphisms (RFLPs) and/or sequencing of the internal transcribed
spacer (ITS) of the ribosomal DNA from individual root tips
(Gardes & Bruns 1996; Begerow et al. 2010) greatly enhanced
the identification of EM species directly on host roots. In a review
on EM community ecology, Horton & Bruns (2001) emphasised
three general features: (i) there is generally a poor correspondence, in terms of species identity and abundance, between
fungi found as above-ground fruitbodies and fungi identified
on EM roots (Gardes & Bruns 1996; Peay et al. 2007); (ii) underground EM communities show high diversity and species often
have patchy distribution (e.g. Richard et al. 2004, 2005); and (iii) at
least some abundant EM species can be shared by different hosts
(including some understory herbs, see Selosse et al. 2004).
Studies of EM populations: evolution of tools.
Historically, a first step in the ecological study of EM fungi
applied the concepts and methods of population genetics (see
Glossary) to EM fungi, in order to characterise the genetic
diversity and distribution of specific species. Early studies
used somatic incompatibility (SI) reactions that had been previously used for many saprotrophic and pathogenic fungi: hyphal
fusion between different mycelia is stable only between genetically similar individuals, while genetically dissimilar ones produce a reaction zone (Worrall 1997; Glass & Fleissner 2006).
Population genetics of EM fungi
571
Fig 1 e The accumulation of papers on EM population genetics over the two last decades is linear (source: ISI web of
knowledge). Grey bars, number of peer-reviewed publications about “ectomycorrhiz* population*” for each year; black line,
accumulation of these publications over time.
Thus, SI operates in the exclusion of non-self, prevents intraspecific parasitism by restricting fusion to kin, and avoids the
spreading of mycoviruses (Glass & Fleissner 2006). Tests on mycelia isolated from fruitbodies delineated individuals in natural
EM populations (e.g. Fries 1987; Dahlberg & Stenlid 1990). However, the isolation step required before SI tests is difficult, and
entails loss of some individuals, and cannot be used on non-cultivable EM species. In addition, results from SI pairings can be
ambiguous, even when a single isolate is paired with itself
(Jacobson et al. 1993), and reveal genetic differences at the loci involved in the reaction (from 1e2 up to 50 loci; Worrall 1997) but
not necessarily across the whole genome. Therefore, although
the alleles of SI recognition can be numerous, lack of SI between
two isolates does not necessarily imply full genetic identity
(Jacobson et al. 1993; Rodrigues et al. 1995). As a result, some features can be overestimated by using SI, such as the size of genets
(¼genetic individuals), or underestimated, such as the genetic
diversity. A major concern arises if spatially close individuals
are genetically related (see below): kinship may entail a lack of
SI and prevent delineation of individuals.
Molecular genotyping, which was introduced later, circumvented the need to obtain pure cultures of EM fungi. Although
more expensive, PCR-RFLP, random amplified polymorphic
DNA (RAPD), amplified fragment length polymorphism
(AFLP), microsatellites and single nucleotide polymorphisms
(SNPs; see Glossary and detailed review below) require less
time and can access genome-wide polymorphisms, whereas
SI reactions involve only alleles associated with recognition/
non-recognition. The latter two methods, most recently introduced, provide polymorphic markers that are very reproducible, co-dominant, and potentially numerous. Moreover, they
can give access to genomic regions that are not under
selection (neutral markers), and therefore may better reflect
the history of the populations. They use species-specific
primers and thus allow genotyping of diverse environmental
samples, including mycorrhizal root tips, which offer a direct
perspective on below-ground populations (e.g. Kretzer et al.
2000; Zhou et al. 2001; Selosse et al. 2002; Lian et al. 2006),
and apply to species that do not fruit or rarely produce fruitbodies (e.g. Jany et al. 2002; El Karkouri et al. 2006).
Studies of EM populations: .changes in focus
Beyond this shift in tools, a review of the literature reveals two
common themes. A majority of the first EM fungal population
genetics studies to date have focused on determining the size
and distribution of fungal genets. As we detail below, these studies have generally been descriptive, but have led to inferences
concerning the reproductive biology and ecological strategies of
EM fungi, e.g. colonization mainly by spores or mycelium (e.g.
Dahlberg & Stenlid 1990; Gherbi et al. 1999), founder effect
(Selosse 2003), phenotypic variability (Redecker et al. 2001),
growth rate (Gryta et al. 2000), adaptation to the host (Gryta et al.
2006; Roy et al. 2008) and fruiting phenology (Selosse et al. 2001).
By contrast, more recent studies have focused on the importance of evolutionary forces such as mating systems, migration, selection, genetic drift, and recombination that
likely shape fungal populations (Milgroom 1996). Molecular
markers made it possible to test hypotheses about mating systems, population subdivision, gene flow, cryptic speciation
and hybridization, with important consequences for conservation biology. Moreover, populations (¼collections of individuals) are defined by the observer, and now range from very
local to global scales: the approach recently enlarged to
572
G. W. Douhan et al.
Fig 2 e Map of the main local population dynamics studies of EM species cited in this paper. Note that this figure does not
reflect the actual ecology of EM fungi: even though most studies focus on populations of the Northern Hemisphere, EM
diversity is not concentrated in these geographic zones; boreal, tropical and subtropical forests also harbour EM species.
1: Rhizopogon vesiculosus, R. vinicolor (Kretzer et al. 2004, 2005), 2: Cantharellus formosus (Dunham et al. 2003, 2006), 3: Russula
brevipes (Bergemann & Miller 2002), 4: R. brevipes (Bergemann et al. 2006), 5: Suillus pungens (Bonello et al. 1998), 6: S. pungens,
Amanita franchetii (Bruns et al. 2002), 7: A. franchetii (Redecker et al. 2001), 8: Suillus granulatus (Jacobson et al. 1993), 9:
Hydnellum peckii, Phellodon tomentosus (van der Linde et al. 2009), 10: Leccinum duriusculum (Selosse 2003), 11: Hebeloma
cylindrosporum (Gryta et al. 1997, 2000; Guidot et al. 2002, 2003a), 12: Tricholoma populinum, T. scalpturatum complex (Gryta
et al. 2006), 13: T. scalpturatum (Carriconde et al. 2008b), 14: Laccaria bicolor (Selosse et al. 1998, 1999), 15: Cenococcum geophilum
(Jany et al. 2002), 16: Laccaria amethystina, Xerocomus chrysenteron, X. pruinatus (Fiore-Donno & Martin 2001), 17: L. amethystina (Gherbi et al. 1999), 18: Suillus luteus (Muller et al. 2004, 2007), 19: L. bicolor (Baar et al. 1994), 20: S. bovinus (Dahlberg &
Stenlid 1990, 1994), 21: S. variegatus (Dahlberg 1997), 22: Laccaria amethystina (Hortal et al. in preparation) 23: Tricholoma
matsutake (Amend et al. 2009), 24: Russula vinosa (Liang et al. 2005), 25: Suillus grevillei (Zhou et al. 1999, 2000, 2001), 26:
L. amethystina, L. laccata (Wadud et al. 2006a, 2006b; Wadud 2007), 27: C. geophilum (Wu et al. 2005), 28: T. matsutake (Lian et al.
2006), 29: Cortinarius rotundisporus (Sawyer et al. 1999), 30: Pisolithus sp. (Anderson et al. 2001), 31: Pisolithus tinctorius
(Anderson et al. 1998), 32: Pisolithus microcarpus (Hitchcock et al. 2010).
biogeography, with a more global view of EM populations
(which will be discussed below).
In this review, we want to show the promising expansion
of population genetics of EM fungi, crosstalk between various
approaches and scales; we concentrate on some of the resulting advances in our understanding of the biology and ecology
of EM fungi. The first section describes the early studies and
discussions about ecological strategies of EM fungi. We then
recall in the second section the challenges and currently available tools for the study of EM populations. In the third section,
we draw a current picture of EM population structure and dynamics at various scales, while the final section proposes
some emerging directions.
EM ecology and population biology, the central
question in pioneering studies
Ecological strategies in EM populations
It is not surprising that plant ecology has influenced fungal
ecology, given that the first textbook devoted to fungal
ecology, The Fungal Community, by Wicklow & Carroll, was
not published until 1981 (Frankland 1998). For example, the
term ‘genet’ itself, used from very early on in the EM literature,
originated in plant ecology (Harper 1977) to represent individuals produced by a given mating event (¼a single zygote).
Mycologists also adopted the concepts of ‘strategies’ from
plant ecology in an attempt to classify fungi into functional
types based on ecophysiology and reproductive biology
(Cooke & Rayner 1984; Andrews 1992).
This illustrates how concepts in ecology should develop
irrespective of the studied groups of organism. Within this
framework, two main types of natural selection have been described (Harper 1977; Andrews 1992). The first type, r-selected
species, has a short life expectancy and commits most of its resources to reproduction. In the case of EM fungi, in accordance
with Dahlberg & Stenlid’s (1990) study and Deacon & Fleming’s
(1992) assumptions, species with numerous small genets are
considered to reproduce primarily from meiotic spores (ascoor basidiospores) and to establish new individuals each year
(Laccaria amethystina, Gherbi et al. 1999, Fiore-Donno & Martin
2001, Hortal et al. in preparation. H. cylindrosporum, Guidot
et al. 2003a; Cantharellus formosus, Dunham et al. 2003;
Study
Fungal
species
Forest
environment
Markers &
populations
Abundance
(fruitbodies/genet)
Growth
(genet sizea)
Persistence
(max. observed)
Ecological
strategyb,c
Dahlberg &
Stenlid 1990
Suillus bovinus
Pinus sylvestris;
12e250 y
(depending on site)
SI; 4 populations
1e14
Mean: 0.7e3.4 m; max.:
4.2 to 30 m (depending
on age of site)
36 y
Baar et al.
1994
Dahlberg &
Stenlid 1994
Laccaria bicolor
P. sylvestris; 17 y
SI; 2 populations
4e8
Max.: 12.5 m
31 y
Competitivec: pioneer
in young stands,
expanding
in late forests
Competitivec
Suillus bovinus
P. sylvestris; 12e250 y
(depending on site)
SI; 5 populations
20e91
35 y
Competitivec
Gryta et al.
1997
Hebeloma
cylindrosporum
Pinus pinaster;
10e60 y (depending
on site); forest or
dune
1e2
(depending
on site)
No data
Ruderalb
Anderson et al.
1998
Bonello et al.
1998
Pisolithus
tinctorius
Suillus
pungens
Sclerophylls stand;
>16 y
Pinus muricata
Mean: <1 m;
max.: 30 m
Max.: 40 m
No data
R, C, S combinationc
40 y
R, C, S combinationb
Gherbi et al.
1999
Laccaria
amethystina
Fagus sylvatica; 150 y
1e5
Mean: 0.3 m;
max.: 2 m
At least 2 y
Ruderal in a late forestb
Sawyer et al.
1999
Cortinarius
rotundisporus
7e9
Max.: 30 m
No data
Competitive or
Stress-tolerantb
Selosse et al.
1999
Gryta et al.
2000
L. bicolor
Ligustrum lucidum,
L. sinense, Lantana
camara
Pseudotsuga menziesii
Single- and
multilocus probed
RFLP, IGS-RFLP,
mtDNA-RFLP; 3
populations
RAPD and RAMS; 1
population
RAPD, AP-PCR; 1
population; 2-y
sampling
RAMS, IGS & ITS; 1
population; 3-y
sampling
MS-PCR, ITS-RFLP; 3
populations
Mean: 0.8e3.5 m;
max.: 1.7e16.5 m
(depending
on age of site)
Max.: 0.1e3.6 m
(depending on site)
5e8
Max.: 3.3 m
At least 3 y
Ruderalb
H. cylindrosporum
P. pinaster; 10e20 y
10e106
Max.: 7 m
At least 5 y
Persistence and rapid
growthb, competitivec
Zhou et al.
1999
Zhou et al.
2000
Suillus grevillei
Larix kaempferii,
P. densiflora; 85 y
Larix kaempferii,
P. densiflora; 35e85 y
1e5
Max.: 6 m
No data
Ruderalb
2e3
Mean: 0.7e2.3 m;
max.: 11 m
At least 2 y
Persistent with erratic
fructificationb
Suillus grevillei
IGS, RAPD; 1
population; 3 y
Mating type, RFLP
single- and
multilocus probed
RFLP, IGS-RFLP,
mtDNA-RFLP, RAPD;
1 population; 5 y
ISSR; 1 population
ISSR; 2 populations;
2y
1.4
3.5
573
(continued on next page)
Population genetics of EM fungi
Table 1 e Main population dynamics estimators (forest environment, study materials: markers & samples, abundance, genet size, persistence capacity observed through
fructification) and ecological strategy (referring to Grime 1977) of local scale studied EM species.
Study
Fungal
species
Forest
environment
Markers &
populations
Abundance
(fruitbodies/genet)
2e3 (P. cf. alba);
18 (P. cf.
marmoratus)
1 (L. amethystina);
39e56 (X. chrysenteron);
1e7 (X. pruinatus)
R, C, S combinationb
(P. cf. alba)
Mean: 1.1 m; max.:
5.4 m (L. amethystina);
110 m (X. chrysenteron);
10 m (X. pruinatus)
At least 2 y (L.
amethystina); at least
3 y (X. chrysenteron
and X. pruinatus)
Max.: 7.3 m
(L. xanthogalactus); 12 m
(R. cremoricolor); 4.7 m
(A. franchetii)
Mean: <3 m; max.:
18 m
Less than 2 y
Ruderal in a late
forestb (L. amethystina);
stress-tolerantc
(X. chrysenteron and
X. pruinatus)
Ruderal in a late forestb
11 y
C, S combinationc
Mean: 0.18e0.48 m
(depending on site)
Mean: 3.2 m; max.:
13 m
2e5 y (depending
on site)
No data
Competitivec (dune)
or ruderalc (forest)
Ruderalc
5e28
Max.: 15 m
At least 3 y
Ruderalc
1e4 (R. vesiculosus);
5 (R. vinicolor)
Max.: 13.5 m
(R. vesiculosus), 2 m
(R. vinicolor)
No data
No data
Ruderal a priori,
invalidated
by resultsb
Persistentb
Sclerophylls stand
ISSR; 2 populations;
3y
Fiore-Donno
&
Martin 2001
L. amethystina,
Xerocomus
chrysenteron,
X. pruinatus
P. abies and
F. sylvatica; 40e150 y
RAPD, IGS, ITS;
1 population; 3 y
Redecker et al.
2001
Lactarius
xanthogalactus,
Russula cremoricolor,
Amanita franchetii
Russula brevipes
P. menziesii,
Lithocarpus densiflora,
P. muricata; 40
and 50 y
Pinus contorta and
Picea sitchensis;
40e100 y
P. pinaster; 10e60 y;
forest or dune
P. menziesii, Tsuga
heterophylla; 40e60 y
ITS-RLFP, AFLP 3, 2
and 1 populations
resp.; 2 y
2 (L. xanthogalactus);
1 (R. cremoricolor);
2 (A. franchetii)
3 microsatellites;
1 population
3e4
IGS-RFLP; 5
populations; 5 y
5 microsatellites,
ITS-RFLP; 18
populations
RAPD, LrDNA, ITS;
3 populations; 3 y
11 microsatellites,
ITS-RFLP; 2
populations
6 microsatellites;
3 populations; 2 y
2e11 (depending
on site)
6
H. cylindrosporum
Selosse 2003
Leccinum
duriusculum
Rhizopogon
vesiculosus,
R. vinicolor
R. brevipes
Kretzer et al.
2004
Bergemann
et al. 2006
Cantharellus
formosus
Populus alba; <20
and 70 y
P. menziesii,
T. heterophylla,
T. plicata; 80 y
Quercus douglasii,
Q. wiziensii, Pinus
sabiniana
Populus nigra; 20
and 25 y
Tricholoma
populinum,
T. scalpturatum
complexd
Lian et al.
2006
Tricholoma
matsutake
P. densiflora; 85 y
Wadud 2007
L. amethystina
and L. laccata
Salix reinii; <300 y
2
At least 2 y
RAPD, ISSR,
IGS2-RFLP; 2
populations; 3 y
3e7 (T. populinum);
1e2 (T. scalpturatum)
Max.: 11.5 m
(T. populinum); 9.5 m
(T. scalpturatum
complexd)
At least 2 y in
undisturbed site
4 microsatellites,
RFLP; 6 populations;
3y
10 and 5
microsatellites;
1 population; 3 y
6
Mean: 2 m; max.:
11.5 m
At least 3 y
13 (L. amethystina)
and 11 (L. laccata)
Mean: 0.4 m, max.:
1.2 m (L. amethystina);
mean: 0.2 m, max.:
1.4 m
(L. laccata)
At least 3 y
(both L. amethystina
and L. laccata)
Persistentb
(T. populinum,
undisturbed site) or
competitiveb
(T. populinum, disturbed
site)
Stress-tolerantc
Ruderal and persistent
in young standb
G. W. Douhan et al.
Gryta et al.
2006
Ecological
strategyb,c
At least 3 y
Pisolithus cf. alba
& P. cf. marmoratus
Guidot et al.
2002
Dunham
et al. 2003
Persistence
(max. observed)
Mean: <1 m; max.:
12 and 30 m
Anderson et al.
2001
Bergemann &
Miller 2002
Growth
(genet sizea)
574
Table 1 (continued)
L. amethystina
Hortal et al.
submitted for
publication
a Genet size: distance between the two most distant fruitbodies of genets represented by more than one fruitbody.
b According to the study’s authors.
c Inferred by the authors of this review from the results of the study.
d A phylogenetic study (Jargeat et al. 2010) revisited species limits of Tricholoma scalpturatum and related species and assigned genetic groups 1 and 2 (as referred to in Carriconde et al. 2008a) of
T. scalpturatum complex to the phylospecies T. argyraceum and T. scalpturatum, respectively.
Ruderal in a late forestb
At least 2 y
Mean: 0.7e1.8 m (based
on fruitbodies) or
0.4e1.0 m (based on EM)
(depending on site)
1e13 (max. 23
mycorrhizal tips/
genet)
8 microsatellites; 4
populations; 2 y
T. scalpturatum
complexb
Carriconde
et al. 2008b
Quercus pubescens,
P. sylvestris,
P. abies; 35 y
Dominant P.
sylvestris and Abies
alba; 180 y
ISSR, IGS-RFLP; 4
populations; 3 y
1e8
Mean: 0.2e0.6 m;
max.: 40 m
Less than 2 y
Ruderalb
Population genetics of EM fungi
575
Tricholoma scalpturatum complex, Carriconde et al. 2008b). The
second type, K-selected species, has a longer life expectancy
and commits more resources to asexual mycelial growth efforts; it thus devotes a smaller portion of resources to reproduction at one time. Species with a few, large genets across
a landscape are considered to be of this type, primarily investing in the growth of a perennial mycelium (Suillus bovinus,
Dahlberg & Stenlid 1994; Suillus variegatus, Dahlberg 1997;
some Laccaria spp., Selosse et al. 1997, 1999; Suillus pungens,
Bonello et al. 1998; Cortinarius rotundisporus, Sawyer et al. 1999;
Xerocomus chrysenteron, Fiore-Donno & Martin 2001). However,
as addressed below, some examples now contradict this view.
Moreover, the reK model was further refined to describe
overlapping domains within three primary strategies R, C
and S (Grime 1977): (i) organisms with competitive strategy
(C) maximise the ability to exploit resources and exclude competitors under low stress and low disturbance conditions; (ii)
ruderal strategy (R) organisms have a short life span with
high reproductive potential and are highly successful in nutrient-rich but disturbed, transiently non-competitive sites
where they arrive first; (iii) stress-tolerant strategy organisms
(S) are adapted to live under continuous environmental stress,
where they avoid competition and do not need to invest a lot
in reproduction. These strategies can overlap in secondary
combinatorial strategies (CeR, CeS, CeSeR). They were
applied to fungi in general in the 80s (Andrews 1992) and
thereafter, to EM fungi in the framework of host forest establishment and ageing. Here, they partly overlap with the socalled “early-stage” or “late-stage” strategies defined for EM
fungi, roughly corresponding to the R and C types, respectively
(Deacon & Fleming 1992), as well as “multi-stage” strategy
(Danielson 1991). Early-stage EM fungi (such as Laccaria bicolor,
de la Bastide et al. 1994; Pisolithus tinctorius, Anderson et al.
1998; Suillus luteus, Muller et al. 2004) are considered as pioneer
colonists in early successional forest stages that primarily colonize by sexual spores and associate with disturbed habitats.
Late-stage species (such as C. rotundisporus, Sawyer et al. 1999;
Russula brevipes, Bergemann & Miller 2002; T. matsutake,
Amend et al. 2009, 2010) are thought to propagate primarily
by mycelial expansion: any disturbance would break down
their hyphae and rhizomorphs, and limit their propagation
(Simard et al. 1997), so that they occupy undisturbed habitats
at the climactic stage. Accordingly, spores of early-stage fungi
germinate easily and react efficiently to host roots, while latestage spores germinate poorly (Nara 2008). Multi-stage species
(e.g. Suillus brevipes, Visser 1995; S. pungens, Bonello et al. 1998
(see below); Leccinum scabrum, Kranabetter 1999) exhibit intermediate characteristics; moreover, many early-stage species
survive in older stands (Visser 1995). These concepts have
been widely used in the EM population literature since their
introduction by Dahlberg & Stenlid (1990, 1994; see below
and Table 1), although, surprisingly, few papers mention
Grime’s (1977) basic paper.
As a good example highlighting how population genetics
can be used to understand ecological features, and the limits
of ReCeS approaches, Gardes & Bruns (1996) were able to
demonstrate that species with the most frequent aboveground fruitbodies were not necessarily the most common
on the root tips in a Pinus muricata forest. S. pungens was the
most frequent fruiter, but accounted for less than 3 % of the
576
EM root tips. They proposed two hypotheses to explain these
results: (i) S. pungens invests fewer resources in vegetative
growth and persistence within this community than other
species, and (ii) S. pungens has more access to carbon or
more efficiently transfers carbon than other dominant species, possibly by mixing biotrophic or saprotrophic abilities.
In a follow-up study, Bonello et al. (1998) suggested that in
the first hypothesis S. pungens would have an R strategy and
would reproduce primarily by basidiospores, resulting in numerous small genets at a given site; conversely, in the second
hypothesis, S. pungens would have a C or S strategy and would
produce large and persistent genets with a competitive advantage due to larger carbon acquisition. Bonello et al. (1998)
found a single S. pungens genet covering at least 300 m2, suggesting vegetative persistence in this species, and several
smaller genets consisting of individual fruitbodies. The genetic structure suggested an intermediate ReC strategy. After
a devastating fire in the same plots, Bruns et al. (2002) found
that the large genets originally detected by Bonello et al.
(1998) had been replaced by numerous small genets, suggesting an R strategy.
Deacon & Fleming (1992) stated that ‘the early stageelate
stage distinction is unlikely to be absolute; it may serve only to identify the ends of a spectrum of behavior, which is also influenced by
other factors.’ As they speculated, results from studies on EM
population structure, as discussed below, have not always followed this conceptual framework: we will now review the link
between genet size and the apparent ‘early’ stage and ‘late’
stage designation, starting with supporting studies and concluding with less supportive ones.
EM genet size versus ecological strategies: a correlation in
some cases
The challenge to maintain a local EM population is to colonize the newly formed tree roots, either by mycelial growth
of resident genets, or by recruitment of new genets. The first
strategy is allowed by the fact that, unlike saprotrophic or
necrotrophic fungi, EM fungi are biotrophic organisms that
do not exhaust their food source, and can persist locally.
For the second strategy, most EM fungi form no asexual
spores (although some Ascomycetes at least may be exceptions; Urban et al. 2004; Egger 2006), nor asexual propagules
(with some exceptions, such as sclerotia in C. geophilum).
Spores are meiotic (asco- or basidiospores), and founding of
a new individual is thus obligately linked to sexual recombination. EM populations therefore result in a trade-off between asexual, vegetative extension of existing mycelia in
soil, extinction and recruitment of dispersed meiotic spores.
In this framework, patterns of establishment and history of
local populations were proposed to be linked to the number
and size of the genets identified within a site. In early studies
(Dahlberg & Stenlid 1990, 1994) of EM population biology, two
common concepts were: (i) species with few large genets
were thought to be maintained by asexual mycelial growth
and were considered as C-type, late-stage species, whereas
species with numerous small genets were thought to reproduce mainly by sexual (meiotic) spores and were considered
as R-type, early-stage fungi; (ii) old and stable forests host
EM species with few large genets (late-stage species),
G. W. Douhan et al.
whereas disturbed ecosystems host EM species recently
established by means of sexual spores, made of numerous
small genets (early-stage species). In other words, genet
size, sexual reproduction versus asexual growth, and adaptation to disturbance are all linked (e.g. Bagley & Orlovich
2004).
Dahlberg & Stenlid (1990) showed that old forests mainly
consist of old, large genets that apparently do not intermingle,
for S. bovinus and, to a lesser extent, Suillus variegatus
(Dahlberg 1997). For example, the maximal genet sizes of S.
bovinus in 15- to 20-y-old Swedish Scots pine forests were
1.7e5.3 m versus 6.8e16.8 m in 70- to 160-y-old forests. After
a disturbance, new genets would establish from basidiospore
inoculum and, over time, many would die or be outcompeted
to the advantage of a few remaining genets covering large
areas (Dahlberg & Stenlid 1990, 1994). This is consistent with
the above-mentioned findings by Bruns et al. (2002) for S. pungens. Accordingly, Sawyer et al. (1999) showed that C. rotundisporus, a late-stage species from an Australian sclerophyll
forest, showed few and large genets (up to 30 m), likely resulting from pluriannual expansion. Fiore-Donno & Martin (2001)
also identified large genets of two Xerocomus species in a 140y-old Picea abies forest and hypothesised that one X. chrysenteron genet may have been established at the same time as
the host trees.
EM genet size versus ecological strategies: no necessary
correlation
Although the previous concepts have held true in some
respects, they remain interpretative, and additional studies
reported a more complex picture (Table 1). H. cylindrosporum
in pine forests shows both non-persistent, small genotypes,
consistent with sexual reproduction and early-stage fungi,
and larger, persistent genotypes, consistent with vegetative
spread and late-stage fungi (Gryta et al. 1997, 2000; Guidot
et al. 2002). Gryta et al. (1997) found that in two out of three investigated sites, no genet exceeded 500 cm2 and that there
was a complete turnover of genotypes observed each year
for three consecutive years. In contrast, one collecting site
contained larger genets that persisted over the 3 y. As further
confirmed by Guidot et al. (2002), these contrasting life strategies correlated with environmental conditions. The high turnover of genotypes was observed in a frequently anthropically
disturbed forest habitat (soil disturbance by parking or camping at tourist sites), where other EM species are regularly eliminated, offering open niches for new genets of the pioneer
H. cylindrosporum, while more persistent genotypes are associated with undisturbed forests with deeply burried roots in
a sand dune habitat. Thus, genet size depends more on the
time elapsed since the last disturbance than simply on the forest age (in other words, there are relevant disturbances other
than the death or replacement of host trees, and this is sometimes overlooked). Other cases of EM strategies affected by environmental factors have been described: Zhou et al. (2000)
studied Suillus grevillei populations in two Larix kaempferi
stands and found that the average genet sizes were different
according to the site (0.7 m versus 2.3 m), and smaller in the
older forest, with a high turnover from one year to another
(Zhou et al. 2001). They suggested that local conditions, such
Population genetics of EM fungi
as the level of animal and anthropic disturbance of the sites
(or competition, see below), might explain these patterns.
Several other studies have challenged the notion that mature, undisturbed forests host primarily late-stage species
with large genets. Equivocally, in 40- to 80-y-old mixed forests,
Kretzer et al. (2004) suggested that Rhizopogon vesiculosus was
a pioneer species, but questioned this after observing despite
unexpectedly large genets (13.5 m wide). The contradiction
was more obvious in a 150-y-old beech forest, where Gherbi
et al. (1999) found up to 134 genets per 100 m2 of the early-stage
species L. amethystina. Most genets were represented by a single
fruitbody and the largest genet only covered 1 m2; moreover,
only 8 out of 388 genets fruited again in the following year
(2 %). Although the RAPD markers used may overestimate the
diversity, a recent study by Wadud (2007) using microsatellites
suggested that only 40 % of the genets fruited again in the following year in a Japanese population of L. amethystina; and the
high genetic diversity at a small scale was also supported in later
studies (Roy et al. 2008; Vincenot et al. submitted for publication).
Similar results were also found in an undisturbed mixed forest
in the Swiss La Chaneaz reserve for L. amethystina (FioreDonno & Martin 2001) e but in all these studies, longer term
monitoring would be necessary to rigorously assess whether
we have here death of the genets or only transient year(s) without fruitbodies as described in some Laccaria spp. (Selosse et al.
2001). Genet sizes of Lactarius xanthogalactus, Russula cremoricolor
and Amanita francheti, all considered to be late-stage species,
were reported to be relatively small (1.1e9.3 m2; Redecker et al.
2001). Bergemann & Miller (2002) found that most genets of R.
brevipes associated with lodgepole pine (100 y old) and Sitka
spruce (40e60 y old) were less than 3 m in diameter, but they
reported fruitbodies up to 18 m apart that formed a single genet
and identified another genet that persisted over an 11-y sampling period.
There are several possible causes for the existence of small
genets in old forests. As stated above, micro-disturbances,
such as soil fungivores or local tree death, may re-open the
possibility for spore settlement (this may apply to Rhizopogon
and Laccaria spp. for example). Amend et al. (2009) revealed
a founder effect and progressive recruitment of genetic diversity over time in a comparison of T. matsutake populations in
50-y-old forests versus old-growth forests, suggesting that
genet settlement is a slow, but continuous process. In taxa
that are susceptible to disturbance (such as many Amanita,
Cortinarius or Russula species), smaller genets may rather represent old, non-growing or slowly growing individuals. In
a comparative study of undisturbed Leccinum duriusculum populations between Populus stands of varying ages, fruitbodies of
the largest genet in a <20-y-old population were 10.4 m away,
while under >70-y-old stands most distal fruitbodies of the
same genet were <6.5 m apart (Selosse 2003). A similar trend
was reported for S. grevilleii under 35-y-old versus >85-y-old
L. kaempferi, with larger genets occurring in the former forest
(Zhou et al. 2000). Such discrepancies could be explained by intraspecific competition (Selosse 2003): lower competition in
newly established stands, due to low genet number, allows
faster size increase, whereas higher competition between
old genets in more ancient stands limits their size, e.g. due
to limited resources and space (roots) availability. Other
factors, like increased interspecific competition or
577
heterogeneities in abiotic conditions, may also limit genet
size in old forests. Moreover, EM genet growth rates can reach
up to a metre per year (Selosse et al. 1999; Selosse 2003 e although most values may be in the centimetre to decimetre
range), possibly due to local mycelial fragmentation and dispersion by various vectors. Thus, growth may not be limiting
for genet size.
The assumption that small genets are short-lived is thus
questionable, especially in most studies that lack surveys
over several years to test for survival. Studies that only monitor two consecutive years are also questionable because (i)
genets may not fruit each year (as shown in Laccaria spp.
over 3 y; Selosse et al. 2001), (ii) smaller genets often produce
fewer fruitbodies (Selosse et al. 2001), and may undergo
more frequent non-fruiting years, and (iii) long-lived genets
may also not fruit regularly and may exhibit years of ‘sexual
dormancy’. Indeed, the more lasting a genet is, the more it
can improve its fitness by diverting resources from sporulation for underground growth and survival. Thus, at least for
some species, size may be more related to population dynamics, life traits and competition, than to reproductive strategy
or age of the forest. Conversely, some early-stage fungi, considered to re-establish frequently by spores, can provide lasting and large genets, such Laccaria laccata and L. bicolor for
which some genets extend to 12.5 m (Baar et al. 1994) and persist over a sampling period of 2e3 y (de la Bastide et al. 1994;
Selosse et al. 2001), or up to decades for artificially inoculated
strains covering even larger areas (Selosse et al. 1997, 1999).
As we will see below, population genetics tools allow more
direct assessments of reproductive strategies. Early studies
describing and mapping genets provided heuristic representations of EM populations and were linked to a progressive
improvement of the molecular tools. They revealed a large diversity of situations, likely reflecting that the EM habit was
convergently acquired in fungal evolution, and that, by evolving over a long time in some lineages, this habit diversified
into various strategies. But inferences on genet size do not allow resolution of the basic ecology and genetics of EM populations; this approach is even biased in most studies because it
is based on fruitbodies only and is thus restricted to the reproductive phase (see below). As we will see now, population
genetics and phylogenetic tools allowed researchers to go beyond a simple description of EM genet sizes, and to shift from
above-ground, reproductive genets to below-ground vegetative populations.
EM population genetics: current challenges
and tools
The importance of EM population genetics
In this section, we discuss the importance of fungal biology in
understanding the particularities of population genetics in EM
fungi and its challenges. Many studies have used molecular
methods as diagnostic tools only, and so have under-utilised
the potential information provided by such techniques.
Beyond the previous interpretations regarding EM population
structure and biology, little is known about the population genetics of many EM species, which is intrinsically tied to how
578
G. W. Douhan et al.
forest ecosystems function and evolve. Moreover, we still do
not understand many of the fundamental life history processes that shape EM populations, such as the respective roles
of mating and mycelial growth, or the importance of inbreeding (the mating between genetically similar individuals) and
its effects on the population structure. EM population genetic
studies can widen the scope of our basic understanding of EM
fungi by characterising gene flow between populations on different scales, differentiation between populations putatively
reflecting environmental variations or barriers, isolation by
distance, etc. By detecting recombination, it can also detect
cryptic speciation, an important phenomenon in fungal evolution (Giraud et al. 2008), and hidden sexuality in putatively
asexual species.
The population structure of plant pathogenic fungi has received considerable attention (e.g. Brown & Hovmøller 2002;
s et al. 2008; Fournier & Giraud 2008; Dilmaghani et al.
Barre
2009), likely because of their economic impact, and information regarding the population biology of plant pathogens can
be used to make decisions on fungicide usage or the deployment of resistance genes (Milgroom & Peever 2003; Gout
et al. 2007). A lot of studies have also been done in saprotrophic
species (e.g. James & Vilgalys 2001; Dettman et al. 2003a, 2003b;
Kauserud & Schumacher 2003a, 2003b; Kauserud et al. 2007;
Liti et al. 2010 e see below ‘population subdivision, gene flow
and isolation by distance’), although no general review is currently available. We argue that knowledge of population structure of EM fungi can help us to better understand these
ecologically important fungi, and guide future forest management and conservation practices. Economically important EM
species, such as those used for the improvement of tree
growth, or for the production of edible fruitbodies, should be
sustainably managed, because they have not been domesticated so far. For example, the routine inoculation of Tuber melanosporum rarely takes into account the origin of the inoculant
in relationship to the outplanting site, whereas EM inoculation
can lead to lasting persistence of inoculant in other EM species
(Selosse et al. 1997, 1999). For T. melanosporum, this has
resulted in the accidental, but potentially disastrous introduction of exotic Tuber indicum strains in European truffle grounds
(Murat et al. 2008). Taking into account a better biological understanding of EM fungi is especially important given the increasing pressures exerted on forest ecosystems by human
activities.
Reproductive biology of EM fungi
A basic understanding of reproductive biology is necessary to
understand population biology of EM fungi, since it regulates
how genes are distributed, moved, and shared among individuals. Since most species investigated so far are Hymenomycetes (with the exception of Cenococcum geophilum and
T. melanosporum, see below), we will focus first on this taxon
(Fig 3) before enlarging the discussion to Ascomycetes. In
the Hymenomycetes, mating systems were first studied in
the genera Schizophyllum and Coprinus (Brown & Casselton
2001), and are now increasingly elucidated in other non-saprotrophic model species, such as L. bicolor (Niculita-Hirzel
et al. 2008).
Mating type loci regulate the assortment of haploid sexual
partners. The standard hymenomycetous life cycle begins
with the fusion of two monokaryotic haploid hyphae into
a long-lived dikaryon, where the two parental haploid nuclei
stably co-exist in a 1:1 ratio without fusion (Fig 3). Dikaryons
are vegetative, and form fruitbodies that bear the meiotic basidiospores: thus, in Hymenomycetes, it is easy to sample
dikaryotic, post-zygotic stages, and haploids are considered
as short-lived (Moore & Novak Frazer 2002), although we
lack field evidence for this (see below). Assortment is controlled by mating type genes (Moore & Novak Frazer 2002;
Fig 3 e The typical life cycle of a heterothallic Hymenomycete (modified from Martin & Selosse 2008).
Population genetics of EM fungi
Fraser et al. 2007): two mating type loci (¼tetrapolar systems,
with multiple alleles for each locus) are the common, plesiomorphic condition, but some secondary situations exist, e.g.
with a single mating type (bipolar system). Some species are
even autogamous, either by directly producing dikaryotic basidiospores (amphithallism or secondary homothallism;
Moore & Novak Frazer 2002; see below) or thanks to mating
re & Noe
€l 1992). Mating type loci act
type switching (Labare
as self-incompatibility systems: for mating compatibility,
interacting hyphae must harbour different alleles. This incompatibility should not be confused with SI (see above),
which regulates fusion/non-fusion between individuals sharing identical alleles during the vegetative stage: in Hymenomycetes, SI is mostly observed in interactions between
dikaryotic mycelia, and not reported to interfere with mating.
Although SI was used in early studies to delineate genets (see
above), little is currently known of its role, if any, in EM populations, which is still to be investigated.
Mating types do not fully prevent selfing when considering
the parental dikaryon: haploids from the same parent, but carrying compatible mating type alleles, can fuse (a given haploid
can fuse with 50 % of all spores from the same fruitbody in a bipolar mating system, and with 25 % in a tetrapolar mating system). This culminates in the production of dikaryotic spores
after meiosis (amphithallism, as stated below, e.g. in the EM
S. pungens, Bonello et al. 1998). Lastly, not all hymenomycetes
are dikaryons: Armillaria species have diploid nuclei (undergoing multiple and complex haploidisation processes in fruitbodies; Selosse 2001b) and many species have multiple
nuclei within each cell, forming so-called heterokaryotic hyphae (e.g. Rhizoctonia spp., Phellinus spp.; Heterobasidion species
possess heterokaryotic hyphae with imbalanced nuclear ratios, a situation viewed to enhance phenotypic plasticity e
James et al. 2008). Whether such complex genetic phenomena
(haploidisation, heterokaryosis) occur in EM fungi and their
potential roles in maintaining EM populations are currently
not known.
Theoretical tools describing population genetic structure
are often based on diploid animal and plant models, so that
some of the assumptions can be violated with respect to fungi,
e.g. due to dikaryosis, and need careful attention. Complex
co-evolution occurs between haploid nuclei in dikaryons,
such as a reciprocal co-adaptation or between-nuclei genetic
exchanges (Clark & Anderson 2004). Another particularity of
dikaryons is that, although they already underwent mating,
they still harbour haploid nuclei (Fig 3) and thus remain able
to mate: the donation of a nucleus by a dikaryon to a monokaryon (also called di-mo mating, or ‘Buller phenomenon’;
Nieuwenhuis et al. 2010) is described in vitro for the EM
L. bicolor (Gardes et al. 1990; de la Bastide et al. 1995) and in
situ for saprotrophic basidiomycetes (Johannesson & Stenlid
2004). Basidiospore discharge near to (or on) the parental
dikaryons may enhance the possibility of inbreeding, and
potentially a more continuous overlap between generations.
The contrast between EM Hymenomycetes and EM Ascomycetes deserves further investigation. EM Ascomycetes
also have SI and two-factor mating types, but are vegetative
at the pre-zygotic haploid stage. Thus SI may be epistatic on
mating systems (but this has not yet been investigated at
the population level in Ascomycetes) and, as expected from
579
non-EM ascomycetes such as Daldinia loculata (Guidot et al.
2003b), one contributing haploid parent in allogamous species
may often be quite distant, and interact by way of micro-conidia (¼spermatia). One parent, investing in providing resources to the fruitbody, is of course closer: it can be
considered as the female mycelium, while the more distal,
which invests less in spore production, is the male one (each
haploid potentially behaves as a male and as a female in different matings, and these behaviours are thus unrelated to
mating types). However, with the possible exception of some
truffles (Urban et al. 2004), spermatia production remains
poorly documented in EM Ascomycetes, but we suspect that
this may change in the future. The asymmetric contribution
of sexual partners to the formation of a given fruitbody may
result in the hiding of one genotype. For example, several Tuber species were long supposed to be autogamous (Bertault
et al. 2001), since DNA extracted from the fruitbody flesh never
revealed heterozygosities. It now turns out that a mix of female and male markers can be obtained when carefully
extracting DNA from ascospores, thus pointing to allogamy
(Paolocci et al. 2006; Riccioni et al. 2008). Truffle flesh, from
which most if not all DNA is extracted by standard protocols,
arises only from the female parent; the male genotype can be
deduced, by difference, from the ascosporal genotype (Rubini
et al. 2011). This asymmetry may not change the fitness, since
each haploid can have a female and a male role in different
matings. However, it may structure parental assortment and
gene flow at the local level. Results reported by Rubini et al.
(2011) suggest that T. melanosporum haploids that colonize all
EM roots locally, and have access to tree photosynthates, act
as the female parent; they open the possibility that males
are smaller, perhaps dominated individuals that co-occur in
soil and are possibly forced to invest less in reproduction.
Such a particularity does not apply to Hymenomycetes dikaryons, and highlights the need for investigation of more Ascomycetes species, since Ascomycetes make up a significant
component of EM communities (Egger 2006; Tedersoo et al.
2006; Grelet et al. 2010), even if most are rare EM species
(with exception of C. geophilum, see below).
The challenges of sampling EM populations
Understanding of the population genetics of EM fungi has
lagged far behind that of fungal pathogens and saprotrophs,
most likely due to the fact that some difficult challenges
have to be overcome to grow EM fungi in culture under laboratory conditions. Although several EM fungi can be cultured in
vitro, all still require a host tree to fruit and the only currently
available model system for in vitro production of fruitbodies is
the EM species H. cylindrosporum (still in the presence of host
seedlings; Debaud & Gay 1987; Marmeisse et al. 2004).
A main problem is adequate in situ sampling for studying
EM populations. Many EM fungi do not form hyphal cords or
rhizomorphs that can be sampled from the soil, and some species are difficult to identify on host roots. The most common
practice is simply to collect fruitbodies, as was done in the
early studies (e.g. Dahlberg & Stenlid 1990, 1994): at least it
gives access to the reproductive genets. A better sampling
would be to collect EM structures (Kretzer et al. 2005; Lian
et al. 2006): however, the resulting disturbance limits the
580
possibility of following the population(s) over several years,
and the high numbers of mycorrhizae (even for a given genet),
as well as their heterogeneous distribution on roots, result in
sampling bias. Typing EM roots additionally requires highly
specific molecular tools, such as Sequence Characterized
Amplified Regions (SCARs), SNPs, and microsatellites (see below), since the DNA extracted is a mixture from various microbes plus host plant. Few studies are based on the
sampling of mycelium or sclerotia (in C. geophilum, LoBuglio
& Taylor 2002; in Laccaria spp., Wadud 2007; or in shiros of
T. matsutake, Amend et al. 2009). Grubisha et al. (2007) used
an original approach to sample the Rhizopogon spp. spore
bank by typing EM roots from bioassay seedlings. However,
most studies still rely on fruitbody sampling, even if authors
are aware that EM genets fruit erratically (Selosse et al. 2001;
Straatsma et al. 2001), and that fruitbodies may underestimate
the genetic diversity (see below).
Developing molecular markers for EM fungi
The first studies of EM population genetics mainly aimed to
discriminate among genets, with the early studies based on
SI and the later studies based on various molecular techniques
(Table 1). With respect to molecular techniques, most early
studies used multilocus, dominant markers such as RAPD
(e.g. Jacobson et al. 1993; de la Bastide et al. 1994; Anderson
et al. 1998; Gryta et al. 2000; Jany et al. 2002) and, in a second
step, AFLP (e.g. Redecker et al. 2001; Bruns et al. 2002; Muller
et al. 2004). A variant of RAPD called inter-simple sequence repeats (ISSRs), using di- or tri-nucleotide repeated PCR primers,
has also been very popular in EM population genetic studies
(e.g. Gherbi et al. 1999; Sawyer et al. 2001; Anderson et al.
2001; Hirose et al. 2004; Liang et al. 2005; Carriconde et al.
2008a; Grelet et al. 2010).
A major drawback of dominant markers is that they cannot
distinguish heterozygous from homozygous loci and, therefore, make it statistically problematic to calculate allele frequencies without assuming HardyeWeinberg equilibrium or
some amount of inbreeding. However, these markers do allow
genetic variation to be calculated by various measurements of
genetic distances, both within and among populations. Since
these methods do not provide allele frequencies, there is no
population genetics theory developed to test hypotheses
regarding genetic structure or to quantify gene flow. The analysis of molecular variance (AMOVA) method (Excoffier et al.
1992; Schneider et al. 2000) can nevertheless be used to estimate levels of population differentiation and gene flow (e.g.
Roy et al. 2008). The use of dominant markers has limited advances in EM fungal population genetics. Paradoxically, their
use on haploid EM Ascomycetes would have been more appropriate, as no dominance problem exists in this case (ironically,
the first study on EM Ascomycetes (truffles; Bertault et al. 1998)
was also the very first to use co-dominant markers for EM
fungi). There have been attempts to turn dominant RAPD fragments into potential co-dominant sequence-characterised
markers allowing direct PCR amplification and sequencing
(SCARs, Weber et al. 2002; Jany et al. 2002; El Karkouri et al.
2006) or to detect Single-Strand Conformation Polymorphisms
(SSCPs, Bonello et al. 1998), but these techniques have not been
widely employed.
G. W. Douhan et al.
Many studies of EM genet size or population genetics have
also relied on co-dominant PCR-RFLP markers derived from
nuclear rDNA, mainly ITS (e.g. Sawyer et al. 1999; Redecker
et al. 2001; Jany et al. 2002; Dunham et al. 2003, 2006; Kretzer
et al. 2004, 2005; Lian et al. 2006; Xu et al. 2008) or intergenic
spacers (IGS; e.g. Selosse et al. 1996, 1999, 2002; Gryta et al.
1997, 2000, 2006; Guidot et al. 1999, 2001, 2002; Hirose
et al. 2004; Murat et al. 2004; Carriconde et al. 2008b; Roy et al.
2008). Although little is known about the nature and origin
of ITS/IGS polymorphism, most observed variation is due to
polymorphisms within the intergenic regions as well as
indels, making this marker potentially co-dominant. In L. bicolor at least, length variation of IGS is due to variable numbers
of repeated motifs (Selosse et al. 1996; Martin et al. 1999). However, this locus was often used in combination with other
markers for multilocus genotyping.
More recent studies have used co-dominant markers from
single loci (microsatellites, SNPs; see Glossary), allowing distinction of homozygous from heterozygous loci. In addition,
these markers are often amplified via PCR with more specificity from the fungal genome, so that they can be used directly
on mycorrhizae. Co-dominant markers are therefore ideal for
analysing genotypic data of dikaryotic EM fungi. Single-locus
co-dominant markers also make it easier to compare data between studies, and have gained recent usage in EM population
genetics studies (Tables 1 and S1). The main disadvantage of
microsatellites and SNPs is the time and cost associated
with developing and collecting the data, although multiplexed
markers and automated analysis on a DNA sequencer reduce
the genotyping time. Up to now, the design of microsatellites
for EM fungi has been hindered by technical difficulties such
as DNA contamination of fruitbodies (since some species are
impossible to isolate and to cultivate) or failures at different
steps of the library procedure (Dutech et al. 2007). Moreover,
microsatellite loci exhibit low allelic diversity in some species,
either from enriched libraries (Kanchanaprayudh et al. 2002;
Muller et al. 2007; Amend et al. 2009) or from Expressed Sequence Tags (ESTs) or genome-based approaches (Jany et al.
2003; Adams et al. 2006; Vincenot et al. submitted for
publication). These obstacles seem to be common in fungal
genomes (Dutech et al. 2007). However, after the first paper
published in 1998 by Bertault et al., the number of studies
based on microsatellites has grown in the last 6 y (Kretzer
et al. 2004, 2005; Rubini et al. 2005; Dunham et al. 2006; Lian
et al. 2006; Grubisha et al. 2007; Muller et al. 2007; Roy et al.
2008; Hitchcock et al. 2010; Vincenot et al. submitted for
publication), and many technical notes have reported the development of microsatellite markers (e.g. Jany et al. 2003;
Rubini et al. 2004; Bergemann et al. 2005; Grubisha et al. 2005;
Adams et al. 2006; Hitchcock et al. 2006; Jany et al. 2006;
€ gberg
Wadud et al. 2006a, 2006b; Hirose & Tokumasu 2007; Ho
et al. 2009; Murat et al. 2011).
The recent and soon-to-be-released EM fungal genomes or
ESTs represent great opportunities to design new markers
(Martin & Selosse 2008) that may reduce the costs of development. For example, SNPs have already been developed thanks
to genomic approaches (on T. melanosporum, Murat et al. 2004;
et al. 2008;
on Amanita sp., Adams et al. 2006; on L. bicolor, Labbe
on T. matsutake, Amend et al. 2009). A caveat is that loci from
ESTs may not be neutral. The rise of a new generation of
Population genetics of EM fungi
sequencing methods (such as pyrosequencing; Shendure &
Ji 2008) makes it easier to obtain genomic data, or to sequence
microsatellite-enriched libraries, so that the use of microsatellites and especially SNPs, as well as the average number
of markers per each study, will likely increase in the coming
years: the present review may be the last of the pre-genomic
area.
EM population genetics: what do we know from
recent advances?
Recombination and mating systems in situ
Although the mating system (existence of recombination,
trend to out- or inbreeding, including autogamy) is of central
importance in population biology, it has not been much studied in field conditions for many Hymenomycetes. Many mating system studies within the Hymenomycetes are based on
in vitro pairings of monokaryons, where formation of clamp
connections is used to check for dikaryon formation (Fig 3;
e.g. Doudrick & Anderson 1989; Gardes et al. 1990). However,
these tests highly depend on in vitro conditions, and may not
reflect how dikaryons are established, selected and maintained under natural field conditions. For example, introduction in European forests of a North American isolate of
L. bicolor that was fully compatible with European strains in
vitro did not lead to detectable hybridization over 10 y at the
introduction site, although both indigenous and introduced
strains formed abundant fruitbodies and meiotic spores
(Selosse et al. 1997, 1998, 1999).
For Ascomycetes, only fruitbodies that grow after mating,
but not haploid fungi on EM roots, allow investigation of the
mating system. In some cases, as stated above for Tuber
spp., the asymmetric contribution of sexual partners may
limit the access to male markers (Paolocci et al. 2006;
Riccioni et al. 2008; Rubini et al. 2011), explaining, as already
mentioned, why early studies wrongly concluded that autogamy was occurring (Bertault et al. 2001). Tuber spp. nevertheless offer access to the genetic identity of both parents: an
indirect reconstruction of the male genotype is possible by
subtracting the female (¼fruitbody flesh) markers from the
zygotic (¼sporal) ones, an easier method than for Hymenomycetes where dikaryons require a laborious de-dikaryotisation
after in vitro isolation (Gryta et al. 2000).
In Hymenomycetes, molecular tools can be used to analyse
the mating system directly on environmental fruitbodies and
EM roots. Conclusions regarding heterozygosity level, and
thus partners’ assortment, are possible by comparison with
the null expectancies of HardyeWeinberg equilibrium. Isozyme markers were the first molecular markers used for EM
fungi (Ho & Trappe 1987; Sen 1990; Keller 1992), but no studies
have used isozymes to examine EM mating systems. Co-dominant markers (microsatellites, SNPs, see Glossary) also distinguish between homo- and heterozygosity in dikaryotic
tissues. Bonello et al. (1998) used 4 random diallelic SSCP
markers to test the mating system of S. pungens, which was
consistent with outbreeding. However, they also found that
1.3e1.4 % of the basidiospores they examined had two binucleate spores, i.e. directly dikaryotised after the meiosis
581
producing the haploid nuclei, suggesting that some inbreeding occurred. Bergemann & Miller (2002), using 3 microsatellite
loci for an R. brevipes population associated with different
hosts, found an excess of heterozygotes, which suggested random mating in a local population associated with Sitka
spruce, whereas a deficiency in heterozygotes indicated nonrandom mating in a population associated with lodgepole
pine. Similarly, Gherbi et al. (1999), using IGS, a rDNA
sequence, on L. amethystina, found an excess of heterozygotes,
also suggesting that monokaryons tend to associate with
diverging partners (more than at random). After these pioneering studies, several reports using higher number of
markers showed no deviation from random mating (e.g.
Bonello et al. 1998; Kretzer et al. 2004; Bergemann et al. 2006;
Amend et al. 2009, 2010), or variable heterozygote deficiency
(e.g. Dunham et al. 2003; Grubisha et al. 2007; Muller et al.
2007; Roy et al. 2008). Heterozygote deficiency can be overestimated by the presence of null (non-amplifying) alleles for
some markers. Often, the presence of null alleles on some
loci is the only way to reconcile different marker loci that
show diverging levels of inbreeding, but even when discarding
biased markers, some inbreeding remains on a local scale (Roy
et al. 2008; Vincenot et al. submitted for publication). In all,
there is a trend to some local inbreeding that was not seen
in the first studies of EM model species, either due to the
markers, or to insufficient sampling effort e a trend that
may result from limited spore dispersal (see below).
Another way of assessing recombination rate is by analysing association of alleles among loci (index of association)
within a population, as originally developed by Burt et al.
(1996) for the human fungal pathogen Coccidioides immitis.
The null hypothesis tested is the signature of recombination,
based on randomisation procedures e absence of recombination entails a linkage between alleles at different loci. Interestingly, this also applies to dominant markers and to haploids,
e.g. in EM Ascomycetes, since observation of post-zygotic diploids is not required. For example, LoBuglio & Taylor (2002)
could not reject the null hypothesis of recombination within
two populations of putatively haploid C. geophilum based on
the analysis of 9 SNPs. Although C. geophilum is not known
ndez-Toira
n & Agueda
to have a sexual state, Ferna
(2007)
claimed to have found fruitbodies, based on morphological,
but not molecular, evidence. Moreover, in Ascomycetes, vegetative recombination may occur out of the meiosis/mating
cycle, between haploid nuclei coexisting after a vegetative fusion (Bennett & Johnson 2003): nevertheless, the so-called
‘parasexual cycle’ has rarely been shown in situ. Douhan
et al. (2007b) supported the results of LoBuglio & Taylor
(2002) on a smaller spatial scale, but their study also revealed
that the results are significantly affected by how species are
defined in C. geophilum sensu lato: pooling isolates that were
clearly distinct phylogenetic species based on 8 polymorphic
loci resulted in rejection of random mating, whereas random
mating could not be rejected when the isolates were treated as
distinct species. Wu et al. (2005) suggested that no evidence of
sexual reproduction occurred in C. geophilum on the slopes of
Mount Fuji based on microsatellite data, but no formal statistical analyses were conducted. Existence of recombination in
C. geophilum, one of the rare putatively asexual EM fungi
(with the possible exception of Meliniomyces variabilis; Grelet
582
et al. 2010) thus deserves further research. A related inter-loci
approach (David et al. 2007) was applied to the hymenomycetous L. amethystina, to circumvent the problem of null alleles in
microsatellites which create an apparent heterozygote deficiency: correlations of heterozygosities between loci confirmed that some inbreeding was indeed occurring at a local
scale (Vincenot et al. submitted for publication).
Population subdivision, gene flow and isolation by distance
Population subdivision, gene flow and the limits at which populations have unrestricted mating and movement of individuals is a central component in the study of population
genetic structure. Due to the absence of asexual propagules
in most EM fungi, gene flow is restricted to displacement of
meiotic spores (Fig 3; although displacement of male gametes
can also occur in Ascomycetes; Guidot et al. 2003b), but local
displacement of mycelial fragments cannot be ruled out
(Selosse et al. 1998). Few studies have specifically focused on
gene flow in EM fungal populations, as compared with plant
pathogenic Ascomycete and Oomycete taxa (Taylor et al.
1999). The main non-EM Hymenomycetes genera studied at
the population level are Agaricus (Xu et al. 1997; Kerrigan
et al. 1998), Armillaria (Saville et al. 1996; Baumgartner et al.
2009), Fomitopsis rosea (Kauserud & Schumacher 2003a),
Heterobasidion annosum (e.g. Johannesson & Stenlid 2004),
Mycena (Boisselier-Dubayle et al. 1996), Phellinus (Kauserud &
Schumacher 2002), Pleurotus (Urbanelli et al. 2003; Kay
& Vilgalys 1992), Schizophyllum (James et al. 1999; James &
Vilgalys 2001), Serpula lacrymans (Kauserud et al. 2007; Engh
et al. 2010) and Trichaptum abietinum (Kauserud &
Schumacher 2003b).
On a very local scale (<100 m), spatial autocorrelation analysis and Mantel tests of isolation by distance (IBD) provide information on the reproduction of EM fungi. For example,
statistically significant but weak spatial autocorrelation was
detected in the two smallest distance classes (25 m) of C. formosus genets, suggesting limited spore dispersal with potential inbreeding (Dunham et al. 2003, 2006). Data reported by
Carriconde et al. (2008b) revealed a similar positive spatial autocorrelation limited to 20.3 m or 6.3 m, according to the study
site for T. scalpturatum complex, and a significant Mantel test
of structure by distance suggested very limited dispersion of
most basidiospores. In H. cylindrosporum, Gryta et al. (2000), trying to explain the genet diversity found in previous studies
(Gryta et al. 1997), showed, thanks to separation of dikaryon
nuclei in protoplasts, that selfing of existing dikaryons by
way of their basidiospores established new genets. Spores falling in the vicinity of parental mycelium may thus create new,
related genets. Although little is known on spore dispersal in
most EM fungi, few spores may escape from the parental
vicinity (Li 2005). Similarly, local nuclear exchanges with haploids were claimed to contribute to genet diversity in the parasitic H. annosum (Johannesson & Stenlid 2004). Further
demonstration of such local patterns requires appropriate
tools: SI is unlikely to detect them (since kinship often entails
somatic compatibility), and a large number of markers may be
required to distinguish genetically close mycelia. Interestingly, if spore deposition proves to be mainly local, this may
account not only for the positive spatial autocorrelation in
G. W. Douhan et al.
EM populations, but also for the well-known patchiness of
species distribution in EM communities (e.g. Richard et al.
2004, 2005). Moreover, the sharing of similar SI alleles by
dikaryons and their progeny may allow hyphal fusions that
could locally enhance the survival of kin genets: such a ‘nursery effect’ may act with the pattern of spore deposition in
shaping positive spatial autocorrelation.
Even if still scarce, studies on gene flow and spatial structure of EM fungal populations on larger scales (0.1e100 km)
are growing in number (Figs 1, 2). In current studies, EM population structures range between two extreme types. EM species with low dispersion, and thus high structure over short
distances (km), are exemplified by species forming hypogeous
fruitbodies dispersed mainly by mycophageous animals (Fig 4,
Table S1). A study of Rhizopogon spp. populations in Oregon
stands of Douglas fir, using microsatellite markers, showed
low but significant differentiation between plots over 5 km
(Kretzer et al. 2005), greater for R. vesiculosus (qST ¼ 0.078)
than for Rhizopogon vinicolor (qST ¼ 0.022). Even greater differentiation was detected between populations of Rhizopogon
occidentalis, with FST values reaching 0.26 over 8.5 km
(Grubisha et al. 2007). Due to their below-ground fruiting, these
species likely accumulate large spore banks in soils, from uneaten fruitbodies, that outcompete migrants; moreover, germination potential of the spores seems to increase over 4 y
at least, suggesting some type of dormancy (Bruns et al.
2008; Nara 2008). Spores of EM species with epigeous fruitbodies dispersed by wind, likely over longer distances, are less
persistent (Ishida et al. 2008). The spore bank is thus less important and, even if most spores land close to parental fruitbodies as previously mentioned, longer travelling distances
are possible and likely reduce founder effects. As a result, differentiation over 100e1000 m scales is lacking: in R. brevipes,
no detectable structure over 230e1090 m was found
(qST ¼ 0.01; Bergemann et al. 2006); in L. amethystina, which exhibits very moderate structure on small scales (FST ranging up
to 0.02 for IGS over 45 m; Gherbi et al. 1999), few instances of
population genetic structure were found in a study of several
French populations sampled over 450 km apart, and FST values
did not correlate with distance (Roy et al. 2008; Vincenot et al.
submitted for publication).
Some studies have investigated EM populations on the
>100 km scale. For example, Roy et al. (2008) and Vincenot
et al. (submitted for publication) genotyped L. amethystina samples from French and European locations and found that the
highest FST values between pairs of populations was 0.13
(across 2000 km), with very little differentiation by distance
over France and from Northern Spain to Southern Finland
(Mantel test over 2900 km: r ¼ 0.10; P ¼ 0.06). Mitigated spatial
genetic structures have been revealed, as for East-Australian
Pisolithus microcarpus: Hitchcock et al. (2010) demonstrated genetic homogeneity over 81 km among four populations, and
a significant, moderate genetic differentiation (FST ¼ 0.08)
between two populations separated by 676 km. Additionally,
a cluster analysis failed to reveal any spatial genetic structure
among all P. microcarpus populations, suggesting a high capacity of spore dispersion by wind. Carriconde et al. (2008a) studied the two biological species of the morphological taxon
T. scalpturatum (recently assigned to phylospecies Tricholoma
argyraceum and T. scalpturatum by Jargeat et al. 2010) and
Population genetics of EM fungi
583
Fig 4 e The spatial genetic structures of EM populations is linked to their dispersal mode. Genetic distances are the values of
differentiation estimators analogous to FST (see Table S1 for reported values); note that geographic distances are on
a logarithmic scale. Each symbol represents a different EM species; studies highlighted in grey report a case of isolation by
distance, demonstrated by a significant Mantel test (i.e. genetic differentiation and genetic distance between populations are
correlated). Supposed dispersal of spores represents the hypothesis proposed by authors to partly explain the main observed
gene flows and IBD patterns (Table S1; note that species with long-distance wind dispersal also show short-distance dispersal).
used AMOVA to test for genetic structure between Western
European populations of each of them, but failed to find a significant correlation with geographic distance for one of them.
For T. matsutake in China, 5 SNPs did not reveal any significant
structure (FST ¼ 0.01) between two sites 70 km apart (Amend
et al. 2010). Some model species with epigeous fruitbodies
show intermediate results, with stronger IBD, such as S. grevillei (FST ¼ 0.02 over 700 m; Zhou et al. 2001) and C. formosus (detectable structure at more than 400 m; Dunham et al. 2006).
Such a null to low structure on the 100 km scale is reminiscent
of the situation reported in wind-dispersed saprotrophic and
parasitic Hymenomycetes (e.g. Kauserud & Schumacher
2003a, 2003b).
On larger, continental scales (>1000 km), all species tend to
show stronger IBD (Fig 4, Table S1). Contrasting with the data
of Amend et al. (2010), Xu et al. (2008), using ITS-RFLP and
20 SNPs on 17 more distant stands in South-Western China,
revealed the strongest differentiation between two populations of T. matsutake sampled 610 km apart (FST ¼ 0.23), and
an IBD over 1050 km (Mantel test: r ¼ 0.32, P ¼ 0.03). Bergemann et al. (2002) reported an FST ¼ 0.43 for R. brevipes over
1500 km in the western USA, but the lack of shared alleles
between the populations may question their conspecificity.
Vincenot et al. (submitted for publication) reported limited
IBD over Europe (3000 km) for L. amethystina, but FST ¼ 0.43
over 10 000 km, between Europe and Japan, and (as for R. brevipes) some microsatellite markers showed limited portability
from European to Japanese populations and vice versa (Donges
et al. 2008; Roy et al. 2008). This is reminiscent of the situation
observed, over the same regions, for the saprotrophic S. lacrymans (Engh et al. 2010). An intriguing possibility is that the
Northern Atlantic may not be a strong barrier to EM gene
flow: there is evidence, from mitochondrial rDNA in Laccaria
spp. (Selosse et al. 1998) and ITS in Tricholoma spp. (Jargeat
et al. 2010), that EM populations may not strongly diverge
between North America and Europe, but this deserves closer
investigation. At the >1000 km scale, allopatric speciation
may occur, and some species may turn into complexes of
cryptic species, as elaborated on below.
In Ascomycetes, only three model species have been investigated: the putatively asexual, non-fruiting C. geophilum, the
hypogeous T. melanosporum and, more recently, the asexual,
sporeless M. variabilis, an ericoid mycorrhizal fungus recently
confirmed to be EM as well (Grelet et al. 2010). C. geophilum first
584
showed a significant correlation between genetic and physical
distances (Mantel test: r ¼ 0.49, P < 0.001 over 250 km) in
French populations, based on ITS-RFLP and a SCAR marker
(Jany et al. 2002), as well as over North America (FST ¼ 0.25;
LoBuglio & Taylor 2002), suggesting that distance does structure C. geophilum populations. At a lower distance, Wu et al.
(2005) showed on Mount Fuji that a population of C. geophilum
was established from genotypes originating from another
population 5 km away, thus giving an indication of the minimal capacity of dispersion in this peculiar ecosystem. Although propagules remain unknown, snow avalanches may
have driven movement of sclerotia. Similarly, in the ascomycetous M. variabilis, no genetic structure was detected by ISSRs
over 40 m or 20 km (Grelet et al. 2010). In this species, we do not
know how genes can move, and this raises the possibility, relevant at least in Ascomycetes, for the production of asexual
propagules. Among truffles, the economically relevant T. melanosporum has been the focus of several studies within Europe.
Populations showed very little structure based on microsatellites and RAPDs (Bertault et al. 2001). However, more recently,
some polymorphism and population structure was found
using SNP markers (FST ¼ 0.20 between Italy and Northern
Spain; Murat et al. 2004) and new microsatellites derived
from the genomic sequence (Murat et al. 2011), highlighting
how different markers might reveal different levels of polymorphism. Although low, the observed structure is congruent
with that observed for the hypogeous Rhizopogon species previously mentioned.
Few studies have addressed the question of factors structuring populations, beyond distance (Fig 4, Table S1). Host species may be one of these: Bergemann & Miller (2002) used allelic
variation at four microsatellite loci to test for population subdivision of R. brevipes sampled in Douglas fir, lodgepole pine, and
Sitka spruce stands in western North America. High estimates
of genetic differentiation suggested that gene flow between
subpopulations was limited, but it was concluded that hosts
do not act as significant barriers to gene flow: rather, populations are geographically structured across western North
America. Populations of L. amethystina were harvested at three
sites in France under various hosts at each site (Abies alba, Castanea europea, Fagus sylvatica, or Quercus spp.) by Roy et al. (2008):
no differentiation by host species was found using 8 microsatellite markers, the IGS1 locus, a polymorphic mitochondrial
marker and dominant markers (Direct Amplification of Length
Polymorphism). Thus, some truly generalist EM fungi exist, for
which the host is not structuring the population. In mixed temperate forests at least, being a generalist may allow better propagation and survival in any forest place (Roy et al. 2008).
However, these studies should be repeated on non-neutral
polymorphic markers, e.g. those involved in the interaction
with the host: in this case, some local selection exerted by
the host may produce a totally different view.
Obviously, geographic barriers can be relevant. Grubisha
et al. (2007) used microsatellites to demonstrate strong IBD
due to such barriers (dry valley without EM host, ocean channel) that limited dispersal on a km scale for R. occidentalis. For
T. matsutake from the mountains of Northwest Yunnan
(China), FST values did not correlate with the Euclidian distance between populations, but with a ‘landscape distance’,
i.e. the shortest distance between populations calculated
G. W. Douhan et al.
below the tree line (i.e. the shortest distance within a continuum of host trees; Amend et al. 2010).
The contrasting patterns of gene flow from one species to
another may thus reflect diverse types of dispersal and geographical barriers (see also anthropic disturbance in the following section, and Fig 4 and Table S1 for a summary of the
different patterns of dispersion). However, differences in
markers, sampling or study scale make comparisons difficult,
and it is still impossible to compare studies and to draw firm
conclusions on general trends: more model species and
homogeneous studies (e.g. all using microsatellites) are required to test how spore vectors affect the dispersal range
and therefore, population structure. Moreover, two main tools
that have not been utilised enough in EM studies may enable
researchers to clarify the dynamics of spatial dispersal and
temporal persistence of spores. First, spore traps (Fierer et al.
2008) could establish and quantify the potential for gene
flow, as well as the actual dispersal of spores, as has been
done for the cosmopolitan saprotrophic Schizophyllum commune (James & Vilgalys 2001) and the EM Amanita muscaria
(Li 2005). A second important issue is spore survival, perhaps
allowing the existence of a ‘spore bank’ in soils, allowing
gene flow ‘in time’, i.e. from previous populations to newly settled ones. An exciting long-term experiment was recently set
up to assess spore survival in Rhizopogon species over a century
(Bruns et al. 2008), but more similar efforts will be needed on
other model species, e.g. to confirm that wind-dispersed
spores persist less efficiently (Ishida et al. 2008). However,
the delicate nature of spores in most epigeous EM taxa and inability to culture most EM homokaryons compared with the
more robust spores of hypogeous fungi such as Rhizopogon
sp., which can be experimentally manipulated, will pose serious challenges to researchers interested in such studies.
Cryptic biological species (CBSs)
As noted above, it is essential to identify the species of interest
when analysing population genetic data, and to avoid mixing
closely related taxa (Douhan et al. 2007a, 2007b). Anyone who
has ever tried to identify mushrooms is familiar with the difficulty of being confident in the identification in some genera,
such as Cortinarius or Sebacina. Species definitions and speciation mechanisms have been thoroughly discussed for the
fungi (e.g. Kohn 2005; Taylor & Berbee 2006; Giraud et al.
2008a). While morphospecies (species based on fruitbody
morphology) are easily tractable in some taxa, no clear species
delineation is currently available for some major EM taxa,
such as Laccaria spp., Thelephoraceae and Sebacinales.
Although there is often good congruence between ITS sequence and morphospecies among EM fungi (Horton 2002),
some morphospecies show ITS polymorphism. In an elegant
investigation on ITS heterozygosities among Hymenomycetes, Hughes et al. (2009) suggested that sequences could
vary up to 3 % within biological species (i.e. groups of individuals that sexually recombine within, but not among groups).
However, this level may well be lower in some taxa (Jargeat
et al. 2010; Begerow et al. 2010), and morphospecies with multiple ITS may sometimes hide complexes of CBSs.
Indeed, recently isolated species can retain ancestral features (Avise 1994), such as similar-looking fruitbody
Population genetics of EM fungi
morphology and close (or even identical) ITS sequence. Morphologies of fungal species display fewer characters than
those of macro-organisms and thus often evolve slower
(Taylor et al. 2006). Molecular markers can detect CBSs that
do not differ morphologically or by ITS sequence. By tracking
indirect evidence of isolation (e.g. the so-called Wahlund effect; Hartl & Clark 2007), molecular markers can untangle genetically isolated sub-groups and circumvent pairing
experiments that are laborious or impossible for uncultivable
taxa. (Conversely, two distinct Californian Russula morphospecies, the white Russula cremicolor and the red R. silvicola,
were shown to have identical ITS sequences and numerous
similarities in AFLP profiles (Redecker et al. 2001), suggesting
that they were a single biological species; however, such taxonomical lumpings of morphospecies remain rare.) Arguments for CBSs have been made recently in various fungal
taxa (Giraud et al. 2008a). Several Hymenomycetes phylogenetically close to EM fungi undergo cryptic speciation, such
as for saprotrophs (e.g. Serpula himantioides; Kauserud et al.
2007) and plant pathogens, for which CBSs often associate
with host specificity, e.g. Armillaria spp. (Coetzee et al. 2000),
H. annosum (Garbelotto et al. 1998; Gonthier et al. 2001), and
Pleurotus eryngii (Zervakis et al. 2001).
Some EM morphospecies also reveal CBSs. At least three
CBSs occur within Paxillus involutus that partly fit into the sexually incompatible groups previously described in the 80s by pairing monokaryons in vitro (Fries 1985; Hedh et al. 2008). CBSs
occur, sometimes in sympatry, within P. tinctorius (Martin et al.
2002; Hitchcock et al. 2003; Jourand et al. 2010), R. vinicolor sensu
lato (Kretzer et al. (2003) distinguished R. vinicolor and R. vesiculosus), C. formosus (Dunham et al. 2003), T. scalpturatum complex
(Gryta et al. 2006; Carriconde et al. 2008a, 2008b; Jargeat et al.
2010), Strobilomyces (Sato et al. 2007; Sato & Murakami 2008)
and Scleroderma species from Africa (Sanon et al. 2009). Although
some EM taxa diversified by specialising on different host species, such as in the genus Leccinum (den Bakker et al. 2004), in
the section Deliciosi among Lactarius (Nuytinck & Verbeken
2007), in the Hebeloma crustuliniforme species complex (Aanen
et al. 2000, 2001), in Strobilomyces species (Sato et al. 2007), or
among the suilloids (Kretzer et al. 1996), host specificity does
not seem so far to be a universal driver for EM CBS, in sharp contrast with parasitic fungi. In P. involutus sensu lato, CBSs show divergent host specificity, but genomic microarrays reveal
divergent gene contents that are not obviously correlated with
host specificity (Hedh et al. 2008); in A. muscaria form Northern
America, CBSs were suggested to correlate with some habitats
and/or biogeographic provinces (Geml et al. 2009); in Pisolithus
albus from New Caledonia, CBSs exist as an adaptation to ultramafic soils (Jourand et al. 2010). Thus, no unique evolutionary
base for CBSs is known for EM fungi.
Taylor et al. (2000) have advocated the analyses of multiple
gene phylogenies to delineate biological species within the
fungi: this method termed genealogical concordance phylogenetic species recognition (GCPSR) has been used extensively to
detect fungal CBSs. Basically, conflicts among independent
gene topologies can be caused by recombination events between individuals within a biological species, while the point
of transition from conflict to concordance in the topology determines the ancestor of each of these biological species.
Thus, phylospecies, i.e. phylogenetically defined species, can
585
be derived that reflect biological species. GCPSR was applied
to C. geophilum, perhaps the most widely distributed (although
not in tropical regions) and most recognisable EM fungus
based on EM root morphology. Recent studies revealed that
C. geophilum sensu lato is a species complex (Douhan & Rizzo
2005; Douhan et al. 2007a, 2007b). Based on fine-scale sampling
and the analysis of four loci, Douhan & Rizzo (2005) found
three phylogenetically distinct lineages of C. geophilum in a single soil sample and between sampling populations 8 m apart.
Using 44 isolates from two of these lineages, Douhan et al.
(2007a) found phylogenetic incongruence for ten loci, and, using six different methods, showed that recombination
occurred between lineages. However, most of the incongruence was caused by a recombination event between the actin
locus and the other loci, perhaps suggestive of a horizontal
gene transfer or of allele sorting from an ancestral polymorphism in the two lineages. Differential ancestral allele sorting
may mimic recombination, and is a limitation of GCPSR approaches. A more global sampling revealed that phylogenetic
resolution of the previously found three lineages broke down
(Douhan et al. 2007a, 2007b), demonstrating that the species
diversity of C. geophilum sensu lato is still not understood. Additionally, two isolates used in previous studies identified as
C. geophilum based on morphology were in fact different genera (Douhan et al. 2007a). These results demonstrate that inferences of population structure are highly dependent upon
how C. geophilum species are interpreted based both on morphology and genetic data. This potential problem also has implications for many other fungal taxa where CBSs have been
found.
The previous examples all imply sympatric (or overlapping) CBSs. Sibling CBSs may exist in allopatric situations, as
a result of IBD: the previously discussed data on IBD on
10 000 km scales may open the way to such a speciation process. In a comparison of species names, Mueller et al. (2007)
showed that 28e63 % of morphospecies were shared between
continental regions. IBD opens the possibility that some of
these species result from a frequent, but biologically improper
re-use of names from other regions for vicariant, phylogenetically close CBSs. The prediction is that intersterility does not
necessarily arise in allopatry, since IBD itself interrupts gene
flow (Brasier 1987). Large-scale IBD is well reported for saprotrophic Hymenomycetes, using phylogenetic reconstructions
(e.g. in Grifola frondosa, Shen et al. 2002; Megacollybia platyphylla,
Hughes et al. 2007; S. lacrymans, Engh et al. 2010): however, few
in vitro intersterility tests (except in some Ascomycetes such
as Neurospora, Dettman et al. 2003a, 2003b) and few studies using multimarkers (but see Kauserud et al. (2007) and Engh et al.
(2010) on S. lacrymans) have been published. For EM fungi, allopatric CBSs may explain the strong population differentiation
previously mentioned for R. brevipes over the Western USA
(Bergemann et al. 2002) or L. amethystina over Eurasia
(Donges et al. 2008; Vincenot et al. submitted for publication).
In the case of L. bicolor, American and European strains differ
genetically (e.g. for RAPD fingerprints), and haploids remain
able to mate in vitro (Selosse et al. 1997, 1998, 1999). EM taxa deserve more studies combining intersterility tests and evaluation of gene flow by tools of population genetics between
potential trans- or inter-continental CBSs. In particular, it is
unclear whether dikaryons produced by mating between
586
allopatric haploids are more or less fit than those issuing from
mating between sympatric haploids: this question, which is
central to gene flow in nature, has received conflicting answers for fungal pathogens (Van Putten et al. 2003; James
et al. 2008) and can be expected to vary among species complexes. Lastly, the distinction between allo- and sympatric
CBSs is not always straightforward: Geml et al. (2006) found
three geographic clades within A. muscaria sensu lato that coexist (perhaps secondarily) in parapatry in Northern America
(Geml et al. 2009); European CBSs detected in T. scalpturatum
complex are also sympatric (Carriconde et al. 2008a, 2008b,
Jargeat et al. 2010). Interestingly, a similar, complex pattern
is currently emerging from genomic sequencing of isolates
of baker’s yeast, Saccharomyces cerevisiae, which mixes geographically isolated lineages and mosaics of these lineages
(Liti et al. 2010).
Human activities have modified gene flow by introducing
species and genets on a worldwide scale. This is well reported
for pathogenic fungi (Brown & Hovmøller 2002), and, to a lesser
extent, for saprotrophic fungi, such as a CBS of S. lacrymans
(Kauserud et al. 2007; Engh et al. 2010) or S. cerevisiae
(Liti et al. 2010). Introduction of EM fungi has also occurred
on large scales, either unintentionally or deliberately, e.g. in
forest nurseries (Vellinga et al. 2009), but little is known about
how this can affect the genetic structure of populations. First,
comparison of genetic structure of introduced fungi at sites of
introduction versus native regions is unknown: for Amanita
muscaria in New Zealand, populations were claimed to involve
large genets (Sawyer et al. 2001; Bagley & Orlovich 2004), a feature common to invasive introduced fungal pathogens (e.g.
€ nwald et al. 2008). However, without comparison with naGru
tive areas, it is difficult to conclude whether introduction has
selected for more inbreeding or more clonality, due to rarity of
sexual partners. Second, introductions often entail a bottleneck (e.g. Kauserud et al. 2007). For example, populations of
Amanita phalloides putatively introduced to North America
showed a genetic bottleneck (Pringle et al. 2009), and this species has strong potential for further studies on genetic structure and founder effects after introduction. Third, the issue
of introgression, whenever indigenous genets are sexually
compatible, is also pending. In one such case, namely the
American strain L. bicolor S238N introduced in France, no introgression was detected in situ, even a decade after the first
introduction (Selosse et al. 1997, 1998, 1999). Although this is
only a short time in forest ecosystems, this introduced strain
is now fully sequenced and could serve to address the questions of persistence and introgression in the future (Martin &
Selosse 2008). More generally, the many EM fungi introduced
in tropical nurseries as inoculant for pines in the last two centuries (Mikola 1970) may also turn out to provide model species for introduction studies. In all, globalisation offers new
avenues for research on EM populations, investigating them
on larger scales and raising the question of anthropic
disturbances.
Biogeography
Biogeographic studies are an extension of population structure analyses that focus more on historical processes.
Although it traditionally uses phylogenies more than
G. W. Douhan et al.
population genetic tools, biogeography focuses on ‘populations’ at the largest geographical and taxonomical scale. Phylogenetic analyses can be used to test hypotheses on the origin
and dispersal of taxa and explain how geological events, such
as Pleistocene glaciations, shaped the distribution of presentday species (Myers & Giller 1988; Riddle 1996). Plant and animal models have been well studied, and biogeographic studies
of microorganisms are now emerging (Martiny et al. 2006;
Ramette & Tiedje 2007). However, little work has been done
on fungi, and even less on EM taxa. We discuss here some notable exceptions.
Wu & Mueller (1997) noted the high similarity of macrofungi between eastern Asia and eastern North America, and
that the disjoint distributions were usually at, or below the
species level designation. They noted, however, that this
was strongly biased by the use of morphospecies. A phylogenetic analysis was thus conducted on ITS rDNA between Eastern Asian and Eastern North American disjointed Suillus
species. Wu et al. (2000) found that morphologically indistinguishable Suillus spraguei specimens from China and North
America were paraphyletic to S. decipiens, a distinct North
American morphospecies, which was a sister taxon to the Chinese S. spraguei. They also noted that S. spraguei was widely
distributed and associated with Pinus subgenus Strobus,
whereas S. decipiens was restricted to the South-Eastern
United States and associated with Pinus subgenus Pinus.
They concluded: “divergence following geographical isolation
from a common ancestor followed different paths, with S. decipiens
changing in morphology, ITS sequence, and host switching to another subgenus while S. spraguei only showed sequence divergence.” This nicely shows how phylogenetic analyses can
address questions of EM host-symbiont evolution. Chapela &
Garbelotto (2004) examined phylogenetic relationships within
the economically important matsutake mushroom, in the genus Tricholoma. ITS and AFLP data showed that these fungi are
of Eocene origin and that the group in the Western United
States that associates with conifers derives from an ancestor
associated with angiosperms. They also concluded that African and European matsutake mushrooms are the most recent
descendants from a westward expansion from North America. In Europe, Murat et al. (2004) studied the post-glacial
recolonisation routes of T. melanosporum from Northern Italy
to Northern France and Northern Spain using ITS and SCAR
markers. They concluded that the oldest haplotypes were
present in Northern Italy, probably from a population that
subsisted in a refugium during the drastic bottleneck of the
last glaciation (20 000e16 000 y BP). T. melanosporum would
then have recolonised northern regions following the postglacial route of recolonisation of oak, its major host. Using 7
microsatellite loci, Rubini et al. (2005) showed that geographic
distribution of Tuber magnatum in Italy also follows the postglacial expansion of its host species, Quercus sp., Corylus sp.
and Tilia sp., which subsisted in Southern and Central Italy
during the last glaciation. Thus, these two truffles operated
a post-glacial recolonisation following that of host trees.
On a global scale, Martin et al. (2002) revised the phylogeny
of the genus Pisolithus using ITS sequences, including new
study zones in Australia, Africa and South America. Some lineages of Pisolithus occurred in restricted geographical regions,
associated with endemic plants, and several lineages
Population genetics of EM fungi
introduced from the Holarctic to the Southern Hemisphere
with their hosts also associated with new regional hosts.
Thus, the lineages of Pisolithus were related to the biogeography of their hosts. Hosaka et al. (2008) found a similar, but
more ancient pattern for the global biogeography of Hysterangiales using a 5-locus phylogeny. They showed that EM lineages of Hysterangiales appeared in Australia or eastern
Gondwana and associated with the Myrtaceae. These trufflelike hymenomycetes then expanded to the Northern Hemisphere via long-distance dispersal events, and shifted many
times to new hosts according to their biogeography. However,
the authors could not date the emergence of EM Hysterangiales and therefore did not explain how and when long-distance dispersal events occurred. Halling et al. (2008)
constructed a phylogeny of the bolete EM fungus Tylopilus balloui all around the Pacific based on loci of Large Subunit (LSU)
of rDNA and RpbI genes. An ancient pangean distribution was
the most parsimonious explanation of the phylogeny observed, and this highlighted again that EM fungi are likely to
have biogeographic patterns linked to their hosts.
Molecular phylogenies can be combined with paleo-biogeography if ages of clades can be calculated. Hypotheses on paleontological events that caused historical clade divergence
could then be tested in fungi, as classically done in other major taxa (e.g. Hedges et al. 1996; Cooper & Penny 1997). This approach has been used for several fungi, e.g. among
saprotrophic Lentinula species, the Old World/New World disjunction was related to the fragmentation of an ancient Laurasian range (Hibbett 2001). To date, few studies have applied
this approach to EM fungi. A molecular clock used on the
ITS and large rDNA subunit was applied to the radiation of
CBSs in A. muscaria sensu lato (Geml et al. 2006), putting
a time frame on its range expansion from a SiberianeBeringian origin to the whole Northern Hemisphere. Den Bakker
et al. (2007) showed that 5.8S-ITS2 in Leccinum sect. Scabra
evolved according to a molecular clock model, but could not
date the Leccinum scabrum and L. rotundifoliae divergence, because they lacked a temporal calibration of their phylogeny.
Chapela & Garbelotto (2004) calibrated their phylogeny of matsutake Tricholoma, following a molecular clock model; they
hypothesised that the shift from an angiosperm-associated
matsutake ancestor to the earliest conifer associates within
the ‘true matsutake’ occurred 55 My ago, when mixed forest
arose in Western North America. This calibration allowed
a time scale for the evolution of vicariance by a migration
through the Bering Strait, and the westward expansion to
Europe and Africa. Matheny & Bougher (2006) made a taxonomic and biogeographic description of the recently identified
genus Auritella from Africa and Australia. They calibrated
their phylogeny on the node Auritella serpentinocystis e A. geoaustralis, two species respectively found in Western and Eastern Australia, hypothesised to be at least 15 My old, when
formation of the Nullarbor Plain in Southern Australia became
an edaphic barrier between the two species. Matheny &
Bougher (2006) then dated the divergence between Australian
and African Auritella to 86 My ago, i.e. in the late Cretaceous,
and concluded the divergence of the two clades was a Gondwanan vicariance. Matheny et al. (2009) then extended the picture to the whole EM family Inocybaceae, and suggested that it
diversified no later than the Cretaceous in the Paleotropics,
587
and later colonised the north and south temperate regions;
as for matsutake, a shift occurred from primary Angiosperm
hosts to conifer, probably during the Paleogene. These examples evidence that the scarcity of fungal fossils limits the calibration to indirect approaches; moreover, as shown by Taylor
& Berbee (2006), dating of fungal clades can be highly variable
depending on the fossils used for the calibration (from 1808 to
400 My ago for the divergence between Ascomycetes and
Basidiomycetes). Taylor & Berbee (2006) concluded that improvement of the time estimates would depend on the discovery of fossil fungi showing recognisable shifts in morphology,
thus providing precise calibration points in the phylogeny.
However, on the scale of 106 y and at family level within the
Asco- and Basidiomycetes, such fossils are unlikely to be
found.
Biogeography is still in its infancy for EM fungi, and historical reconstructions are limited to a small number of model
taxa, mostly centred on temperate regions (where most
research teams are situated, as reflected in Fig 2). Especially,
the role of hybridization after secondary contacts between allopatric species that remain interfertile, and the potential for
reticulated evolution in EM fungi deserve further study, since
possible speciation by ancient hybridization was found in Tricholoma spp. (Jargeat et al. 2010). A global pattern will not be
reached until more samples from tropical regions and the
Southern Hemisphere are taken into account (Matheny et al.
2009; Tedersoo et al. 2010). Moreover, EM population genetics
is still only rarely involved in global analysis so far (with the
few exceptions revealing CBSs mentioned above, e.g.
Vincenot et al. submitted for publication), whereas it could
be of help at least in phylogenetically related species with partial portability of markers. Biogeographic studies, combined
with population genetics, will undoubtedly shed light on basic
taxonomic confusions in the EM fungi, and speciation processes. This may finally lead to a general picture of how the
EM associations repeatedly evolved and diversified to reach
their extant distributions.
EM population genetics: emerging directions
Soil sampling and the below-ground picture
We have already emphasised the discrepancy between aboveand below-ground species composition at community level
(Horton & Bruns 2001). Therefore, the ability to genotype individual mycorrhizal root tips or mycelium from soil should also
be considered in EM population genetics. Probably, a significant portion of genotypes found in soil only will never reproduce, and thus may have no role in evolution and gene flow
(except perhaps as donors of haploid nuclei; e.g. in truffles,
Rubini et al. 2011). However, they are of interest to depict
and analyse the pattern of the total genetic diversity as well
as for various functional questions, because they contribute
to the local soil processes. Newly available tools allow genotyping of roots without the risk of cross-amplification, e.g.
SCARs, SNPs and microsatellites. Analyses of EM root tips
have been accomplished, especially to assess the persistence
of inoculated strains in plantations (e.g. El Karkouri et al.
2006; Selosse et al. 2000). Zhou et al. (2001) used microsatellite
588
markers to demonstrate that EM root tips and extra-radical
mycelium of the same genet could be found directly under
fruitbodies, but below-ground distribution was not centred
on the fruitbodies and the amount of subterranean presence
of each genet did not always correlate with the actual fruitbody abundance. Hortal et al. (in preparation) also showed
a spatial coincidence between above- and below-ground
genets of L. amethystina, using microsatellites, whenever the
same genets were found in fruitbodies and mycorrhizae. Using a specific IGS region, Guidot et al. (2001) found a similar
congruence of above- and below-ground distribution for H.
cylindrosporum, and in this case the same genets tended to be
dominant above and below ground. Guidot et al. (2003a) then
used competitive PCR based on the same marker and were
able to detect genets in the soil under fruitbodies, and showed
that genets disappeared below ground a year or more after
fruitbody disappearance.
More quantitative approaches were developed, but here,
the detection remains at the species, not the genet, level.
van der Linde et al. (2008, 2009) proposed a quantitative PCRbased method to detect the mycelia of EM stipitate hydnoid
fungi in soil. They developed species-specific primers in the
ITS region for 12 hydnoid fungal species, with positive results
on amplification, up to 40 cm away from sporocarps. Hortal
et al. (2008, 2009) investigated the field persistence of inoculated Lactarius deliciosus by real-time PCR analysis of soil, and
found a correlation between the percentage of EM tips and
mycelial abundance in soil, suggesting that quantifying mycelium in soils is a non-destructive proxy of the colonization
level in their system. In the future, more similar studies using
species-specific (or even genet-specific) primers to explore diversity in the soil may test further the congruence between
the below- and above-ground pictures, and investigate persistence and distribution of EM mycelia themselves.
The very fine-scale soil picture
Although most of the first studies were descriptive, we still
have a limited view of the three-dimensional distribution
of genets in soil on a very fine scale (with a few exceptions
listed above). Indeed, analysis of the fine-scale distribution
of EM mycelia associated with pine at EM species level
(Genney et al. 2006) revealed very different patterns across
soil profiles for the different species. Within populations,
how is the genet diversity structured in soil, especially in 3
dimensions? Is there some intermingling between genets?
In case of persistence, does the genet distribution change?
More studies on a centimetre scale are required, especially
in ascomycetes where the distribution of parental, haploid
mycelia can be compared with that of the resulting fruitbodies (as was recently done for truffles, Rubini et al. 2010; see
above). Moreover, the exact shape of ramets and the temporal evolution of mycelial connectivity within a genet remain
elusive and appropriate tools to determine ramets, such as
diffusible dyes or labeled compounds, have not been
employed hitherto.
Analyses on a very fine scale may be an indirect way to
determine if EM fungi form mycorrhizal networks between
plants. A particular EM fungus may be found on different
host species, and therefore may be involved in nutrient
G. W. Douhan et al.
exchange or interactions between plants, from the same or
different species e and this represents an exciting perspective
in plant ecology (Selosse et al. 2006, Vincenot et al. 2008). Although this feature has important ecological consequences,
few studies have demonstrated that a single genet is connected between two plants. Two reports using microsatellites
investigated links within a single EM tree population. Investigating the dense mats formed by T. matsutake (the so-called
shiros), Lian et al. (2006) showed that each genet colonised multiple pine trees (three to seven trees per genet). Beiler et al.
(2010) showed that an EM network formed by R. vesiculosus
and R. vinicolor was present in a 30 30 m Douglas fir stand.
They identified 13 and 14 genets, respectively, each connected
to up to 19 trees within the stand; 55 of the 56 trees from the
stand were linked to at least one other tree, i.e. they shared
at least one fungal genet. Three reports investigated links between plant species. Based on rDNA polymorphism, i.e. a very
limited tool for genet typing, some heterotrophic plants were
suggested to harbour the same fungal genet as surrounding
trees, which are the likely origin of the carbon conveyed by
their mycorrhizal fungi (Taylor & Bruns 1997; Selosse et al.
2002). Among green plants, Grelet et al. (2010) showed that
the same genets of Rhizoscyphus ericae aggregate (mostly
from the Melinomyces variabilis clade), as recognised by ISSRs
after in vitro isolation, formed EM on Pinus sylvestris and ericoid
mycorrhizae on Vaccinium vitis-idaea: here, the small genet
sizes (<13 cm) were counterbalanced by root intermingling.
The three later studies contribute to the idea that some EM
fungi act as ericoid or orchid mycorrhizal fungi as well
(Selosse et al. 2004). The common EM Sebacinales were also
suggested to behave as plant endophytes on herbs (Selosse
et al. 2009): thus, soil sampling may consider, for some species
at least, more than EM root tips.
Mycelial links, which may allow cooperation among EM
host populations, e.g. between adult trees and seedlings or understory and canopy plants, are probably of major importance
in forest ecosystems (Selosse et al. 2006). Population genetic
analyses of mycorrhizal root tips, along with methodologies
to trace movement of nutrients, such as radioactive labels or
stable isotopes, will probably help to determine the shape, importance and functioning of EM networks in natural
ecosystems.
Where are the haploid monokaryons?
In the future, it may also be possible to detect alleles from
monokaryons of hymenomycetes in the soil. Almost nothing
is known about the actual role or prevalence of monokaryotic
hyphae in soil. This aspect of the life cycle of most hymenomycetes fungi remains a mystery in situ. Appropriate molecular
tools could really add to our understanding, but disentangling
them from homozygous dikaryons may be difficult. Moreover,
the role, if any, that monokaryons play as mycorrhizal symbionts is also unknown, although some monokaryons can form
mycorrhizae ex situ (e.g. Gardes et al. 1990).
Recent work on Hymenomycetes suggests that, in a given
in vitro mating, haploids may receive and donate haploid nuclei (true hermaphroditism), or only donate or only receive
(James et al. 2008; Nieuwenhuis et al. 2010). The latter situations are, respectively, a male-like role, with minor
Population genetics of EM fungi
investment in post-mating mycelium, and a female-like role,
with larger investment in post-mating mycelium. If this occurs in situ and, in these conditions, impacts the fitness of haploid nuclei, it opens the way to potential sexual selection in
fungi, with competition among male haploids and choice of
mating partners by the female haploids (Nieuwenhuis et al.
2010): such a phenomenon remains to be investigated in EM
populations.
New sequencing technologies and ‘omics’ approaches
Current technological advances that enable the sequencing of
complete genomes, or of thousands of sequences in a few
days, may improve the robustness of EM population genetic
analyses (Martin & Selosse 2008). The fundamental use of
markers and statistical analyses will not change, but genotyping could be greatly improved, thanks to markers that are genetically or chromosomally unlinked, an assumption in most
population genetic analyses that is usually not verified. Pyrosequencing allows the sequencing of fungal genomes at
a cost that is decreasing over time and could be useful in developing new microsatellites or SNP markers, with primers directly designed from the genomic sequence. Even though
a single run of pyrosequencing remains expensive, it could
soon be efficient in comparison with the time and money
spent in ‘traditional’ strategies of development of molecular
markers, i.e. the creation of enriched libraries for microsatellites, cloning, sequencing, and the uncertainty in actually
finding useful markers.
All reported studies have used putatively neutral markers to
investigate EM population genetics, even if this assumption is
often not validated. For example, the discrepancy between
early, microsatellite-based, population analysis of T. melanosporum, suggesting homogeneity over Europe (Bertault et al.
1998), and later work reporting SNP polymorphisms (Murat
et al. 2004; C. Murat, pers. com.) may have been due to the
fact that these markers were in coding regions that could
have been under directional selection. The use of markers
potentially under selection, which are available through genome sequencing, is an interesting prospect from a different
point of view (Martin & Selosse, 2008). The availability of genomic resources offers opportunities to investigate functional
variation in populations, by looking at potentially selected
genes. For example, Roy et al. (2008) used microsatellites to
show that the genetic diversity of L. amethystina could not be
explained by a specialisation to the host. However, an unexplored possibility is that several genes involved in interaction
with the host may show a specialisation to the locally available
hosts. Similarly, using AFLP and thereafter microsatellites,
Muller et al. (2004 and 2007, respectively) showed that there is
extensive gene flow between S. luteus populations from heavy
metal-polluted and nearby non-polluted soils, although individuals varied in their adaptation to heavy metals. Although
this pollution had a limited effect on neutral markers, one
may suppose that some selection occurs at non-neutral loci involved in adaptation to heavy metals (conversely, in P. albus,
CBSs exist on ultramafic versus non-ultramafic soils; Jourand
et al. 2010). More generally, a part of the local genetic variability
of EM populations could be driven by environmental factors
(host species or ages, edaphic or climatic conditions, etc.) in
589
some parts of the genome. Additionally, other selection pressures may also arise from within the fungal population itself:
for example, mating type alleles showed balanced selection
in populations of the saprotrophic Coprinus cinereus (May et al.
1999). In the near future, identifying genes under selection
and their pattern of distribution within populations may allow
investigation of local adaptation and could be crossed with
gene expression in soil or EM root tips.
Lastly, phylogenetic and phylogeographic studies can be
improved thanks to phylogenomics: as highlighted by Delsuc
et al. (2005), sequencing whole genomes or the whole transcriptomes of non-model species provides relevant nucleotide
variations for phylogenetic purposes. Moreover, in terms of
studies of diversity, metagenomics could be applied to the exploration of below-ground diversity. For example, Zinger et al.
e et al. (2009) propose to use new sequencing
(2007) and Bue
technologies for high-throughput studies of fungal communities by treating high numbers of soil samples where species
can be identified with ‘barcodes’ of ITS regions.
Toward the emergence of conservation concerns
As mentioned in the Introduction, several edible EM fungi
have high economic values, such as Cantharellus spp., Tuber
spp., Boletus spp. and T. matsutake (Yun & Hall 2004). Their
easily perceived importance for humans, associated with
poorly understood cases of fructification decrease (Jaenike
1991; Straatsma et al. 2001), currently raises concerns about
anthropic influence and conservation of EM species, part of
which can be addressed by means of population genetics
studies. A major concern is whether mushroom picking
could impair future harvests, although there is evidence
that it has little influence in the short term (Egli et al. 2006).
Another concern deals with inoculation of exotic strains
that may influence the local EM genetic makeup, e.g. upon
introduction of Chinese truffles in Europe (Murat et al. 2008)
or introduction of strains beneficial for tree growth. In the
only case investigated (L. bicolor inoculated in France;
Selosse et al. 1998, 1999), no effect on local genet diversity
was found over a decade, but more analyses, especially
over the long term, are required. As noted in the
Biogeography section, ancient introductions from Europe to
the previous tropical colonies may offer interesting cases of
introgression. Physicochemical disturbance of habitats is another major concern, e.g. nitrogen deposition entails changes
in EM species diversity (Cox et al. 2010). In general, disturbance does influence local structure of EM populations (e.g.
physical disturbance, Guidot et al. 2003a; Gryta et al. 2006),
but few authors have explored this in the long run, or in
the context of new anthropic pressures.
Harvesting fruitbodies may also impact diversity out of
the stand, by limiting gene flow, and the ability to disseminate and reproduce, even if few effects are noticeable within
the short term at a community level (Straatsma et al. 2001).
Lian et al. (2006) and Xu et al. (2008) focused on the declining,
non-cultivable and highly prized Asian T. matsutake, in order
to evaluate the genetic diversity and potential of its populations in Japanese and South-Western Chinese forests, respectively. High local genetic diversities were shown
within the matsutake shiros, and the authors insist on the
590
crucial importance of gene flow within and among populations, locally and over distances of hundreds of km. They
thus suggested potentially adapted conservation procedures
to limit the harvest of fruitbodies before their maturity, enabling zoochorous and anemochorous dissemination of sufficient amounts of basidiospores to maintain intra- and
inter-population genetic resources. This is especially challenging, since we currently do not know what level of spore
flow is required (usually low on the 100 km scale) for spatial
structuring.
Currently, with few exceptions such as Tuber spp. (Rubini
et al. 2010) and Laccaria spp. (Selosse et al. 1997, 1998, 1999),
management of EM genetic resources and populations is nonexistent. By understanding how EM fungi complete their lifecycles in time and space, for different population sizes, we
may uncover the critical stages and bottlenecks in local and
global diversity dynamics. This could, in the future, allow
some management of EM fungi in situ.
Final remarks
Since the pioneering work of Dahlberg & Stenlid (1990, 1994),
significant progress has been made in the population genetics
of EM fungi, especially due to incorporation of modern molecular markers and specific hypotheses on various aspects of EM
population dynamics, mating and dispersion. Several phylogenetically unrelated EM fungal species have been investigated and, although sampling designs and genotyping
methods hitherto often limit comparisons between studies
(Table 1), there is a need to maintain this diversity if we are
to answer the important question: ‘what is specific to the EM
lifestyle?’ This question requires us to go beyond the characteristics of each species, and the repeated evolution of the
EM lifestyle offers evolutionarily independent replicates of
EM syndromes, from which common features specific to EM
lifestyle could be derived. It is currently too early to compare
EM populations to saprotrophic or parasitic fungal populations, even if for some traits (CBS and IBD in EM Hymenomycetes; asymmetric mating in ascomycetes) they look to be
similar to their phylogenetic relatives.
It is to be hoped that publications will continue to accrue
on EM population genetics (Fig 1), perhaps at an increased
rate due to the easier design of markers by using sequenced
genomes (Martin & Selosse, 2008). Future studies in the population genetics of EM fungi simultaneously based on aboveand below-ground perspectives will ultimately lead to a better understanding of these important symbiotic organisms,
and possibly of the way of managing them. There is, also,
a real need for more theoretical work on population genetics
to incorporate the life histories of fungi in general (and Ascomycetes versus Hymenomycetes especially), so that analytical methods can be developed that more realistically fit
EM models. In particular, beyond demographic and biogeographic history, more functional approaches e implying
crosstalk with studies analysing gene expression in situ e
may enrich the approach. Lastly, we call for a change in
scales in time (spore survival, genet survival, reaction to disturbances such as fire, drought, global change. over the
long term) and in space (more global investigations, more
G. W. Douhan et al.
model species from the tropics), in order to reach the full
picture for EM fungi.
Acknowledgments
We thank David Rizzo, François Rousset, Annette Kretzer and
Tom Gordon for corrections to early versions of this manuscript,
and David Marsh for English corrections. We also warmly thank
two anonymous referees for helpful corrections and additions.
Support for this work was provided by an NSF Biocomplexity
grant (DEB 9981711) and by financial support of the Agricultural
Experiment Station, University of California Riverside to G. D.
te
Française d’Orchidophilie and
and by the CNRS, the Socie
the Agence Nationale de la Recherche to M.-A. S.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.funbio.2011.03.005.
references
Aanen DK, Kuyper TW, Hoekstra RF, 2001. A widely distributed
ITS polymorphism within a biological species of the ectomycorrhizal fungus Hebeloma vetulipes. Mycological Research 105:
284e290.
Aanen DK, Kuyper TW, Mes THM, Hoekstra RF, 2000. The evolution of reproductive isolation in the ectomycorrhizal Hebeloma
crustuliniforme aggregate (Basidiomycetes) in northwestern
Europe: a phylogenetic approach. Evolution 54: 1192e1206.
Adams RI, Hallen HE, Pringle A, 2006. Using the incomplete genome of the ectomycorrhizal fungus Amanita bisporigera to
identify molecular polymorphisms in the related Amanita
phalloides. Molecular Ecology Notes 6: 218e220.
Amend A, Keeley S, Garbelotto M, 2009. Forest age correlates with
fine-scale spatial structure of Matsutake mycorrhizas. Mycological Research 113: 541e551.
Amend A, Garbelotto M, Fang Z, Keeley S, 2010. Isolation by
landscape in populations of the prized edible mushroom Tricholoma matsutake. Conservation Genetics 11: 795e802.
Agerer R, 1991. Characterization of ectomycorrhiza. Methods in
Microbiology 23: 23e73.
Agerer R, 1995. Index of unidentified ectomycorrhizae. 4. Names
and identifications published 1993e1994. Mycorrhiza 5:
449e450.
Anderson IC, Chambers SM, Cairney JWG, 1998. Use of molecular
methods to estimate the size and distribution of mycelial individuals of the ectomycorrhizal basidiomycete Pisolithus
tinctorius. Mycological Research 102: 295e300.
Anderson IC, Chambers SM, Cairney JWG, 2001. Distribution and
persistence of Australian Pisolithus species genets at native
sclerophyll forest field sites. Mycological Research 108: 971e976.
Andrews JH, 1992. Fungal life-history strategies. In: Carroll GC,
Wicklow DT (eds), The Fungal Community e Its Organization and
Role in the Ecosystem. Dekker, New York, USA.
Avise JC, 1994. Molecular Markers, Natural History and Evolution.
Chapman & Hall, New York.
Baar J, Ozinga WA, Kuyper TW, 1994. Spatial distribution of Laccaria bicolor genets reflected by sporocarps after removal of
litter and humus layers in a Pinus sylvestris forest. Mycological
Research 98: 726e728.
Population genetics of EM fungi
Bagley SJ, Orlovich DA, 2004. Genet size and distribution of Amanita muscaria in a suburban park, Dunedin, New Zealand. New
Zealand Journal of Botany 42: 939e947.
s B, Halkett F, Dutech C, Andrieux A, Pinon J, Frey P, 2008.
Barre
Genetic structure of the poplar rust fungus Melampsora laricipopulina: evidence for isolation by distance in Europe and recent founder effects overseas. Infection, Genetics and Evolution
8: 577e587.
Baumgartner K, Grubisha LC, Fujiyoshi P, Garbelotto M,
Bergemann SE, 2009. Microsatellite markers for the diploid
basidiomycete fungus Armillaria mellea. Molecular Ecology Resources 9: 943e946.
Begerow D, Nilsson H, Unterseher M, Maier W, 2010. Current
states and perspectives of fungal DNA barcoding and rapid
identification procedures. Applied Microbiology and Biotechnology 87: 99e108.
Beiler KJ, Durall DM, Simard SW, Maxwell SA, Kretzer AM, 2010.
Architecture of the wood-wide web: Rhizopogon spp. genets
link multiple Douglas-fir cohorts. New Phytologist 185:
543e553.
Bennett RJ, Johnson AD, 2003. Completion of a parasexual cycle in
Candida albicans by induced chromosome loss in tetraploid
strains. EMBO Journal 22: 2505e2515.
Bergemann SE, Miller SL, 2002. Size, distribution, and persistence
of genets in local populations of the late-stage ectomycorrhizal fungus basidiomycete, Russula brevipes. New Phytologist
156: 313e320.
Bergemann SE, Miller SL, Garbelotto M, 2005. Microsatellite loci
from Russula brevipes, a common ectomycorrhizal associate of
several tree species in North America. Molecular Ecology Notes
5: 472e473.
Bergemann SE, Douhan GW, Garbelotto M, Miller SL, 2006. No
evidence of population structure across three isolated subpopulations of Russula brevipes in an oak/pine woodland. New
Phytologist 170: 177e184.
Bertault G, Raymond M, Berthomieu A, Callot G, Fernandez D,
1998. Triffling variation in truffles. Nature 394: 734.
Bertault G, Rousset F, Fernandez D, Berthomieu A, Hochberg ME,
Callot G, Raymond M, 2001. Population genetics and dynamics of
the black truffle in a man-made truffle field. Heredity 86: 451e458.
re J, 1996.
Boisselier-Dubayle MC, Perreau-Bertrand J, Lambourdie
Genetic variability in wild populations of Mycena rosea. Mycological Research 100: 753e758.
Bonello P, Bruns TD, Gardes M, 1998. Genetic structure of a natural population of the ectomycorrhizal fungus Suillus pungens.
New Phytologist 138: 533e542.
Brasier CM, 1987. The dynamics of fungal speciation. In: Evolutionary Biology of the Fungi. Cambridge University Press.
Brundrett MC, 2002. Coevolution of roots and mycorrhizas of land
plants. New Phytologist 54: 275e304.
Brown AJ, Casselton LA, 2001. Mating in mushrooms: increasing
the chances but prolonging the affair. Trends in Genetics 17:
393e400.
Brown JKM, Hovmøller MG, 2002. Aerial dispersal of pathogens on
the global and continental scales and its impact on plant
disease. Science 297: 537e541.
Bruns TD, Peay KG, Boynton PJ, Grubisha LC, Hynson NA,
Nguyen NH, Rosenstock NP, 2008. Inoculum potential of Rhizopogon spores increases with time over the first 4 yr of a 99-yr
spore burial experiment. New Phytologist 181: 463e470.
Bruns T, Tan J, Bidartondo M, Szaro T, Redecker D, 2002. Survival
of Suillus pungens and Amanita francheti ectomycorrhizal genets
was rare or absent after a stand-replacing wildfire. New Phytologist 155: 517e523.
e M, Reich M, Murat C, Morin E, Nilsson RH, Uroz S, Martin F,
Bue
2009. 454 pyrosequencing analyses of forest soils reveal an
unexpectedly high fungal diversity. New Phytologist 184:
449e456.
591
Burt A, Carter DA, Koenig GL, White TJ, Taylor JW, 1996. Molecular
markers reveal cryptic sex in the human pathogen Coccidioides
immitis. Proceedings of the National Academy of Sciences of the
United States of America 93: 770e773.
Carriconde F, Gardes M, Jargeat P, Heilmann-Clausen J,
Mouhamadou B, Gryta H, 2008a. Population evidence of cryptic species and geographical structure in the cosmopolitan
ectomycorrhizal fungus, Tricholoma scalpturatum. Microbial
Ecology 56: 513e524.
Carriconde F, Gryta H, Jargeat P, Mouhamadou B, Gardes M, 2008b.
High sexual reproduction and limited contemporary dispersal
in the ectomycorrhizal fungus Tricholoma scalpturatum: new
insights from population genetics and autocorrelation analysis. Molecular Ecology 17: 4433e4445.
Chapela IH, Garbelotto M, 2004. Phylogeography and evolution in
matsutake and close allies inferred by analyses of ITS sequences and AFLPs. Mycologia 96: 730e741.
Clark T, Anderson J, 2004. Dikaryons of the basidiomycete fungus
Schizophyllum commune: evolution in long-term culture. Genetics 167: 1663e1675.
Coetzee MPA, Wingfield BD, Harrington TC, Dalevi D,
Coutinho TA, Wingfield MJ, 2000. Geographical diversity of
Armillaria mellea s. s. based on phylogenetic analysis. Mycologia
92: 105e113.
Cooke RC, Rayner ADM, 1984. Ecology of Saprotrophic Fungi. Longman Inc., New York.
Cooper A, Penny D, 1997. Mass survival of birds across the CretaceouseTertiary boundary: molecular evidence. Science 275:
1109e1113.
Cox F, Barsoum N, Lilleskov EA, Bidartondo MI, 2010. Nitrogen
availability is a primary determinant of conifer mycorrhizas
across complex environmental gradients. Ecology Letters 13:
1103e1113.
Dahlberg A, 2001. Community ecology of ectomycorrhizal fungi:
an advancing interdisciplinary field. New Phytologist 150:
555e562.
Dahlberg A, Stenlid J, 1990. Population structure and dynamics in
Suillus bovinus as indicated by spatial distribution of fungal
clones. New Phytologist 115: 487e493.
Dahlberg A, Stenlid J, 1994. Size, distribution and biomass of genets
in populations of Suillus bovinus (L.: Fr.) Roussel revealed by
somatic incompatibility. New Phytologist 128: 225e234.
Dahlberg A, Stenlid J, 1995. Spatiotemporal patterns in ectomycorrhizal populations. Canadian Journal of Botany 73:
S1222eS1230.
Dahlberg A, 1997. Population ecology of Suillus variegatus in old
Swedish Scots pine forests. Mycological Research 101: 47e54.
Danielson RM, 1991. Temporal changes and effects of amendments on the occurrence of sheating (ecto-) mycorrhizas of
conifers growing in oil sands tailings and coal spoil. Agriculture, Ecosystems and Environment 35: 261e281.
David P, Pujol B, Viard F, Castella V, Goudet J, 2007. Reliable selfing
rate estimates from imperfect population genetic data. Molecular Ecology 16: 2474e2487.
Y, 1994. Spatial distribution and
de La Bastide PY, Kropp BR, Piche
temporal persistence of discrete genotypes of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton. New Phytologist 127:
547e556.
Y, 1995. Population structure
de La Bastide PY, Kropp BR, Piche
and mycelial phenotypic variability of the ectomycorrhizal
basidiomycete Laccaria bicolor (Maire) Orton. Mycorrhiza 5:
371e379.
Deacon JW, Fleming LV, 1992. Interactions of ectomycorrhizal
fungi. In: Allen MF (ed.), Mycorrhizal Functioning: an Integrated
PlanteFungal Process. Chapman & Hall, London, pp. 249e300.
Debaud JC, Gay G, 1987. In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum
associated with Pinus pinaster. New Phytologist 105: 429e435.
592
Delsuc F, Brinkmann H, Philipp H, 2005. Phylogenomics and the
reconstruction of the tree of life. Nature Reviews Genetics 6:
361e375.
den Bakker HC, Zuccarello GC, Kuyper TW, Noordeloos ME, 2004.
Evolution and host specificity in the ectomycorrhizal genus
Leccinum. New Phytologist 163: 201e215.
den Bakker HC, Zuccarello GC, Kuyper TW, Noordeloos ME, 2007.
Phylogeographic patterns in Leccinum sect. Scabra and the
status of the arctic-alpine species L. rotundifoliae. Mycological
Research 111: 663e672.
Dettman JR, Jacobson DJ, Taylor JW, 2003a. A multilocus genealogical approach to phylogenetic species recognition in
the model eukaryote Neurospora. Evolution 57: 2703e2720.
Dettman JR, Jacobson DJ, Turner E, Pringle A, Taylor JW, 2003b.
Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in a model
eukaryote. Evolution 57: 2721e2741.
€ m E, Le Tacon F,
Di Battista C, Selosse M-A, Bouchard D, Stenstro
1996. Variations in symbiotic efficiency, phenotypic characters
and ploidy level among different isolates of the ectomycorrhizal basidiomycete Laccaria bicolor strain S238. Mycological
Research 100: 1315e1324.
Dilmaghani A, Balesdent MH, Didier JP, Wu C, Davey J, Barbetti MJ,
Li H, Moreno-Rico O, Philipps D, Despeghel JP, Vincenot L,
Gout L, Rouxel T, 2009. The Leptosphaeria maculans e Leptosphaeria biglobosa species complex in the American continent.
Plant Pathology 58: 1044e1058.
Donges K, Schlobinski D, Cremer E, Rexer KH, Kost G, 2008. Six
newly developed microsatellite markers of Laccaria amethystina, using an improved CSSR approach. Mycological Progress 7:
285e290.
Doudrick RL, Anderson NA, 1989. Incompatibility factors and mating
competence of 2 Laccaria spp. (Agaricales) associated with black
spruce in Northern Minnesota. Phytopathology 79: 694e700.
Douhan GW, Rizzo DM, 2005. Phylogenetic divergence in a local
population of the ectomycorrhizal fungus Cenococcum geophilum. New Phytologist 166: 263e271.
Douhan GW, Huryn KL, Douhan LI, 2007a. Significant diversity
and potential problems associated with inferring population
structure within the Cenococcum geophilum species complex.
Mycologia 99: 812e819.
Douhan GW, Martin DP, Rizzo DM, 2007b. Using the putative
asexual fungus Cenococcum geophilum as a model to test how
species concepts influence recombination analyses using
sequence data from multiple loci. Current Genetics 52:
191e201.
Dunham SM, Kretzer A, Pfrender E, 2003. Characterization of Pacific
golden chanterelle (Cantharellus formosus) genet size using codominant microsatellite markers. Molecular Ecology 12: 1607e1618.
Dunham MS, O’Dell TE, Molina R, 2006. Spatial analysis of withinpopulation microsatellite variability reveals restricted gene
flow in the Pacific golden chanterelle (Cantharellus formosus).
Mycologia 98: 250e259.
s B, Carlier J,
Dutech C, Enjalbert J, Fournier E, Delmotte F, Barre
Tharreau D, Giraud T, 2007. Challenges in microsatellite isolation in fungi. Fungal Genetics and Biology 44: 933e949.
Egli S, Peter M, Buser C, Stahel W, Ayer F, 2006. Mushroom picking
does not impair future harvests e results of a long-term study
in Switzerland. Biological Conservation 129: 271e276.
Egger KN, 2006. The surprising diversity of ascomycetous mycorrhizas. New Phytologist 170: 421e423.
El Karkouri K, Selosse MA, Mousain D, 2006. Molecular markers
detecting an ectomycorrhizal Suillus collinitus strain on Pinus
halepensis roots suggest successful inoculation and persistence
in Mediterranean nursery and plantation. FEMS Microbiology
and Ecology 55: 146e158.
€ gberg N, Doi S, Kauserud H, 2010.
Engh IB, Carlsen T, Sætre GP, Ho
Two invasive populations of the dry rot fungus Serpula
G. W. Douhan et al.
lacrymans show divergent population genetic structures. Molecular Ecology 19: 706e715.
Excoffier L, Laval G, Schneider S, 1992. Arlequin (version 3.0): an
integrated software package for population genetics data
analysis. Evolutionary Bioinformatics 1: 47e50.
ndez-Toira
n LM, Agueda
Ferna
B, 2007. Fruitbodies of Cenococcum
geophilum. Mycotaxon 100: 109e114.
Fierer N, Liu ZZ, Rodriguez-Hernandez M, Knight R, Henn M,
Hernandez MT, 2008. Short-term temporal variability in airborne bacterial and fungal populations. Applied and Environmental Microbiology 74: 200e207.
Fiore-Donno A, Martin F, 2001. Populations of ectomycorrhizal
Laccaria amethystina and Xerocomus spp. show contrasting
colonization patterns in a mixed forest. New Phytologist 152:
533e542.
Fournier E, Giraud T, 2008. Sympatric genetic differentiation of
a generalist pathogenic fungus, Botrytis cinerea, on two different host plants, grapevine and bramble. Journal of Evolutionary
Biology 21: 122e132.
Frankland JC, 1998. Fungal succession e unraveling the unpredictable. Mycological Research 102: 1e15.
Fraser JA, Hsueh Y-P, Findley KM, Heitman J, 2007. Evolution of
mating-type locus: the basidiomycetes. In: Heitman J,
Kronstad JW, Taylor JW, Casselton LA (eds), Sex in Fungi: Molecular Determination and Evolutionary Implications. ASM Press,
Washington, DC, pp. 19e34.
Fries N, 1985. Intersterility groups in Paxillus involutus. Mycotaxon
24: 403e410.
Fries N, 1987. Somatic incompatibility and field distribution of the
ectomycorrhizal fungus Suillus luteus, Boletaceae. New Phytologist 107: 735e740.
Garbelotto M, Otrosina WJ, Cobb FW, Bruns TD, 1998. The European S and F intersterility groups of Heterobasidion annosum
may represent sympatric protospecies. Canadian Journal of
Botany 76: 397e409.
Gardes M, Wong KKY, Fortin JA, 1990. Interactions between
monokaryotic and dikaryotic isolates of Laccaria bicolor on
roots of Pinus banksiana. Symbiosis 8: 233e250.
Gardes M, Bruns TD, 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest e above and below-ground views. Canadian Journal of Botany 74:
1572e1583.
Geml J, Laursen GA, O’Neill K, Nusbaum HC, Taylor DL, 2006.
Beringian origins and cryptic speciation events in the fly agaric (Amanita muscaria). Molecular Ecology 15: 225e239.
Geml J, Laursen GA, Timling I, McFarland JM, Booth MG,
Lennon N, Nusbaum C, Taylor DL, 2009. Molecular phylogenetic biodiversity assessment of arctic and boreal ectomycorrhizal Lactarius Pers. (Russulales: Basidiomycota) in Alaska,
based on soil and sporocarp DNA. Molecular Ecology 18:
2213e2227.
Genney DR, Anderson IC, Alexander IJ, 2006. Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. New Phytologist 170: 381e390.
Gherbi H, Delaruelle C, Selosse MA, Martin F, 1999. High genetic
diversity in a population of the ectomycorrhizal basidiomycete Laccaria amethystina in a 150-year-old beech forest. Molecular Ecology 8: 2003e2013.
gier G, Le Gac M, de Vienne DM, Hood ME, 2008.
Giraud T, Refre
Speciation in fungi. Fungal Genetics and Biology 45: 791e802.
Glass NL, Fleissner A, 2006. Re-wiring the network: understanding
the mechanism and function of anastomosis in filamentous
€ es U, Fischer R (eds), The Mycota I.
ascomycete fungi. In: Ku
Springer, Berlin.
Gonthier P, Garbelotto M, Varese GC, Nicolotti G, 2001. Relative
abundance and potential dispersal range of intersterility
groups of Heterobasidion annosum in pure and mixed forests.
Canadian Journal of Botany 79: 1057e1065.
Population genetics of EM fungi
Gout L, Kuhn ML, Vincenot L, Bernard-Samain S, Cattolico L,
Barbetti M, Moreno-Rico O, Balesdent M-H, Rouxel T, 2007.
Genome structure impacts molecular evolution at the AvrLm1
avirulence locus of the plant pathogen Leptosphaeria maculans.
Environmental Microbiology 9: 2978e2992.
Grelet GA, David J, Vralstad T, Alexander IJ, Anderson IC, 2010.
New insights into the mycorrhizal Rhizoscyphus ericae aggregate: spatial structure and co-colonization of ectomycorrhizal
and ericoid roots. New Phytologist 188: 210e222.
Guidot A, Lumini E, Debaud JC, Marmeisse R, 1999. The nuclear
ribosomal intergenic DNA as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete
Hebeloma cylindrosporum directly on Pinus roots system. Applied
and Environmental Microbiology 65: 903e909.
Guidot A, Debaud JC, Marmeisse R, 2001. Correspondence between genet diversity and spatial distribution between aboveand below-ground populations of the ectomycorrhizal fungus
Hebeloma cylindrosporum. Molecular Ecology 10: 1121e1131.
re F, Debaud JC, Marmeisse R, 2002.
Guidot A, Gryta H, Gourbie
Forest habitat characteristics affect balance between sexual
reproduction and clonal propagation of the ectomycorrhizal
fungus Hebeloma cylindrosporum. Oikos 99: 25e36.
Guidot A, Debaud JC, Effosse A, Marmeisse R, 2003a. Belowground distribution and persistence of an ectomycorrhizal
fungus. New Phytologist 161: 539e547.
Guidot A, Johannesson H, Dahlberg A, Stenlid J, 2003b. Parental
tracking in the postfire wood decay ascomycete Daldinia loculata using highly variable nuclear gene loci. Molecular Ecology
12: 1717e1730.
Guidot A, Verner MC, Debaud JC, Marmeisse R, 2005. Intraspecific
variation in use of different organic nitrogen sources by the
ectomycorrhizal fungus Hebeloma cylindrosporum. Mycorrhiza
15: 167e177.
Grime JP, 1977. Evidence for the existence of three primary
strategies in plants and its relevance to ecological and evolutionary theory. American Naturalist 111: 1169e1194.
Grubisha LC, Bergemann SE, Bruns TD, 2007. Host islands within
the California Northern Channel Islands create fine-scale genetic structure in two sympatric species of the symbiotic ectomycorrhizal fungus Rhizopogon. Molecular Ecology 16: 1811e1812.
Grubisha LC, Kretzer A, Bruns TD, 2005. Isolation and characterization of microsatellite loci from the truffle-like ectomycorrhizal fungi Rhizopogon occidentalis and Rhizopogon vulgari.
Molecular Ecology Notes 5: 608e610.
€ nwald NJ, Goss EM, Press CM, 2008. Phytophtora ramorum:
Gru
a pathogen with a remarkably wide host range causing sudden oak death on oaks and ramorum blight on woody ornamentals. Molecular Plant Pathology 9: 729e740.
Gryta H, Carriconde F, Charcosset JY, Jargeat P, Gardes M, 2006.
Population dynamics of the ectomycorrhizal fungal species
Tricholoma populinum and Tricholoma scalpturatum associated
with black poplar under differing environmental conditions.
Environmental Microbiology 8: 773e786.
Gryta H, Debaud JC, Effosse A, Gay G, Marmeisse R, 1997. Finescale structure of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune ecosystem forest. Molecular
Ecology 6: 353e364.
Gryta H, Debaud JC, Marmeisse R, 2000. Population dynamics of
the symbiotic mushroom Hebeloma cylindrosporum: mycelial
persistence and inbreeding. Heredity 84: 294e302.
Halling RE, Osmundson TW, Neves MA, 2008. Pacific boletes: implication for biogeographic relationships. Mycological Research
112: 437e447.
Harper JL, 1977. Population Biology of Plant. Academic Press,
London.
Hedges SB, Parker PH, Sibley CG, Kumar S, 1996. Continental
breakup and the ordinal diversification of birds and mammals.
Nature 381: 226e229.
593
Hedh J, Samson P, Erland S, Tunlid A, 2008. Multiple gene genealogies and species recognition in the ectomycorrhizal fungus
Paxillus involutus. Mycological Research 112: 965e975.
Hibbett DS, 2001. Shiitake mushrooms and molecular clocks:
historical biogeography of Lentinula. Journal of Biogeography 28:
231e241.
Hibbett DS, Matheny PB, 2009. The relative ages of ectomycorrhizal mushrooms and their plant hosts estimated using Bayesian relaxed molecular clock analyses. BMC
Biology 7: 13.
Hirose D, Kikuchi J, Kanzaki M, Futai K, 2004. Genet distribution of
sporocarps and ectomycorrhizas of Suillus pictus in a Japanese
white pine plantation. New Phytologist 164: 527e541.
Hirose D, Tokumasu S, 2007. Microsatellite loci from the ectomycorrhizal basidiomycete Suillus pictus associated with the genus
Pinus subgenus strobus. Molecular Ecology Notes 7: 854e856.
Hitchcock CJ, Chambers SM, Anderson IC, Cairney JWG, 2003.
Development of markers for simple sequence repeat-rich regions that discriminate between Pisolithus albus and P. microcarpus. Mycological Research 107: 699e706.
Hitchcock CJ, Chambers SM, Cairney JWG, 2006. Development of
polymorphic simple sequence repeat markers for Pisolithus
macrocarpus. Molecular Ecology Notes 6: 443e445.
Hitchcock CJ, Chambers SM, Cairney JWG, 2010. Genetic population structure of the ectomycorrhizal fungus Pisolithus macrocarpus suggests high gene flow in south-eastern Australia.
Mycorrhiza. doi:10.1007/s00572-010-0317-3.
Ho I, Trappe JM, 1987. Enzymes and growth substances of Rhizopogon species in relation to mycorrhizal hosts and infrageneric
taxonomy. Mycologia 79: 553e558.
€ gberg N, Guidot A, Jonnson M, Dahlberg A, 2009. Microsatellite
Ho
markers for the ectomycorrhizal basidiomycete Lactarius
mammosus. Molecular Ecology Resources 9: 1008e1010.
J, 2008. Tracking mycorrhizas and extraHortal S, Pera J, Parlade
radical mycelium of the edible fungus Lactarius deliciosius under field competition with Rhizopogon spp. Mycorrhiza 18:
69e77.
J, 2009. Field persistence of the edible
Hortal S, Pera J, Parlade
ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics.
Mycorrhiza 19: 167e177.
e M, Chybicki I, Trojankiewicz
Hortal S, Trocha LK, Murat C, Bue
M, Burczyk J, Martin F. Above and below-ground population
structure of the ectomycorrhizal fungus Laccaria amethystina in
a mature beech forest, in preparation.
Horton TR, 2002. Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale. Plant and Soil
244: 29e39.
Horton TR, Bruns TD, 2001. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology
10: 1855e1871.
Hosaka K, Castellano MA, Spatafora JW, 2008. Biogeography of
Hysterangiales (Phallomycetidae, Basidiomycota). Mycological Research 112: 448e462.
Hughes KW, Petersen RH, Mata JL, Psurteva NV, Kovalenko AE,
Morozova OV, Lickey EB, Cifuentes Blanco J, Lewis DP,
Nagasawa E, Halling RE, Takehashi S, Aime MC, Bau T,
Henkel T, 2007. Megacollybia (Agaricales). Reports of the Tottori
Mycological Institute 45: 1e57.
Hughes KW, Petersen RH, Lickey EB, 2009. Using heterozygosity to
estimate a percentage DNA sequence similarity for environmental species’ delimitation across basidiomycete fungi. New
Phytologist 182: 795e798.
Hynes MM, Smith ME, Zasoski RJ, Bledsoe CS, 2010. A molecular
survey of ectomycorrhizal hyphae in a California QuercusePinus woodland. Mycorrhiza 20: 265e274.
Ishida TA, Nara K, Tanaka M, Kinoshita A, Hogetsu T, 2008. Germination and infectivity of ectomycorrhizal fungal spores in
594
relation to their ecological traits during primary succession.
New Phytologist 180: 491e500.
Jacobson KM, Miller OK, Turner BJ, 1993. Randomly amplified
polymorphic DNA markers are superior to somatic incompatibility tests for discriminating genotypes in natural populations of the ectomycorrhizal fungus Suillus granulatus.
Proceedings of the National Academy of Sciences of the United States
of America 90: 9159e9163.
Jaenike J, 1991. Mass extinction of European fungi. Trends in Ecology & Evolution 6: 174e175.
James TY, Porter D, Hamrick JL, Vilgalys R, 1999. Evidence for
limited intercontinental gene flow in the cosmopolitan
mushroom, Schizophyllum commune. Evolution 53: 1665e1677.
James TY, Stenlid J, Olson A, Johannesson H, 2008. Evolutionary
significance of imbalanced nuclear ratios within heterokaryons of the basidiomycete fungus Heterobasidion parviporum. Evolution 62: 2279e2296.
James TY, Vilgalys R, 2001. Abundance and diversity of Schizophyllum commune spore clouds in the Caribbean detected by
selective sampling. Molecular Ecology 10: 471e479.
A, Khasa DP, 2006. Simple sequence
Jany JL, Bousquet J, Gagne
repeat (SSR) markers in the ectomycorrhizal fungus Laccaria
bicolor for environmental monitoring of introduced strains and
molecular ecology applications. Mycological Research 110:
51e59.
Jany JL, Garbaye L, Martin F, 2002. Cenococcum geophilum populations show a high degree of genetic diversity in beech forests.
New Phytologist 154: 651e659.
Jany JL, Guidot A, Jonnson M, Dahlberg A, Bousquet J,
Khasa DP, 2003. Microsatellite markers for Hebeloma species developed from expressed sequence tags in the ectomycorrhizal fungus Hebeloma cylindrosporum. Molecular
Ecology Notes 3: 659e661.
Jargeat P, Martos F, Carriconde F, Gryta H, Moreau PA, Gardes M,
2010. Phylogenetic species delimitation in ectomycorrhizal
fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota). Molecular Ecology
19: 5216e5230.
Johannesson H, Stenlid J, 2004. Nuclear reassortment between
vegetative mycelia in natural populations of the basidiomycete Heterobasidion annosum. Fungal Genetics and Biology 41:
563e570.
Jourand P, Ducousso M, Loulergue-Majorel C, Hannibal L,
Santoni S, Prin Y, Lebrun M, 2010. Ultramafic soils from New
Caledonia structure Pisolithus albus in ecotypes. FEMS Microbiology Ecology 72: 238e249.
Hartl DL, Clark A, 2007. Principles of Population Genetics, 4th Edition.
Sinauer Associates, Sundreland.
Kanchanaprayudh J, Lian C, Zhou Z, Hogetsu T, Sihanonth P, 2002.
Polymorphic microsatellite markers of a Pisolithus sp. from
a Eucalyptus plantation. Molecular Ecology Notes 2: 263e264.
Kauserud H, Schumacher T, 2002. Population structure of the
endangered wood decay fungus Phellinus nigrolimitatus (Basidiomycota). Canadian Journal of Botany 80: 597e606.
Kauserud H, Schumacher T, 2003a. Genetic structure of Fennoscandian populations of the threatened wood-decay fungus
Fomitopsis rosea (Basidiomycota). Mycological Research 107:
155e163.
Kauserud H, Schumacher T, 2003b. Regional and local population
structure of the pioneer wood-decay fungus Trichaptum abietinum. Mycologia 95: 416e425.
Kauserud H, Svegarden IB, Saetre GP, Knudsen H, Stensrud O,
Schmidt O, Doi S, Sugiyama T, Hogberg N, 2007. Asian origin
and rapid global spread of the destructive dry rot fungus Serpula lacrymans. Molecular Ecology 16: 3350e3360.
Kay E, Vilgalys R, 1992. Spatial distribution and genetic relationships among individuals in a natural population of the oyster
mushroom Pleurotus ostreatus. Mycologia 84: 173e182.
G. W. Douhan et al.
Keller G, 1992. Isozymes in isolates of Suillus species from Pinus
cembra L. New Phytologist 120: 351e358.
Kerrigan RW, Carvalho DB, Horgen PA, Anderson JB, 1998. The
indigenous coastal Californian population of the mushroom
Agaricus bisporus, a cultivated species, may be at risk of extinction. Molecular Ecology 7: 35e45.
Kohn LM, 2005. Mechanisms of fungal speciation. Annual Review of
Phytopathology 43: 279e308.
Kranabetter JM, 1999. The effect of refuge trees on a paper birch
ectomycorrhiza community. Canadian Journal of Botany 77:
1523e1528.
Kretzer AM, Dunham S, Molina R, Spatafora JW, 2004. Microsatellite markers reveal the below ground distribution of genets
in two species in Rhizopogon forming tuberculate ectomycorrhizas on Douglas fir. New Phytologist 161: 313e320.
Kretzer AM, Dunham S, Molina R, Spatafora JW, 2005. Patterns of
vegetative growth and gene flow in Rhizopogon vinicolor and R.
vesiculosus (Boletales, Basidiomycota). Molecular Ecology 14:
2259e2268.
Kretzer A, Li Y, Szaro T, Bruns TD, 1996. Internal transcribed
spacer sequences from 38 recognized species of Suillus sensu
lato: phylogenetic and taxonomic implications. Mycologia 88:
776e785.
Kretzer AM, Luoma DL, Molina R, Spatafora JW, 2003. Taxonomy of
the Rhizopogon vinicolor species complex based on analysis of ITS
sequences and microsatellite loci. Mycologia 95: 480e487.
Kretzer AM, Molina R, Spatafora JW, 2000. Microsatellite markers
for the ectomycorrhizal basidiomycete Rhizopogon vinicolor.
Molecular Ecology 9: 1190e1191.
re J, Noe
€l T, 1992. Mating type switching in the tetrapolar
Labare
Basidiomycete Agrocybe aegerita. Genetics 131: 307e319.
J, Zhang X, Yin T, Schmutz J, Grimwood J, Martin F, Tuskan GA,
Labbe
Le Tacon F, 2008. A genetic linkage map for the ectomycorrhizal
fungus Laccaria bicolor and its alignment to the whole-genome
sequence assemblies. New Phytologist 180: 316e328.
Le Tacon F, Alvarez IF, Bouchard D, Henrion B, Jackson RM, Luff S,
JI, Pera J, Stenstro
€ m E, Villeneuve N, Walker C, 1992.
Parlade
Variations in field response of forest trees to nursery ectomycorrhizal inoculation in Europe. In: Read DJ, et al. (eds),
Mycorrhizas in Ecosystems. CAB International, Wallingford, UK.
Li DW, 2005. Release and dispersal of basidiospores from Amanita
muscaria var. alba and their infiltration into a residence. Mycological Research 109: 1235e1242.
Lian C, Narimatsu M, Nara K, Hogetsu T, 2006. Tricholoma matsutake in a natural Pinus densiflora forest: correspondence between above- and below-ground genets, association with
multiple host trees and alteration of existing ectomycorrhizal
communities. New Phytologist 171: 825e836.
Liang Y, Guo LD, Ma KP, 2005. Population genetic structure of an
ectomycorrhizal fungus Amanita manginiana in a subtropical
forest over two years. Mycorrhiza 15: 137e142.
Liti G, Carter DM, Moses AM, Warringer J, Parts L, James SA,
Daveys RP, Roberts IN, Burt A, Koufopanou V, Tsai IJ,
Bergman CM, Densasson D, O’Kellys MJT, van
Oudenaardens A, Barton DB, Bailes E, Nguyen Ba AN, Jones M,
Quai M, Goodhead I, Sims S, Smith F, Blomberg A, Durbin R,
Louis EJ, 2010. Population genomics of domestic and wild
yeasts. Nature 458: 337e341.
LoBuglio KF, Taylor JW, 2002. Recombination and genetic differentiation in the mycorrhizal fungus Cenococcum geophilum Fr.
Mycologia 94: 772e780.
Marmeisse R, Guidot A, Gay G, Lambilliotte R, Sentenac H,
Combier JP, Melayah D, Fraissinet-Tachet L, Debaud JC, 2004.
Hebeloma cylindrosporum e a model species to study ectomycorrhizal symbioses from gene to ecosystem. New Phytologist
163: 481e498.
Martin F, Selosse MA, 2008. The Laccaria genome: a symbiont
blueprint decoded. New Phytologist 180: 296e310.
Population genetics of EM fungi
Martin F, Diez J, Dell B, Delaruelle C, 2002. Phylogeography of the
ectomycorrhizal Pisolithus species as inferred from nuclear
ribosomal DNA ITS sequences. New Phytologist 153: 345e357.
Martin F, Selosse MA, Le Tacon F, 1999. The nuclear rDNA intergenic spacer of the ectomycorrhizal basidiomycete Laccaria
bicolor: structural analysis and allelic polymorphism. Microbiology-SGM 145: 1605e1611.
Martiny JBH, Bohannan BJM, Brown JH, Colwell RK, Fuhrman JA,
Green JL, Horner-Devine MC, Kane M, Adams Krumins J,
Kuske CR, Morin PJ, Naeem S, Øvre
as L, Reysenbach AL,
Smith VH, Staley JT, 2006. Microbial biogeography: putting microorganisms on the map. Nature Reviews Microbiology 4: 102e112.
Matheny PB, Aime MC, Bougher NL, Buyck B, Desjardin DE, Horak E,
Kropp BR, Lodge DJ, Soytong K, Trappe JM, Hibbett DS, 2009. Out
of the paleotropics? Historical biogeography and diversification
of the cosmopolitan ectomycorrhizal mushroom family Inocybaceae. Journal of Biogeography 36: 577e592.
Matheny PB, Bougher ML, 2006. The new genus Auritella from
Africa and Australia (Inocybaceae, Agaricales): molecular
systematics, taxonomy and historical biogeography. Mycological Progress 5: 2e17.
May G, Shaw F, Badrane H, Vekemans X, 1999. The signature of
balancing selection: fungal mating compatibility gene evolution. Proceedings of the National Academy of Sciences of the United
States of America 96: 9172e9179.
Mikola P, 1970. Mycorrhizal inoculation in afforestation. International Review of Forestry Research 3: 123e196.
Milgroom MG, 1996. Recombination and the multilocus structure
of fungal populations. Annual Review of Phytopathology 34:
457e477.
Milgroom MG, Peever TL, 2003. Population biology of plant pathogens: the synthesis of plant disease epidemiology and population genetics. Plant Disease 87: 608e617.
Moore D, Novak Frazer L, 2002. Essential Fungal Genetics. Springer
Verlag, New York.
Mueller GM, Schmit JP, Leacock PR, Buyck B, Cifuentes J,
Desjardin DE, Halling RE, Hjortstam K, Iturriaga T, Larsson KH,
Lodge DJ, May TW, Minter D, Rajchenberg M, Redhead SA,
Ryvarden L, Trappe JM, Watling R, Wu QW, 2007. Global diversity and distribution of macrofungi. Biodiversity and Conservation 16: 37e48.
Muller LAH, Lambaerts M, Vangronsveld J, Colpaert JV, 2004.
AFLP-based assessment of the effects of environmental heavy
metal pollution on the genetic structure of pioneer populations of Suillus luteus. New Phytologist 164: 297e303.
Muller LAH, Vangronsveld J, Colpaert JV, 2007. Genetic structure
of Suillus luteus populations in heavy metal polluted and
nonpolluted habitats. Molecular Ecology 16: 4728e4737.
C, Chevalier G, Bonfante P,
Murat C, Dıez J, Luis P, Delaruelle C, Dupre
Martin F, 2004. Polymorphism at the ribosomal DNA ITS and its
relation to postglacial re-colonization routes of the Perigord
truffle Tuber melanosporum. New Phytologist 164: 401e411.
Murat C, Zampieri E, Vizzini A, Bonfante P, 2008. Is the Perigord
black truffle threatened by an invasive species? We dreaded it
and it has happened!. New Phytologist 178: 699e702.
J, Morin E, TisMurat C, Riccioni C, Belfiori B, Cichocki N, Labbe
serant E, Rubini A, Paolocci F, Martin F. Distribution of microsatellites in the Perigord black truffle genome and
identification of new molecular markers. Fungal Genetics and
Biology 48: 592e601.
Myers AA, Giller PS, 1988. Analytical Biogeography. An integrated
Approach to the Study of Animal and Plant Distributions. Chapman
& Hall, London.
Nara K, 2008. Spores of ectomycorrhizal fungi: ecological strategies for germination and dormancy. New Phytologist 181:
245e248.
J, Kohler A, Le Tacon F, Martin F,
Niculita-Hirzel H, Labbe
€ es U, 2008. Gene organization of the mating type
Sanders IR, Ku
595
regions in the ectomycorrhizal fungus Laccaria bicolor reveals
distinct evolution between the two mating type loci. New
Phytologist 180: 329e342.
Nieuwenhuis BPS, Debets AJM, Aanen DK, 2010. Sexual selection
in mushroom-forming basidiomycetes. Proceedings of the Royal
Society B. doi:10.1098/rspb.2010.1110.
Nuytinck J, Verbeken A, 2007. Species delimitation and phylogenetic relationships in Lactarius section Deliciosi in Europe.
Mycological Research 111: 1285e1297.
€ gberg O, K
Nylund JE, Dahlberg A, Ho
aren O, Grip K, Jonsson L,
1995. Methods for studying species composition of mycorrhizal fungal communities in ecological studies and environmental monitoring. In: Stocchi V, et al. (eds), Biotechnology of
Ectomycorrhizae. Plenum Press, New York.
Paolocci F, Rubini A, Riccioni C, Arcioni S, 2006. Reevaluation of
the life cycle of Tuber magnatum. Applied and Environmental
Microbiology 72: 2390e2393.
Peay KG, Bruns TD, Kennedy PG, Bergemann SM, Garbelotto M,
2007. A strong speciesearea relationship for eukaryotic soil
microbes: island size matters for ectomycorrhizal fungi. Ecology Letters 10: 470e480.
Pringle A, Adams RI, Cross HB, Bruns TD, 2009. The ectomycorrhizal fungus Amanita phalloides was introduced and is expanding its range on the west coast of North America.
Molecular Ecology 18: 817e833.
Ramette A, Tiedje JM, 2007. Biogeography: an emerging cornerstone for understanding prokaryotic diversity, ecology, and
evolution. Microbial Ecology 53: 197e207.
Redecker D, Szaro TM, Bowman RJ, Bruns TD, 2001. Small genets
of Lactarius xanthogalactus, Russula cremoricolor and Amanita
francheti in late-stage ectomycorrhizal successions. Molecular
Ecology 10: 1025e1034.
Riccioni C, Belfiori B, Rubini A, Passeri V, Arcioni S, Paolocci F,
2008. Tuber melanosporum outcrosses: analysis of the genetic
diversity within and among its natural populations under this
new scenario. New Phytologist 180: 466e478.
Richard F, Millot S, Gardes M, Selosse MA, 2005. Diversity and
specificity of ectomycorrhizal fungi retrieved from an oldgrowth Mediterranean forest dominated by Quercus ilex L. New
Phytologist 166: 1011e1023.
Richard F, Moreau PA, Selosse MA, Gardes M, 2004. Diversity and
fruiting patterns of ectomycorrhizal and saprobic fungi in an
old-growth Mediterranean forest dominated by Quercus ilex L.
Canadian Journal of Botany 82: 1711e1729.
Riddle BR, 1996. The molecular phylogeographic bridge between
deep and shallow history in continental biotas. Trends in Ecology & Evolution 11: 207e211.
Rinaldi AC, Comadini O, Kuyper TW, 2008. Ectomycorrhizal fungal diversity: separating the wheat from the chaff. Fungal Diversity 33: 1e45.
Rodrigues KF, Petrini O, Leuchtmann A, 1995. Variability among
isolates of Xylaria cubensis as determined by isozyme analysis
and somatic incompatibility tests. Mycologia 87: 592e596.
Roy M, Dubois MP, Proffit M, Vincenot L, Desmarais E, Selosse MA,
2008. Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont. Molecular Ecology 17: 2825e2838.
Rubini A, Topini F, Riccioni C, Paolocci F, Arcioni S, 2004. Isolation
and characterization of polymorphic microsatellite loci in
white truffle (Tuber magnatum). Molecular Ecology Notes 4:
116e118.
Rubini A, Paolocci F, Riccioni C, Vendramin GG, Arcioni S, 2005.
Genetic and phylogeographic structures of the symbiotic
fungus Tuber magnatum. Applied and Environmental Microbiology
71: 6584e6589.
Rubini A, Belfiori B, Riccioni C, Arcioni, Martin F, Paolocci F, 2011.
Tuber melanosporum: mating type distribution in a natural
plantation and dynamics of strains of different mating types
596
on the roots of nursery-inoculated host plants. New Phytologist
183: 723e735.
Sanon KB, Ba AM, Delaruelle C, Duponnois R, Martin F, 2009.
Morphological and molecular analyses in Scleroderma species
associated with some Caesalpinioid legumes, Dipterocarpaceae and Phyllanthaceae trees in southern Burkina Faso. Mycorrhiza 19: 571e584.
Sato H, Yumoto T, Murakami N, 2007. Cryptic species and host
specificity in the ectomycorrhizal genus Strobilomyces (Strobilomycetaceae). American Journal of Botany 94: 1630e1641.
Sato H, Murakami N, 2008. Reproductive isolation among cryptic
species in the ectomycorrhizal genus Strobilomyces: population-level CAPS marker-based genetic analysis. Molecular
Phylogenetics and Evolution 48: 326e334.
Saville BH, Yoell H, Anderson JB, 1996. Genetic exchange and
recombination in populations of the root-infecting fungus
Armillaria gallica. Molecular Ecology 5: 485e497.
Sawyer NA, Chambers SM, Cairney JWG, 1999. Molecular investigation of genet distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests.
New Phytologist 142: 561e568.
Sawyer NA, Chambers SM, Cairney JWG, 2001. Distribution and
persistence of Amanita muscaria genotypes in Australian Pinus
radiata plantations. Mycological Research 105: 966e970.
Schneider S, Roessli D, Excoffier L, 2000. Arlequin: a Software for
Population Genetics Data Analysis. User Manual Ver 2.000. Genetics and Biometry Lab., Dept. of Anthropology, University of
Geneva, Geneva.
es re
centes dans l’e
tude des
Selosse MA, 2001a. Avance
s et des populations ectomycorhiziennes. Lejeucommunaute
nia 165: 1e108.
Selosse MA, 2001b. Adding pieces to fungal mosaics. New Phytologist 149: 159e162.
Selosse MA, 2003. Founder effect in a young Leccinum duriusculum
(Schultzer) Singer population. Mycorrhiza 13: 143e149.
Selosse MA, Bouchard D, Martin F, Le Tacon F, 2000. Effect of
Laccaria bicolor strains inoculated on Douglas fir (Pseudotsuga
menziesii) several years after nursery inoculation. Canadian
Journal of Forest Research e Revue Canadienne de Recherche
Forestiere 30: 360e371.
Selosse MA, Costa G, Di Battista C, Le Tacon F, Martin F, 1996.
Meiotic segregation and recombination of the intergenic
spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor. Current Genetics 30: 332e337.
Selosse MA, Faccio A, Scappaticci G, Bonfante P, 2004. Chlorophyllous and achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with
ectomycorrhizal septomycetes, including truffles. Microbial
Ecology 47: 416e426.
Selosse MA, Jacquot D, Bouchard D, Martin F, Le Tacon F, 1997.
Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete
Laccaria bicolor in French plantations. Molecular Ecology 7:
561e574.
Selosse MA, Le Tacon F, 1998. The land flora: a phototroph-fungus
partnership? Trends in Ecology and Evolution 13: 15e20.
Selosse MA, Martin F, Bouchard D, Le Tacon F, 1999. Structure
and dynamics of experimentally introduced and naturally
occurring Laccaria sp. discrete genotypes in a Douglas fir
plantation. Applied and Environmental Microbiology 65:
2006e2014.
Selosse MA, Martin F, Le Tacon F, 1998. Survival of an introduced
ectomycorrhizal Laccaria bicolor strain in an European forest
plantation confirmed by mitochondrial analysis. New Phytologist 140: 753e761.
Selosse MA, Martin F, Le Tacon F, 2001. Intraspecific variation
in fruiting phenology in an ectomycorrhizal Laccaria population under Douglas fir. Mycological Research 105: 524e531.
G. W. Douhan et al.
Selosse MA, Richard F, He XH, Simard SW, 2006. Micorrhizal
netwoks: des liaisons dangereuses? Trends in Ecology & Evolution 21: 621e628.
Selosse MA, Weiß M, Jany JL, Tillier A, 2002. Communities and
populations of sebacinoid basidiomycetes associated with the
achlorophyllous orchid Neottia nidus-avis (L.) L.C.M. Rich. and
neighbouring tree ectomycorrhizae. Molecular Ecology 11:
1831e1844.
Selosse MA, Dubois M-P, Alavrez N, 2009. Do Sebacinales commonly associate with plant roots as endophytes? Mycological
Research 113: 1062e1069.
Sen R, 1990. Intraspecific variation in 2 species of Suillus from scots
pine (Pinus sylvestris L.) forests based on somatic incompatibility
and isozyme analyses. New Phytologist 114: 607e616.
Shen Q, Geiser DM, Royse DJ, 2002. Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition
separating eastern North American and Asian isolates. Mycologia 94: 472e482.
Shendure J, Ji HL, 2008. Next-generation DNA sequencing. Nature
Biotechnology 26: 1135e1145.
Simard SW, Perry DA, Smith JE, Molina R, 1997. Effects of soil
trenching on occurrence of ectomycorrhizas on Pseudotsuga
menziesii seedlings grown in mature forests of Betula papyrifera
and Pseudotsuga menziesii. New Phytologist 136: 327e340.
Smith SE, Read DJ, 2008. Mycorrhizal Symbiosis, 3rd edn. Academic
Press, London.
Straatsma G, Ayer F, Egli S, 2001. Species richness, abundance,
and phenology of fungal fruit bodies over 21 years in a Swiss
forest plot. Mycological Research 105: 515e523.
Taylor AFS, 2002. Fungal diversity in ectomycorrhizal communities:
sampling effort and species detection. Plant and Soil 144: 19e28.
Taylor DL, Bruns TD, 1997. Population, habitat and genetic correlates of mycorrhizal specialization in the ‘cheating’ orchids
Corallorhiza maculata and C. mertensiana. Molecular Ecology 8:
1719e1732.
Taylor JW, Berbee ML, 2006. Dating divergences in the fungal tree
of life: review and new analyses. Mycologia 98: 838e849.
Taylor JW, Geiser DM, Burt A, Koufopanou V, 1999. The evolutionary biology and population genetics underlying fungal
strain typing. Clinical Microbiology Reviews 12: 126e146.
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS,
Fisher MC, 2000. Phylogenetic species recognition and species
concepts in fungi. Fungal Genetics and Biology 31: 21e32.
Taylor JW, Turner E, Towsend JP, Dettman JR, Jacobson D,
2006. Eukaryotic microbes species recognition and the geographic limits of species: examples from the kingdom
Fungi. Philosophical Transactions of the Royal Society B 361:
1947e1963.
Tedersoo L, Perry BA, Kjøller R, 2006. Molecular and morphological diversity of pezizalean ectomycorrhiza. New Phytologist
170: 581e596.
Tedersoo L, May TW, Smith ME, 2010. Ectomycorrhizal lifestyle in
fungi: global diversity, distribution, and evolution of phylogenetic lineages. Mycorrhiza 20: 217e263.
Urban A, Neuner-Plattner I, Krisai-Greilhuber I, Haselwandter K,
2004. Molecular studies on terricolous microfungi reveal novel
anamorphs of two Tuber species. Mycological Research 108:
749e758.
Urbanelli S, Della Rosa V, Fanelli C, Fabbri AA, Reverberi M, 2003.
Genetic diversity and population structure of the Italian fungi
belonging to the taxa Pleurotus eryngii (DC.: Fr.) Quel and
P. ferulae (DC.: Fr.) Quel. Heredity 90: 253e259.
van der Linde S, Alexander I, Anderson I, 2008. A PCR-based
method for detecting the mycelia of stipitate hydnoid fungi in
soil. Journal of Microbiological Methods 75: 40e46.
van der Linde S, Alexander IJ, Anderson IC, 2009. Spatial distribution of sporocarps of stipitate hydnoid fungi and their below-ground mycelium. FEMS Microbiology Ecology 69: 344e352.
Population genetics of EM fungi
Van Putten WF, Biere A, Van Damme JMM, 2003. Intraspecific
competition and mating between fungal strains of the anther
smut Microbotryum violaceum from the host plants Silene latifolia and S. dioica. Evolution 57: 766e776.
Vellinga EC, Wolfe BE, Pringle A, 2009. Global patterns of ectomycorrhizal introductions. New Phytologist 181: 960e973.
~ ljalg U,
Vincenot L, Tedersoo L, Richard F, Horcine H, Ko
Selosse MA, 2008. Fungal associates of Pyrola rotundifolia,
a mixotrophic Ericaceae, in two Estonian boreal forests. Mycorrhiza 19: 15e25.
J, Dubois MP, Tedersoo L, Nara K,
Vincenot L, Sthultz C, Labbe
Martin F, Selosse MA. Extensive gene flow over Europe
and possible speciation over Eurasia in the ectomycorrhizalbasidiomycete Laccaria amethystina, submitted for publication.
Visser S, 1995. Ectomycorrhizal fungal succession in jack pine
stands following wildfire. New Phytologist 129: 389e401.
Wadud MA, Lian CL, Nara K, Ishida TA, Hogetsu T, 2006a. Development of microsatellite markers from an ectomycorrhizal
fungus, Laccaria amethystina, by a dual-suppression-PCR technique. Molecular Ecology Notes 6: 130e132.
Wadud MA, Lian CL, Nara K, Ishida TA, Hogetsu T, 2006b.
Isolation and characterization of five microsatellite loci in an
ectomycorrhizal fungus Laccaria laccata. Molecular Ecology Notes
6: 700e702.
Wadud MA, 2007. Reproduction ecology of pioneer ectomycorrhizal fungi, Laccaria amethystina and L. laccata, in the volcanic
desert on Mount Fuji. Ph.D. thesis, the University of Tokyo,
Tokyo, Japan.
Weber J, Diez J, Selosse MA, Tagu D, Le Tacon F, 2002. SCAR
markers to detect mycorrhizas of an American Laccaria bicolor
strain inoculated in European Douglas-fir plantations. Mycorrhiza 12: 19e27.
Worrall JJ, 1997. Somatic incompatibility in basidiomycetes. Mycologia 89: 24e36.
Wu B, Nara K, Hogetsu T, 2005. Genetic structure of Cenococcum
geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers. New
Phytologist 165: 285e293.
Wu QX, Mueller GM, 1997. Biogeographic relationships between
the macrofungi of temperate eastern Asia and eastern North
America. Canadian Journal of Botany 75: 2108e2116.
Wu QX, Mueller GM, Lutzoni FM, Huang YQ, Guo SY, 2000. Phylogenetic and biogeographic relationships of Eastern Asian
and Eastern North American disjoint Suillus species (fungi) as
inferred from nuclear ribosomal RNA ITS sequences. Molecular
Phylogenetics and Evolution 17: 37e47.
Xu J, 2006. Fundamentals of fungal molecular population genetic
analyses. Current Issues in Molecular Biology 8: 75e90.
Xu J, Kerrigan RW, Callac P, Horgen PA, Anderson JB, 1997.
Genetic structure of natural populations of Agaricus bisporus,
the commercial button mushroom. Journal of Heredity 88:
482e488.
Xu J, Sha T, Li YC, Zhao ZW, Yang ZL, 2008. Recombination and
genetic differentiation among natural populations of the ectomycorrhizal mushroom Tricholoma matsutake from Southwestern China. Molecular Ecology 17: 1238e1247.
Yun W, Hall IR, 2004. Edible ectomycorrhizal mushrooms: challenges and achievements. Canadian Journal of Botany 82:
1063e1073.
Zervakis GI, Venturella G, Papadopoulou K, 2001. Genetic
polymorphism and taxonomic infrastructure of the Pleurotus
eryngii species-complex as determined by RAPD analysis, isozyme profiles and ecomorphological characters. MicrobiologySGM 147: 3183e3194.
Zhou Z, Miwa M, Hogetsu T, 1999. Analysis of genetic structure of
a Suillus grevillei population in a Larix kaempferi stand by
polymorphism of inter-simple sequence repeat (ISSR). New
Phytologist 144: 55e63.
597
Zhou Z, Miwa M, Hogetsu T, 2000. Genet distribution of ectomycorrhizal fungus Suillus grevillei populations in two Larix
kaempferi stands over two years. Journal of Plant Research 113:
365e374.
Zhou Z, Miwa M, Matsuda Y, Hogetsu T, 2001. Spatial distribution
of the subterranean mycelia and ectomycorrhizae of Suillus
grevillei. Journal of Plant Research 114: 179e185.
Zinger L, Gury J, Giraud F, Krivobok S, Gielly L, Taberlet P,
Geremia RA, 2007. Improvements of polymerase chain reaction and capillary electrophoresis single-strand conformation
polymorphism methods in microbial ecology: toward a highthroughput method for microbial diversity studies in soil. Microbial Ecology 54: 203e216.
Glossary. Major genetic markers for EM population structure
analysis (see also Xu 2006).
Amplified fragment length polymorphism (AFLP): Multilocus, dominant fingerprint, where polymorphism arises from length variation and enzymatic digestion of non-characterised, amplified
DNA fragments.
Intergenic spacer (IGS)Single-locus, co-dominant polymorphism,
consisting of variations in the length of nuclear DNA fragments
between the 18S and 5S rDNA (IGS1) or the 5S and 25S rDNA (IGS2).
Internal transcribed spacer (ITS): Single-locus, co-dominant polymorphism, consisting of variations in the length, sequence and
enzymatic digestion of nuclear DNA fragments located between
the sequences of 18S and 25S rDNA (thus encompassing the 5.8S
rDNA gene), commonly used as a “barcode” for identification and
phylogeny of fungal species.
Isozymes: Single- to multilocus co-dominant polymorphism, consisting of variation in the electrophoretic properties of a target
enzyme.
Random amplification of polymorphic DNA (RAPD): Multilocus, dominant fingerprint obtained after random amplification of noncharacterised DNA fragments using short primers, often very dependent on PCR conditions.
Restriction fragment length polymorphism (RFLP): Single-locus, codominant polymorphism, based on length variation of amplified
(sometimes then enzyme-digested) target DNA fragments.
Sexual incompatibility: Single- or two-gene system of sexual incompatibility (¼mating types), blocking the karyogamy and further sexual cycle between two monokaryotic mycelia in the case of
allelic similarity for the corresponding loci (homogenic
incompatibility).
Microsatellite: Single-locus, co-dominant polymorphism issuing
from variations in repeat numbers of short di-, tri- or quadrinucleotide repeats. Often assumed to be neutral, they may, however,
occur in coding regions. Also called simple sequence repeats
(SSRs). Confusion with ISSRs (inter-simple sequence repeats),
a kind of RAPD using repeated primers, should be avoided.
Single nucleotide polymorphism (SNP): Single-locus, co-dominant
polymorphism point mutation of the DNA, offering a neutral or
selected marker (depending on the position).
Somatic incompatibility (SI): Single- or (often) multigene system of
vegetative incompatibility, physically blocking the mycelial fusion
(plasmogamy) in the case of allelic dissimilarity for at least one
locus (heterogenic incompatibility); creates a rejection zone that
can be more or less easily observed in vitro.