Seminar for Cornell Dept of Plant Pathology

1. EVOLUTION AND
EXPLORATION OF THE
TRANSCRIPTIONAL
LANDSCAPE IN TWO
FILAMENTOUS FUNGI,
COCCIDIOIDES AND
NEUROSPORA
Jason Stajich
University of California, Riverside

2. Studying Evolution with
Comparative Genomics
Reconstructing evolution from inferred changes DNA sequences.
From populations to species to kingdoms
How do mutations arise, become fixed (in a population),
influence phenotypic change, and influence formation of new
species?
Genome sequencing provides the data from extant (living) species.
Comparative genomics to identify genome changes and establish
hypotheses about importance of changes.

3. How old are the fungi?
Opisthokont
Animal/Fungi Ancestor
Taylor and Berbee,
Mycologia 2006
Opisthokont Ancestor ~1Bya
- Problems:
Few collected fossils; dates are still very debated

4. BALDAUF. 2003.
SCIENCE

5. Genome samples from fungi
Dictyostelium
Monosiga Choanoflagellida
Caenorhabditis
Metazoa
Drosophila
Homo
Batrachochytrium ‘Chytrid’ Chytrid 5
Spiromyces Zygomycota
Opisthokont ‘Chytrid’
Olpidium
Rhizopus Mucormycotina Muromycotina 3
Fungi Glomus Glomeromycota Glomeromycota 1
Puccinia
Cryptococcus Basidiomycota Basidiomycota 31
Coprinopsis
Schizosaccharomyces Taphrinomycotina Taphrinomycotina 4
Yarrowia
Saccharomyces
Saccharomycotina
Saccharomycotina > 20
Ascomycota Candida
Morchella
Cochliobolus
Cladonia
Pezizomycotina Aspergillus
Coccidioides
Magnaporthe
Pezizomycotina >55
100+ Genomes Neurospora
Fusarium
Botryotinia
Tree Based on James TY et al. 2006. Nature.
http://fungalgenomes.org/wiki/Fungal_Genome_Links

6. Evolution of Fungal Diversity
Cryptococcus neoformans X. Lin Coprinopsis cinerea Ellison & Stajich Aspergillus niger. N Read Glomus sp. Univ Sydney Rozella allomycis. James et al
Puccinia graminis J. F. Hennen Batrachochytrium dendrobatidis
Laccaria bicolor Martin et al. Neurospora crassa. Hickey & Reed Phycomyces blakesleansus T. Ootaki
J. Longcore
Ustilago maydis Kai Hirdes Amanita phalloides. M Wood Xanthoria elegans. Botany POtD Rhizopus stolonifera. Rhizophydium. The Fifth Kingom

7. Phylogeny from genome sequences
!"#$%&'()*+',-./$!+'('0/!"##$%!!&'' ())*&++,,,-./0123425)678-401+9:;9<"9:=+$+''
100s-1000s of gene Zygomycota
Basidiomycota
Rhizopus oryzae
Ustilago maydis
phylogenies, 100
100
Cryptococcus neoformans
Phanerochaete chrysosporium
Hymenomycetes 100 Coprinus cinereus
Concatenated and Schizosaccharomyces pombe
Histoplasma capsulatum
100
Uncinocarpus reesii
individual gene trees and Eurotiomycetes
100
100
100
Coccidioides immitis
Aspergillus nidulans
Aspergillus fumigatus
consensus Pezizomycotina
100
100
Aspergillus oryzae
Aspergillus terreus
100 100 Stagonospora nodorum
Magnaporthe grisea
100
Generally recapitulate 100 100
Neurospora crassa
Podospora anserina
Sordariomycetes 100
100 Chaetomium globosum
what is found from multi- 100
100
100
Trichoderma reesei
Fusarium verticillioides
100 Fusarium graminearum
locus studies of 2-4 genes Ascomycota
Leotiomycetes
100 Botrytis cinerea
Sclerotinia sclerotiorum
Yarrowia lipolytica
Candida lusitaniae
Candida guilliermondii
Identify conflicting 100
Saccharomycotina
CTG
100
100
60 Debaryomyces hansenii
Candida parapsilosis
Candida tropicalis
topologies to find 100
100
100
Candida dubliniensis
Candida albicans
100
Saccharomyces castellii
Incomplete lineage WGD
100
Candida glabrata
Saccharomyces bayanus
90
sorting, bad orthology 100
100
60
Saccharomyces kudriavzevii
Saccharomyces mikatae
100 Saccharomyces cerevisiae
assignment, or potential 70
100 Saccharomyces paradoxus
Kluyveromyces waltii
Saccharomyces kluyveri
lateral transfer events 70
100
Ashbya gossypii
Kluyveromyces lactis
0.1
Fitzpatrick, Logue, Stajich, and Butler 2006. BMC Evol Biol

8. Phylogeny from genome sequences
!"#$%&'()*+',-./$!+'('0/!"##$%!!&'' ())*&++,,,-./0123425)678-401+9:;9<"9:=+$+''
100s-1000s of gene Zygomycota
Basidiomycota
Rhizopus oryzae
Ustilago maydis
phylogenies, 100
100
Cryptococcus neoformans
Phanerochaete chrysosporium
Cryptococcus
4 spp
Hymenomycetes 100 Coprinus cinereus
Concatenated and Schizosaccharomyces pombe
Histoplasma capsulatum
100
Uncinocarpus reesii
individual gene trees and Eurotiomycetes
100
100
100
Coccidioides immitis
Aspergillus nidulans
Coccidioides
13 strains, 2 spp
Aspergillus fumigatus
consensus Pezizomycotina
100
100
Aspergillus oryzae
Aspergillus terreus
100 100 Stagonospora nodorum
Magnaporthe grisea
100
Generally recapitulate 100 100
Neurospora crassa
Podospora anserina
Neurospora
3 spp
Sordariomycetes 100
100 Chaetomium globosum
what is found from multi- 100
100
100
Trichoderma reesei
Fusarium verticillioides
100 Fusarium graminearum
locus studies of 2-4 genes Ascomycota
Leotiomycetes
100 Botrytis cinerea
Sclerotinia sclerotiorum
Yarrowia lipolytica
Candida lusitaniae
Candida guilliermondii
Identify conflicting 100
Saccharomycotina
CTG
100
100
60 Debaryomyces hansenii
Candida parapsilosis
Candida tropicalis
topologies to find 100
100
100
Candida dubliniensis
Candida albicans
100
Saccharomyces castellii
Incomplete lineage WGD
100
Candida glabrata
Saccharomyces bayanus
90
sorting, bad orthology 100
100
60
Saccharomyces kudriavzevii
Saccharomyces mikatae Saccharomyces
100 Saccharomyces cerevisiae 37+ strains
assignment, or potential 70
100 Saccharomyces paradoxus
Kluyveromyces waltii
2 spp
Saccharomyces kluyveri
lateral transfer events 70
100
Ashbya gossypii
Kluyveromyces lactis
0.1
Fitzpatrick, Logue, Stajich, and Butler 2006. BMC Evol Biol

9. Marcet-Hoube and Gabaldón
PLoS One 2009.

10. College,
Gradschool
xkcd.org

11. Building Comparative Genomics Tools
BioPerl - Perl language programming tools for bioinformatics
(Stajich et al 2002).
Parsing sequences, alignments, report output (BLAST,
HMMER, PAML, CLUSTALW, PHYLIP)
My development focuses on Sequence, Phylogenetics, and
Molecular Evolution analyses tools
Gbrowse - Genome Browser (Stein et al 2002)

12. Genome Browser data integration
Ncra_OR74A_chrIV_contig7.20
300k 310k 320k 330k
DNA_GCContent
% gc
NCBI genes (Broad called)
NCU04433 NCU04430 NCU04426
sulfate permease II CYS-14 related to aminopeptidase Y precursor; vacuolar related to cyclin-supressing protein kinase
NCU04432 NCU04429 NCU04425
hypothetical protein conserved hypothetical protein putative protein
NCU04431 NCU04428 NCU04424
related to endo-1; 3-beta-glucanase related to spindle assembly checkpoint protein related to regulator of chromatin
NCU04427
conserved hypothetical protein
PASA updated NCBI/Broad genes
NCU04433 NCU04432
[pasa:asmbl_9429,status:12],[pasa:asmbl_9430,status:12] [pasa:asmbl_9440,status:12],[pasa:asmbl_9441,status:12],[pasa:asmbl_9442,status:12]
[pasa:asmbl_9431,status:12],[pasa:asmbl_9432,status:12] [pasa:asmbl_9443,status:12],[pasa:asmbl_9444,status:12]
[pasa:asmbl_9433,status:12],[pasa:asmbl_9434,status:12],[pasa:asmbl_9435,status:12]
[pasa:asmbl_9436,status:12],[pasa:asmbl_9437,status:12],[pasa:asmbl_9438,status:12],[pasa:asmbl_9439,status:12]
[pasa:asmbl_9445,status:12],[pasa:asmbl_9446,status:12]
NCU04424
Named Genes (Radford laboratory)
cys-14 gh16-3
tRNA{phe}-9
miRNA Solexa histogram
miRNA
K4dime ChIP-Seq histogram (SOAP)
K4dime_Solexa
K9met3 ChIP-Seq histogram (SOAP) Stajich et al, unpublished
K9met3 Smith, Freitag, et al unpublished

13. Synteny Browser

14. Comparative Genomics and
Evolution
Population genomic inference of migration
and hybridization
Genomics approaches to finding genes
underlying adaptation
Deeper divergences: What makes a fungus?

15. Comparative Genomics and
Evolution
Population genomic inference of migration
and hybridization
Comparative genomic approaches to finding
genes underlying adaptation
Deeper divergences: What makes a fungus?

16. Human pathogen Coccidioides
Coccidioides (Valley fever)
Is a primary human pathogen - infects healthy people - most
human pathogenic fungi are opportunistic.
Endemic in US Southwest, Mexico
Requires laboratory BSL3 and is a Select Agent
Comparative analyses of Coccidoides and related species to attempt
to understand how a pathogen evolved
Comparative genomics, Population genomics, and Transcriptional
Profiling

17. Human pathogen Coccidioides
Development
S/
Hypha Spherule Endospores

18. Coccidioides life cycle
Short Life
Granuloma
D octorfungus. com
M. McGinnis
Spherule
Endospores Long Life

19. Comparative & Population Genomics
of a human pathogenic fungus
Genomes from 2 species of Coccidioides diverged ~5 Mya. (dS 0.023)
Population Genomics
Genomes of 13 strains of Coccidioides
Evidence for introgression?
Differences in population size between species?
Strain variation in virulence and distribution
Molecular basis for virulence? Evolutionary signature?

20. Two species of Coccidioides
C.immitis
C.posadasii
EVOLUTION
Fisher et al, 2000

21. Aspergillus clavatus
Aspergillus fumigatus
Aspergillus flavus Animal Pathogen
Aspergillus oryzae (Opportunistic)
Aspergillus terreus
Eurotiales Aspergillus niger Animal Pathogen
Aspergillus nidulans (Primary)
Penicillium marneffei
Blastomyces dermatitidis
Eurotiomycetes Plant Pathogen
Histoplasma capsulatum 186AR
Histoplasma capsulatum 217B
Histoplasma capsulatum WU24
Paracoccidioides brasiliensis
Onygenales
Coccidioides immitis
Coccidioides posadasii
Uncinocarpus reesii
Fusarium graminearum
Sclerotinia sclerotiorum
200 100 0
Mya

22. Phylogeny of 13 99
CP RMSCC 2133
sequenced Coccidioides CP RMSCC 3700
CP RMSCC 1037
genomes
CP RMSCC 3488
CP CPA 0001
100 C. posadasii
CP CPA 0020
95
CP1
100
CP RMSCC 1038
CPS2
99 99
CP CPA 0066
CI RMSCC 2394
100
CI RMSCC 3703 C. immitis 73 kb CDS
CIH1
ML, HKY + Γ
CI2 0.01 D. Neafsey, et al unpublished

23. Population Genomics
660 000 filtered SNPs across the 13 strain genomes (~28Mb
genome).
4 sampled from C. immitis, 9 from C. posadasii
Effective population size - but can we still estimate with different
sample numbers?
Testing for selective sweeps in region of the genome
Hybridization and Migration inferred from via FST

24. C. immitis has smaller effective
population size
C. posadasii
simulated
(N=4)
C. immitis C. posadasii actual
(N=4) (N=9)
Whiston, Stajich

25. Genome-wide summary statistics
4151/3'!67
,-==>%"$ C. immitis RS Chrom 3
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26. Genome-wide summary statistics
4151/3'!67
,-==>%"$ C. immitis RS Chrom 3
$<"! =1:5.M:3M/.+2.+8J9+A+4.0:-8/
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$#"%
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Tajima's D: Positive values indicate excess of both high
!"!&
!"!)
!"!!
frequency and low frequency alleles consistent with balancing
! # % $ )
selection or decrease in population size. Negative values
indicate excess of low frequency polymorphisms and
potential population size expansion or negative selection.

27. Testing for evidence of hybridization
0.8
1 1
0.8
0.6 0.6
0.4 0.4
FST is a measure of 0.2 0.2
separation between 0 0
populations. -0.2
0.005 1.045 2.055 3.055 4.07
-0.2
0.005
Contig3 pos (Mb)
FST 1 is complete separation,
0 is no separation
1 1
Applied to whole genome
0.8 0.8
can estimate when regions 0.6 0.6
diverged and if there has 0.4 0.4
been recent hybridization 0.2 0.2
(migration of alleles). 0 0
-0.2
Neafsey, Barker, Rounsley
-0.2

28. Coccidioides population genomics
C. immitis is endemic to Central and Southern
California, mountain ranges likely block its
migration into Arizona.
Smaller effective population size consistent
with smaller geographic range or perhaps the
fission of the population due to introduced
geographic barrier.
There is evidence of inter-species
hybridization events (introgression) and
bidirectional exchange of alleles.
Some evidence for selective sweeps as well
based on populations, ongoing work to verify
and validate these observations.

29. Comparative Genomics and
Evolution
Aspergillus clavatus
Aspergillus fumigatus
Aspergillus flavus
Aspergillus oryzae
Aspergillus terreus
Population genomic inference of migration Aspergillus niger
Aspergillus nidulans
and hybridization Penicillium marneffei
Blastomyces dermatitidis
Histoplasma capsulatum 186AR
Histoplasma capsulatum 217B
Comparative genomic approaches to finding Histoplasma capsulatum WU24
Paracoccidioides brasiliensis
genes underlying adaptation Coccidioides immitis
Coccidioides posadasii
Uncinocarpus reesii
Deeper divergences: What makes a fungus? Fusarium graminearum
Sclerotinia sclerotiorum
200 100 0

30. Aspergillus clavatus
Aspergillus fumigatus
Aspergillus flavus Animal Pathogen
Aspergillus oryzae (Opportunistic)
Aspergillus terreus
Eurotiales Aspergillus niger Animal Pathogen
Aspergillus nidulans (Primary)
Penicillium marneffei
Blastomyces dermatitidis
Eurotiomycetes Plant Pathogen
Histoplasma capsulatum 186AR
Histoplasma capsulatum 217B
Histoplasma capsulatum WU24
Paracoccidioides brasiliensis
Onygenales
Coccidioides immitis
Coccidioides posadasii
Uncinocarpus reesii
Fusarium graminearum
Sclerotinia sclerotiorum
200 100 0
Mya

31. Evolution of a pathogen
Comparing sequences from two Coccidioides species, closely
related outgroup, and many related species.
Are there genes with signatures of positive selection that may
distinguish pathogen from non-pathogen?
Are there differences in presence-absence of genes or sizes of gene
families that suggest differences in pathogen?

32. Testing of directional natural
selection
Evaluate patterns of molecular substitution in protein-coding
genes.
Ratio of replacement (dN) to silent substitutions (dS)
Ser Cys Gly
> 1 Positive
1 TCT TGT GGT replacement
= 1 Neutral
2 TCA TGC CGT silent
< 1 Purifying
Ser Cys Arg

33. Rapidly Evolving Coccidioides Proteins
Pairwise Orthologs between C. immitis and C. posadasii with Ka/Ks » 1
Coccidioides immitis
Ka/Ks p* Annotation
48 1.22 0.003 Basic salivary proline-rich protein 1 precursor -related
23 1.74 0.035 hypothetical protein
Coccidioides posadasii
74 1.79 0.049 hypothetical protein
54 1.87 0.045 hypothetical protein Uncinocarpus reesii
29 1.89 0.044 "SUA5 protein, putative"
84
91
1.90
1.93
0.006
0.049
ankyrin repeat containing protein
hypothetical protein
57 genes in Pairwise dN/dS
04 1.95 0.029 hypothetical protein
00 2.16 0.043 hypothetical protein
63
42
2.19
2.23
0.042
0.042
MT-A70 family protein
hypothetical protein
GO enrichment metabolic
92 2.24 0.018 Major Facilitator Superfamily protein
84 2.36 0.050 hypothetical protein process, phosphorylation, and
07 2.40 0.032 U1 zinc finger family protein
01
10
2.41
2.53
0.048
0.014
hypothetical protein
hypothetical protein
S-adenosylmethionine-
87 2.53 0.041 hypothetical protein
97 2.80 0.017 F-box domain containing protein dependent methyltransferase
63 2.85 0.030 Glycosyl hydrolases family 31 protein
84
11
2.88
3.20
0.016
0.048
hypothetical protein
hypothetical protein
activity
64 3.24 0.018 hypothetical protein
51 3.25 0.009 hypothetical protein
25
39
3.49
3.62
0.039
0.040
GDP dissociation inhibitor family protein
"Sterol 24-C-methyltransferase, putative"
60 genes in 3-way relative
75 3.91 0.004 hypothetical protein
13 4.01 0.008 hypothetical protein rates test
40 4.69 0.019 hypothetical protein
10 5.37 0.014 hypothetical protein
37 5.37 0.014 hypothetical protein
94 5.37 0.014 hypothetical protein GO enrichment biopolymer
69 99.00 0.026 hypothetical protein
58
10
99.00
99.00
0.032
0.040
Mitochondrial ATP synthase g subunit family protein
hypothetical protein
and RNA metabolic processes
10 99.00 0.043 TPR Domain containing protein
14 99.00 0.039 hypothetical protein Sharpton, Stajich, et al, in revision

34. Gene family changes
A mechanism for adaptation may be changes in copy number of a
gene family
Gene duplication is a source of novelty allowing for changes in
the function of one copy if the other maintains original
function
Expansions of copy number may also be an easy way to get
more protein for a particular process
Gene family losses may represent unneeded processes
Loss requires appropriate sampling to polarize changes

35. Aspergillus clavatus
Aspergillus fumigatus
Aspergillus flavus Animal Pathogen
Aspergillus oryzae (Opportunistic)
Aspergillus terreus
Eurotiales Aspergillus niger Animal Pathogen
Aspergillus nidulans (Primary)
Penicillium marneffei
Blastomyces dermatitidis
Eurotiomycetes Plant Pathogen
Histoplasma capsulatum 186AR
Histoplasma capsulatum 217B
Histoplasma capsulatum WU24
Paracoccidioides brasiliensis
Onygenales
Coccidioides immitis
Coccidioides posadasii
Uncinocarpus reesii
Fusarium graminearum
Sclerotinia sclerotiorum
200 100 0
Mya

36. Coccidioides expansions
Peptidase_M35
Peptidase_M36
Peptidase_S8
Pec_lyase_C
Subtilisin_N
Cellulase
Cutinase
Tannase
CBM_1
NPP1
APH
Anid 6 6 6 2 4 13 2 3 3 0 9
Afum 17 5 5 2 5 10 2 5 2 1 9
Ater 15 6 6 2 8 13 2 6 2 1 29
Hcap 0 0 0 0 2 2 2 6 1 0 20
Uree 0 0 0 0 1 2 15 19 4 2 33
Cimm 0 0 0 0 1 1 13 16 7 2 38
Cpos 0 0 0 0 1 1 14 16 7 2 32
Ncra 18 1 1 4 3 6 3 6 2 0 6
Fgra 12 7 9 9 12 8 11 24 1 1 15
Sharpton, Stajich, et al, in revision

37. Keratinases in Onygenales
SignalP
Subtilisin_N
Onygenales are Keratinophilic
Domains: Peptidase S8, Subtilisin domains
Large expansion of putative keratinases in Onygenales

38. Keratinase expansion
I in Onygenales
14 copies in Coccidioides
1 in Histoplasma
II
III
Sharpton, Stajich, et al, in revision

39. Keratinase expansion
I in Onygenales
14 copies in Coccidioides
1 in Histoplasma
II
III
Sharpton, Stajich, et al, in revision

40. Onygenales contractions
Peptidase_M35
Peptidase_M36
Peptidase_S8
Pec_lyase_C
Subtilisin_N
Loss of plant
Cellulase
Cutinase
Tannase
CBM_1
saprophytic
NPP1
APH
enzymes
Anid 6 6 6 2 4 13 2 3 3 0 9
Afum 17 5 5 2 5 10 2 5 2 1 9
Ater 15 6 6 2 8 13 2 6 2 1 29
Hcap 0 0 0 0 2 2 2 6 1 0 20
Uree 0 0 0 0 1 2 15 19 4 2 33
Cimm 0 0 0 0 1 1 13 16 7 2 38
Cpos 0 0 0 0 1 1 14 16 7 2 32
Ncra 18 1 1 4 3 6 3 6 2 0 6
Fgra 12 7 9 9 12 8 11 24 1 1 15
Sharpton, Stajich, et al, in revision

41. Towards genes underlying adaptation

42. Towards genes underlying adaptation
Coccidioides is found in desert soil and associated with animals

43. Towards genes underlying adaptation
Coccidioides is found in desert soil and associated with animals
Loss of genes involved in plant product metabolism suggests
nutritional shift in Onygenales from relatives in Eurotiales

44. Towards genes underlying adaptation
Coccidioides is found in desert soil and associated with animals
Loss of genes involved in plant product metabolism suggests
nutritional shift in Onygenales from relatives in Eurotiales
Expansion of a few gene families, may be involved in metabolism -
none are Coccidioides specific though.

45. Towards genes underlying adaptation
Coccidioides is found in desert soil and associated with animals
Loss of genes involved in plant product metabolism suggests
nutritional shift in Onygenales from relatives in Eurotiales
Expansion of a few gene families, may be involved in metabolism -
none are Coccidioides specific though.
Sampling of a closer non-pathogenic outgroup can help polarize
recent changes. Expression analyses may help assign function to
some of genes with positive selection signatures

46. Comparative Genomics and
Evolution
Population genomic inference of migration
and hybridization
Comparative genomic approaches to finding
genes underlying adaptation
Comparisons of deeply diverged lineages:
What makes a fungus?

47. Making sense of differences when
comparing deep divergences
Nucleotide substitutions have been saturated (turned over enough
times we cannot reconstruct their history)
Proteins sequences evolve more slowly than DNA and homology
can be assessed across great evolutionary distances
Simple comparisons of gene content useful for gleaning high level
differences

48. Evolution of early Fungi
Physcomitrella patens
Dictyostelium discoideum
Monosiga brevicollis
Trichoplax adhaerens
Nematostella vectensis
Batrachochytrium Takifugu rubripes Animals
Homo sapiens
Drosophila melanogaster
Caenorhabditis elegans
Batrachochytrium dendrobatidis JEL423
Batrachochytrium dendrobatidis JAM81
Rhizopus oryzae
Ustilago maydis
Rhizopus Candida
Cryptococcus neoformans
Coprinopsis cinerea
Schizosaccharomyces pombe
Yarrowia lipolytica
Saccharomyces cerevisiae
Thick branches have Neurospora crassa
Bayesian (MrBayes Magnaporthe grisea
and PhyloBayes)
Aspergillus fumigatus
posterior of 1 and
Coprinopsis 100% ML (RAxML) Coccidioides immitis
Neurospora
0.1
bootstrap support. 32487 filtered concatenated amino-acid positions

49. Zoospore Young sporangia
Sporangia discharging
zoospores
Batrachochytrium dendrobatidis, ‘Chytridiomycota’
Amphibian Pathogen
2 sequenced strains, JEL423 (Sierras, USA) and JAM81 (Panama) by Joint
Genome Institute (JGI) and Broad respectively.
Rosenblum, EB Stajich JE, Maddox N,
~24 Mb genome, ~8000 genes EIsen MB, PNAS 2008
Stajich, et al, in prep

50. Batrachochytrium dendrobatidis
growing in a frog
Speare, Berger, Hyatt et al. 1999!
Daszak et al. 1999. EID !
James Cook University, Townsville, Australia! http://www.jcu.edu.au/school/phtm/PHTM/frogs/chpr1/fc13.htm!

51. Exploring deep divergences with
phylogenetic profiling
Classification of gene content
For each gene in a genome.
Identify which other species have a homlog.
Consolidate this per Clade (i.e. Animals, Plants, Ascomycetes,
etc)
For Chytrid comparison: Compare 7 Clades across 40 genomes
Pairwise similarities (BLAST) refined with shuffled Smith-
Waterman alignment for empirical pairwise E-value
Interact with results via Web Browser

52. B.dendrobatidis Phylogenetic Profile

53. B.dendrobatidis Phylogenetic Profile
Basidiomycota Fungi
1.5% 3.3%
.7% 7.5% 6.9% 1.5%
58% 46%
1.5% 4.9%
2%
Zygomycota Ascomycota
1550 (19.2%) Chytrid specific genes Animal Plant

54. B.dendrobatidis Phylogenetic Profile

55. B.dendrobatidis Phylogenetic Profile

56. Shared Genes

57. Shared Genes

58. Detailed investigation

59. Detailed investigation

60. Detailed investigation

61. FUNGAL CELL WALL
EVOLUTION

62. Yeast Cell Wall
Mannoprotein
β-Glucan
β-Glucan + Chitin
Mannoprotein
Membrane

63. Ascomycete hyphal walls
Cell Wall Component Amount
Mannan-proteins 50%
β 1,6 glucans 5%
β 1,3 glucans 40%
Chitin 1-3%
Plasma Membrane Ergosterol

64. B. dendrobatidis cell wall
biosynthesis missing genes
No 1,3-beta Glucan synthetase genes (2 genes)
No1,3-beta-glucanase genes (4 genes)
No KTR family genes (mannosyltranserase) (8 genes)
Need enzyme assay to assess cell wall composition ...

65. B. dendrobatidis cell wall
biochemical analysis
β (1,3)- β(1,6)- α(1,3)-
Cellulose Chitin
glucan glucan glucan
X X X ✓ ✓
Currently No cellulose synthase gene found in genome
based on genes defined in bacteria, oomyctes, or plants.
Stajich et al, in prep. with JP Latgé

66. Nuclear Division differs across
the fungal kingdom
Chytrids and Animals have centrioles and basal bodies for
microtuble attachment
Ascomycetes and Basidiomycetes have Spindle Pole Bodies for
microtuble attachment
Not homologous to centrioles

67. Paired Centrioles in
Animal and Chytrid centrosome homology
the Centrosome!
Metaphase in Newt cells! Centric division in a Chytrid with highlighted centrioles!
Centrosome!
McNitt 1973 Taken from Introductory Mycology!
Rieder and Khodjakov 2003. Science!

68. Chytrid and Animal centrioles
are homologous
Zoospore of a chytrid showing
two kinetocores (basal bodies).
Longcore 1995. Mycologia

69. Missing chromosome segregation and
mitosis related genes in chytrid
genome (S.cerevisiae names)
MSP3, KAR1, KAR2 for nuclear membrane fusion during
karyogamy and Spindle-body duplication
SPC42 - central plaque component of spindle pole bodies
CEP3 - essential kinetochore protein
CIN2 - Tubulin folding protein
REC8 for sister chromatid cohesion
DASH Complex for kinetochores coupling during mitosis (10
genes)

70. Ancient Photoreceptors?
Rhodopsin molecule implicated in zoospore
phototaxis in A!omyces
Sequence similarity identifies a candidate 7-
transmembrane protein found in both Bd and
A!omyces (draft).
Some critical residues and changes in helix 6 are
are different
insertion?

71. Transitions inferred from genomes
AFTER CHYTRID SPLIT Monosiga brevicollis
Loss of:
• Flagella, centrioles Homo sapiens
• 1,4 Beta-glucan Batrachochytrium dendrobatidis
synthesis
Gain of: Rhizopus oryzae
• STE50 adaptor
• Some glucan synthase Cryptococcus neoformans
and transferases
• Rhodopsin-like Coprinopsis cinerea
7TM
Dikarya Laccaria bicolor
IN DIKARYA, Gain of: Schizosaccharomyces pombe
• Spindle-pole bodies
• 1,3 Beta glucan synthase Saccharomyces cerevisiae
• Thiamine biosynthesis
• STE3 pheremone receptor
Neurospora crassa
• DASH complex Aspergillus nidulans
• KTR mannosylphosphate family
Loss of: Coccidioides immitis
• Chitosanase

72. Comparative genomics to build
evolutionary hypotheses
Population genomics approaches to studying differences between
species and populations identified evidence of recent
hybridization between species
Working to identify loci under directional selection.
Comparative genomics of recently diverged species suggests
differences in nutritional shift in the human pathogen Coccidioides
Comparisons between distantly related species can identify large
pathway or morphological differences to test
Reconstructing the ancestral fungus through inference of order
of evolutionary events

73. Acknowledgements
John Taylor, UC Berkeley Genome sequencing at
Broad Institute, JCVI,
Thomas Sharpton, Emily Genoscope, and DOE Joint
Whiston [Coccidiodies] Genome Institute
Erica Rosenblum (U Idaho),
JP Latgé (Pasteur Inst)
Christina Cuomo (Broad Inst)
[Batrachochytrium]
Dan Neafsey, (Broad Inst)
Steve Rounsley, Bridget
Barker, Marc Orbach (U
Arizona) [Coccidioides]

74. Acknowledgements
John Taylor, UC Berkeley Genome sequencing at
Broad Institute, JCVI,
Thomas Sharpton, Emily Genoscope, and DOE Joint
Whiston [Coccidiodies] Genome Institute
Erica Rosenblum (U Idaho), News about fungi and their genomes,
JP Latgé (Pasteur Inst) genome browsers, and community wiki
Christina Cuomo (Broad Inst) http://fungalgenomes.org
[Batrachochytrium]
Dan Neafsey, (Broad Inst)
Steve Rounsley, Bridget
Barker, Marc Orbach (U
Arizona) [Coccidioides]

75. Fungal Genomics @UCR
My lab will be starting at
University of California, Riverside
July 2009
Interested in research in fungal genomics?
Evolution of fungal development
Post-transcriptional gene regulation and
small RNAs
Fungal cell evolution in early branching
fungi
Bioinformatics and genome informatics

76. Fungal Genomics @UCR
My lab will be starting at
University of California, Riverside
July 2009
Interested in research in fungal genomics?
Evolution of fungal development
Post-transcriptional gene regulation and
small RNAs
Fungal cell evolution in early branching
fungi
Bioinformatics and genome informatics