Evolution of the Aspergilli: stuff for genomicists to think about David M. Geiser Penn State University, USA [email protected].

Evolution of the Aspergilli:stuff for genomicists to think about

David M. Geiser

Penn State University, USA

[email protected]

Aspergillus evolution

• What is Aspergillus?

• The major players in the genus and how are they related

• Interesting comparative questions about Aspergillus

• Studying evolution using genomics and studying genomics using evolution

What is Aspergillus?

• Aspergillus is a Eurotiomycete (or “Plectomycete”)• Aspergillus species appear to have a common evolutionary

origin– this is a weak inference

• Aspergillus species are diverse in their life histories– Homothallic– Heterothallic– Not seen to undergo sex

• Nine different described sexual stages (Emericella, Eurotium, Neosartorya, Petromyces, Fennellia, Sclerocleista, Warcupiella, Hemicarpenteles, Chaetosartorya)

Aspergillus is a EurotiomyceteAscosphaera apis

Eremascus albusAuxarthron zuffianumMalbranchea dendritica

Coccidioides immitisUncinocarpus reesiiRenispora flavissimaCtenomyces serratus

Trichophyton rubrumGymnoascoideus spetalosporusBlastomyces dermatididusChrysosporium pavumHistoplasma capsulatum

Elaphomyces maculatusElaphomyces leveillei

Emericella nidulansEupenicillium javanicum

Eurotium rubrumNeosartorya fischeriMerimbla ingelheimenseMonascus purpureusTalaromyces bacillisporusTrichocoma paradoxaCapronia pilosellaCapronia mansonii

Lecanora dispersa

Cladia aggregataCladonia bellidiflora

Solorina acroceum

Microascus cirrosusNeurospora crassaOphiostoma ulmi

Leptosphaeria doliolumPleopsora herbarum

Cochliobolus sativus

Dothidea hippophaeosAureobasidium pullulans

Sclerotinia sclerotiorum

Cudonia confusaSpathularia flavida

Blumeria graminis

Morchella elataPlectania nigrella

Saccharomyces cerevisiae

Neolecta vitellina

Byssochlamys nivea

Ascosphaerales

Chaetothyriales

Lecanorales

Peltigerales

Dothideales

Pleosporales

MicroascalesSordariales

OphiostomatalesScletotiniales

Erysiphales

Pezizales

93

98

100

95

98

100

59

82 94

100

71

99

69

89

9674

100

69

87

92

57

7160 54

Helotiales

10064

Eurotiomycetes

Schizosaccharomyces pombe

Onygenales

Eurotiales

Pezizomycotina

Geiser andLo Buglio2001

18S rDNA

Eurotiomycetes• Thallic (Ascosphaerales, Onygenales) and Phialidic (Eurotiales)

anamorphs• Asci produced naked, in wefts of hyphae (gymnothecia) or in a solid

enclosed fruiting body (cleistothecia)• Cleistothecia may be borne in a stroma• Many human and animal pathogens

– Onygenales: Cocci, Histo, Blasto, dermatophytes– Eurotiales: Aspergillus, P. marneffei

• Very chemically creative• An ectomycorrhizal fungus! (Elaphomyces)• NO LICHENIZED FUNGI• Osmophiles, thermophiles• Industrial fungi

Conidiation in the Plectomycetes• thallic (T): Onygenales, Ascosphaerales

• blastic (B): Eurotiales

B

BB

B

T

T

T

Geiser and LoBuglio 2001

biseriate

uniseriate

Ascomatal types in the Eurotiomycetes• cleistothecia borne in a stroma (C/S)

• cleistothecia not borne in a stroma (C)

• stroma, with no cleistothecia (S)

• no cleistothecia and no stroma (N)

C/SC/S

C/S

S

S

CC

CN

nutriocyte

Geiser and LoBuglio 2001

Emericella

Petromyces

Onygena

Trichocoma

Eupenicillium

Eurotium

Gymnoascus

MonascusByssochlamys

Ascosphaera

Sexual stages associated phylogenetically with important

Aspergillus spp.• Emericella and Aspergillus nidulans

– True cleistothecium surrounded by a stroma of hülle cells

• Petromyces and A. flavus/oryzae/parasiticus/niger (?)– Cleistothecia embedded in a stroma (sclerotia)

• Neosartorya and A. fumigatus– Cleistothecia/gymnothecia with wall composed of

cottony hyphae.

Aspergillus phylogenetics

• Not a lot of data useful for inferring large-scale relationships

• Take many of the trees I show with a grain of salt…

Aspergillus -- monophyletic?

Tamura, M.Kawahara, K.Sugiyama, J.2000.

18S rDNA

Aspergillus 25S rDNA tree

• Based on a single locus, ~600 bp

• Backbone of tree gets poor support

• Major traditional species groups are supported

• The morphologists did/are doing a pretty good job

Petromycessexual stagemultiloculate stroma

Emericella sexual stagecleistothecium surroundedby hülle cells (stroma)

Neosartorya sexual stagecleistothecium composedof cottony hyphae

Eurotium sexual stagecleistothecium, nostroma

Fennellia sexual stage

Chaetosartorya sexual stage

WarcupiellaSclerocleista

psychrotoleranceXerophilicty/toleranceheat-resistant ascospores

Thermotoleranceheat-resistant ascospores

SubgenusCircumdati:Association with plants,seeds

The players in Aspergillus genomics

• Aspergillus nidulans– “Emericella clade”– Aspergillus nidulans group– Aspergillus section Nidulantes

• Aspergillus fumigatus

• Aspergillus oryzae

Emericella clade

• A. nidulans• A. sydowii - coral

pathogen• Hülle cells• Homothallics• Putative asexuals• Heterothallic

NJ analysis of Steve Peterson’s 25S data

A sydowii NRRL 4768A caesiellus NRRL a14879

A versicolor NRRL 4838Em unguis NRRL 5041A mellinus NRRL 6328Em unguis NRRL 2393

A caespitosus NRRL1929Em heterothallica NRRL 5097Em astellata NRRL 2397Em variecolor NRRL 4736

A eburneocremeus NRRL 4773A crustosus NRRL 4988

A aeneus NRRL 4769Em spectabilis NRRL 6363

Em bicolor NRRL 6364A egyptiacus NRRL 5920Em desertorum NRRL 5921

Emericella nidulans NRRL 4266Em dentata NRRL 4908Em echinulata NRRL 2395

A recurvatus NRRL 4902Em violacea NRRL 4178Em rugulosa NRRL 4581Em quadrilineata NRRL 4992Em acristata NRRL A9919Em parvithecia NRRL 4904

Em fruticulosa NRRL 4903A multicolor NRRL 4775

A puniceus NRRL 4991A pseudodeflectus NRRL 6135

A granulosus NRRL1932A spelunceus NRRL 4990A aureolatus NRRL 5126

A asperescens NRRL 5036A varians NRRL 4793

A cavernicola NRRL 6327A amylovorus NRRL 5813

A subsessilis NRRL 3752A ivoriensis NRRL22883

A raperi NRRL 5039A deflectus NRRL 4993

A lucknowensis NRRL3491A elongatus NRRL 5176

Em aurantiobrunnea NRRL 4545A bisporus NRRL 3693

A silvaticus NRRL2398

92

100

93

70

77

79

98

92

87

100

95

Aspergillosis of sea fan corals

Kiho Kim

Aspergillus sydowiiin culture: a relative ofA. nidulans!

Aspergillosis of sea fan coral

Geiser, D.M.Ritchie, K.Smith, G.Taylor, J.W.Nature, 1998

Sexual versus asexual strategies• Aspergillus heterothallicus - described by June

Kwon-Chung (Raper and Fennell 1965)• One of three known heterothallic species (other two

are Neosartoryas, clademates with A. fumigatus)• Known asexuals should be predicted to be

recombining:– Population genetic evidence: a rich literature

• Taylor et al. 2000, Annu. Rev. Phytopath.

– MAT locus molecular genetics/phylogenetics• Turgeon, Berbee and others

• Homothallics do not necessarily outcross less than heterothallics!

Known heterothallics

Early eventsin ascus

developmentin the

Eurotiales:designed for

homothallism?

Benjamin, 1955


• Aspergillus nidulans

• Aspergillus fumigatus– Neosartorya clade– Aspergillus fumigatus group– Aspergillus section Fumigati

• Aspergillus oryzae

N. fischeriA. fumigatusA. fumigatus

N. spinosaA. viridi-nutans

N. aureolaN. spathulata AN. spathulata a

N. quadricinctaA. brevipes

A. duricaulisN. hiratsukae

N. glabra

N. fennelliae AN. fennelliae a

N. aurataN. stramenia

A. unilateralis

N. pseudofischeriA. clavatus

var. ellipticus100

9788

99

90

100

100

100

78

combined analysis1 of 9 MP trees

length 695 steps

99

9

9

9

6

3

3

5 11

1

1

11

11

Geiser, D.M.Frisvad, J.Taylor, J.W.Mycologia,1998

Beta-tubulin +rodA hydrophobin

heterothallic

heterothallic

N. fischeriN. spinosa

A. brevipes A. duricaulis

A. unilateralisA. fumigatus

A. fumigatusvar. ellipticusA. viridi-nutans

N. aureolaN. spathulata a N. spathulata A

N. hiratsukaeN. glabra

N. fennelliae A N. fennelliae a


N. quadricinctaN. pseudofischeri

A. clavatus

N. fischeri

N. spinosa

A. brevipesA. duricaulis

A. unilateralis

A. fumigatusA. fumigatus

var. ellipticusA. viridi-nutans

N. aureolaN. spathulata aN. spathulata A




N. quadricincta

N. pseudofischeri A. clavatus

A. Meiosis lost once B. Heterothallism gained once C. Heterothallism ancestral

1 MP tree length 738 steps

1 MP treelength 698 steps

1 MP treelength 709 steps

N. fischeri

N. spinosa


A. unilateralis

A. fumigatusA. fumigatus

var. ellipticus A. viridi-nutans

N. aureola

N. spathulata aN. spathulata A

N. hiratsukae

N. glabra



N. quadricincta N. pseudofischeri

A. clavatus

significantly longer than MP tree

significantly longer than MP tree

not significantly longerthan MP tree

Geiser, D.M.Frisvad, J.Taylor, J.W.Mycologia 1998


Geiser, D.M.Frisvad, J.Taylor, J.W.Mycologia 1998


54

8790

59

5489

55

N. fischeriA. fumigatus

A. fumigatus var. ellipticus

N. spinosaA. viridi-nutans

N. aureolaN. spathulata m.t. aN. spathulata m.t. A

N. quadricinctaA. brevipes

A. duricaulisN. hiratsukae

N. glabraA. unilateralis


N. fennelliae m.t. aN. fennelliae m.t. A

N. pseudofischeriA. clavatus

sec. met. datastrict consensusof 19 MP treeslength 95 steps

combined molecular datastrict consensus

of 9 MP treeslength 695 steps

10097

90

99

100

78

100

100

A. B.N. fischeriA. fumigatus

A. fumigatus var. ellipticus

N. spinosa

A. viridi-nutans

N. aureola

N. spathulata m.t. aN. spathulata m.t. A

N. quadricincta



A. unilateralis

N. aurata

N. stramenia

N. fennelliae m.t. aN. fennelliae m.t. A

N. pseudofischeri

A. clavatus

Evolution in Aspergillus fumigatus group

• Monophyletic• A. fumigatus and N. fischeri are close

relatives• Heterothallism derived?• Secondary metabolite data contrasts with

benA/rodA phylogeny• Pringle, Taylor and others: asking the

question whether it’s asexual


• Aspergillus nidulans

• Aspergillus fumigatus

• Aspergillus oryzae– Petromyces clade– Aspergillus flavus group– Aspergillus Section Circumdati

A. flavus, A. oryzae, A. niger and relatives (Petromyces)

A ochraceus NRRL4565A bridgeri NRRL13078A sclerotiorum NRRL 4901A sulph var crass NRRL 5584

A sulphureus NRRL 6161A auricomus NRRL397

A elegans NRRL 4850A insulicola NRRL 6138A ochraceopetaliformis NRRL 47A ostianus NRRL423A melleus NRRL 5103A petrakii NRRL 4748

A tamarii NRRL20818A flavofurcatus NRRL 4911A terricola NRRL 426A tamariiA caelatusA sp. NRRL 25517

A oryzae NRRL 506A sojae NRRL1988A thomiiA kambarensis NRRL3751A oryzae var effusus NRRL 1958A oryzae NRRL 458A flavus var columnaris NRRL 4A subolivaceus NRRL 4998A parasiticusA flavusA kambarensis NRRL 3751

P albertensis NRRL 20602P alliaceusP alliaceus NRRL 5108A lanosus NRRL 3648

A leporis NRRL 6599A bombycis

A nomiusA avenaceus NRRL 517

A phoenicis NRRL1956A niger NRRL 363A carbonarius NRRL 4849

A ellipticus NRRL 5120A japonicus NRRL A661A aculeatus NRRL 360

A. ochraceus and relatives

A. niger and relatives

A. flavus, A. parasiticus,A. oryzae and relatives

6574

7485

8699

100

73

70

79

76

71

77

100100

9897

NJ analysis of Steve Peterson’s 25S data

B1DFB2

PEIJ

KLMOCA. oryzae 469

AGHN

A. parasiticus CA1-05A. parasiticus CA3-01

amdS121 of 2 MP trees

CI = 0.9625RI = 0.9730

RC = 0.9365

B1D

EB2IKMOP

A

F

G

JL

HN

CA. oryzae 469

A. parasiticus CA1-05


benA561 of 6 MP trees

CI = 1.000RI = 1.000

RC = 1.000

B1EB2LC

FJ

OP

DI

KM

AGHN

A. oryzae 447, 448, 449, 469


B1B2OIM

KC

DE

J

PF

LA

HGN

A. oryzae 469



pecA121 of 30 MP trees

CI = 0.9048RI = 0.9245

RC = 0.8365

100

73

B1D

B2L

MCEF

JKO

PI

AGHN

A. oryzae 469


trpC131 MP tree

CI = 0.9167RI = 0.9412

RC = 0.8627

100

95

A. flavus Group I A. flavus Group II

omt121 of 4 MP trees

CI = 0.9036RI = 0.9623

RC = 0.8695

100

100

98

100

100

80

77

87

86

96

9872

100

100

G

H

N

A

Q*B

C*

D

EF

I JK

LM

O

P

parsimony analysis 11-locus RFLP haplotypes

Geiser, DMPitt, JITaylor JWPNAS1998

A55A120A399tx-18-11Stx19-21Stx20-32Stx21-1S1-2612-37-27-4A111A130A150A. oryzae NRRL 448A. oryzae NRRL 449A. oryzae NRRL 469F14F601-91-295-111-417-4F15F353-27-314-114-2tx12-10-2S4-212-413-4NRRL A-116121-22A. parasiticus CA1-05A. parasiticus CA3-01

Group II

Group I

California

Texas

Australia

California

Georgia

Australia

Georgia

Australia

Texas

AustraliaNigeria

AustraliaCalifornia

60

6263

100

90

92

86

84

61

93

94

98

99

100

Small sclerotia High B-aflatoxin No G-aflatoxin

Large sclerotia No aflatoxin

Large sclerotia High B aflatoxin No G aflatoxin

Small sclerotia Low B-aflatoxin Low G-aflatoxin Southern Hemisphere?

3-gene combined genealogy of Aspergillus flavus

I-A

I-B

I-C

(most of data come from omt12: a toxin locus!)

= A. flavus var. parvisclerotigenus?

Geiser, DMHorn, BWDorner, JWTaylor JWFG&B2001

Using evolution to do good genomics

• Comparative genomics IS evolutionary biology

• Conservation of function is an adaptive process

• Clark et al. 2003. Inferring non-neutral evolution from human-chimp-mouse orthologous gene trios. Science 302; 1960-62.

The future

• Genomics helps traditional evolutionary biologists to do their jobs because it provides tools

• Understanding how genes evolve provides a framework for thinking about how they might function

Evolution of the Aspergilli: stuff for genomicists to think about David M. Geiser Penn State University, USA [email protected].

Documents

aspergillus evolution

important aspergillus

aspergillus phylogenetics

evolution slide

chaetosartorya slide

asexual slide

s data slide

s rdna slide