Fungal Diversity

, Volume 51, Issue 1, pp 279–296 | Cite as

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
Article

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 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, 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 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.
Table 1

Collections of Agaricus and closely related genera

a

Sample

Identificationb

Country

Genbank

Collectorc

Date

Locationd

Habitat

Herbariume

COLLECTIONS FROM TEMPERATE AREAS (grouped by section)

 

Section Bivelares

        

28

RWK1462

A. bitorquis

USA

AF432898

  

From GenBank

  

29

MATA681

A. tlaxcalensis T

Mexico

EU363033

  

From GenBank

  
 

Section Chitonioides

        

30

PEAR83340

A. pearsonii

France

JF797186

AM

11/1983

Perpignan

 

LIP

31

CA684

A. sp.

Australia

JF797187 

PW

22/12/2008

Canberra, Capital Territory

Lawn, Acacia

CGAB

32

ARP173

A. bernardi

USA

AF432880

  

From GenBank

  

33

CA387

A. gennadii

France

JF797188 

ORo

2005

Seine-Maritime, Tancarville

On roadside

CGAB

 

Section Xanthodermatei

        

34

CA217

A. phaeolepidotus

France

DQ185552

  

From GenBank

  

35

RWK1938

A. hondensis

USA

DQ182513

  

From GenBank

  

36

CA186

A. freirei

France

DQ185553

  

From GenBank

  

40

CA160

A. xanthodermulus T

France

AY899273

  

From GenBank

  

48

CA15

A. xanthodermus

France

AY899271

  

From GenBank

  
 

Section Sanguinolenti

        

49

WC913

(A. fuscofibrillosus)

USA

AY484684

  

From GenBank

  

50

LAPAG341

A. sylvaticus

Spain

JF797178

LP

06/11/2004

Madrid, Parque del Retiro

Under Cedrus

LAPAG

51

LAPAG283

A. benesii

Spain

JF797179

LP

13/11/2003

Burgos, Barrio de Cortes

Under Pinus

LAPAG

52

LAPAG531

A. bohusii

Czech Repub.

JF797180

OJ

15/09/2002

Tremosnice near Caslav

Under Carpinus

LAPAG

55

RWK1415

A. pattersonae

USA

AY943974

  

From GenBank

  

56

CA123

A. boisseletii

France

DQ182531

  

From GenBank

  
 

Section Agaricus

        

57

CA87

A. cupreobrunneus

France

DQ182532

  

From GenBank

  

58

RWK1917

A. campestris

USA

AF432877

  

From GenBank

  

59

LAPAG141

A. langei

Spain

JF797181

LP

27/10/1999

Gumiel de Mercado

Grass, oak

LAPAG

 

Section Spissicaules

        

72

CA486

A. sp.

France

JF797189 

JG, AG

23/09/2006

Gironde, Illats

Under Robinia

CGAB

74

CA406

A. lanipes

France

JF797190

anonymous

13/10/2004

Paris, Salon MNHN

 

CGAB

76

CA279

A. sp.

France

JF797191

TB

27/08/2004

Dordogne Brouchau Thenon

Forest edge

CGAB

78

CA829

A. litoralisf

France

JF727867

JG, AG

09/10/2010

Gironde, Préchac

In grassland

CGAB

79

RWK1940

A. subrutilescens

USA

AY943973

  

From GenBank

  

80

CA583

A. aff.impudicus

France

JF797192

JG

13/10/2007

Gironde, Léognan

In grassland

CGAB

88

CA177

A. bresadolanus

France

DQ185570

  

From GenBank

  
 

Section Arvenses

        

94

CA590

A. augustus

France

JF797193

PC,JG

28/05/2008

Gironde, Labrède

Under Cedrus

CGAB

97

WC777

A. fissuratus

Denmark

AY484683

  

From GenBank

  

99

ECVel2339

A. inapertus

USA

AF482834

  

From GenBank

  

98

CA640

A. arvensis

France

JF797194

JG

16/10/2008

Gironde, Villenave d’Ornon

Under Pinus

CGAB

 

Section Minores

        

103

CA101

A. aridicola

France

JF797195

JG

05/11/1997

Oléron Island

In dune

CGAB

118

Horak68/79

A. viridopurpurascens T

New Zealand

JF514525

EH

12/02/1968

Kowai, Monts Grey

Nothofagus

BR

121

LAPAG77

A. pseudolutosus

Spain

JF727868

LP

06/11/1997

Honrubia de la Cuesta

In meadow

LAPAG

122

CA490

A. brunneolusg

France

JF797203

JG

30/09/2006

Gironde, Blanquefort

Glade of oak

CGAB

123

LAPAG339

A. comtulus

France

JF715065

LP-RK-PC

15/10/2003

Yvelines, Mantes la Jolie

In lawn

LAPAG

125

GAL9420

A. campbellensis T

New Zealand

DQ232644

  

From GenBank

  

124

LAPAG111

A. heinemannianus

Spain

JF797182

LP

19/11/1999

Segovia, Pradales

Under Pinus

LAPAG

Collections from tropical or subtropical areas (sorted by number of species; species 1 to 4 belong to genera closely related to Agaricus)

1

ZRL3043

cf. He. splendidissimus

Thailand

JF691559

ZR

10/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

1

ZRL3062

cf. He. splendidissimus

Thailand

 

DD

13/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

2

LAPAF9

Hy. ardosiicolor

Togo

JF727840

LP

21/05/2010

Lomé, University Campus

Azadirachta

LAPAF

2

LAPAF14

Hy. ardosiicolor

Togo

 

LP

21/05/2010

Lomé, University Campus

Azadirachta

LAPAF

3

CA833

 

Thailand

JF727858

JG-GB

25/07/2010

Chiang Mai, University P

In forest

 

4

CA801

 

Thailand

JF727859

JG-GB

25/07/2010

Chiang Mai, University P

In grassland

 

4

ZRL3103

 

Thailand

 

ZR

15/09/2006

Chiang Rai, Ob Luang P

In forest

BBH

5

Goossens5066

A. heterocystis T

RDCongo

JF514522

MG

11/1948

Panzi, Kivu

Grazed bush

BR

5

NTF9

 

Thailand

 

NT

25/06/2010

Chiang Mai, Mae Taeng

In forest

 

6

ZRL10.072

 

China

JF514543

ZR

25/07/2010

Yunnan, Xishuangbana

In forest

 

7

CA819

 

Thailand

JF727860

JG-GB-SK

27/07/2010

Chiang Mai, University P

In grassland

 

8

CA799

 

Thailand

JF727863

JG-GB

24/07/2010

Chiang Mai, University P

In forest

 

9

CA820

 

Thailand

JF727861

JG-GB-SK

27/07/2010

Chiang Mai, University P

In grassland

 

10

ADK4732

A. subsaharianus T

Burkina-Faso

JF440300

EM

25/07/2004

Ouagadougou

In urban park

BR

11

NTT117

 

Thailand

JF514534

SK-KW

27/07/2010

Chiang Mai, Mae Taeng

In forest

 

12

CA800

 

Thailand

JF727862

JG-GB

25/07/2010

Chiang Mai, University P

In grassland

 

13

NTS116

 

Thailand

JF514532

SK-JG

27/07/2010

Chiang Mai, University P

In grassland

 

13

NTS115

 

Thailand

 

SK-JG

27/07/2010

Chiang Mai, University P

In grassland

 

14

NT019

 

Thailand

JF727844

SK-KW

22/0720/09

Chiang Rai, Khun Kone W

In forest

 

15

NTS113

 

Thailand

JF514531

SK-JG

27/07/2010

Chiang Mai, University P

In grassland

 

16

ADK2564

(A. brunneopictus)

Bénin

JF514518

AK

08/06/1999

Niaouli, Plateau

Old termite hill

BR

17

ZRL3031

 

Thailand

JF691550

DD

07/06/2006

Chiang Mai, DSPNP

In forest

BBH

17

ZRL3064

 

Thailand

 

TO

13/06/2006

Chiang Mai, DSPNP

In forest

BBH

17

ZRL3041

 

Thailand

 

ZR

08/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

18

NTT34

 

Thailand

JF514536

SK

19/06/2010

Chiang Mai, Mae Taeng

In forest

 

19

LAPAF1

A. inoxydabilis

Togo

JF727841

LP

12/05/2010

Ola

In corn field

LAPAF

20

ZRL4017

 

Thailand

JF691549

PS

15/05/2007

Chiang Mai, Mae Taeng

In forest

BBH

20

NTT118

 

Thailand

 

SK-KW-JG

27/07/2010

Chiang Mai, University P

In grassland

 

20

ZRL3005

 

Thailand

 

ZR

26/05/2006

Chiang Mai, Mae Taeng

In forest

BBH

21

CA856

 

Thailand

JF797202

JG

25/07/2010

Chiang Mai, University P

In grassland

 

22

ZRL2043

 

Thailand

JF691553

JK

26/06/2005

Chiang Mai, Mae Taeng

In forest

BBH

22

ZRL3086

 

Thailand

 

ZR

22/07/2006

Chiang Mai, Chiang Dao

In forest

BBH

22

ZRL2085

 

Thailand

 

TB

03/07/2005

Chiang Mai, Mae Taeng

In forest

BBH

23

LAPAF2

A. campestroides

Togo

JF727842

LP

12/05/2010

Ola-Okpa-Fou

Path in forest

LAPAF

24

ZRL3099

 

Thailand

JF691556

ZR

05/09/2006

Chiang Mai, Mae Taeng

In forest

BBH

25

ZRL2132

 

Thailand

JF691558

ZR

21/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

26

LAPAF4

A. trisulphuratus

Togo

JF727843

LP

12/05/2010

Ola-Okpa-Fou

Path in forest

LAPAF

27

ZRL2123

A. aff. trisulphuratus

Thailand

JF691557

KH

11/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

27

NTS118

A. aff. trisulphuratus

Thailand

 

SK-JG

27/07/2010

Chiang Mai, University P

In grassland

 

27

ZRL2128

A. aff. trisulphuratus

Thailand

 

EG

18/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

27

ZRL3014

A. aff. trisulphuratus

Thailand

 

ZR

03/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

37

Grinling70109

A. microvolvatulus T

Congo Braz.

JF514524

KG

15/01/1967

Brazzaville

In forest edge

BR

37

NTT84

 

Thailand

 

KW

07/10/2010

Chiang Mai, Doi Suthep

In forest

 

37

NTT38

 

Thailand

 

KW

20/06/2010

Chiang Mai, Doi Suthep

In forest

 

37

NTS117

 

Thailand

 

SK-JG

27/07/2010

Chiang Mai, University P

In grassland

 

38

ZRL3044

 

Thailand

JF691555

TO

10/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

39

NTF61

 

Thailand

JF514528

NT

16/07/2010

Chiang Mai, Kiewtubyoung

In forest

 

41

F2530

(A. caribaeus)

Fr. Martinique

JF727856

JF

15/04/2002

Tartane, pointe rouge

In forest

LIP

42

F2715

 

Fr. Martinique

JF727847

JF

06/11/2002

Tartane, pointe rouge

In forest

LIP

43

ZRL3095

A. aff. endoxanthus

Thailand

JF691554

ZR

13/08/2006

Chiang Mai, Mae Taeng

In forest

BBH

44

Goossens5415

A. xanthosarcus T

RDCongo

JF514523

MG

12/1954

Panzi, Kivu

Coffee plantation

BR

44

Hendrickx515

 

RDCongo

 

FH

15/04/1939

Mulungu

 

BR

44

NTT50

 

Thailand

 

KW

24/06/2010

Chiang Mai, New waterfall

In forest

 

45

NTS7

 

Thailand

JF514533

SK

11/05/2010

Chiang Rai, Khun Kone W

In forest

 

45

ZRL3094

 

Thailand

 

ZR

09/08/2006

Chiang Mai, Mae Taeng

In forest

BBH

45

NTT95

 

Thailand

 

SK

17/07/2010

Chiang Rai, Doi Tung

In forest

 

45

NT007

 

Thailand

 

SK

15/08/2009

Chiang Rai, Khun Kone W

In forest

 

46

F2767

A. aff. volvatulus

Fr. Martinique

JF727848

JF

19/06/2003

Grand rivière

In forest

LIP

46

ADK2785

A. aff. volvatulus

Bénin

 

AK

13/06/2000

Niaouli, Plateau

In forest

BR

47

NTF58

 

Thailand

JF514527

NT

10/07/2010

Chiang Mai, DSPNP

In forest

 

53

ZRL3012

 

Thailand

JF691551

ZR

03/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

54

ZRL2136

 

Thailand

JF691552

TBa

07/06/2005

Chiang Mai, DSPNP

In forest

BBH

54

ZRL2109

 

Thailand

 

ZR

02/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

60

F2047

A. aff. argyropotamicus

Fr. Martinique

JF727849

JF

28/06/1999

Fort-de-France

In lawn

LIP

61

F2272

 

Fr. Martinique

JF727850

JF

12/07/2000

Lamentin

 

LIP

61

LD026

 

Thailand

 

JC

25/07/2010

Chiang Rai, MFL University

In grassland

 

61

F3109

(A. aff. argyropotamicus)

Fr. Martinique

 

JF

   

LIP

62

NTS05

 

Thailand

JF514530

SK

29/04/2010

Chiang Rai, Muang

In forest

 

63

F2389

(A. magnivelaris)

Fr. Martinique

JF727851

JF

02/06/2001

Gros-Morne, Rivière Rouge

In forest

LIP

64

F2187

 

Fr. Martinique

JF727852

JF

   

LIP

65

NT020

 

Thailand

JF797197

SK

21/08/2009

Chiang Mai, Mae Taeng

In forest

 

66

MATA816

 

Mexico

JF727870

GM

07/2010

Veracruz, Coatepec

 

XAL

67

F1779

(A. johnstonii)

Fr. Martinique

JF727853

JF

06/07/1998

Prêcheur, Anse Couleuvre

In forest

LIP

68

JH1

A. cf. floridanus

USA

JF896226

JH

07/2006

Suffolk Co., New York

Mulch

SFSU

69

LAPAM1

A. aff. rufoaurantiacus

Venezuela

JF797183

LP

01/04/2006

Choroni

 

LAPAM

70

CL/GUAD05.099

A. aff. rufoaurantiacus

Fr. Guadeloupe

JF727857

CLu

29/11/2005

Sainte-Rose, Trace de Sofaïa

 

LIP

71

CJL090302-05

 

Fr. Guiana

JF727869

JLC

02/03/2009

Cayenne, La Mirande

  

73

ADK2171

A. cf. goossensiae

Benin

JF514517

AK

20/06/1998

Wari Maro, Borgou

On soil

BR

75

F2255

(A. parasilvaticus)

Fr. Martinique

JF797198

JF

02/04/2000

Pointe banane

In forest

LIP

75

F2039

 

Fr. Martinique

 

JF

27/06/1999

Saint-Esprit, Bois la Charles

In forest

LIP

77

F2301

 

Fr. Martinique

JF727854

JF

10/10/2000

Case-Pilote, Morne Rose

 

LIP

81

RC/GUY07.019

 

Fr. Guiana

JF797199

CL

27/02/2007

Sinnamary, Parcelle Guyaflux

Litter, humus

LIP

82

NYS122

(A. cf augustus)

Benin

JF514540

SY

11/07/1999

Wari Maro, Borgou

 

BR

83

NTT42

 

Thailand

JF514538

KW

20/06/2010

Chiang Mai, Doi Suthep

In forest

 

83

ZRL10.071

 

China

 

ZR

25/07/2010

Yunnan, Xishuangbannan

In forest

 

84

LAPAF3

A. aff. impudicus

Togo

JF797184

LP

12/05/2010

Ola

In wood litter

LAPAF

85

ZRL3093

 

Thailand

JF691548

OM

08/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

86

Rammeloo5756

A. kivuensis

Burundi

JF514541

JR

17/11/1978

Mugara

Oil palm plantation

BR

86

Goossens5323

A. kivuensis T

RDCongo

 

MG

11/1953

Panzi, Kivu

Grevillea

BR

86

Goossens5406

A. kivuensis T

RDCongo

 

MG

12/1954

Panzi, Kivu

Coffee plantation

BR

87

F2467

(A. porosporus)

Fr. Martinique

JF797200

JF

26/12/2001

Trinité, La Caravelle

In forest

LIP

89

LAPAM4

 

Brazil

JF797185

JB

17/04/2005

Rio de Janeiro

In forest edge

LAPAM

90

DeMeijer1904

(A. argyropotamicus)

Brazil

JF797196

AMe

02/04/1991

Paraná, Paranagua

In dune

BR

91

RWK2019

A. deserticola

USA

JF896228

JS

07/2007

Bernalillo Co., New Mexico

Sandy soil

SFSU

92

F2285

A. fiardiih

Fr. Martinique

JF797201

JF

26/09/2000

Sainte-Anne

In forest

LIP

92

RM05/156

A. fiardiih

Fr. Martinique

 

RC

27/08/2005

Trinité, La Caravelle

In forest

LIP

92

F2286L

A. fiardiih

Fr. Martinique

 

JF

29/09/2000

Saint-Anne, Morne Manioc

In forest

LIP

93

NTF67

A. sp.h

Thailand

JF514529

SK

17/08/2010

Chiang Rai, Muang

In forest

 

93

ZRL2036

A. sp.h

Thailand

 

RL

25/06/2005

Chiang Mai, Mok Fa W

In forest

BBH

93

NT001

A. sp.h

Thailand

 

SK

150/8/2009

Chiang Rai, Khun Kone W

In forest

 

93

ZRL2134

A. sp.h

Thailand

 

KH

28/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

95

Thoen7297

 

Senegal

JF514542

DT

14/10/1984

Dakar

In market

BR

96

ZRL2127

 

Thailand

JF691547

KH

16/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

96

OR71

 

Thailand

 

OR

03/07/2010

Chiang Mai, Mae Taeng

In forest

 

96

ZRL3028

 

Thailand

 

ZR

05/06/2006

Chiang Mai, Doi Inthanon

In forest

BBH

96

CA798

 

Thailand

 

JG-SK

28/07/2010

Chiang Mai, Nat.Park

In forest

 

96

ZRL2053

 

Thailand

 

DD

27/06/2005

Chiang Mai, Doi Inthanon

In forest

BBH

100

F2815

A. martinicensis

Fr. Martinique

JF727855

JF-RC-CLu

02/09/2003

Prêcheur, Anse Couleuvre

In forest

LIP

100

CL/MART03.055

A. martinicensis

Fr. Martinique

 

CL

02/09/2003

Prêcheur, Anse Couleuvre

In forest

LIP

100

F2343

A. martinicensis

Fr. Martinique

 

JF

19/11/2000

Prêcheur, Anse Lévrier

In forest

LIP

101

SP307818

A. martinezianus

Brazil

JF896227

UP

14/12/2001

Sao Paulo

On soil

 

102

NTT37

 

Thailand

JF514537

KW

20/06/2010

Chiang Mai, Mae Taeng

In forest

 

102

ZRL2110

 

Thailand

 

ZR

03/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

102

ZRL3039

 

Thailand

 

TBa

08/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

104

CA848

 

Thailand

JF727864

JG-GB

25/07/2010

Chiang Mai, University P

In forest

 

105

ZRL3088

 

Thailand

JF691543

ZR

02/08/2006

Chiang Rai, Pamae Lao P

In forest

BBH

105

NTSCR1

 

Thailand

 

SK

08/03/2010

Chiang Rai, Muang

In forest

 

106

NTT33

 

Thailand

JF514535

KW

18/06/2010

Chiang Mai, DSPNP

In forest

 

106

NTS73

 

Thailand

 

SK

30/06/2010

Chiang Mai, Mae Taeng

In forest

 

107

NTF26

 

Thailand

JF514526

NT

29/06/2010

Chiang Mai, Doi Suthep

In forest

 

108

CA846

 

Thailand

JF727865

JG-GB

25/07/2010

Chiang Mai, University P

In forest

 

109

ZRL2044

 

Thailand

JF691540

ZR

26/06/2005

Chiang Mai, Mae Taeng

In forest

BBH

109

ecv3614

 

Thailand

HM488742

EV

 

From GenBank

  

110

NT055

 

Thailand

JF727846

SK

15/08/2009

Chiang Mai, DSPNP

In forest

 

111

ADK2905

(A. cf bulbillosus)

Benin

JF514520

AK

19/09/2000

Wari Maro, Borgou

In sandy soil

BR

111

CA847

 

Thailand

 

JG-GB

25/07/2010

Chiang Mai, University P

In forest

 

112

ZRL3101

 

Thailand

JF691544

ZR

13/09/2006

Chiang Mai, Mae Taeng

In forest

BBH

113

NT62

 

Thailand

JF727845

SK

20/08/2009

Chiang Mai, Mae Taeng

In forest

 

114

NTT72

 

Thailand

JF514539

SK

07/03/2010

Chiang Mai, Mae Taeng

In forest

 

115

CA843

 

Thailand

JF727866

JG-GB

04/08/2010

Hua-Hin, Golf

In grassland

 

116

ZRL3080

 

Thailand

JF691542

ZR

10/07/2006

Chiang Mai, Mae Taeng

In forest

BBH

116

NTF063

 

Thailand

 

NT

28/07/2010

Chiang Mai, Mae Taeng

In forest

 

117

ZRL3091

 

Thailand

JF691546

ZR

02/08/2006

Chiang Rai, Pamae Lao P

In forest

BBH

119

MATA774

 

Mexico

JF727871

GM-PC

29/11/2007

Veracruz, San Andrés Tuxtla

In dune

XAL

120

ADK2751

A. goossensiae

Benin

JF514519

AK

05/06/2000

Calavi Campus, Atlantique

In sandy soil

BR

126

ZRL3056

 

Thailand

JF691541

ZR

12/06/2006

Chiang Mai, Mae Taeng

In forest

BBH

127

ZRL3102

 

Thailand

JF691545

ZR

15/09/2006

Chiang Mai, Ob Luang P

In forest

BBH

128

LD030

 

Thailand

JF514521

JC

03/08/2010

Chiang Rai, MFL University

On litter

MFLU

128

ZRL2124

 

Thailand

 

KH

12/08/2005

Chiang Mai, Mae Taeng

In forest

BBH

128

NTS106

 

Thailand

 

SK

24/07/2010

Chiang Mai, Mae Taeng

In forest

 

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

hA. subrufescens complex

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 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 8.0 100 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% ß-mercaptoethanol 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 ITS1-Rev323, Kerrigan 2007) and ITS4 for the latter.

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 T-Coffee 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 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 non-Agaricus 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 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.

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.
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

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 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.
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

Number of tropical species

Species found in:

Identified tropical species

2 con-tinents

ASb

AFb

AMb

(see also Fig. 1 and Table 1)

Species of other genera in a single clade

100/100–100

1–4

4

0

3

1

0

cf. He. splendidissimus, Hy. ardosiicolor

Sections based on temperate species

Bivelares

98/98–100

28–29

0

0

0

0

0

 

Chitonioides

81/55–98

30–33

0

0

0

0

0

 

Xanthodermatei

85-

34–48

10

3

7

3

3

A. microvolvatulus, A. xanthosarcus

        

A. aff. endoxanthus, A. aff. volvatulus

Sanguinolenti I + II + III (3 clades)

84–87, 83–56, 89/62–100

49–56

2

0

2

0

0

 

Agaricus

92–99

57–62

3

1

2

0

2

A. aff. argyropotamicus

Spissicaules (polytomies with 3 clades)

95/86–100, 88–70, 99/93–100

72–81

3

0

0

1

2

 

Arvenses

99/87–100

92–99

4

0

2

1

1

A. fiardii

Minores

89–51

115–128

8

0

6

1

1

A. Goossensiae

Isolated branch (A. aridicola)

 

103

0

0

0

0

0

 

Tropical branches and clades of presumed species found in Africa and/or in Asia (with the exception of species 88 in clade TR III)

Tropical clade TR I

91–82

11–20

10

0

8

2

0

A. inoxydabilis

Tropical clade TR III

100/100–100

83–88

6

0

2

3

1

A. kivuensis, A. aff. impudicus

Tropical clade TR V

93/89–100

104–107

4

0

4

0

0

 

Tropical clade TR VI

93/55–92

108–111

4

1

4

1

0

 

Tropical clade TR VII

99/91–100

112–113

2

0

2

0

0

 

Tropical clade TR a (poorly supported)

59–60

8–9

2

0

2

0

0

 

Tropical clade TR b (poorly supported)

82-

22–27

6

0

4

2

0

A. campestroides, A. trisulphuratus

Isolated tropical branches

 

5, 6, 7, 10, 21

8

1

7

2

0

A. heterocystis, A. subsaharianus

  

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

A. aff. rufoaurantiacus, A. cf. floridanus

Tropical clade TR IV

97/96–100

89–91

3

0

0

0

3

A. deserticola

Tropical clade TR c (poorly supported)

85–61

63–64

2

0

0

0

2

 

Tropical clade TR d (poorly supported)

67-

100–101

2

0

0

0

2

A. martinicensis, A. martinezianus

Isolated branch

 

82

1

0

0

0

1

 

Total for tropical species of Agaricus

 

5–128

86

6

52

16

24

 

In sections

  

30 (35%)

4

19

6

9

 

Out of sections

  

56 (65%)

2

33

10

15

 

a 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

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 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 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.

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 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 (23-24) 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 and their circumscription need consideration. Only one-third 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 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.

Notes

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 DEB-0118776 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.

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Copyright information

© Kevin D. Hyde 2011

Authors and Affiliations

  • Ruilin Zhao
    • 1
  • Samantha Karunarathna
    • 2
  • Olivier Raspé
    • 3
  • Luis A. Parra
    • 4
  • Jacques Guinberteau
    • 5
  • Magalie Moinard
    • 5
  • André De Kesel
    • 3
  • Gérard Barroso
    • 5
  • Régis Courtecuisse
    • 6
  • Kevin D. Hyde
    • 2
    • 7
  • Atsu K. Guelly
    • 8
  • Dennis E. Desjardin
    • 9
  • Philippe Callac
    • 5
  1. 1.Key Laboratory of Forest Disaster Warning and Control in Yunnan Province, Faculty of Conservation BiologySouthwest Forestry UniversityKunmingChina
  2. 2.School of ScienceMae Fah Luang UniversityChiang RaiThailand
  3. 3.National Botanic Garden of BelgiumMeiseBelgium
  4. 4.Aranda de DueroSpain
  5. 5.INRAVillenave d’OrnonFrance
  6. 6.Département de botanique, Faculté des sciences pharmaceutiques et biologiquesLilleFrance
  7. 7.Botany and Microbiology Department, College of ScienceKing Saud UniversityRiyadhSaudi Arabia
  8. 8.Département de Botanique, Faculté des Sciences, Université de LoméLoméTogo
  9. 9.Department of BiologySan Francisco State UniversitySan FranciscoUSA

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