MicrobialReproductive

ModesFungal Reproduction

Week 12, PMB 220

J. Taylor

The cost of sex is two-fold.Clonal progeny have twiceas many parental genes.

Lewis CarrollJohn Tenniel (illus)1872.

The Value of Sex? The Red Queen Hypothesis."Now, here, you see, it takes all the runningyou can do, to keep in the same place."

The Value of Sex?

Muller’s Ratchet

H. Muller, 1964

Goddard,GodfrayBurt. 2005Nature434:636-640

Goddard,GodfrayBurtNature434:636-402005

Goddard,GodfrayBurtNature434:636-402005

Benignenvironment

Harshenvironment

Aydin Örstan

http://users.unimi.it/ricci/html/bdelloid.htm

Sex is nearly ubiquitous.Only the bdelloid rotifers have beenclaimed to be an old asexual group.

Numbers of Species of Fungi

Ascomycota 32,267 46.0%Lichenized fungi 13,500 18.7%Basidiomycota 22,244 32.0%Chytridiomycota 793 1.0%Zygomycota 1,056 1.5%

Mitosporic fungi 14,104 19.6%

Dictionary of the Fungi, Hawksworth et al. 1996

What is aspecies?

How do they reproduce?

Determining thereproductive mode

of Microbes:Recombination v. clonality

Clonal:Associationof Alleles.

Recombining: Lack of Association

Testing for reproductive mode.

Tree Length Test

00110011001

10110100101

01010101010

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

Individuals

OBSERVED

00110011001

10110100101

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

Individuals

SCRAMBLING

0

0

1101

0 101

0

00110011001

10110100101

110100110

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

Individuals

SCRAMBLED

0

0

a b c

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11Loci Loci Loci

Compter sex: Resampling without replacement.

TreeLengthTest

Index of Association

Clonal Reproduction

1 2 3 4 5 6 71 A - 0.14286 0.14286 0.57143 0.71429 0.85714 0.285712 B 1 - 0.28571 0.71429 0.85714 1.00000 0.428573 C 1 2 - 0.42857 0.57143 0.71429 0.142864 D 4 5 3 - 0.14286 0.28571 0.285715 E 5 6 4 1 - 0.14286 0.428576 F 6 7 5 2 1 - 0.571437 G 2 3 1 2 3 4 -

Distance Matrix

Recombining Reproduction

1 2 3 4 5 6 71 A - 0.28571 0.57143 0.57143 0.42857 0.71429 0.428572 B 2 - 0.57143 0.28571 0.14286 0.71429 0.428573 C 4 4 - 0.28571 0.42857 0.71429 0.428574 D 4 2 2 - 0.14286 0.42857 0.428575 E 3 1 3 1 - 0.57143 0.571436 F 5 5 5 3 4 - 0.571437 G 3 3 3 3 4 4 -

Distance Matrix

Distancematrix

IA

.

CONCORDANCE

CONSENSUS

A B C D W X Y ZRECOMBINING

CLONAL

GeneGenealogyConcordance

Computer sex:Shuffling variable nucleotidesamong genes.

Aspergillus flavus

You have to knowthe species before

you can studyreproductive mode.

Example:Coccidioides immitisVasso KoufopanouAustin BurtMat Fisher

Distributionof

Coccidioidesimmitis

Rippon, 1988

Phylogenetic Species in C. immitisS

AZ1

AZ2

TX1

MX2

MX1, AG1-5

TX2

CA1

CA2

CA3

CA4

CA5

17 fixed sites

C. immitis C. posadasii

Finding Single Nucleotide PolymorphismsAgarose and SSCP gels of PCR products

Coccidioides immitis: multilocus genotypesas single nucleotide polymorphisms (SNPs)

Locus Isolate Size(bp) Polymorphismabcdefghijklmnopqrstuvwxy

a1 0000111000010100011100101 NS1 ITS5 286 144C/Ta2 0000001000010000000000000 NS1 ITS5 154 40T/Ce2 0000000000100010000000000 NS23 ITS5 183 108TT/C-k 0010100001000111010010001 NS2 NS22 590 189G/A

q2 0000001000000000010000000 CNS19 ITS4 163 20C/Tz 0111101110111101111100011 NS24 CNS19 260 13A/T

aa 0111111110111101111100000 NS24 CNS25 207 11A/Gam 0000100000001010000000000 NS23 ML5.1 309 66G/A;97C/Tbg 0101000000000001110000000 ITS2 ML3.5 165 34CTC/---bk 0000000000000000100100000 ITS4 ML5.1 369 146C/Abl 0001000010000000000111000 NS26 MS1 822 233T/C;733A/-bq 0011000000000100100001000 ML5.1 MS1 433 170A7/A8,9;235A/G;260T/C;293A/Gaf 0000100010000101110101001 ITS4 ITS1 630 46T/Acs 0000000011000000010101011 CS2 CS4A 260 53G/A

Primers

Parsimony analysis:Consensus of 62 most parsimonious trees.

Test of association of alleles: Index of Association

Phylogenetic Species in C. immitisS

AZ1

AZ2

TX1

MX2

MX1, AG1-5

TX2

CA1

CA2

CA3

CA4

CA5

17 fixed sites

C. immitis C. posadasii

91

98

Isolate

20052267

20122013

2275

22812270

22712273227422762277

2002

20042006

2007

200820092010201420152017264922682269

2278

2279

22803255325732583262326428082610435201110363272

EpidemicCA

NCA

IA, P = 0.28

PTLPT, P = 0.09

CaliforniaCoccidioides

Fisher et al. 2000

Likelihood ratio tests

Kishino-HasegawaTwo different topologies

Shimodaira-HasagawaMultiple topologies

Lichens

Trebouxia and Letharia

Scott Kroken

Question: Are there twospecies of Letharia, one

sexual and the other not?

Letharia columbiana

Letharia vulpina

Letharia vulpina• always produces soredia• apothecia are rare

Letharia columbiana • always produces apothecia• sometimes produces isidia

Are they a “species pair” and how do they reproduce?

Distribution of Letharia species

XeroxPARCmap

Thomas Nash

Apothecia, filledwith meioticascospores

www.lichen.com

Soredia, algalcells wrapped

in hyphae

Sexual Asexual

Letharia 6 loci51 individuals

6 species suggested

Kroken and Taylor. 2000. Mycologia 93:38-53

Question: does the lichenoutbreed or inbreed?

Paternity analysis of lichen apothecia

Fertilization

Spermagonium--produces spermatia

Trichogyne--fuses with spermatium

Parent and progeny

Letharia “lupina” paternity analysis

All 36 apothecia in both speciesare the result of outcrossing

‘lupina’ locus CS EarI ‘lupina’ ITS 1F/ 2 SacI

Mom1 and 7 kids Mom2 and 6 kids

Mom2 and 6 kidsMom1 and 7 kids

Kroken and Taylor 2001 Fungal Genetics & Biology 34:83-92

Outbreeding andseparate fertilizations

Dispersal of Letharia vulpina with its alga

XeroxPARCmap

Högberg et al. 2002. Molecular Ecology 11:1191-1196

Recombining:North Americansorediate

Recombining:North Americanapotheciate

Clonal: European and North African sorediate Letharia species

Recombining and Clonal in Letharia

Daubin et al. 2003. Science 301:829-832

More polymorphism?

Microsatellites orShort Tandem Repeats

Molecular markers- Microsatellites

• Dinucleotide repeats randomly dispersed through the genome

ctgcgtgtgacatACACACACACACACActgtatgtgatc

• Highly polymorphic and multialleleic due to polymerase slippage during strand replication

Cocci_1 ctgcgtgtgacatACACACACACACACA--------ctgtatgtCocci_2 ctgcgtgtgacatACACACACACACACACA------ctgtatgtCocci_3 ctgcgtgtgacatACACACACACACACACACA----ctgtatgtCocci_4 ctgcgtgtgacatACACACACACACACACACACACActgtatgt

0

0.1

0.2

0.3

208

209

210

211

212

213

214

215

216

217

218

219

220

227

228

229

231

232

233

235

240

241

247

248

249

250

GA37

0

0.25

0.5

0.75

1

397

399

400

401

414

416

418

419

420

422

424

426

621

0

0.25

0.5

0.75

1

206

216

218

220

222

224

226

228

Allele size (bp)

GAC2

0

0.1

0.2

0.3

0.4

0.5

0.6234

252

253

254

255

256

258

259

260

261

262

263

264

Allele size (bp)

GA1

0

0.2

0.4

0.6

0.8

186

187

188

202

203

204

206

207

208

209

210

212

227

ACJ

0

0.25

0.5

0.75

1

220

226

228

229

237

238

239

240

241

243

249

252

253

255

257

258

259

KO3

0

0.25

0.5

0.75

1

262

292

293

294

295

296

297

299

301

Allele size (bp)

KO7

0

0.2

0.4

0.6

0.8229

231

233

234

235

237

238

239

241

243

245

246

247

KO1

0

0.1

0.2

0.3

0.4

0.5

141

143

145

147

149

150

151

152

153

154

156

160

162

163

164

166

168

KO9

Microsatellites

Microsatellite distance Flanking-sequence distance

These trees have the same topology (Kishino-Hasegawa test non-significant)THE MICROSATELLITE MARKERS ARE GOOD

(Fisher et al. Mol. Biol. Evol. 2000)

100

64

70

61

69

5999

68

58

CA

non-CA

CentralCalif.

SouthernCalif.

Arizona

Texas

California- Coccidioides immitis Rixford and Gilchrist 1896

non-California- Coccidioides posadasii after Alejandro Posadas

Renaming the species

All populations

Arizona

Texas/ South America

North America/ Mexico C. immitis ( ) and C. posadasii ( )show isolation by distance...

0

5

10

15

20

25

30

35

40

45

50

0 200 400 600 800 1000 1200 1400 1600 1800

Geographical distance (miles)

r = 0.905

r = 0.694**

05

101520253035404550

0 1000 2000 3000 4000 5000 6000Geographical distance (miles)

r = 0.023

r = 0.694**

...but NOT if South American isolates (all C. posadasii) are included…

• S. American isolates contain 6% of the variation found in N. America• 28% of loci are in linkage disequlibrium (7% in N. America)

• This is a bottlenecked population

…and is descended from the TEXAS ! population of Coccidioides posadasii

Genetic dating show that South American populations were founded from those in North America between 9,000 - 140,000 BP (the Pleistocene)

Migration of Homo sapiens into South America by 10,000 BC

Jared Diamond ‘Guns, Germs and Steel’, 1997

How did non-CA C. immitis arrive in South America?

Host-pathogen dispersal:

• 9,000 year old bones of Bison antiquus from Nebraska contain C. immitis spherules. Demonstrates potential for long-distance dispersal with a host

• Human infections are viable for more than 12 years

• Ancient Amerindian middens contain high concentrations of C. immitis

• Documented invasion of South America by the Amerindians 12,500 yrs bp.

Does the present distribution of C. posadasii reflect the co-dispersal of a host and its pathogen?

Recombining Recombining

Clonal (C. posadasii in Latin Amer?)

Recombining and Clonal in Coccidioides

Daubin et al. 2003. Science 301:829-832 Fig 1

Daubin et al.2003. Science301:829-832Fig 2

Daubin et al.2003. Science301:829-832Fig 3