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Aspergillus evolution

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					Evolution of the Aspergilli: stuff for genomicists to think about

David M. Geiser Penn State University, USA dgeiser@psu.edu

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 Eurotiomycete
93

Eurotiomycetes
82

100 94

Ascosphaera apis Ascosphaerales Eremascus albus Auxarthron zuffianum Malbranchea dendritica Coccidioides immitis Uncinocarpus reesii Renispora flavissima Onygenales Ctenomyces serratus Trichophyton rubrum Gymnoascoideus spetalosporus Blastomyces dermatididus Chrysosporium pavum Histoplasma capsulatum Byssochlamys nivea Elaphomyces maculatus Elaphomyces leveillei

100

95

71

18S rDNA
69 98 60

59

99

Emericella nidulans
Eupenicillium javanicum 54 71

100 98

89

Pezizomycotina

57

100 64 74

96 92

100 69

87

Merimbla ingelheimense Monascus purpureus Talaromyces bacillisporus Trichocoma paradoxa Capronia pilosella Chaetothyriales Capronia mansonii Cladia aggregata Cladonia bellidiflora Lecanorales Lecanora dispersa Solorina acroceum Peltigerales Dothidea hippophaeos Dothideales Aureobasidiumpullulans Cochliobolus sativus Leptosphaeria doliolum Pleosporales Pleopsora herbarum Microascus cirrosus Microascales Neurospora crassa Sordariales Ophiostoma ulmi Ophiostomatales Sclerotinia sclerotiorum Scletotiniales Blumeria graminis Erysiphales Cudonia confusa Helotiales Spathularia flavida Morchella elata Pezizales Plectania nigrella Saccharomyces cerevisiae Schizosaccharomyces pombe Neolecta vitellina

Eurotium rubrum Neosartorya fischeri

Eurotiales

Geiser and Lo Buglio 2001

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

biseriate B

T

T

B

B B uniseriate

T

Geiser and LoBuglio 2001

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)
Trichocoma

Emericella Petromyces

Eupenicillium

C/S
Gymnoascus

C/S C/S
Onygena

S S

Monascus
Byssochlamys Ascosphaera Eurotium

C
Geiser and LoBuglio 2001

C

C

nutriocyte

N

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

18S rDNA

Aspergillus -monophyletic?

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

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

Petromyces sexual stage multiloculate stroma

Emericella sexual stage cleistothecium surrounded by hülle cells (stroma)

Sclerocleista Warcupiella

Eurotium sexual stage cleistothecium, no stroma

Neosartorya sexual stage cleistothecium composed of cottony hyphae Fennellia sexual stage Chaetosartorya sexual stage

Subgenus Circumdati: Association with plants,seeds

psychrotolerance Xerophilicty/tolerance heat-resistant ascospores Thermotolerance heat-resistant ascospores

The players in Aspergillus genomics
• Aspergillus nidulans
– “Emericella clade” – Aspergillus nidulans group – Aspergillus section Nidulantes

• Aspergillus fumigatus • Aspergillus oryzae

Emericella clade
NJ analysis of Steve Peterson’s 25S data

• A. nidulans • A. sydowii - coral pathogen • Hülle cells • Homothallics • Putative asexuals • Heterothallic

A sy dowii NR RL 47 68 92
1 00 87 A ca es ie ll s NR RL a1 4 87 9 u Em u ng u s N RR L 6 3 28 A me lln u is N RR L 5 0 41 i

A v er sic olorNR RL 48 38

1 00 Em u ng u is N RR L 2 3 93 A ca es p itos u s N RR L1 9 29 Em heter othallica NR RL 50 97

Em a ste llata NR RL 2 39 7 Em v ar ie co lo r N RR L 4 7 36 A eb ur n e oc r em eu s NR RL 47 7 3 A cr u sto su s NR RL 49 8 8 A ae ne u s N RR L 4 7 69 Em s pe c tab ilis N RR L 6 3 63 Em b ico lor N RR L 6 3 64 95 A eg yp tia cu s N RR L 59 2 0 Em d es e rto r um NR RL 5 92 1

Em d ene ch in ula ta N 90 8L 2 3 95 a Em ta t N RR L 4 RR A re c ur v atu s N RR L 49 0 2 Em v iola ce a NR RL 41 7 8 Em r ug u los a NR RL 45 8 1 Em q ua d rilin ea ta N R RL 49 9 2 is tata RL 7 7 Em a cr v ithe cNRNR RA9 9 19 4 Em p ar a i L 4 90 Em fr utic ulo s a N RR L 4 9 03 A mu ltc o lor NR RL 4 77 5 i A pu nic e us NR RL 4 99 1 A ps eu d od e flec tus NR R L 6 13 5 A gr a nu los u s N RR L 19 3 2 A sp elu n ce u s N RR L 4 9 90 9 2 A au r eo latu s NR RL 51 2 6 A 7 9 A va r ia n s N RR L 4 7 93 as pe r e sc e ns NR RL 5 03 6 A ca ve r n i o la N RR L 6 3 27 c A am ylo v or u s N RR L 5 8 13 93 A su bs e ss ilis N RR L 3 7 52 A i o rie n sis NR RL 2 28 8 3 v A ra p er i NR RL 5 03 9 70 A de flec tus N RR L 4 99 3 A l c kn o we n sis NR RL 3 49 1 u 98 A elo ng a tus NR RL 5 17 6 Em a ur a ntio b ru n ne a N R RL 45 4 5 A bis po r us N RR L 3 69 3 A silv atic us NR RL 2 39 8

Em er ice lla nidula ns NR RL 42 66

Aspergillosis of sea fan corals

Aspergillus sydowii in culture: a relative of A. nidulans!

Kiho Kim
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 events in ascus development in the Eurotiales: designed for homothallism?

Benjamin, 1955

The players in Aspergillus genomics
• Aspergillus nidulans • Aspergillus fumigatus
– Neosartorya clade – Aspergillus fumigatus group – Aspergillus section Fumigati

• Aspergillus oryzae

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

Beta-tubulin + rodA hydrophobin

N. fischeri • 9 A. fumigatus 88 A. fumigatus 100 90 3 var. ellipticus N. spinosa 6 A. viridi-nutans N. aureola 99 • 9 N. spathulata A heterothallic 100 N. spathulata a 1 5 1 N. quadricincta 1 78 A. brevip es 1 A. durica ulis N. hiratsukae 1 1 N. glabra 1 1 A. unila teralis • 9 N. fennelliae A heterothallic N. fennelliae a 100 • 9 N. aurata 100 N. stramenia N. pseud ofischeri A. clavatus combine d analysis 1 of 9 M P tre e s le ngth 6 95 ste ps • 9 3 97

A. M e iosis lost once
N. fisch eri N. spi nosa

B. He te rothallism gaine d once He te rothallism ance str al C.
N. fisch eri A. fumi gatus A. fumi gatus va r. el l ip ti cu s N. spi nosa A. vi rid i-nu ta ns N. aureol a N. spathul ata a N. spathul ata A N. fenn el li aeA N. fenn el li aea N. hi ratsukae N. gl ab ra A. un il ateral i s N. au ra ta N. stramen ia N. qu adri ci ncta A. brevip es A. du ri ca ul is N. pseud ofisch eri A. cl avatus

A. brevip es A. du ri ca ul is A. un il ateral i s A. fumi gatus A. fumi gatus A. vi rid i-nu ta ns va r. el l ip ti cu s N. aureol a N. spathul ata a N. spathul ata A N. hi ratsukae N. gl ab ra N. fenn el li aeA N. fenn el li aea N. au ra ta N. stramen ia N. qu adri ci ncta N. pseud ofisch eri A. cl avatus

N. fisch eri A. fumi gatus A. fumi gatus va r. el l ip ti cu s N. spi nosa A. vi rid i-nu ta ns N. aureol a N. pseud ofisch eri N. qu adri ci ncta A. brevip es A. du ri ca ul is N. au ra ta N. stramen ia N. gl ab ra A. un il ateral i s N. hi ratsukae N. fenn el li aeA N. fenn el li aea N. spathul ata a N. spathul ata A A. cl avatus

1 MP tre e le ngth 73 8 ste ps

1 MP tre e le ngth 69 8 ste ps

1 MP tre e le ngth 70 9 ste ps

significantly longe r than M P tr e e
Geiser, D.M. Frisvad, J. Taylor, J.W. Mycologia 1998 Beta-tubulin + rodA hydrophobin

not significantly longe r than M P tr e e

significantly longe r than M P tr e e

A.
54 55

N. fischeri A. fumigatus A. viridi-nutans N. pseud ofischeri N. fennelliae m.t. a N. fennelliae m.t. A N. quadricincta N. spinosa N. spathulata m.t. a N. spathulata m.t. A N. hiratsukae N. glabra A. brevip es A. durica ulis N. aurata N. aureola N. stramenia A. unila teralis A. clavatus

B.

N. fischeri A. fumigatus
100

97 90 99

A. fumi gatus va r. el l ip ti cu s A. fumi gatus va r. el l ip ti cu s

90 87 59 54 89

N. spinosa A. viridi-nutans N. aureola N. spathulata m.t. a N. spathulata m.t. A N. quadricincta A. brevip es A. durica ulis N. hiratsukae N. glabra A. unila teralis N. aurata N. stramenia N. fennelliae m.t. a N. fennelliae m.t. A N. pseud ofischeri A. clavatus

100

78

100 100

Geiser, D.M. Frisvad, J. Taylor, J.W. Mycologia 1998 Beta-tubulin + rodA hydrophobin

sec. met. data strict con sensus of 19 MP trees length 95 steps

combined molecular data strict con sensus of 9 MP trees length 695 steps

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

The players in Aspergillus genomics
• Aspergillus nidulans • Aspergillus fumigatus • Aspergillus oryzae
– Petromyces clade – Aspergillus flavus group – Aspergillus Section Circumdati

A. flavus, A. oryzae, A. niger and relatives (Petromyces)
85 74 65 100
99
A b rid g erN RR L1 3 07 8 i A s cle r otio r um RR L 90 1 N 4 A s ulp h ar c ra s s RR L 58 4 v N 5 A s ulp h ur e us RR L 16 1 N 8 6 A a ur ic om uN RR L3 9 7 6 s A e leg a ns RR L 85 0 N 4 A in su lico la RR L 13 8 N 6 A o ch r ac e op e talifo rm is N RR L 7 4 A o stia nu s RR L4 2 3 N A m elle usN RR L 10 3 5 A p etr a kii RR L 74 8 N 4

74

A ochra ce us NR RL45 65

A. ochraceus and relatives

76

A flav o fur c atuNR RL20 81 8 tam ar ii s RR L 91 1 A N 4
A te rr ic ola RR L 26 N 4 A ta ma r i A c ae la t s u A s p.N RR L 55 1 7 2

A oryza e NR RL 50 6 Ath om iiae NR RL19 88 s oj A

79

77 70 73 98 100
97

A oryza e NR RL 45 8 A flav u s ar c olu mn a r i RR L v s N 4 A ub o RR 4 7 1A spar liv a ce uNsus L 99 8 as itic A flav am b ar e ns is RR L 75 1 A k us N 3

A k am b ar e ns is RR L3 7 51 N A o ry z ae ar e ff s usN RR L 95 8 v u 1

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

100

A niger NR RL ar b on a r iuN RR L 84 9 A c 36 3 s 4 A e llp ticu s RR L 12 0 i N 5 A ja po n i u s RR L c N A66 1 A a cu le atu s RR L 60 N 3

A a ve n ac e us RR L 17 N 5 A p ho e nic is RR L1 9 56 N

A bombycis A nomius

P a lbe r ten s i RR L 06 0 2 s N 2 P a lla c eu s i P a lla c eu s RR L 10 8 i N 5 A la no s us RR L 64 8 N 3 A le po r is RR L 59 9 N 6

A. niger and relatives

NJ analysis of Steve Peterson’s 25S data

amdS12 1 of 2 MP trees
CI = 0.9625 RI = 0.9730 RC = 0.9365 80

100

100 77 100 A. parasiticus CA1-05 A. parasiticus CA3-01

B1 D F B2 P E I J K L M O C A. oryzae 469 A G H N

B1 D

benA56 1 of 6 MP trees
CI = 1.000 RI = 1.000 RC = 1.000 100

87

A H N J L F

E B2 I K M O P G 72

86 96

B1 E B2 L C F J O 98 P

omt12 1 of 4 MP trees
CI = 0.9036 RI = 0.9623 RC = 0.8695 D

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

C A. oryzae 469

I K M 98 A. oryzae 447, 448, 449, 469 A G 100 H N A. parasiticus CA1-05 A. parasiticus CA3-01 100

pecA12 1 of 30 MP trees
CI = 0.9048 RI = 0.9245 RC = 0.8365 73 100 G N

B1 B2 O I M K C D E J A. oryzae 469 P F L A H

B1 D

trpC13 1 MP tree
CI = 0.9167 RI = 0.9412 RC = 0.8627 95 100

B2 L M C E F J K O P I A. oryzae 469

E D C* B

F

I

J

K

L M O P

Geiser, DM Pitt, JI Taylor JW PNAS 1998
G

Q*

A G H N

H A parsimony analysis N 11-locus RFLPhaplotypes

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

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

A. flavus Group I

A. flavus Group II

I-A 100 Group I 90

60 62

63 I-B 92

98 84 I-C 94 100 Group II 93

86 61

99

A5 5 A1 2 0 A3 9 9 tx-1 8-11 S tx1 9-2 1S tx2 0-3 2S tx2 1-1 S 1 -2 6 1 2-3 7 -2 7 -4 A1 1 1 A1 3 0 A1 5 0 A. oryza e N RR L 44 8 A. oryza e N RR L 44 9 A. oryza e N RR L 46 9 F14 F60 1 -9 1 -2 9 5 -1 1 1-4 1 7-4 F15 F35 3 -2 7 -3 1 4-1 1 4-2 tx1 2-1 0-2 S 4 -2 1 2-4 1 3-4 NR RL A-11 61 2 1 -2 2 A. par as itic us CA1 -05 A. par as itic us CA3 -01

Califor nia Texas

Small scle rotia High B-aflatoxin No G-aflatoxin

Australia Califor nia

Large scle rotia No afla toxin

Geor gia

Geiser, DM Horn, BW Dorner, JW Taylor JW FG&B 2001

Australia

Geor gia

Large scle rotia High B aflatoxin No G aflatoxin

Australia

Small scle rotia Low B-aflatoxin = A. flavus Australia ? Low G-aflatoxin v ar. parv isclerotige nus Niger ia Southe r n He misphe re ? Australia
Texas Califor nia

3-gene combined genealogy of Aspergillus flavus
(most of data come from omt12: a toxin locus!)

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; 196062.

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


				
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