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Probing early eukaryotic evolution using phylognetic methods . The Universal SSU rRNA Tree Wheelis et al. 1992 PNAS 89: 2930. The SSU Ribosomal RNA Tree for Eukaryotes. Mitochondria?. Prokaryotic outgroup. Archezoa. The Archezoa Hypothesis T. Cavalier-Smith (1983).

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slide3

The SSU Ribosomal RNA Tree for Eukaryotes

Mitochondria?

Prokaryotic outgroup

Archezoa

the archezoa hypothesis t cavalier smith 1983
The Archezoa HypothesisT. Cavalier-Smith (1983)

“Archezoa are eukaryotes which primitively lack mitochondria”

  • The nucleus was invented before the mitochondrion was acquired
  • The first eukaryotes were anaerobes
  • Archezoans might provide insight into the nature of ancestral eukaryotic genomes and biology
the archezoa hypothesis t cavalier smith 19831
The Archezoa HypothesisT. Cavalier-Smith (1983)
  • The Archezoa hypothesis would fall if:
    • Find mitochondrial genes on archezoan genomes
    • Find mitochondrion-derived organelles in archezoans
    • Find that archezoans branch among aerobic species with mitochondria
is trichomonas an archezoan

Chaperonin60/GroEL phylogeny

pyruvate ferredoxin oxidoreductase

PFO

Pyruvate Acetyl CoA

Fe-hydrogenase

2H+ H2

Pi + ADP ATP

Is Trichomonas an Archezoan?
trichomonas chaperonin 60 shares common ancestry with mitochondrial chaperonins
Trichomonas chaperonin 60 shares common ancestry with mitochondrial chaperonins

Mitochondria

alpha - proteobacteria

mitochondrial genes in archezoa
Mitochondrial genes in Archezoa

Archezoa

Proteins of mitochondrial origin*

Giardia /

Spironucleus

Trichomonas

Microsporidia

Heat shock 70, Chaperonin 60

Heat shock 70, Chaperonin 60

Heat shock 70

*defined as forming a monophyletic group with mitochondrial homologues in a non-controversial species phylogeny

chaperonin 60 protein maximum likelihood tree protml roger et al 1998 pnas 95 229
Chaperonin 60 Protein Maximum Likelihood Tree(PROTML, Roger et al. 1998, PNAS 95: 229)

Note 100% Bootstrap support

A case of

Eukaryote Eukaryote

HGT?

long branches may cause problems for phylogenetic analysis

A

A

B

p

p

D

q

q

q

C

C

D

B

Long branches may cause problems for phylogenetic analysis
  • Felsenstein (1978) made a simple model phylogeny including four taxa and a mixture of short and long branches

TRUE TREE

WRONG TREE

p > q

  • Methods which assume all sites changeat the same rate (e.g. PROTML) may be particularly sensitive to this problem
chaperonin 60 protein maximum likelihood tree protml roger et al 1998 pnas 95 2291
Chaperonin 60 Protein Maximum Likelihood Tree(PROTML, Roger et al. 1998, PNAS 95: 229)

Longest branches

a simple experiment
A simple experiment:
  • Does the Cpn60 tree topology change:
    • If we remove long-branch outgroups
    • If we remove sites where every species has the same amino acid
slide14

Competing Hypotheses for Microsporidia

Microsporidia + Fungi

Tubulin, mitHSP70

“Microsporidia Early”

SSU rRNA, EF-1 alpha

EF-2

  • HGT from Fungi to Microsporidia? (Sogin, 1998)
  • Another artefact of the method of analysis?
microsporidia have a number of unusual features
Microsporidia have a number of unusual features
  • Absence of mitochondria and peroxisomes
  • 70s ribosomes - most eukaryotes have 80S
  • 5.8S and 23S rRNA genes are fused - like in some prokaryotes
  • Lack 9 + 2 microtubule structures
alternative explanations of microsporidia unusual features
Alternative explanations of Microsporidia unusual features
  • Retention of ancestral features of the eukaryote cell at an early stage of evolution?

Or are they

  • Adaptations to an obligate intracellular lifestyle?
elongation factor 2 protein ml tree protml hashimoto et al 1997 arch protist 148 287
Elongation Factor 2 protein ML tree (PROTML)(Hashimoto et al. 1997 Arch. Protist. 148:287)

Entamoeba

Dictyostelium

88

Eukaryote root

75

Microsporidia

Archaebacteria outgroups

Also note that in PROTML the amino acid substitution process is assumed to be homogeneous across the tree

slide18
Shared nucleotide or amino acid composition biases can also cause problems for phylogenetic analysis

Aquifex

Thermus

Aquifex (73%)

Bacillus (50%)

True tree

Wrong tree

16S rRNA

Thermus

(72%)

Bacillus

Deinococcus

Deinococcus

(52% G+C)

Aquifex

The correct tree can be obtained if a model is used which allows base/aa composition to vary between sequences -LogDet/Paralinear Distances

Heterogeneous Maximum Likelihood

Bacillus

Thermus

Deinococcus

logdet paralinear distances for ef 2 dna variable sites codon positions 1 2
LogDet/Paralinear distances for EF-2 DNA variable sites codon positions 1+2

Animals

Chlorella

Note that root has changed

70

Trypanosoma

Trichomonas

60

Giardia

Dictyostelium

25

Entamoeba

Sacharomyces

76

45% G+C

Microsporidia

Cryptosporidium

Sulfolobus

Archaebacteria outgroups

Methanococcus 44%

Halobacterium 58%

slide20
A combination of factors (outgroup GC content and site rate heterogeneity) influence the EF-2 DNA tree

Methanococcus outgroup

(low G+C)

Halobacterium outgroup

Higher G+C

100

100

80

80

LogDet

Bootstrap values

ML estimate

60

60

40

40

20

20

0

0

0

20

40

60

80

100

0

20

40

60

80

100

Fraction of constant sites removed

(Microsporidia, outgroup)

slide21

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

100

100

80

80

Bootstrap values

60

60

40

40

20

20

0

0

0

20

40

60

80

100

0

20

40

60

80

100

Fraction of constant sites removed

(Microsporidia, outgroup)

(Microsporidia, Fungi)

a combination of factors outgroup gc content site rate heterogeneity influence the ef 2 dna tree
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

100

100

80

80

Bootstrap values

60

60

40

40

20

20

0

0

0

20

40

60

80

100

0

20

40

60

80

100

Fraction of constant sites removed

(Giardia, Trichomonas, outgroup)

slide23

Competing hypotheses for Microsporidia

Microsporidia + Fungi

Tubulin, RNA polymerase,

LSU rRNA, HSP70,

TATA binding protein,

EF-2, EF-1 alpha

“Microsporidia Early”

SSU rRNA

The best supported hypothesis for Microsporidia is a relationship to fungi - why does SSU rRNA place them deep?

summary i
Summary I
  • 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
    • Biases need not be large if phylogenetic signal is weak
summary ii
Summary II
  • Are Archezoa ancient offshoots?
    • Microsporidia are related to fungi
    • Evidence for Giardia and Trichomonas branching deeper than other eukaryotes is based on trees made using unrealistic assumptions
  • PLUS
    • For the same reasons we don’t know where the root lies on the eukaryote tree
    • So arguments about early or late branching are probably premature anyway
can we make a robust unrooted tree for eukaryotes
Can we make a robust unrooted tree for eukaryotes?
  • Combining different genes in a single analysis may provide a more robust eukaryotic tree
  • One argument is that phylogenetic signal should be additive whereas gene-specific “noise” will pull in different directions
slide27
DNA ML tree found using a model which allows both base composition and site rates to vary across the tree

Animals + fungi + slime moulds

Ciliates plus apicomplexa

Giardia and Trichomonas

Red and green algae/plants

Actin+tubulin+EF-2

hydrogenosomes
Hydrogenosomes
  • Strange anaerobic eukaryotic organelles which make hydrogen
slide29

12um

Methanocorpusculum

(Uses H2 and CO2)

Metopus endosymbiont

origin s of hydrogenosomes
Origin(s) of Hydrogenosomes
  • Is a 2 part problem
    • The organelle (the bag)
    • The biochemistry to produce hydrogen particularly hydrogenase
slide33

CO2

hsp70

Protein import

ME

Malate

Pyruvate

cpn60

Transit

peptides

AAC

NAD(P)H

NAD(P)+

ATP

N

ADP

NAD(P)-FO

[Fe]Hyd

H2

2Fd

2Fd-

2H+

ASCT

Acetate

Acetyl-CoA

PFO

Succinate

Succinyl-CoA

CO2

Double membrane

CoASH

STK

Fungi and Trichomonas

ADP + Pi

ATP

Enzyme found also in mitochondria

Alpha-proteobacterial ancestry

Schematic Map of Hydrogenosomes

(after Muller 1993)

Unknown ancestry

a likelihood ratio test of monophyly huelsenbeck hillis nielson 1996
A likelihood ratio test of monophyly (Huelsenbeck, Hillis & Nielson 1996)

The Test Statistic (d) = lnL1 - lnL0

  • Where lnL1 is the likelihood of the best tree and lnL0 is the likelihood of the best monophyly tree
  • The null (eukaryote monophyly) distribution of d is generated by simulation under an appropriate model (parametric bootstrapping)
parametric bootstrapping to estimate a test distribution
Parametric Bootstrapping to estimate a test distribution

What might the test statistic distribution look like if the Fe hydrogenases were monophyletic?

Estimate ML model parameters using original data

Simulate 1000 new sequence data sets using this model over the best monophyly tree

Calculate d for original data and compare to distribution - if it falls outside of the 95% interval it is bigger than expected by chance and monophyly can be rejected

For each new data set estimate L0 and L1 using ML, with model re-optimised each time

Plot d for each of the 1000 data sets to give the test distribution and the 95%confidence interval

the likelihood ratio test rejects the hypothesis that eukaryotic hydrogenases are monophyletic
The likelihood ratio test rejects the hypothesis that eukaryotic hydrogenases are monophyletic

d= lnL1 - lnL0

d for original data

95%

d Original data

95%

9.64

d (lnL1 - lnL0) distribution from 1000 simulations of the [Fe] hydrogenase data on the best monophyly tree

slide41

Iron hydrogenase ML tree

Eukaryotic compartment

Trichomonas Hydrogenosome

Cytosolic?

Plastid

Ciliate Hydrogenosome

conclusions i
Conclusions I
  • Hydrogenosomes share common ancestry with mitochondria
  • Hydrogenase has been acquired at least twice and can be targeted to different cell compartments in different eukaryotes
    • Humans, plants and fungi also contain remnants of iron hydrogenases
  • There is no evidence from phylogenetic analysis that the “bag” and hydrogenase share a common origin from the mitochondrion endosymbiont
conclusions ii
Conclusions II
  • Phylogeny is hard, there are lots of potential problems with data, so we need to be careful in our interpretations of what trees mean - includes inferences of HGT
  • Better methods hold promise of more reliable trees (allowing re-analysis of SSUrRNA data)
  • Archezoa contain genes which originated with mitochondrion endosymbiont and the jury is still out on whether former archezoa have lost the mitochondrial bag
  • We don’t know which eukaryotes are early branching - for this we need a rooted tree
the mitosome a novel organelle related to mitochondria in entamoeba histolytica
The mitosome, a novel organelle related to mitochondria in Entamoeba histolytica

Tovar et al., 1999.

Slide shows epitope tagged recombinant cpn60 localised to mitosome

are there still organelles of common ancestry with mitochondria in giardia and microsporidia
Are there still organelles of common ancestry with mitochondria in Giardia and Microsporidia?
  • Giardia:
    • “What are the ovoid pellicular bodies (in Giardia)? The study made suggests that they might be nothing but changed mitochondria with a few crysts or tubules”
    • “The ultrastructure of mitochondria may be related with the oxygen deficiency in Lamblia environment”

(Cheissen, 1965)

  • Microsporidia:
    • “There are reports of mitochondria-like structures in several microsporidia”

(Vavra, 1976)