Fungal Diversity (2011) 51:279–296
DOI 10.1007/s13225-011-0136-7
Major clades in tropical Agaricus
Ruilin Zhao & Samantha Karunarathna & Olivier Raspé & Luis A. Parra &
Jacques Guinberteau & Magalie Moinard & André De Kesel & Gérard Barroso &
Régis Courtecuisse & Kevin D. Hyde & Atsu K. Guelly & Dennis E. Desjardin &
Philippe Callac
Received: 17 February 2011 / Accepted: 3 September 2011 / Published online: 18 September 2011
# Kevin D. Hyde 2011
Abstract Agaricus (Basidiomycota) is a genus of saprobic
fungi that includes edible cultivated species such as
Agaricus bisporus, the button mushroom. There has been
considerable ecological, nutritional and medicinal interest
in the genus, yet the extent of its diversity remains poorly
known, particularly in subtropical and tropical areas.
Classification of tropical species has for a large part
followed the classification of temperate species. The
objective of our study was to examine to what extent this
system of classification is appropriate for tropical Agaricus
species. Species from temperate sections were therefore
compared to the major clades of tropical species using a
phylogenetic approach. ITS1+2 sequence data from 128
species were used in the phylogenetic analysis. Specimens
included four species of genera closely related to Agaricus,
38 temperate species representing the eight classical
sections of the genus, and 86 putative species of Agaricus
from tropical areas of Africa, Asia and the Americas.
Bayesian and maximum likelihood analyses produced
relatively congruent trees and almost identical clades. Our
data show that (i) only about one-third of tropical species
belong to the classical sections based on temperate species;
the systematics of the genus therefore needs to be
expanded; (ii) among the remaining two-thirds of tropical
species, those from the Americas and those from Africa
and/or Asia group in distinct clades, suggesting that
secondary diversification occurred in these two areas; (iii)
in contrast, several clades of classical sections contain
R. Zhao
Key Laboratory of Forest Disaster Warning and Control
in Yunnan Province, Faculty of Conservation Biology,
Southwest Forestry University,
Kunming 650224, China
R. Courtecuisse
Département de botanique,
Faculté des sciences pharmaceutiques et biologiques,
59006 Lille, France
S. Karunarathna : K. D. Hyde
School of Science, Mae Fah Luang University,
Chiang Rai 57100, Thailand
K. D. Hyde
Botany and Microbiology Department, College of Science,
King Saud University,
Riyadh 11442, Saudi Arabia
O. Raspé : A. De Kesel
National Botanic Garden of Belgium,
Domein van Bouchout,
1860 Meise, Belgium
L. A. Parra
Avda. Padre Claret 7, 5º G,
09400 Aranda de Duero, Burgos, Spain
J. Guinberteau : M. Moinard : G. Barroso : P. Callac (*)
INRA,
UR1264, Mycologie et Sécurité des Aliments,
33883 Villenave d’Ornon, France
e-mail: callac@bordeaux.inra.fr
A. K. Guelly
Département de Botanique,
Faculté des Sciences,
Université de Lomé,
B.P. 1515,
Lomé, Togo
D. E. Desjardin
Department of Biology, San Francisco State University,
San Francisco, CA 94132, USA
280
American and African+Asian species along with temperate
species. In this study, we used approximately 50 distinct
species from a small area of northern Thailand, most
probably being novel species. This diversity indicates that
Agaricus is a species-rich genus in the tropics as well as in
temperate regions. The number of species and the hypothetical paleotropical origin of the genus are discussed.
Keywords Agaricus . Basidiomycota . Tropical
biodiversity . Biogeography . ITS . Phylogeny
Introduction
Agaricus L. is a genus of basidiomycetes that includes
numerous species of which about 200–250 are presently
known. Previous estimates of species are 300–400 worldwide, but the number is probably closer to 400 according to
Bas (1991). Species live in various climates on all
continents, with the exception of Antarctica. Agaricus
species are saprobic and generally humicolous. Several
species are collected (A. campestris L. : Fr.) or cultivated
(A. bisporus (J.E. Lange) Imbach, the button mushroom)
for consumption or for medicinal use (A. subrufescens
Peck; Angeli et al. 2006; Bernarshaw et al. 2007). Despite
their ecological and economical interest, the diversity of
species remains poorly known, particularly in subtropical
and tropical areas. Numerous species of Agaricus have
however, been described from tropical areas over the last
century (Baker and Dale 1951; Berkeley and Broome 1871;
Heinemann 1956a, b, c, 1957, 1961, 1962a, 1962b, 1962c,
1971, 1978, 1980, 1982, 1990, 1993; Murrill 1918, 1942,
1945, 1946; Pegler 1966, 1968, 1969, 1977, 1983, 1986;
Pegler and Rayner 1969; Peterson et al. 2000; and Rick
1906, 1919, 1920, 1930, 1939, 1961). Agaricus species
have a limited number of characteristic phenotypic traits,
and therefore identification of species can be challenging
due to environmental effects and intraspecific variability; it
is therefore often difficult to correctly identify specimens in
the field.
In temperate areas and particularly in Europe and North
America, this situation is changing because of recent
progress in the classification facilitated by molecular
characterization and phylogeny. The genus Agaricus has
been shown to be monophyletic (Vellinga et al. 2011).
Among the eight sections recognized in the subgenus
Agaricus (Parra 2008), Bivelares (Kauffman) L.A. Parra
and Xanthodermatei Singer have been phylogenetically
reconstructed (Challen et al. 2003; Kerrigan et al. 2006;
Kerrigan et al. 2008) and others are under investigation.
Circumscription of the species and sections has been
improved, but phylogenetic reconstruction for groups of
temperate species is far from complete. For example,
Fungal Diversity (2011) 51:279–296
section Sanguinolenti Jul. Schäff. & Møller ex L.A. Parra
seems polyphyletic and the circumscription of species as
frequently encountered as A. campestris remains obscure.
Species identification, as well as description of new species,
should take characteristic traits into consideration, not only
of the species but also of the phylogenetic group to which
they belong. However, different traits can be selected to
characterize the groups. For instance, the yellow vs. red
discolouration of the flesh of the sporophore that was
considered a major trait 50 years ago, although still
important, should not be heavily weighted, since two
non-related sections, Arvenses Konrad & Maubl. and
Xanthodermatei, share this trait (Parra 2008). In contrast,
certain odours appear to be synapomorphic and are crucial
taxonomic traits, at least for temperate species (Parra
2008).
Despite the number of species described, mainly
between 1956–1993 by P. Heinemann and D.N. Pegler
from tropical and subtropical areas, many species remain
poorly known, and many remain to be described judging
from the specimens we have collected and sequenced from
these areas within our project of the European Distributed
Institute of Taxonomy (EDIT). Similarly, tropical specimens are often difficult to identify or describe as novel
species for two main reasons: (i) literature is scattered and
type specimens are not easily available for comparison; and
(ii) description and classification of tropical species, despite
the introduction of the tropical subgenera Conioagaricus
Heinem. and Lanagaricus Heinem. and the tropical section
Brunneopicti Heinem & Gooss.-Font. (Heinemann 1956a),
has been globally based on traditional systematics of
temperate species, and therefore on the traits characterizing
the temperate sections. Sequence data for less than ten
identified tropical species are currently available in GenBank; therefore in this study we mostly use sequences from
our collections in the analyses.
The understanding of temperate sections has conceptually changed in recent years (Parra 2008), however the
changes may neither be appropriate or sufficient to
incorporate tropical diversity. The objective of the present
study was to assess whether tropical Agaricus species are
distributed in the same clades as the temperate species of
the genus and to establish if there are any exclusively
tropical clades. Our approach was to use a limited number
of temperate species assigned to the different sections and a
large sample of tropical species. We used tropical in the
broad sense to include “tropical and subtropical” samples:
most samples however were collected in tropical regions,
but some were collected at high altitudes in Mexico, or
outside the “tropics” on the western coast of the USA or in
Brazil where climates can be considered as subtropical. We
have chosen at this stage to obtain a better understanding of
the phylogeny of the genus and its sections and have not
Fungal Diversity (2011) 51:279–296
attempted to carry out monographic work. However, we
have incorporated some named species as well as some
type specimens of tropical taxa.
The phylogeny is inferred via the analysis of the DNA
sequences of ITS1 and ITS2 (internal transcribed spacers 1
and 2) separating the rRNA genes and that have already
been largely used for the temperate species of this genus.
This study will facilitate future taxonomic work on tropical
Agaricus species and allow evolutionary considerations and
biogeographical analyses.
Materials and methods
Fungal material Among 178 sequenced samples listed in
Table 1, 156 have been collected and sequenced by the
authors or are from their Herbaria. MATA816 provided by
Gérardo Mata is an exception. The remaining sequences
(22) were provided by Richard W. Kerrigan (SP307818,
RWK2019, JH1) or were downloaded from GenBank.
The 178 sequenced specimens included seven collections belonging to genera closely related to Agaricus
(Heinemannomyces, Hymenagaricus, or unidentified), 38
temperate species of Agaricus, and 133 tropical collections
of Agaricus from Africa, Asia, and the Americas. The
seven collections of genera closely related to Agaricus have
been used in preliminary analyses with species of other
genera such as Hymenagaricus, Micropsalliota and Lepiota.
Since they consistently formed a sister clade or one of the
most closely related clades to the monophyletic genus
Agaricus, they were selected as outgroups for the analyses.
ZRL43, has a sequence similar to ecv3586 which Vellinga et
al. (2011) identified as Heinemannomyces splendidissimus
Watling and also found to be closely related to Agaricus. The
38 temperate species were chosen among about 100
sequenced species to represent all the major phylogenetic
clades or taxonomic subgroups of the eight presently
recognized sections of subgenus Agaricus (Challen et al.
2003; Kerrigan et al. 2006; Kerrigan et al. 2008; Parra 2008;
and unpublished data). The type species, or in section
Chitonioides Romagn. one of its most closely related species,
was included for each section: A. bitorquis (Quèl.) Sacc. in
section Bivelares, A. gennadii Chatin & Boud. in section
Chitonioides, A. xanthodermus Genev. in section Xanthodermatei, A. sylvaticus Schaeff. in section Sanguinolenti, A.
campestris in section Agaricus, A. litoralis (Wakef. & A.
Pearson) Pilát in section Spissicaules (Heinem.) Kerrigan, A.
arvensis Schaeff. in section Arvenses, and A. comtulus Fr. in
section Minores Fr.. Four temperate samples (WC913,
CA279, CA486 and CA684) were not formally identified and could represent new species, however, they
were assigned to sections based on their morphology.
The remaining 133 tropical collections of Agaricus were
281
deliberately not assigned to any section since the objective
of the study was to clarify the classification of the tropical
species and to establish to what extent the temperate
Agaricus classification is useful for accommodating
tropical species.
DNA isolation Three DNA isolation methods were used
depending on the laboratory. At the Institut National de la
Recherche Agronomique (INRA), DNA was isolated
following a CTAB protocol (Saghai-Maroof et al. 1984;
Doyle and Doyle 1987) with ethanol precipitation and
modified as follows: approximately 25 mg of dried
mushroom were ground to a fine powder in liquid nitrogen.
The samples were transferred into 2 mL reaction tubes.
700 μL of hot extraction buffer (CTAB 2% w/v; NaCl
1.4 M; Tris pH 8.0100 mM; EDTA 10 mM; ß-mercaptoethanol 2% v/v) were added. After 20 minutes of incubation
at 56°C, cell debris, polysaccharides and proteins were
separated from aqueous DNA portions through two
purification steps with 700 μL chloroform:isoamylalcohol
(24:1). DNA was washed with 700 μL precipitation buffer
(CTAB 1% w/v; Tris pH 8.0 50 mM; EDTA 10 mM). The
pellet was resuspended in 500 μL NaCl 1 M. DNA was
precipitated with the addition of two volumes of absolute
ethanol. The DNA pellet was washed 3 times in 1 mL 70%
ethanol, air-dried and resuspended in 50 μL sterile H2O.
At the National Botanic Garden of Belgium (BR), a
similar protocol was used except that only ca. 10 mg of
tissue was ground with a Retsch 300 mill; only 0.2% ßmercaptoethanol was added to the lysis buffer; samples
were lysed for 1 hour at 60°C; proteins and polysaccharides
were removed by two consecutive extractions with chloroform:isoamylalcohol (24:1), after which DNA was immediately precipitated by the addition of 0.8 volume
isopropanol to the aqueous phase; the pellet was washed
once in 600 μL 70% ethanol, air-dried, and resuspended in
100 μL TE pH 8.0; RNA was then digested with RNase A.
DNA was isolated mostly from dry specimens, but in a few
cases from CTAB-preserved tissue samples.
At San Francisco State University (SFSU) and Hong
Kong University (HKU), genomic DNA of the samples
collected in 2004–2007 were isolated from dried fungal
specimens using the E.Z.N.A. Forensic DNA Extraction Kit
(Omega Bio-Tek, Norcross, GA, U.S.A.).
PCR, primer design and sequencing PCR amplification
was accomplished mainly with primers ITS5 and ITS4
(White et al. 1990). Because some specimens were old or
because they exhibited length heteromorphisms, it was
sometimes necessary to sequence ITS1 and ITS2 separately,
with primers ITS5 and ITS2 (White et al. 1990) for the
former, and ITS-PM (reverse complement of primer ITS1Rev323, Kerrigan 2007) and ITS4 for the latter.
282
Table 1 Collections of Agaricus and closely related genera
N°a
Sample
Identificationb
Country
Collectorc
Date
AF432898
EU363033
JF797186
JF797187
AF432880
JF797188
DQ182532
AF432877
JF797181
Habitat
Herbariume
Lawn, Acacia
LIP
CGAB
On roadside
CGAB
Under Cedrus
Under Pinus
Under Carpinus
LAPAG
LAPAG
LAPAG
Grass, oak
LAPAG
Under Robinia
Forest edge
In grassland
CGAB
CGAB
CGAB
CGAB
In grassland
CGAB
Under Cedrus
CGAB
From GenBank
From GenBank
AM
PW
11/1983
22/12/2008
ORo
2005
DQ185552
DQ182513
DQ185553
AY899273
AY899271
AY484684
JF797178
JF797179
JF797180
AY943974
DQ182531
Locationd
Perpignan
Canberra, Capital Territory
From GenBank
Seine-Maritime, Tancarville
From
From
From
From
From
LP
LP
OJ
06/11/2004
13/11/2003
15/09/2002
LP
27/10/1999
JF797189
JF797190
JF797191
JF727867
AY943973
JF797192
DQ185570
JG, AG
anonymous
TB
JG, AG
23/09/2006
13/10/2004
27/08/2004
09/10/2010
JG
13/10/2007
JF797193
PC,JG
28/05/2008
GenBank
GenBank
GenBank
GenBank
GenBank
From GenBank
Madrid, Parque del Retiro
Burgos, Barrio de Cortes
Tremosnice near Caslav
From GenBank
From GenBank
From GenBank
From GenBank
Gumiel de Mercado
Gironde, Illats
Paris, Salon MNHN
Dordogne Brouchau Thenon
Gironde, Préchac
From GenBank
Gironde, Léognan
From GenBank
Gironde, Labrède
Fungal Diversity (2011) 51:279–296
COLLECTIONS FROM TEMPERATE AREAS (grouped by section)
Section Bivelares
28
RWK1462
A. bitorquis
USA
29
MATA681
A. tlaxcalensis T
Mexico
Section Chitonioides
30
PEAR83340
A. pearsonii
France
31
CA684
A. sp.
Australia
32
ARP173
A. bernardi
USA
33
CA387
A. gennadii
France
Section Xanthodermatei
34
CA217
A. phaeolepidotus
France
35
RWK1938
A. hondensis
USA
36
CA186
A. freirei
France
40
CA160
A. xanthodermulus T
France
48
CA15
A. xanthodermus
France
Section Sanguinolenti
49
WC913
(A. fuscofibrillosus)
USA
50
LAPAG341
A. sylvaticus
Spain
51
LAPAG283
A. benesii
Spain
52
LAPAG531
A. bohusii
Czech Repub.
55
RWK1415
A. pattersonae
USA
56
CA123
A. boisseletii
France
Section Agaricus
57
CA87
A. cupreobrunneus
France
58
RWK1917
A. campestris
USA
59
LAPAG141
A. langei
Spain
Section Spissicaules
72
CA486
A. sp.
France
74
CA406
A. lanipes
France
76
CA279
A. sp.
France
78
CA829
A. litoralis f
France
79
RWK1940
A. subrutilescens
USA
80
CA583
A. aff.impudicus
France
88
CA177
A. bresadolanus
France
Section Arvenses
94
CA590
A. augustus
France
Genbank
N°a
Sample
Identificationb
Country
AY484683
AF482834
JF797194
JF797195
JF514525
JF727868
JF797203
JF715065
DQ232644
JF797182
species 1 to 4
JF691559
JF727840
JF727858
JF727859
JF514522
JF514543
JF727860
JF727863
JF727861
JF440300
JF514534
JF727862
JF514532
JF727844
JF514531
JF514518
JF691550
Collectorc
Date
Locationd
Habitat
Herbariume
JG
16/10/2008
From GenBank
From GenBank
Gironde, Villenave d’Ornon
Under Pinus
CGAB
JG
EH
LP
JG
LP-RK-PC
05/11/1997
12/02/1968
06/11/1997
30/09/2006
15/10/2003
In dune
Nothofagus
In meadow
Glade of oak
In lawn
CGAB
BR
LAPAG
CGAB
LAPAG
Under Pinus
LAPAG
In forest
In forest
Azadirachta
Azadirachta
In forest
In grassland
In forest
Grazed bush
In forest
In forest
In grassland
In forest
In grassland
In urban park
In forest
In grassland
In grassland
In grassland
In forest
In grassland
Old termite hill
In forest
In forest
In forest
BBH
BBH
LAPAF
LAPAF
Oléron Island
Kowai, Monts Grey
Honrubia de la Cuesta
Gironde, Blanquefort
Yvelines, Mantes la Jolie
From GenBank
LP
19/11/1999 Segovia, Pradales
belong to genera closely related to Agaricus)
ZR
10/06/2006 Chiang Mai, Mae Taeng
DD
13/06/2006 Chiang Mai, Mae Taeng
LP
21/05/2010 Lomé, University Campus
LP
21/05/2010 Lomé, University Campus
JG-GB
25/07/2010 Chiang Mai, University P
JG-GB
25/07/2010 Chiang Mai, University P
ZR
15/09/2006 Chiang Rai, Ob Luang P
MG
11/1948
Panzi, Kivu
NT
25/06/2010 Chiang Mai, Mae Taeng
ZR
25/07/2010 Yunnan, Xishuangbana
JG-GB-SK
27/07/2010 Chiang Mai, University P
JG-GB
24/07/2010 Chiang Mai, University P
JG-GB-SK
27/07/2010 Chiang Mai, University P
EM
25/07/2004 Ouagadougou
SK-KW
27/07/2010 Chiang Mai, Mae Taeng
JG-GB
25/07/2010 Chiang Mai, University P
SK-JG
27/07/2010 Chiang Mai, University P
SK-JG
27/07/2010 Chiang Mai, University P
SK-KW
22/0720/09 Chiang Rai, Khun Kone W
SK-JG
27/07/2010 Chiang Mai, University P
AK
08/06/1999 Niaouli, Plateau
DD
07/06/2006 Chiang Mai, DSPNP
TO
13/06/2006 Chiang Mai, DSPNP
ZR
08/06/2006 Chiang Mai, Mae Taeng
BBH
BR
BR
BR
BBH
BBH
BBH
283
WC777
A. fissuratus
Denmark
ECVel2339
A. inapertus
USA
CA640
A. arvensis
France
Section Minores
103 CA101
A. aridicola
France
118 Horak68/79
A. viridopurpurascens T
New Zealand
121 LAPAG77
A. pseudolutosus
Spain
122 CA490
A. brunneolusg
France
123 LAPAG339
A. comtulus
France
125 GAL9420
A. campbellensis T
New Zealand
124 LAPAG111
A. heinemannianus
Spain
Collections from tropical or subtropical areas (sorted by number of species;
1
ZRL3043
cf. He. splendidissimus
Thailand
1
ZRL3062
cf. He. splendidissimus
Thailand
2
LAPAF9
Hy. ardosiicolor
Togo
2
LAPAF14
Hy. ardosiicolor
Togo
3
CA833
Thailand
4
CA801
Thailand
4
ZRL3103
Thailand
5
Goossens5066
A. heterocystis T
RDCongo
5
NTF9
Thailand
6
ZRL10.072
China
7
CA819
Thailand
8
CA799
Thailand
9
CA820
Thailand
10
ADK4732
A. subsaharianus T
Burkina-Faso
11
NTT117
Thailand
12
CA800
Thailand
13
NTS116
Thailand
13
NTS115
Thailand
14
NT019
Thailand
15
NTS113
Thailand
16
ADK2564
(A. brunneopictus)
Bénin
17
ZRL3031
Thailand
17
ZRL3064
Thailand
17
ZRL3041
Thailand
97
99
98
Genbank
Fungal Diversity (2011) 51:279–296
Table 1 (continued)
284
Table 1 (continued)
Sample
18
19
20
20
20
21
22
22
22
23
24
25
26
27
27
27
27
37
37
37
37
38
39
41
42
43
44
44
44
45
45
45
45
46
46
47
NTT34
LAPAF1
ZRL4017
NTT118
ZRL3005
CA856
ZRL2043
ZRL3086
ZRL2085
LAPAF2
ZRL3099
ZRL2132
LAPAF4
ZRL2123
NTS118
ZRL2128
ZRL3014
Grinling70109
NTT84
NTT38
NTS117
ZRL3044
NTF61
F2530
F2715
ZRL3095
Goossens5415
Hendrickx515
NTT50
NTS7
ZRL3094
NTT95
NT007
F2767
ADK2785
NTF58
Identificationb
A. inoxydabilis
A. campestroides
A.
A.
A.
A.
A.
A.
trisulphuratus
aff. trisulphuratus
aff. trisulphuratus
aff. trisulphuratus
aff. trisulphuratus
microvolvatulus T
(A. caribaeus)
A. aff. endoxanthus
A. xanthosarcus T
A. aff. volvatulus
A. aff. volvatulus
Country
Genbank
Collectorc
Date
Locationd
Habitat
Thailand
Togo
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Togo
Thailand
Thailand
Togo
Thailand
Thailand
Thailand
Thailand
Congo Braz.
Thailand
Thailand
Thailand
Thailand
Thailand
Fr. Martinique
Fr. Martinique
Thailand
RDCongo
RDCongo
Thailand
Thailand
Thailand
Thailand
Thailand
Fr. Martinique
Bénin
Thailand
JF514536
JF727841
JF691549
SK
LP
PS
SK-KW-JG
ZR
JG
JK
ZR
TB
LP
ZR
ZR
LP
KH
SK-JG
EG
ZR
KG
KW
KW
SK-JG
TO
NT
JF
JF
ZR
MG
FH
KW
SK
ZR
SK
SK
JF
AK
NT
19/06/2010
12/05/2010
15/05/2007
27/07/2010
26/05/2006
25/07/2010
26/06/2005
22/07/2006
03/07/2005
12/05/2010
05/09/2006
21/08/2005
12/05/2010
11/08/2005
27/07/2010
18/08/2005
03/06/2006
15/01/1967
07/10/2010
20/06/2010
27/07/2010
10/06/2006
16/07/2010
15/04/2002
06/11/2002
13/08/2006
12/1954
15/04/1939
24/06/2010
11/05/2010
09/08/2006
17/07/2010
15/08/2009
19/06/2003
13/06/2000
10/07/2010
Chiang Mai, Mae Taeng
Ola
Chiang Mai, Mae Taeng
Chiang Mai, University P
Chiang Mai, Mae Taeng
Chiang Mai, University P
Chiang Mai, Mae Taeng
Chiang Mai, Chiang Dao
Chiang Mai, Mae Taeng
Ola-Okpa-Fou
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Ola-Okpa-Fou
Chiang Mai, Mae Taeng
Chiang Mai, University P
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Brazzaville
Chiang Mai, Doi Suthep
Chiang Mai, Doi Suthep
Chiang Mai, University P
Chiang Mai, Mae Taeng
Chiang Mai, Kiewtubyoung
Tartane, pointe rouge
Tartane, pointe rouge
Chiang Mai, Mae Taeng
Panzi, Kivu
Mulungu
Chiang Mai, New waterfall
Chiang Rai, Khun Kone W
Chiang Mai, Mae Taeng
Chiang Rai, Doi Tung
Chiang Rai, Khun Kone W
Grand rivière
Niaouli, Plateau
Chiang Mai, DSPNP
In forest
In corn field
In forest
In grassland
In forest
In grassland
In forest
In forest
In forest
Path in forest
In forest
In forest
Path in forest
In forest
In grassland
In forest
In forest
In forest edge
In forest
In forest
In grassland
In forest
In forest
In forest
In forest
In forest
Coffee plantation
JF797202
JF691553
JF727842
JF691556
JF691558
JF727843
JF691557
JF514524
JF691555
JF514528
JF727856
JF727847
JF691554
JF514523
JF514533
JF727848
JF514527
In
In
In
In
In
In
In
In
forest
forest
forest
forest
forest
forest
forest
forest
Herbariume
LAPAF
BBH
BBH
BBH
BBH
BBH
LAPAF
BBH
BBH
LAPAF
BBH
BBH
BBH
BR
BBH
LIP
LIP
BBH
BR
BR
BBH
LIP
BR
Fungal Diversity (2011) 51:279–296
N°a
Sample
53
54
54
60
61
61
61
62
63
64
65
66
67
68
69
70
71
73
75
75
77
81
82
83
83
84
85
86
86
86
87
89
90
91
92
92
ZRL3012
ZRL2136
ZRL2109
F2047
F2272
LD026
F3109
NTS05
F2389
F2187
NT020
MATA816
F1779
JH1
LAPAM1
CL/GUAD05.099
CJL090302-05
ADK2171
F2255
F2039
F2301
RC/GUY07.019
NYS122
NTT42
ZRL10.071
LAPAF3
ZRL3093
Rammeloo5756
Goossens5323
Goossens5406
F2467
LAPAM4
DeMeijer1904
RWK2019
F2285
RM05/156
Identificationb
A. aff. argyropotamicus
(A. aff. argyropotamicus)
(A. magnivelaris)
(A. johnstonii)
A. cf. floridanus
A. aff. rufoaurantiacus
A. aff. rufoaurantiacus
A. cf. goossensiae
(A. parasilvaticus)
(A. cf augustus)
A. aff. impudicus
A. kivuensis
A. kivuensis T
A. kivuensis T
(A. porosporus)
(A. argyropotamicus)
A. deserticola
A. fiardiih
A. fiardiih
Country
Genbank
Collectorc
Date
Locationd
Habitat
Herbariume
Thailand
Thailand
Thailand
Fr. Martinique
Fr. Martinique
Thailand
Fr. Martinique
Thailand
Fr. Martinique
Fr. Martinique
Thailand
Mexico
Fr. Martinique
USA
Venezuela
Fr. Guadeloupe
Fr. Guiana
Benin
Fr. Martinique
Fr. Martinique
Fr. Martinique
Fr. Guiana
Benin
Thailand
China
Togo
Thailand
Burundi
RDCongo
RDCongo
Fr. Martinique
Brazil
Brazil
USA
Fr. Martinique
Fr. Martinique
JF691551
JF691552
ZR
TBa
ZR
JF
JF
JC
JF
SK
JF
JF
SK
GM
JF
JH
LP
CLu
JLC
AK
JF
JF
JF
CL
SY
KW
ZR
LP
OM
JR
MG
MG
JF
JB
AMe
JS
JF
RC
03/06/2006
07/06/2005
02/08/2005
28/06/1999
12/07/2000
25/07/2010
Chiang Mai, Mae Taeng
Chiang Mai, DSPNP
Chiang Mai, Mae Taeng
Fort-de-France
Lamentin
Chiang Rai, MFL University
In
In
In
In
BBH
BBH
BBH
LIP
LIP
29/04/2010
02/06/2001
Chiang Rai, Muang
Gros-Morne, Rivière Rouge
In forest
In forest
21/08/2009
07/2010
06/07/1998
07/2006
01/04/2006
29/11/2005
02/03/2009
20/06/1998
02/04/2000
27/06/1999
10/10/2000
27/02/2007
11/07/1999
20/06/2010
25/07/2010
12/05/2010
08/06/2006
17/11/1978
11/1953
12/1954
26/12/2001
17/04/2005
02/04/1991
07/2007
26/09/2000
27/08/2005
Chiang Mai, Mae Taeng
Veracruz, Coatepec
Prêcheur, Anse Couleuvre
Suffolk Co., New York
Choroni
Sainte-Rose, Trace de Sofaïa
Cayenne, La Mirande
Wari Maro, Borgou
Pointe banane
Saint-Esprit, Bois la Charles
Case-Pilote, Morne Rose
Sinnamary, Parcelle Guyaflux
Wari Maro, Borgou
Chiang Mai, Doi Suthep
Yunnan, Xishuangbannan
Ola
Chiang Mai, Mae Taeng
Mugara
Panzi, Kivu
Panzi, Kivu
Trinité, La Caravelle
Rio de Janeiro
Paraná, Paranagua
Bernalillo Co., New Mexico
Sainte-Anne
Trinité, La Caravelle
In forest
JF727849
JF727850
JF514530
JF727851
JF727852
JF797197
JF727870
JF727853
JF896226
JF797183
JF727857
JF727869
JF514517
JF797198
JF727854
JF797199
JF514540
JF514538
JF797184
JF691548
JF514541
JF797200
JF797185
JF797196
JF896228
JF797201
forest
forest
forest
lawn
In grassland
LIP
In forest
Mulch
On soil
In forest
In forest
Litter, humus
In forest
In forest
In wood litter
In forest
Oil palm plantation
Grevillea
Coffee plantation
In forest
In forest edge
In dune
Sandy soil
In forest
In forest
LIP
LIP
XAL
LIP
SFSU
LAPAM
LIP
BR
LIP
LIP
LIP
LIP
BR
LAPAF
BBH
BR
BR
BR
LIP
LAPAM
BR
SFSU
LIP
LIP
285
N°a
Fungal Diversity (2011) 51:279–296
Table 1 (continued)
286
Table 1 (continued)
Sample
Identificationb
92
93
93
93
93
95
96
96
96
96
96
100
100
100
101
102
102
102
104
105
105
106
106
107
108
109
109
110
111
111
112
113
114
115
116
116
F2286L
NTF67
ZRL2036
NT001
ZRL2134
Thoen7297
ZRL2127
OR71
ZRL3028
CA798
ZRL2053
F2815
CL/MART03.055
F2343
SP307818
NTT37
ZRL2110
ZRL3039
CA848
ZRL3088
NTSCR1
NTT33
NTS73
NTF26
CA846
ZRL2044
ecv3614
NT055
ADK2905
CA847
ZRL3101
NT62
NTT72
CA843
ZRL3080
NTF063
A.
A.
A.
A.
A.
A.
A.
A.
A.
fiardiih
sp.h
sp.h
sp.h
sp.h
martinicensis
martinicensis
martinicensis
martinezianus
(A. cf bulbillosus)
Country
Fr. Martinique
Thailand
Thailand
Thailand
Thailand
Senegal
Thailand
Thailand
Thailand
Thailand
Thailand
Fr. Martinique
Fr. Martinique
Fr. Martinique
Brazil
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Benin
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Genbank
JF514529
JF514542
JF691547
JF727855
JF896227
JF514537
JF727864
JF691543
JF514535
JF514526
JF727865
JF691540
HM488742
JF727846
JF514520
JF691544
JF727845
JF514539
JF727866
JF691542
Collectorc
Date
Locationd
Habitat
Herbariume
JF
SK
RL
SK
KH
DT
KH
OR
ZR
JG-SK
DD
JF-RC-CLu
CL
JF
UP
KW
ZR
TBa
JG-GB
ZR
SK
KW
SK
NT
JG-GB
ZR
EV
SK
AK
JG-GB
ZR
SK
SK
JG-GB
ZR
NT
29/09/2000
17/08/2010
25/06/2005
150/8/2009
28/08/2005
14/10/1984
16/08/2005
03/07/2010
05/06/2006
28/07/2010
27/06/2005
02/09/2003
02/09/2003
19/11/2000
14/12/2001
20/06/2010
03/08/2005
08/06/2006
25/07/2010
02/08/2006
08/03/2010
18/06/2010
30/06/2010
29/06/2010
25/07/2010
26/06/2005
Saint-Anne, Morne Manioc
Chiang Rai, Muang
Chiang Mai, Mok Fa W
Chiang Rai, Khun Kone W
Chiang Mai, Mae Taeng
Dakar
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Chiang Mai, Doi Inthanon
Chiang Mai, Nat.Park
Chiang Mai, Doi Inthanon
Prêcheur, Anse Couleuvre
Prêcheur, Anse Couleuvre
Prêcheur, Anse Lévrier
Sao Paulo
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Chiang Mai, University P
Chiang Rai, Pamae Lao P
Chiang Rai, Muang
Chiang Mai, DSPNP
Chiang Mai, Mae Taeng
Chiang Mai, Doi Suthep
Chiang Mai, University P
Chiang Mai, Mae Taeng
From GenBank
Chiang Mai, DSPNP
Wari Maro, Borgou
Chiang Mai, University P
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
Hua-Hin, Golf
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
In forest
In forest
In forest
In forest
In forest
In market
In forest
In forest
In forest
In forest
In forest
In forest
In forest
In forest
On soil
In forest
In forest
In forest
In forest
In forest
In forest
In forest
In forest
In forest
In forest
In forest
LIP
15/08/2009
19/09/2000
25/07/2010
13/09/2006
20/08/2009
07/03/2010
04/08/2010
10/07/2006
28/07/2010
In
In
In
In
In
In
In
In
In
forest
sandy soil
forest
forest
forest
forest
grassland
forest
forest
BBH
BBH
BR
BBH
BBH
BBH
LIP
LIP
LIP
BBH
BBH
BBH
BBH
BR
BBH
BBH
Fungal Diversity (2011) 51:279–296
N°a
N°a
Sample
117
119
120
126
127
128
128
128
ZRL3091
MATA774
ADK2751
ZRL3056
ZRL3102
LD030
ZRL2124
NTS106
Identificationb
A. goossensiae
Country
Genbank
Collectorc
Date
Locationd
Habitat
Herbariume
Thailand
Mexico
Benin
Thailand
Thailand
Thailand
Thailand
Thailand
JF691546
JF727871
JF514519
JF691541
JF691545
JF514521
ZR
GM-PC
AK
ZR
ZR
JC
KH
SK
02/08/2006
29/11/2007
05/06/2000
12/06/2006
15/09/2006
03/08/2010
12/08/2005
24/07/2010
Chiang Rai, Pamae Lao P
Veracruz, San Andrés Tuxtla
Calavi Campus, Atlantique
Chiang Mai, Mae Taeng
Chiang Mai, Ob Luang P
Chiang Rai, MFL University
Chiang Mai, Mae Taeng
Chiang Mai, Mae Taeng
In forest
In dune
In sandy soil
In forest
In forest
On litter
In forest
In forest
BBH
XAL
BR
BBH
BBH
MFLU
BBH
Fungal Diversity (2011) 51:279–296
Table 1 (continued)
a
Number of species (as numbered in the phylogenetic tree); when there are redundant samples belonging the same presumed species, the first one appearing in the list was used in the phylogenetic
analyses and the genbank accession number of its ITS1+2 sequence is given in the Table
b
T, Type (holotype, paratype or isotype); the species names in brackets require complementary studies of the collection to be confirmed or not.
c
AG, A. Guinberteau; AK, A. De Kesel; AM, A. Marchand; AMe, A. De Meijer; CL, C. Lechat; CLu, C. Lecuru; DD, D. Desjardin; DT, D. Thoen; EH, E. Horak; EG, E. Grand; EM, E. Maes;
EV, E. C. Vellinga; FG, K. Grinling; FH, F. Hendrickx; GB, G. Barroso; GM, G. Mata; JB, J. Borovicka; JC, J. Chen; JF, J.P. Fiard; JG, J. Guinberteau; JH, J. Horman; JK, J. Kerekes; JLC, J.-L.
Cheype; JR, J. Rammeloo; JS, J. W. Sparks II; KH, K. D. Hyde; KW, K. Wisitrassameewong; LP, L. Parra; MG, M. Gossens-Fontana; NT, N. Thongklong; OJ, O. Juhasz; OM, O. Myo Aung; OR,
O. Raspé; ORo, O. Roblot; PC, P. Callac; PS, P. Sysouphanthong; PW, P. Wenzel; R. Courtecuisse; RK, R.W. Kerrigan; SK, S. Karunarathna; SY, S. Yorou Norou; TB, T. Boonpratuang; TBa, T.
Baroni; TBo, T. Bouchara; UP, U.C. Peixoto; ZR, R.L. Zhao;
d
P, Park or National Park; W, Waterfall; MNHN, Museum National d’Histoire Naturelle; DSPNP, Doi Suthep Pui National Park.
e
SFSU: Harry D. Thiers Herbarium, Department of Biology, San Francisco State University. USA; BBH: Biotec Bandkok Herbarium, National Science and Technology Development Agency,
Klong Luang, Pathumthani, Thailand; LIP: Herbier du Département de Botanique, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Lille, Lille, France; BR : National Botanic
Garden of Belgium Herbarium, Meise, Belgium; CGAB:Collection du germoplasme des agarics à Bordeaux, INRA, Bordeaux, France; XAL : Herbario del Instituto de Ecología, A.C., Xalapa,
Veracruz, Mexico; LAPAM, LAPAF, LAPAG: Luis Alberto Parra private herbarium for America, Africa and Europe respectively, Aranda de Duero, Burgos, Spain. MFLU: Mae Fah Luang
University, Chiang Rai Prov., Thailand.
f
syn. A. spissicaulis
g
syn. A. porphyrizon
h
A. subrufescens complex
287
288
At INRA, the reaction mix (final volume 25 μL)
contained 5 μL PCR GO Taq buffer (5X, Promega),
2.5 μL dNTP mix (1.2 mM, Eurobio), 0.5 μL BSA
(10 mg/mL, Promega); 1 μl of each primer (25 μM);
0.2 μL Taq polymerase (5 U/μL, Go Taq Promega); 1 μL
DNA extract; ddH2O up to 25 μL. The PCR profile was
5 min at 95°C; 35 cycles (1 min at 94°C, 1.5 min at 55°C,
1.5 min at 72°C); 5 min at 72°C. At BR, amplifications
were usually performed in 50 μL reactions using DreamTaq DNA polymerase (Fermentas), according to manufacturer’s instructions, except that reactions contained
0.2 mg/mL BSA (Fermentas) and 0.25 μM of each primer.
Cycling conditions were as follows: 3 min at 95°C; 35
cycles of 1 min at 94°C, 1.5 min at 55°C, 1.5 min at 72°C;
5 min at 72°C. At SFSU and HKU, the thermal cycles
consisted of 3 min at 94°C, 30–35 cycles of 1 min at 94°C,
50 sec at 52°C and 1 min at 72°C, with a final extension step
of 72°C for 10 min.
Sequencing was performed on ABI Prism Genetic
analysers (Applied Biosystems) at the following institutions: Beckman Coulter Genomics, England; Macrogen,
Republic of Korea; Genome Research Centre of the
University of Hong Kong; Department of Biology, San
Francisco State University.
Phylogenetic analyses Based on our previous knowledge
on the circumscription of species in Agaricus, sequences
were considered as redundant in the three following cases:
they were identical; they differed only by heteromorphisms
and shared presumed alleles; they differed at a single
polymorphic position and possibly by heteromorphisms at
others. The entities defined above must be considered as
presumed species because such a method might in some
cases be too stringent for taxonomic purposes.
After alignment using T-Coffee ver 8.99 (Notredame et
al. 2000), corrections were made by hand, firstly because TCoffee did not interpret the heteromorphisms, secondly to
suppress highly variable or ambiguous positions.
The maximum likelihood (ML) analysis was performed
on the ATGC bioinformatics platform. The phylogenetic
tree was constructed using the ML method implemented
in the PhyML ver 3.0 aLRT (Guindon and Gascuel 2003;
Anisimova and Gascuel 2006). This method does not allow
partitions. The GTR substitution model was selected with
an estimated proportion of invariable sites of 0.438 and
assuming 4 gamma-distributed rate categories to account
for rate heterogeneity across sites. The gamma shape
parameter was estimated directly from the data (gamma=
0.782), and the likelihood was increased by using the SPR
tree improvement. Reliability of internal branches was
assessed using the approximate Likelihood-Ratio test
(aLRT) which assesses that the branch being studied
provides a significant likelihood gain, in comparison with
Fungal Diversity (2011) 51:279–296
the null hypothesis that involves collapsing that branch but
leaving the rest of the tree topology identical (Anisimova
and Gascuel 2006). The analysis included 100 bootstrap
replicates. We also performed a Bayesian analysis with
MrBayes 3.1 (Huelsenbeck and Ronquist 2001; Ronquist
and Huelsenbeck 2003). We first estimated the best
evolutionary model for ITS1 and ITS2 separately with
jModeltest (Posada 2008), using the Bayesian information
criterion, and restricting the explored evolutionary models
to the ones implemented in MrBayes. The best models
were GTR + Γ + I and HKY + Γ + I, respectively. We
partitioned the data and set the evolutionary models
accordingly in MrBayes. The model parameters were
unlinked across partitions. We ran two parallel analyses,
each with one cold and three heated chains, with tree
sampling every 200th generation. Since the two runs
converged slowly (the average deviation of split frequencies decreased slowly), we ran the analyses for 10×106
generations, and when summarizing the tree samples the
first 12,000 trees were discarded. The burnin was
determined by inspecting the log-likelihood by generation
plot generated with Tracer ver 1.5 (Rambaut and Drummond
2007). Graphical representation and editing of the phylogenetic tree were performed with TreeDyn ver.198.3 (Chevenet
et al. 2006).
Results
Sampling for analyses using ITS1+2 sequences
We obtained complete sequence data from the 178 samples
presented in Table 1. Ten of these were from types
specimens, 51 tropical samples were collected in 2010,
mainly in Thailand but also in China, Togo and Mexico; the
oldest herbarium collection was from 1939. Among the 178
samples, 89 were from Asia, 37 from the Americas, 28
from Europe, 21 from Africa, and 3 from Oceania.
Complete and non-redundant sequences for 128 samples,
representing 124 presumed Agaricus species and four
species of closely related genera, were used in the
phylogenetic analyses; these were numbered from 1
through to 128 according to their phylogenetic placement.
A total of 110 new sequences have been deposited in
GenBank. The final 590-character alignment has been
deposited in TreeBase (ID number 11249). Four nonAgaricus species represented the outgroups. Among the
124 Agaricus collections included in the study, 38 were
from temperate areas and 86 from tropical or subtropical
areas. Among the 86 tropical collections of Agaricus plus
four collections of related genera, 25 have been identified
to species (Table 1). Limited but important data concerning
Fungal Diversity (2011) 51:279–296
289
redundancy can be summarized from this Table, as follows:
first, pairwise sequence comparisons indicated that 60
putative species from the tropics were found once, 15 two
times, 10 three times, and 5 four times or more. Second, for
six presumed species of tropical Agaricus, redundant
sequences from different continents were found: four in
Africa and Asia (5: A. heterocystis Heinem. & Gooss.-Font.;
37: A. microvolvatulus Heinem.; 44: A. xanthosarcus
Heinem. & Gooss.-Font.; and 111), one in Africa and
America/Caribbean (46: A. aff. volvatulus Heinem. &
Gooss.-Font.), and one in Asia and America/Caribbean
(61). The 80 remaining species of Agaricus were found on
a single continent: 47 in Asia, 22 in the Americas, and 11 in
Africa; some of them have nearly but not entirely redundant
sequences and belong to presumed closely related species, e.
g. 26 and 27 (A. trisulphuratus Berk. group), or 92 and 93
(A. subrufescens complex); further studies are necessary to
clarify their taxonomic status.
Fig. 1 Most likely ML phylogram based on ITS1+2 sequences of
124 species of Agaricus. The SH-like branch support values above
50%, the bootstrap support values above 50% (after slash) and the
posterior probabilities of 50% majority rule consensus Bayesian tree
(in bold type) are shown. Red and blue branches refer to tropical and
temperate species of Agaricus, respectively. Branch label indications
are: the number of the species (outgroups from 1 to 4; Agaricus
species from 5 to 128), the continent (Africa AF, the Americas AM,
Asia AS, Europe EU, and Oceania OC), the section for the temperate
species only (Bivelares BIV, Chitonioides CHI, Sanguinolenti SAN,
Agaricus AGA, Spissicaules SPI, Arvenses ARV, Minores MIN), and
the species name when known, T meaning type specimen. Sections are
indicated as above and major tropical clades are numbered from TR I
to TR VII for the well-supported ones and TR a to TR d for the others
Phylogenetic results
The most likely ML tree obtained by analysis is presented
in Fig. 1. The branches having SH-like support values
lower than 50% are collapsed. SH-like support values and
Bayesian posterior probabilities of the 50% majority rule
consensus tree are showed. Bootstrap support values were
mostly lower than 50%; those above 50% are showed.
Most of the major clades were similar and relatively well
supported in the ML and Bayesian analyses with SH-like
support values and posterior probabilities greater than
80%, while the bootstrap support values were sometimes much lower. Despite numerous polytomies and
290
Fungal Diversity (2011) 51:279–296
Fig. 1 (continued)
topological differences, the ML and Bayesian trees
remain congruent enough to have most of their major
clades globally appearing in the same order.
Because deep branches, except the branch bearing
species 66 to 128, are very short, we preferred to
emphasise the content of the clades (detailed in Table 2)
rather than phylogenetic relationships between clades.
One consequence of these short branches is the low
bootstrap support values that we obtained, while aLRT
SH-like values are less sensitive to the shortness of the
branches.
Phylogenetic distribution of the temperate species
Temperate species, except A. aridicola Geml, Geiser &
Royse (103), were distributed into eight accepted taxonomic sections of subgenus Agaricus.
The sections Bivelares, Chitonioides, Xanthodermatei,
Sanguinolenti and Agaricus are related. In both trees, they
are monophyletic, except the section Sanguinolenti which
is paraphyletic and constituted three clades. Among the
seven clades of these five sections, four are entirely
temperate and the three remaining ones have an early
temperate branch.
The section Spissicaules which is a less well-known
section of the subgenus, is constituted by three clades and
two branches that form a large polytomy with clade TR II
and clade TR III in the Bayesian tree. In the ML tree, this
section is paraphyletic with clade TR III. The possible
polyphyly of section Spissicaules and the phylogenetic
relationships with the closely related clades TR II and TR
III remain unresolved.
The sections Arvenses and Minores with the exception of
the secotioid species A. aridicola are monophyletic in the
Bayesian tree and in the ML tree.
Twelve temperate species that have not been included in
previous phylogenetic analyses were also taxonomically
distributed in the eight sections. Temperate species, except
A. aridicola and two species of the section Spissicaules
which is not clearly resolved, are distributed in 12 clades
(Table 2). All of these clades have SH-like support values
greater than 80%, eight of them have Bayesian posterior
Fungal Diversity (2011) 51:279–296
291
Table 2 Phylogenetic and geographic distribution of 90 tropical species into tropical clades and sections based on temperate species
Sections, clades and
branches
ML/Bayesian branch supporta Species as
numbered in
the tree
Species of other genera in a
single clade
Sections based on temperate species
Bivelares
Chitonioides
Xanthodermatei
Sanguinolenti I+II+III
(3 clades)
Agaricus
Spissicaules (polytomies
with 3 clades)
Arvenses
Number
of
tropical
species
Species found in:
2 continents
ASb AFb AMb (see also Fig. 1
and Table 1)
100/100–100
1–4
4
0
3
1
0
98/98–100
81/55–98
85-
28–29
30–33
34–48
0
0
10
0
0
3
0
0
7
0
0
3
0
0
3
84–87, 83–56, 89/62–100
49–56
2
0
2
0
0
92–99
95/86–100, 88–70, 99/93–100
57–62
72–81
3
3
1
0
2
0
0
1
2
2
A. aff. argyropotamicus
99/87–100
92–99
4
0
2
1
1
A. fiardii
1
0
1
0
A. Goossensiae
Minores
89–51
115–128
8
0
6
Isolated branch
103
0
0
0
(A. aridicola)
Tropical branches and clades of presumed species found in Africa and/or in Asia (with the exception of
Tropical clade TR I
91–82
11–20
10
0
8
Tropical clade TR III
100/100–100
83–88
6
0
2
Tropical clade TR V
Tropical clade TR VI
Tropical clade TR VII
Tropical clade TR a
(poorly supported)
Tropical clade TR b
(poorly supported)
Isolated tropical branches
Identified tropical species
A. microvolvatulus, A.
xanthosarcus
A. aff. endoxanthus, A. aff.
volvatulus
species 88 in clade TR III)
2
0 A. inoxydabilis
3
1 A. kivuensis, A. aff.
impudicus
0
0
1
0
0
0
0
0
93/89–100
93/55–92
99/91–100
59–60
104–107
108–111
112–113
8–9
4
4
2
2
0
1
0
0
4
4
2
2
82-
22–27
6
0
4
2
0
5, 6, 7, 10, 21
8
1
7
2
0
65, 102, 114
Tropical branches and clades of presumed species found in the Americas
Tropical clade TR II
96–99
66–71
6
0
0
0
6
Tropical clade TR IV
Tropical clade TR c
(poorly supported)
Tropical clade TR d
(poorly supported)
Isolated branch
Total for tropical species
of Agaricus
In sections
Out of sections
cf. He. splendidissimus,
Hy. ardosiicolor
97/96–100
85–61
89–91
63–64
3
2
0
0
0
0
0
0
3
2
67-
100–101
2
0
0
0
2
82
5–128
1
86
0
6
0
52
0
16
1
24
30 (35%)
56 (65%)
4
2
19
33
6
10
9
15
a
A. campestroides, A.
trisulphuratus
A. heterocystis, A.
subsaharianus
A. aff. rufoaurantiacus, A.
cf. floridanus
A. deserticola
A. martinicensis, A.
martinezianus
SH-like branch support values above 50%, bootstrap support values above 50% (after a slash), and posterior probabilities of 50% majority rule
consensus Bayesian tree (after a dash)
b
AS=Asia, AF=Africa, AM=the Americas; the six species found in two continents are counted two times
292
probabilities greater than 80%, and four of the eight also
have bootstrap support values greater than 80%.
Phylogenetic and geographic distributions of the tropical
species
The four non-Agaricus species representing outgroups,
formed a clade sister to Agaricus in both trees; although it
was not necessary to impose them as outgroups in the ML
analysis. This clade was supported at 100% by all the
methods. These outgroups are from tropical Africa or Asia.
Thirty five percent of the tropical species of Agaricus
(30 species) were distributed amongst six of the eight
accepted sections of the subgenus Agaricus (Table 2). In
other terms, about one-third of tropical (including subtropical) Agaricus species nested in seven of the 12 clades
based on temperate species. The 56 remaining tropical
species were distributed in eleven exclusively tropical
clades and in nine isolated branches detailed in Table 2.
Among the eleven tropical clades detected in the analyses,
seven that are well supported by both analyses are
numbered as TR I to TR VII. Both SH-like branch support
and Bayesian posterior probability were greater than 95%
for four of these seven clades, greater than 90% for two,
and greater than 80% for the remaining clade. Moreover,
the four clades III, IV, V, and VII have high bootstrap
support values of 100, 96, 89, and 91 respectively.
Geographic information appears highly correlated with
the phylogenetic data although African, Asian and American samples were not equally represented. Firstly, all
basally joined species including those of the closely related
genera, from 1 to 20, are tropical and were found in Africa
and/or in Asia, but not in the Americas. Secondly, among
the 11 tropical clades, three contained eight species
collected in Asia, three contained 20 species found only
in Africa and/or Asia, four contained 13 species found only
in the Americas, and one contained six species from the
three continents with a single species from the Americas
(87 from Martinique). Samples from Africa were underrepresented, thus exclusively African clades possibly exist.
Likewise, it is possible that the exclusively Asian clades
would be in fact African and Asian. In Table 2, tropical
clades and isolated branches of Africa and Asia are
separated from those of the Americas.
The separation between the American and the African+
Asian tropical species is much less pronounced in the
sections based on temperate species than in the tropical
clades although the tropical species are 50% less numerous
in the former than in the latter. Five sections contain both
American and African+Asian tropical species: the four
monophyletic sections Xanthodermatei, Agaricus, Arvenses,
Minores, and the possibly polyphyletic section Spissicaules.
Section Sanguinolenti contains tropical species from Asia
Fungal Diversity (2011) 51:279–296
only and the two remaining sections Bivelares and
Chitonioides have no tropical species. Moreover, the only
two species for which samples were found in the Americas
and in Africa+Asia (46 and 61) belong to clades of the
monophyletic sections Xanthodermatei and Agaricus.
Discussion
The number of recognized Agaricus species lies between
200 and 250 according to Bas (1991) and is estimated as ca
200 mostly temperate species in Kirk et al. (2008). These
estimations are lower than our own calculation of 386
recognized species among which 203 are temperate and 183
are tropical. This estimation is updated with the most
recently described species but remains approximate because
some synonymies have not been detected between species
described on different continents or climates. Thus, the
numbers of tropical and temperate recognized species are
quite similar. In our analyses, we used sequences from 86
tropical species and 38 temperate species carefully selected
among about 100 species to represent their distribution in
the eight presently recognized temperate sections of
subgenus Agaricus (Challen et al. 2003; Kerrigan et al.
2006; Kerrigan et al. 2008; Parra 2008). Tropical and
temperate samples are approximately equally represented.
Concerning the number of species in existence, Bas (1991)
recognized 200-250 described species in Agaricus, but
estimated there were 300–400 species worldwide. We
believe this is also an underestimate since it is similar to
the number of presently recognized species and since
probably most of the species collected in Thailand in the
present study are new. The total number of species collected
in only a small part of northern Thailand during the last five
years, including the about 50 used in the present study, is
likely to be more than the 70 species recognized in Europe
over one century. From the present study, we have also
established that a large part of the American samples are
taxonomically different from those of Africa and Asia. The
number of tropical species is certainly much greater than the
number of temperate species and the total number of species
in existence should be therefore much greater than 400.
In the genus Agaricus ITS sequences were used for
species characterization or for phylogenetic analyses of the
sections of the genus; however, at the scale of the entire
genus, it appears that most of the deep branches are short
suggesting that major evolutionary radiations would have
occurred in a relatively short time. In other respects, the low
bootstrap support values of the deep branches are partly due
to their shortness. The present analysis was not aimed at
establishing a very strong phylogeny for the genus, but
identifies the major clades and the distribution of the
tropical species among these clades.
Fungal Diversity (2011) 51:279–296
There is no major change in the classification of the
temperate species into eight sections with the inclusion of
tropical species plus an additional dozen temperate species
never included in previous phylogenetic analyses (except
for the secotioid species A. aridicola). The nLSU rDNA
sequence of our collection CA101 of A. aridicola has
previously been obtained by Moncalvo et al. (2002) and
also used by Geml et al. (2004) and Capelari et al. (2006) in
analyses. This species, exhibiting a positive Schaeffer
reaction (orange, red or violaceous-purple discoloration
when aniline and nitric acid are consecutively applied on
the same spot of a sporophore) and an odor of almond, was
primarily assigned to section Minores according to the data
of Geml et al. (2004). However, tropical samples were
mostly absent in this study and our present data better agree
with the study of Capelari et al. (2006) who found that A.
aridicola was more closely related to A. martinezianus than
to sections Arvenses and Minores. With caution, we do not
include A. aridicola in the clade TR V which would be
much less supported with this species. In other respect, the
‘temperate’ status of this secotioid species is doubtful since
it is rare in France but abundant in Israel according to
Wasser (2002). To classify the species in climatic groups is
not easy: the geographical range of some temperate species
such as A. bisporus and A. bitorquis extends into tropical
areas and, reciprocally, the tropical species A. subrufescens
exists also in Europe (Kerrigan 2005). Moreover, some
species such as the tropical species A. endoxanthus Berk. &
Broome are sometimes found in greenhouses (Parra et al.
2002) in Europe and are suspected to have been introduced
with plants.
Our analyses are similar to those of Geml et al. (2004)
and show that the three secotioid species, A. aridicola, A.
deserticola G. Moreno, Esqueda & Lizárraga, and A.
inapertus Vellinga, do not share a common ancestor. The
secotioid form, considered as an adaptation to arid
environment, has therefore evolved independently several
times in a large clade that includes sections Arvenses and
Minores, and five related tropical clades (samples from 89
to 128).
Two-thirds (56/86) of the tropical or subtropical species
did not cluster in the recognized sections based on
temperate species. Nine species occurred on isolated
branches, 12 form poorly supported clades, TR a to TR d,
and 35 belong to seven well-supported clades, I to VII. We
accept these seven tropical clades of which four are more
strongly supported in the analyses (III, IV, V, and VII).
Characterization of these tropical clades will be challenging. There are at least four examples where tropical species
share some characteristics with the recognized temperate
sections, although they do not belong to these sections. (i)
Two samples both having affinity with A. impudicus (Rea)
Pilát, a species of section Spissicaules, belong to two
293
different clades: the temperate species (81) to a clade of the
section Spissicaules as expected, but the tropical species
(85) to the most closely related tropical clade TR III. (ii)
The sample of A. subsaharianus L.A. Parra, Hama & De
Kesel (10) has traits that, until now characterized only
sections Spissicaules, Minores and Arvenses (Hama et al.
2010), but A. subsaharianus is not related to these sections.
(iii) A. heterocystis is not in section Arvenses as stated by
Heinemann (1978) but on a non-related isolated branch.
(iv) A. inoxydabilis Heinem. is not in section Sanguinolenti
as stated by Heinemann (1980) but a member of clade TRI.
Specimens belonging to the tropical clades can be used
to identify these clades to the tropical section Brunneopicti
or the subgenus Lanagaricus proposed by Heinemann
(1956a). For the section Brunneopicti, the clade TR III is
a plausible candidate since it contains A. kivuensis Heinem.
& Gooss.-Font (87) that Heinemann included in this clade,
but clade TR I could be the best candidate because it
contains a specimen identified as A. brunneopictus Heinem.
& Gooss.-Font (16), the type species of the section.
Unfortunately we were unable to confirm this identification
as the specimen we examined is immature. Further studies
will be necessary to clarify what clade represents this
section and how this section mainly based on the
morphology of the cap can be better characterized.
Heinemann acknowledged that this section was not well
characterized (“Brunneopicti mais cette section est mal
caracterisée”; Heinemann 1984). A sample of A. trisulphuratus (26), the type species of subgenus Lanagaricus was
used in our analysis. It clustered in clade TR b which in our
ML tree belongs to a subclade of subgenus Agaricus since
it also includes sections Bivelares, Chitonioides and
Xanthodermatei. In Vellinga et al. (2011) A. trisulphuratus
also clusters in a clade of the subgenus Agaricus;
unfortunately, in both cases the phylogenetic position of
the clade is not well-supported. The confirmation of such a
clade into subgenus Agaricus would imply the inclusion of
all sections of subgenus Lanagaricus in subgenus Agaricus
and disappearance of subgenus Lanagaricus. In our
analysis, the clade TR b is poorly supported, but it contains
two strongly supported African+Asian sister clades: one
(25-26-27) with A. trisulphuratus and the other clade (2324) with A. campestroides Heinem.(23). The hypothesis
that the whole clade TR b would represent the section
Lanagaricus is reinforced by the uncertain classification of
A. campestroides: this species was initially placed in
section Agaricus (Heinemann 1956a) and later combined
in genus Micropsalliota (Heinemann 1988). However,
according to our microscopic studies it can be neither a
member of section Agaricus nor Micropsalliota (data not
shown). Although some of the tropical clades from the
present study will probably use the section names or
subgenera described by Heinemann, their characterization
294
and their circumscription need consideration. Only onethird of tropical Agaricus species are distributed in the
recognized infrageneric organization of the genus based on
temperate species. Although some species among the
remaining two-thirds could be included in the tropical
sections proposed by Heinemann, it appears necessary to
expand the number of sections in the genus.
The fact that numerous clades are exclusively tropical
indicates that geography and climate have had a major
impact on the evolution of the genus. Adaptation to
temperate climates has never occurred in certain genera of
Agaricaceae such as Micropsalliota (Zhao et al. 2010). A
relative tolerance to cold is required in temperate climates
and the ability to fruit at ambient temperatures that is
required under warm climates is not advantageous in
temperate regions because the summer is not the wettest
season and consequently the most favorable season for
fruiting. Largeteau et al. (2011) showed that the rate of wild
isolates able to fruit at 25°C is 100% in A. bisporus var.
burnettii Kerrigan & Callac, a variety that lives in hot
climates, while it is only about 50% in the populations of A.
bisporus var. bisporus living in temperate climates.
Our data also indicate that clades of four of the eight
recognized temperate sections include both American and
African+Asian tropical species. To our knowledge, all
temperate sections contain North American and Eurasian
temperate species, and the distribution range of numerous
temperate species also extends to both continents (Challen
et al. 2003; Kerrigan et al. 2006; Kerrigan et al. 2008).
These species probably migrated via land before the
complete separation of these continents. Moreover, for
some species, such as A. bisporus and A. bitorquis,
subpopulations that adapted to (sub)tropical climates in
Africa or in North America are known. For example, the
geographic range of A. bisporus extends from the boreal
region of Alaska (Geml et al. 2008) to the equatorial
climate of Congo (Heinemann 1956a) in Africa and to the
hot and dry climate of the Sonoran Desert of California in
America (Callac et al. 1993); the ancestral condition of such
species, however, remains unknown. Finally, a similar
hypothesis for the amphiatlantic distribution of the temperate species and for the American and African+Asian
distribution of the tropical species belonging to the same
clades as the temperate species needs consideration.
Forty-seven tropical species are distributed in 11 tropical
clades. Seven of these clades contain species exclusively from
Africa+Asia with the exception of one species in clade III,
while the four remaining clades contain species exclusively
from the Americas. The different phylogenetic distribution of
the American species versus the African+Asian species
appears to be a reliable pattern since four tropical clades
contained only species found in America although the Asian
samples were overrepresented compared to the American
Fungal Diversity (2011) 51:279–296
samples. The distinct African+Asian and American tropical
clades also suggest that species diversification probably
occurred independently in Africa+Asia and in the Americas.
Moreover, some clades belonging to the African+Asian group
are not phylogenetically closely related, indicating that species
diversification occurred several times on these continents. This
is also possible in the Americas but less evident from the data.
One of the four species used in the outgroup and that
does not belong in Agaricus, was identified as Hymenagaricus ardosiicolor (Heinem.) Heinem. while another shares
some characteristic traits with Heinemannomyces splendidissimus but others with Hymenagaricus and Xanthagaricus.
However, when other GenBank sequences of Hymenagaricus species were included in the analyses (data not shown),
they diverged more from the Agaricus clade than the four
species that we used, with the exception of Hymenagaricus
epipastus (Berk. & Broome) Heinem. & Little Flower
recently reported from Thailand by Zhao et al. (2010).
Further studies will be necessary to clarify the taxonomic
rank of these taxa. In Vellinga et al. (2011) one of the two
species Heinemannomyces splendisissimus or Clarkeinda
trachodes (Berk.) Singer is sister to the monophyletic genus
Agaricus depending on the analysis. Hymenagaricus
epipastus, Hymenagaricus ardosiicolor, Heinemannomyces
splendisissimus, Clarkeinda trachodes and two unidentified
species in our outgroups are from tropical Africa or Asia.
The geographical origin of Agaricus is unknown,
however, the following evidences suggest a paleotropic
origin. (i) Species of genera known to be the most closely
related to Agaricus species are from tropical Africa or Asia.
(ii) All the early branches and clades of our trees (from
species 1 to 20) contain species exclusively from tropical
Africa or Asia; and (iii) the first African+Asian clades
appeared before the first American ones. This hypothesis
remains to be confirmed because these basal branches and
clades are not strongly supported in the analyses (Fig. 1).
The analyses should also be extended to include species
from tropical Australia and temperate areas of Asia and the
South Hemisphere to better understand the biogeography of
the genus and possibly correlate migrations with the known
periods of connection between continents.
Acknowledgments The authors are grateful to Jean-Pierre Fiard,
Else Vellinga, Gerardo Mata, Marina Capelari, Marc-André Lachance,
Rick Kerrigan, Peter Wenzel, Dario De Franceschi, Vincent Lefort,
Stephane Welti and Saturnino (Nino) Santamaría. This work was
supported by an Integration Research Grant from the European
Distributed Institute of Taxonomy (EDIT).
The National Science Foundation (USA) (PEET-grant DEB0118776 to Desjardin), the National Natural Science Foundation of
China (Project ID: 31000013), and the project “value added products
from Basidiomycetes: Putting Thailand’s biodiversity to use”
(BRN049/2553) are thanked for providing partial support to this
research. The Global Research Network for Fungal Biology and King
Saud University are also thanked for support.
Fungal Diversity (2011) 51:279–296
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