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Probing early eukaryotic
evolution using phylogenetic
methods
The SSU Ribosomal RNA Tree for Eukaryotes
Animals
The Universal SSU rRNA Tree
Wheelis et al. 1992 PNAS 89: 2930
The Archezoa Hypothesis - primitively
amitochondriate eukaryotes
(Tom Cavalier-Smith 1983)
Fungi
Choanozoa
Trichomonas
Plants / green algae
Mitochondria?
Microsporidia
Ciliates + Apicomplexa
Stramenopiles
Red algae
Dictyostelium
Entamoebae
Prokaryotic
outgroup
Euglenozoa
Physarum
Percolozoa
Trichomonas
Giardia
Microsporidia
Archezoa
‘Tree’ courtesy of W. Ford Doolittle
Giardia
Entamoeba
The Archezoa Hypothesis
T. Cavalier-Smith (1983)
• The Archezoa hypothesis would fall if:
– Find mitochondrial genes on archezoan
genomes
– Find that archezoans branch among
aerobic species with mitochondria
– Find mitochondrion-derived organelles
in archezoans
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Mitochondrial HSP 70 is encoded by the
host nucleus but is of endosymbiotic origin
Targeting
sequences
ATPase DOMAIN
PEPTIDE BINDING
DOMAIN
Retention
sequences
N
C
Cytosolic HSP70
mitochondrial HSP70
RER HSP70
alpha-proteobacteria
Endosymbiotic
origin
other proteobacteria
chloroplasts
Gram +ve & Archaea
cyanobacteria
Mitochondrial genes in Archezoa
Archezoa
Proteins of mitochondrial origin*
Giardia /
Spironucleus
Heat shock 70, Chaperonin 60
Trichomonas
Heat shock 70, Chaperonin 60
Microsporidia
Heat shock 70
Entamoeba
Heat shock 70, chaperonin 60
Chaperonin 60 Protein Maximum Likelihood Tree
(PROTML, Roger et al. 1998, PNAS 95: 229)
Note 100% Bootstrap support
*defined as forming a monophyletic group with mitochondrial
homologues in a non-controversial species phylogeny
Long branches may cause problems for
phylogenetic analysis
A case of
Eukaryote
Eukaryote
HGT?
Chaperonin 60 Protein Maximum Likelihood Tree
(PROTML, Roger et al. 1998, PNAS 95: 229)
• Felsenstein (1978) made a simple model phylogeny including four
taxa and a mixture of short and long branches
A
p
TRUE TREE
A
B
p
q
q
q
C
D
D
WRONG TREE
p>q
Longest
branches
C
B
• Methods which assume all sites change at the same rate
(e.g. PROTML) may be particularly sensitive to this
problem
2
Cpn-60 Protein ML tree (PROTML) from variable
sites with outgroups removed
A simple experiment:
Giardia
• Does the Cpn60 tree topology change:
– If we remove long-branch outgroups
– If we remove sites where every species
has the same amino acid
Trichomonas
30
Euglena &
Trypanosoma
31
Animals & Fungi
Apicomplexa
Plants
Dictyostelium
Entamoeba
The Archezoa Hypothesis
T. Cavalier-Smith (1983)
• The Archezoa hypothesis would fall if:
– Find mitochondrial genes on archezoan
genomes
– Find that archezoans branch among
aerobic species with mitochondria
– Find mitochondrion-derived organelles
in archezoans
a-tubulin trees place Microsporidia with Fungi
Edlind TD, et al. (1996). Mol Phylog Evol 5 :359-67
Keeling PJ, Doolittle WF. (1996). Mol Biol Evol 13 :1297-305.
Microsporidia
65
Fungi
Microsporidia are primitive and early
branching eukaryotes or relatives of fungi?
• Microsporidia branch deep in some trees but not in others
– Deep: SSU rRNA, EF-2
– With fungi: tubulin and HSP70
• Microsporidia contain a mitochondrial HSP70
– They once contained the mitochondrial endosymbiont and thus are
not Archezoa sensu Cavalier-Smith
– Hirt et al., 1997
Other eukaryotes
– Or they obtained this gene (and the one for tubulin) from fungi via
HGT
– Sogin, 1997 Curr. Op. Genet. Dev. 7: 792-799
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Maximum likelihood and phylogenetic
inference
• The maximum likelihood tree is the one with
The tree shown
is the maximum
likelihood tree
obtained using a
program called
PROTML
the highest probability of giving rise to the
data under a particular model
88
• It is not the probability that it is the “true”
83
75
tree
Assumptions underpinning the
PROTML model
LogDet/Paralinear distances for EF-2 DNA
variable sites codon positions 1+2
Animals
• Assumes all sites in the molecule can change
• Assumes that all sequences are evolving in
the same way
70
CAGGAATCACCTACGATCCTCGGCGGCGTTACGAACCGAA
CAGGAATCACTTACGATCTTAAGCGGCGTTACGAACAGAA
CAGGAATCACCTACGATCCTCAGCGGCGCTACGAACCGGC
CAGGAAACACTTTCGATCTTGTTCGGAGTAATTCTGCGGC
CAGGAATCACTTTCGATCCTGAGCGGCGATATGAACCGGC
CAGGAATCACTTACGATCCTAAGCGGAGTAACGAACAGCC
CAGGAATCACTTACGATCCTGCGCGGCGTTACGAACCGGC
CAGGAATCACCTACGATCCTAAGCGGCGTTACGAACCGCC
CAGGAATCACCTACGATCCTAAGCGGCGTTACGAACCGCC
CAGGAATCACCTACGATCCTTCGCGGCGTTACGAACCGCA
CAGGAAAGACCTACGATGCTGATCGGAGTAATGAACCGGA
CAGGAAACACACAGGAACCTGTGCGGCTGCTTGATACTTC
CAGGAAACACTTTCGAAATTTTGCGGCTGCCTGATTGAGA
CAGGAAACACCTACGAAACTACGCGGCTGCATGAACGAGA
Methanococcus outgroup
(low G+C)
100
25
76
Entamoeba
Microsporidia
+ fungi!
Saccharomyces
Glugea
Cryptosporidium
54%G+C
54%G+C
53%G+C
45%G+C
43%G+C
44%G+C
58%G+C
Sulfolobus
Methanococcus
Halobacterium
Archaebacteria
outgroups
A combination of factors (outgroup GC content & site
rate heterogeneity) influence the EF-2 DNA tree
Methanococcus outgroup
(low G+C)
Halobacterium outgroup
Higher G+C
80
60
ML
estimate
60
40
40
20
20
Halobacterium outgroup
Higher G+C
100
100
80
Bootstrap
values
80
60
60
40
40
20
20
0
0
Giardia
Dictyostelium
100
80
LogDet
Bootstrap
values
60
53%G+C
46%G+C
A combination of factors (outgroup GC content and
site rate heterogeneity) influence the EF-2 DNA tree
Trypanosoma
Trichomonas
• EF-2 violates both of these assumptions:
Human
Saccharomyces
Chlorella
Dictyostelium
Trypanosoma
Entamoeba
Cryptosporidium
Tritrichomonas
Tritrichomonas
Giardia
Glugea
Sulfolobus
Methanococcus
Halobacterium
Chlorella
0
20
40
60
80
100
0
0
20
40
60
80
Fraction of constant sites removed
(Microsporidia, outgroup)
100
0
20
40
60
80
100
0
0
20
40
60
80
100
Fraction of constant sites removed
(Microsporidia, outgroup)
(Microsporidia, Fungi)
4
A combination of factors (outgroup GC content & site
rate heterogeneity) influence the EF-2 DNA tree
Methanococcus outgroup
(low G+C)
Bootstrap
values
Halobacterium outgroup
Higher G+C
100
100
80
80
60
60
40
40
20
0
20
40
60
80
100
0
• Probably not:
• Microsporidia are related to fungi (Tubulin, RNA
polymerase,LSU rRNA, HSP70, TATA binding protein,
EF-2, EF-1 alpha, SSU rRNA)
• Evidence for Giardia and Trichomonas branching deeper
than other eukaryotes is based on trees made using
unrealistic assumptions (often PROTML)
20
0
Are the former Archezoa really
ancient offshoots?
• There is plenty of room for new hypotheses
0
20
40
60
80
100
Fraction of constant sites removed
(Giardia, Trichomonas, outgroup)
If former archezoa contain
genes from the mitochondrial
endosymbiont what happened to
the organelle?
Microsporidia are obligate
intracellular parasites
Life cycle of microsporidia
mtHsp70 has diverse roles in mitochondria
Pfanner and Geissler 2001 Nature Reviews 2: 339-344
Tree of Mitochondrial Hsp70
mtHsp70
mtHsp70 also plays a role in assembly of Fe-S clusters
- an essential function for yeast mitochondria (Lill & Kispal, 2000)
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Localisation of a mtHsp70 in Trachipleistophora
Infected
cells
Rabbit
Confocal
microscopy
using an
antibody to
Th-mtHsp70
Spores
Microsporidial HSP70 have no obvious
N-terminal targeting signal
Western blot using an antibody to Th-mtHsp70
The Hsp70 protein is
localised to membrane
bound or electron dense
structures.
Microsporidian
Microsporidian
‘mitochondrial remnant’
Host cell mitochondrion
Traditional fixation
methods show a double
membrane
Williams et al. 2002 Nature 418: 865-869
Trees are a good way of exploring the
history of genes but care is needed!
• Making trees is not easy:
– Among-site rate heterogeneity, “fast
clock” species, shared nucleotide or amino
acid composition biases
– Different data sets may be affected by
individual phenomena to different degrees
Phylogenetic analysis requires
careful thought
• Phylogenetic analysis is frequently treated as a
black box into which data are fed (often gathered
at considerable cost) and out of which “The Tree”
springs
• (Hillis, Moritz & Mable 1996, Molecular Systematics)
– Biases need not be large if phylogenetic
signal is weak
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