Microbial Reproductive Modes Fungal Reproduction Week 12, PMB 220 J. Taylor.

transcript

MicrobialReproductive

ModesFungal

ReproductionWeek 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

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

Clonal 1234567----------A 0101111B 0101011C 0111111D 1011110E 1010110F 1010100G 1111111

LOCI 1234567---------A 0001110B 0011111C 1111010D 1111111E 1011111F 1100101G 0110110

RecombiningLOCI

Testing for reproductive mode.

Tree Length Test

A 0101111B 0101011C 0111111D 1011110E 1010110F 1010100G 1111111

2, 746

Clonal

Trees: 1Length: 7 stepsConsistency Index: 1.0Homoplasy Index: 0.0

Recombining A 0001110B 0011111C 1111010D 1111111E 1011111F 1100101G 0110110Trees: 9Length: 11 stepsConsistency Index: 0.63Homoplasy Index: 0.36

00110011001

10110100101

01010101010

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

OBSERVED

00110011001

10110100101

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

SCRAMBLING

00110011001

10110100101

110100110

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

SCRAMBLED

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 11

Loci Loci Loci

Compter sex: Resampling without replacement.

Observed

Recombining p< 0.996

Observed

Clonal p< 0.002

A 0101111B 0101011C 0111111D 1011110E 1010110F 1010100G 1111111

2, 746

Clonal

Trees: 1Length: 7 stepsConsistency Index: 1.0Homoplasy Index: 0.0

Recombining A 0001110B 0011111C 1111010D 1111111E 1011111F 1100101G 0110110Trees: 9Length: 11 stepsConsistency Index: 0.63Homoplasy Index: 0.36

TreeLengthTest

Clonal 1234567----------A 0101111B 0101011C 0111111D 1011110E 1010110F 1010100G 1111111

LOCI 1234567---------A 0001110B 0011111C 1111010D 1111111E 1011111F 1100101G 0110110

RecombiningLOCI

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

.1 .2 .3 .4 .5 .6 .7 .8 .9 1 1.1

Distance

Variance = 7.3 x 10-2

.1 .2 .3 .4 .5 .6 .7 .8

Distance

Variance = 2.7 x 10-2

MeanMean

Dist ribut ion of Dist ances Dist ribut ion of Dist ances

-.8 -.6 -.4 -.2 0 .2 .4 .6 .8 1 1.2 1.4

Observed1.187

p = 0.003

-.8 -.6 -.4 -.2 0 .2 .4 .6 .8 1 1.2 1.4

Observed-0.153 p = 0.785

CONCORDANCE

CONSENSUS

ABCDWXYZGeneGenealogyConcordance

00110011001

10110100101

01010101010

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

LociOBSERVED

10110100101

11100100010

LociSCRAMBLING

00111010001

11100100010

01010101010

00110011001

10110100101

01010101010

10101010101

11100100010

10110100101

00111010001

11100100010

10101010101

00110011001

01110101110

LociSCRAMBLED

1 2 43 51 2 431 2 43Gene A Gene B Gene C

Computer sex:Shuffling variable nucleotidesamong genes.

40 45 50 55 60

actual summedtree lengths = 42

summed tree lengths

Aspergillus flavus

You have to know the

species before you can study reproductive

Example:Coccidioides immitis

Vasso KoufopanouAustin BurtMat Fisher

Distributionof

Coccidioidesimmitis

Rippon, 1988

Phylogenetic Species in C. immitis

MX1, AG1-5

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/T

a2 0000001000010000000000000 NS1 ITS5 154 40T/C

e2 0000000000100010000000000 NS23 ITS5 183 108TT/C-

k 0010100001000111010010001 NS2 NS22 590 189G/A

q2 0000001000000000010000000 CNS19 ITS4 163 20C/T

z 0111101110111101111100011 NS24 CNS19 260 13A/T

aa 0111111110111101111100000 NS24 CNS25 207 11A/G

am 0000100000001010000000000 NS23 ML5.1 309 66G/A;97C/T

bg 0101000000000001110000000 ITS2 ML3.5 165 34CTC/---

bk 0000000000000000100100000 ITS4 ML5.1 369 146C/A

bl 0001000010000000000111000 NS26 MS1 822 233T/C;733A/-

bq 0011000000000100100001000 ML5.1 MS1 433 170A7/A8,9;235A/G;260T/C;293A/G

af 0000100010000101110101001 ITS4 ITS1 630 46T/A

cs 0000000011000000010101011 CS2 CS4A 260 53G/A

Primers

a b c d e f g i h j k l m n o p q r s t u v w x y

Parsimony analysis:Consensus of 62 most parsimonious trees.

10 15 20 25 30 35 40 45

MinimumPossible

Observed38

Tree Length

Test of association of alleles: Index of Association

Phylogenetic Species in C. immitis

MX1, AG1-5

17 fixed sites

C. immitis C. posadasii

I s o l a t e

20052267

20122013

22712273227422762277

20042006

20082009201020142015

2017264922682269

22803255325732583262326428082610

435201110363272

E p i d e m i c

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 two species of

Letharia, one sexual and the other not?

Letharia columbiana

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, filled with

meiotic ascospores

www.lichen.com

Soredia, algal cells wrapped

in hyphae

SexualAsexual

sorediate

apotheciate

sorediate

601 of 4608 treesCIre=.57202 steps 1 step

Letharia 6 loci51 individuals

6 species suggested

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

Question: does the lichen outbreed 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 and separate 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

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)

non-CA

CentralCalif.

SouthernCalif.

Arizona

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...

0 200 400 600 800 1000 1200 1400 1600 1800

Geographical distance (miles)

r = 0.905

r = 0.694**

0 1000 2000 3000 4000 5000 6000

Geographical 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

Microbial Reproductive Modes Fungal Reproduction Week 12, PMB 220 J. Taylor.

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