Fungal Phylogenetics:
How to find the missing link
Ringvorlesung Genetik – Biologisch-Pharmazeutische Fakultät
Jena, April 2009
Martin Eckart – Fungal Reference Center Jena – Institute
of Microbiology (2009)
Agenda

Introduction: terms in phylogenetics
Data: how to find a phylogenetic marker
Reconstruction: planting phylogenetic trees
Applied Phylogenetics:
Reconstruction of evolution of basal fungi
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Terms in phylogenetics

Phylogeny: study of evolution
Cladistics: hypothesis of phylogeny

Plesiomorphic (original) characters
Apomorphic (derived) characters
Synapomorphies proving
Evolution
P
S
A
S
Phylogenetic course 2009 (Voigt, Hoffmann, Eckart)
3
Example

Plesiomorphy: a tail
Synapomorphy: loss of tail
(Aut-)Apomorphy: to speak
P
S1
A1
S1 A2
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
4
Para- und Monophyly

Paraphyletic groups are identified by
plesiomorphic characters
no closed/complete evolutionary community

Monophyletic groups are defined by
Apomorphies
always closed/complete evolutionary
community
Phylogenetic course 2009 (Voigt, Hoffmann, Eckart)
5
Example:
para- & monophyletic groups
P
S1
A1
S1 A2
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Homology & Analogy
Both terms are describing similar characters!
Homologous characters originate from a
single evolutionary point
structure of arms by Mammalia

Analogous characters evolve independently
or convergent
flying animals
Phylogenetic course 2009 (Voigt, Hoffmann, Eckart)
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Example:
Homology & Analogy
Homologous character:
Structures of arms of
Mammalia
Phylogenetic course 2009 (Voigt, Hoffmann, Eckart)
Analogous character:
flying animals
8
Kind of Homologies

orthologous genes:
same Gene, other Spezies
Specifications are helpful redrawing the
evolution of characters
e.g.: Actin, Cytochrome C
paralogous genes:
same Species, other (duplicated) Gene
phylogeny of paralogous genes is leading to
errors in reconstruction
e.g.: Hämoglobin (α, β, γ), MADS box gene
Phylogenetic course 2009 (Voigt, Hoffmann, Eckart)
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Generating Data
Phylogenetic marker
Phenologic
characters
biochemical
Ontogenesis
Moleculargenetic
characters
morphological
physiological
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
Protein
DNA
10
Kerstin Hoffmann, 2008
http://de.wikipedia.org/wiki/Darwinfinken
Phenologic characters:
non-molecular-genetic criteria

Morphological characters

Physiological characters

Color, size, shape, etc.
e.g. optimal temperature of
growth,
nutritions, oxygen, etc.
Biochemical characters

Metabolism pathways,
biochemical
production
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
11
Moleculargenetic character:
DNA or Protein?

DNA

Relatively easy to extract
Universal code
Triple information
non-coding sequences, no posttranscriptional loss of
information

FRC: 18S rDNA, 28S rDNA, { ITS1 & 2, 5.8S rDNA }
Protein

Codon Position
More substitution models
Information beyond the sequence: Function of AA, structure,
group of function

FRC: actin, tef 1-alpha, btub, (rpb1, rpb2, tdh)
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Actin

Bild: http://de.wikipedia.org/wiki/Actin
Moleculargenetic character
Structure protein of cytoskeleton in
eucaryotic cells
Coding sequence
Highly conserved gene (15% divergency
between algae and human)
ITS (internal transcribed spacer)

Non-coding sequence between rDNA
High copy number
Not under selective pressure
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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(Dis)Advantages
pro
contra
Morphological
characters
Easy
to discover
Low cost
Fossils
„Visible“ facts
Same
Moleculargenetic
characters
Comparable
Limited
Same
procedure
Limited data
Easy storage
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
level?
Inaccurate/misinterpretion
Evolut. rate not definable
Not comparable
No limited data
Preservation/ conservation
possibilities of fossils
Error due procedures
14
Taxon sampling & Alignment
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Methods of reconstruction

Distance based phylogeny

Parsimony

Heuristic, weighted, unweighted
Likelihood

UPGMA, Neighbor Joining, …
Maximum Likelihood
Bayesian inference
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Substitution model

4 nucleotides = 4 state of characters
DNA vs. Protein
how to calculate? Why not GTR+G+I?

1. time consuming 2. possible boost of errors
no substitution
reverse subst.
convergent subst.
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
parrallel subst.
17
Level of phylogeny
ATTACGGCA
ATTACGGCA
ATTACGCCA
AATAGGGCA
conserved
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
ATCACGCCA
ATTAGGGCA
ATTACGCCA
AATAGGGTT
variable
18
Methods to combine data

SuperMatrix alias multi-gene-analysis

alignments of single markers
final alignment is the combination of sets
missing characters are filled with gaps
SuperTree

informal Supertree

taxonomic substitution
formal SuperTree

algorithm-based coding
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Methods: Supermatrix

Combination of partitions with different
types of characters
Partition 1 Partition 2 Partition 3
partitions include:

morphological data
DNA sequence data
protein sequence data
Taxa

Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Methods: Informal Supertrees

“taxonomic substitution”
hierarchically nested
non-overlapping source
trees
still in use
e.g., Tree of Life
Bininda-Emonds 2008
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Methods: Formal Supertrees
E F GH J KL
Direct
AB C D E F GH I J K L
consensus-like techniques
A B C K L
MRP Matrix
C DE H I K
optimization
criterion
coding
technique
Indirect
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
Bininda-Emonds 2008
22
How to interprete a tree?
http://blogs.nature.com/news/thegreatbeyond/2008/04/jellies_were_first_animal.html
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Insecta
Applied phylogenetics
Plantae
Vertebrata
Metazoa
The study of evolutionary relations among
all living (and dead) organisms

Tree of Life – Project
AFTOL –Archaea
A fungal tree of Protozoa
life
The FRC Jena: basal fungi
Eukaryota
Zygomycetes
and Chytridiomycetes
Eubacteria
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
http://www.tolweb.org/tree/
Fungi
24
Phylogeny of the kingdom Fungi
Past till Present
Basidiomycota
Ascomycota
Glomeromycota
Schüßler et al. 2001
Blastocladiomycota
„Zygomycota“
James et al. 2006
Hibbett et al. 2007
Chytridiomycota
Neocallimastigomycota
© Addison Wesley 1999
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
Microsporidia
James et
al. 2006
Keeling et al. 2000
25
Hibbett et al. 2007
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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Characteristics of
chytridiomycetes
chytridium: unreleased spores
chytridion: ("little pot“) [Greek]

predominantly aquatic,
also terrestric
flagellated gametes
and zoospores
chitin (cellulose) in cell walls
mostly saprobic, also parasitic
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
Allomyces sp. (Tom Volk, Univ. of Wisconsin-LC)
27
Traditional classification
based on habitate, zoospore characterization, life cycles
Chytridiomycota
Chytridiomycetes
Chytridiales (Schröter 1892)
Blastocladiales (Fitzpatrick 1930)
Monoblepharidales (Sparrow 1942)
Spizellomycetales (Barr 1980)
Neocallimastigales (Li et al. 1993)
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
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super tree – all data set
based on 4 source trees
29
Questions?
“Whenever a theory appears to you as the only possible one, take this as a sign that
you have neither understood the theory nor the problem which it was intended to solve”
Karl Popper (1972)
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
30
Thank you for your attention
With Many thanks to

PD Dr. K. Voigt
K. Hoffmann
Dr. K . Fliegerova
Prof. Dr. O. Bininda-Emonds
Dr. Alexandro Stamatikis
Malte Brinkmeyer
Prof. Dr. S. Böcker
Thasso Griebel
Christoph Thieme
Matthias Gube
… and for the support at the Group Microbial Phytopathology Jena
Martin Eckart – Fungal Reference Center Jena – Institute of Microbiology (2009)
31
Literatur

Books:

Knoop/ Müller
Gene und Stammbäume
Jean-Michel Ph. D. Claverie
Bioinformatics for Dummies
Paper:

W.M. Fitch (2000) Homology
Trends in Genetics 16:227

PubMed...
Phylogenetic course 2009 (Voigt, Hoffmann, Eckart)
32
Phylogenetische Rekonstruktion der
Evolution basaler Pilze
Kerstin Voigt & Martin Eckart
Friedrich-Schiller-Universität Jena, Pilz-Referenz-Zentrum
http://www.prz.uni-jena.de
1. Deep-level phylogeny
3. Supertrees & -matrices
http://www.prz.uni-jena.de
2. Zygomycetes
4. Chytridiomycetes
Zygomycetes – Characteristics
Sexual Reproduction – Gametangiogamy
Copulation of non-differentited, coenocytic gametangia leads to the formation of
zygotes (zygospores) which are associated with two yoke-shaped suspensors
Absidia glauca
Syzygites
megalocarpus
Phycomyces
blakesleeanus
100 μm
Mucorales – Parasitism
Sikyospore versus Galls
Parasite
Parasite
Host
10 μm
Host
10 μm
Parasitella parasitica (-) parasitises on
Gilbertella persicaria (+)
Chaetocladium brefeldii (+) parasitises on
Mucor hiemalis forma hiemalis (-)
Foto: Abdel-Rahman Saleem (2002)
Foto: Reinicke M, Eckart M & Voigt K (2000)
Mucorales (Fungi)
Gall-mediated Mycoparasitism
Host
Parasit
Parasite
Absidia parricida x Mucor rouxii
Sikyotic Mycoparasitism
Host
Parasite
Parasitella parasitica x Thamnidium elegans
Sikyotic Mycoparasitism
Host
Parasite
Parasitella parasitica x Mucor rouxii
Zygomycetes – Characteristics
Asexual reproduction via non-motile mitospores
Sporangium
Sporangiolum
Merosporangium
multispored
uni- or fewspored
elongated sporangiolum
producing uniserate spores
50 µm
Absidia corymbifera
Syzygites megalocarpus
Syncephalastrum racemosum
Mucorales
Mortierellales
Kickxellales
20 μm
25 μm
Mucor
Mortierella
Kickxella
Coenocytic
Irregularly septated
Regularly septated
Micropores
Micropores
Dolipores
Sporangia
Sporangiola
Merosporangia
Sporangia
Soil fungi
Soil fungi
Merosporangia
Soil fungi
Zygomycota – Traditional Classification
Basidiomycota
Phylum
Class
Order
Zygomycota
Trichomycetes
Class
Order
Zygomycetes
Mucorales
Mortierellales
Kickxellales
Ascomycota
Harpellales
Asellariales
Eccrinales
Glomeromycota
Blastocladiomycota
Zygomycota
Chytridiomycota
Neocallimastigomycota
Zoopagales
Dimargaritales
Endogonales
Entomophthorales
Basidiobolales
(Alexopoulos et al. 1996)
Alexopolous, C.J.; Mims, C.W.; Blackwell, M. 1996.
Introductory Mycology. NY: John Wiley & Sons
Zygomycota – Novel Classification
No rank
Phylum
Class
Order
Zygomycota
Trichomycetes
Class
Order
Zygomycetes
Mucorales
Mortierellales
Kickxellales
Harpellales
Asellariales
Eccrinales
Zoopagales
Dimargaritales
Endogonales
Entomophthorales
Basidiobolales
Fungi incertae sedis
Basal fungal linages
Subphylum
Entomophthoramycotina
Order
Entomophthorales
Family
Basidiobolaceae
Subphylum
Order
Kickxellomycotina
Harpellales
Asellariales
Dimargaritales
Kickxellales
Subphylum
Order
Mucoromycotina
Mucorales
Mortierellales
Endogonales
Subphylum
Order
Zoopagomycotina
Zoopagales
(Alexopoulos et al. 1996)
No rank Fungi/Metazoa incertae sedis
Class
Mesomycetozoa
Order
Eccrinales
Alexopolous, C.J.; Mims, C.W.; Blackwell, M. 1996. Introductory Mycology
Hibbett et al. 2007 Mycol Res. 111: 509-47
(Hibbett et al. 2007)
Zygomycetes represent the most basal group of terrestric fungi
Chytridiomycota
+ Actin (273 aa)
+ β-tub (435 aa)
Mucorales
´Zygomycota´
+ EF-1α (458 aa)
Σ 1,166 aa
MYCOTA
Maximum Parsimony
8 trees
4,018 steps
CI = 0.58
HI = 0.42
RI = 0.59
Ascomycota
Basidiomycota
Metazoa
Choanoflagellida
Microsporidia
BP > 60 %
10 changes
Mycetozoa
Entamoebidae
Diplomonadida
Oomycota
Viridiplantae
Euglenozoa
Heterolobosea
Mucorales (Fungi)
Zygomycetes – Importance
Biotechnology
biotransformations
carotene
lycopene
chitosan
fatty acids
Food production
cheese
tempeh
soy souce
Agriculture
plant pathogens
food spoilage
post-harvest pathogens
Medicine
human, animal pathogens
(zygomycoses and
entomophthoromycoses)
Rhizopus
Circinella
Pilobolus
Syzygites
Columella
The trade-mark of the Mucorales is a swollen extension of the sporangiophore
called a columella, which protrudes like a balloon into the sporangium.
Columella
a bulbous
vesicle at the
sporangiophore apex
50 µm
Family structure of the Mucorales
Aktin & EF-1α
18S & 28S rDNA
Σ 4,151 bp
82 Taxa
Gewichtete
Maximum-Parsimony
2 trees
12,046 steps
CI = 0.27
HI = 0.73
RI = 0.62
Mucoraceae – Thermophile Absidia spp.
Thamnidiaceae
Mucoraceae – Mesophile Absidia spp.
Cunninghamellaceae
Mucoraceae
Parasitella parasitica
Chaetocladiaceae
Thamnidiaceae
Mycotyphaceae
Gilbertellaceae
Choanephoraceae
Pilobolaceae
Mucoraceae
Saksenaeaceae incl. Apophysomyces
Phycomycetaceae incl. Spinellus
Syncephalastraceae
Umbelopsis spp.
100 changes
Mortierellales
Voigt & Wöstemeyer (2001) Gene 270: 113-120.
Spinellus fusiger classifies within the
Phycomycetaceae
Proposed by
von Arx
(1983)
Sydowia
Evidenced
by Voigt et
al. (2008)
Sporangia of Spinellus fusiger (Mucorales) parasitic on fruitbodies of the
mushroom Mycena pura. (© Malcolm Storey 2004, from www.bioimages.org.uk).
The Intron Structure of the Actin Gene
1412
1
Act-1
I
II
501-668
Max. Intron Length [bp]
Intron
Group
805-969
167
164
Intron Presence
III
IV
V
1122-1267
1351-1411
1278-1342
145
64 65
Organism Group
I
II
III
IV
V
Higher Taxon
0
1
+
-
-
-
-
Mortierellales
Mortierella
Mortierellales
2
+
+
-
-
-
3
-
+
-
-
-
Thermophilic Absidiagroup
and all others
Mesophilic Absidiagroup
4
-
-
+
-
-
5
6
+
+
+
+
-
+ - +
Mortierella, Umbelopsis,
Micromucor
Absidia, Rhizomucor,
Thermomucor
and all others
Absidia, Chlamydoabsidia,
Cunninghamella,
Halteromyces
Fennellomyces,
Phascolomyces,
Thamnostylum, Zychaea
Rhizomucor miehei
Thamnidiaceae
Syncephalastraceae
Act-4R
Genera and Species
Syncephalastrum
Family structure of the Mucorales based on a Five-Locus-Phylogeny
Neighbor-Joining
(similar topology: MP, ML)
actin & EF-1α & βtub & 18S & 28S rDNA
5,312 nt
35 Taxa
Weighted
BP > 70
Mortierellales
Chaetocladium brefeldii
Syzygites megalocarpus
Rhizopus microsporus
Rhizopus oryzae
Cokeromyces recurvatus
Blakeslea trispora
Choanephora infundibulifera
Poitrasia circinans
Gilbertella persicaria
Mucor mucedo
Pilaira anomala
Zygorhynchus moelleri
Actinomucor elegans
Mucor hiemalis
Utharomyces epallocaulus
Parasitella parasitica
Ellisomyces anomalus
Mycotypha afriana
Mucor racemosus
Radiomyces spectabilis
Absidia glauca
Chlamydoabsidia padenii
Halteromyces radiatus
Absidia corymbifera
Phycomyces blakesleeanus
Spinellus fusiger
Fennellomyces linderi
Thamnostylum piriforme
Syncephalastrum racemosum
Gongronella butleri
Umbelopsis ramanniana
Umbelopsis isabellina
0.1
substitutions per site
Morphology: sporangia versus sporangiola
Sporangia
Rhizopus microsporus
Rhizopus oryzae
Blakeslea trispora
Choanephora infundibulifera
Poitrasia circinans
Gilbertella persicaria
Mucor mucedo
Pilaira anomala
Zygorhynchus moelleri
Actinomucor elegans
Mucor hiemalis
Utharomyces epallocaulus
Parasitella parasitica
Ellisomyces anomalus
Mucor racemosus
Absidia glauca
Chlamydoabsidia padenii
Halteromyces radiatus
Absidia corymbifera
Phycomyces blakesleeanus
Spinellus fusiger
Fennellomyces linderi
Thamnostylum piriforme
Umbelopsis ramanniana
Umbelopsis isabellina
Mortierellales
Gongronella butleri
Morphology: sporangia versus sporangiola
Sporangiola
Chaetocladium brefeldii
Syzygites megalocarpus
Cokeromyces recurvatus
Blakeslea trispora
Choanephora infundibulifera
Ellisomyces anomalus
Mycotypha afriana
Radiomyces spectabilis
Fennellomyces linderi
Thamnostylum piriforme
Syncephalastrum racemosum
Morphology: appendaged, striated sporangiospores and longitudinal
suture in sporangial wall
Blakeslea trispora
Choanephora cucurbitarum
Poitrasia circinans
Gilbertella persicaria
Poitrasia circinans
Choanephora fruit (wet) rot on yellow
straight neck squash (Cucurbita pepo)
caused by Choanephora cucurbitarum.
Photo: Gerald J. Holmes, Dept. Plant
Pathology, North Carolina State University
Raleigh, NC 27695
Voigt K & Olsson, L (2008) Acta Biologica Hungarica 59 (3): 365383.
Take Home Message – Criteria of Phylogenetic Relevance
Monophyletic:
1.
Columella
2.
Sporangiospores: appendaged, striated
3.
Longitudinal suture in sporangial wall
4.
Growth temperature
Polyphyletic:
1.
Spore number (Sporangia, sporangiola)
2.
Spore shape
3.
Apophysis
4.
Sporangial or sporangiolar wall (persistent, deliquescent, evanescent)
5.
Rhizoids
6.
Zygospore morphology (appendaged, opposed-apposed suspensors)
7.
Mycoparasitism
Mucorales – the genus Absidia
-pyriform, apophysate sporangia
-polyphyletic (mesophilic, thermotolerant, parasitic species)
spores
columella
apophyse
20 µm
Hoffmann et al. (2007) Mycological Research 111 (10): 1169-1183.
Mucorales – the genus Absidia
0.2
mesophilic
thermotolerant
growthrate [mm/h]
mycoparasitic
0.15
0.1
0.05
0
18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
temperature [°C]
Hoffmann et al. (2007) Mycological Research 111 (10): 1169-1183.
Splitting the genus Absidia
Mortierellales
100
Umbelopsidaceae
Absidia zychae
Absidia parricida
94
100
Lentamyces
Hoffmann & Voigt 2008
99
Circinella umbellata
100
100
Lichtheimia spp.
79
Lichtheimiaceae
Hoffmann, Walther & Voigt 2009
100
Dichotomocladium elegans
Dichotomocladium hesseltinei
Dichotomocladium robustum
100
100
61
98
100
91
0.1 substitutions/site
100
Absidia spp.
Absidiaceae s. str.
von Arx 1982
We thank our collaborators…
Institute of Systematic Zoology and Evolutionary
Biology with Phyletic Museum, University Jena
Lennart Olsson
Nadine Piekarski
Jena Bioinformatics Centre, University Jena
Stefan Schuster
Christoph Thieme
Institute of Microbiology, University
Innsbruck
Martin Kirchmair
Sigrid Neuhauser
Reinhold Pöder
Institute of Applied Microbiology, University
of Nat. Resources and Applied Life Sciences
Vienna
Hansjörg Prillinger
Centraalbureau voor Schimmelcultures
Utrecht
Grit Walther
Sybren deHoog
Dept. of Forest Resources, University of
Idaho, Moscow
George Newcombe
Grants:
Taif University, Saudi Arabia
Youssuf Gherbawy
Institute of Microbiology, University Szeged
Tamás Papp
Csaba Vágvölgyi
Fungal Reference Centre (Pilz-Referenz-Zentrum) Jena
10.000 fungal
cultures (active
and cryo)
Kerstin Hoffmann
Martin Eckart
Claudia Kesselboth
Lysette Wagner
Gisela Baumbach
...and many helpful hands from volunteer students