Probing early eukaryoticevolution using phylognetic

methods

The Universal SSU rRNA TreeWheelis et al. 1992 PNAS 89: 2930

Archezoa

The SSU Ribosomal RNA Tree for Eukaryotes

Mitochondria?

Prokaryoticoutgroup

AnimalsFungi

Ciliates + ApicomplexaStramenopiles

Euglenozoa

GiardiaTrichomonas

Plants / green algae

Red algae

Entamoebae

Choanozoa

Dictyostelium

Physarum

Microsporidia

Percolozoa

The Archezoa HypothesisT. Cavalier-Smith (1983)

“Archezoa are eukaryotes which primitively lackmitochondria”

– The nucleus was invented before themitochondrion was acquired

– The first eukaryotes were anaerobes

– Archezoans might provide insight into thenature of ancestral eukaryotic genomes andbiology

The Archezoa HypothesisT. Cavalier-Smith (1983)

• The Archezoa hypothesis would fall if:– Find mitochondrial genes on archezoan

genomes

– Find mitochondrion-derived organelles inarchezoans

– Find that archezoans branch amongaerobic species with mitochondria

Is Trichomonas an Archezoan?

Chaperonin60/GroEL phylogeny

mitochondria

alpha-proteobacteria

Clostridia

other bacteria

Pyruvate Acetyl CoA

2H+ H2

Pi + ADP ATP

pyruvate ferredoxin oxidoreductasePFO

Fe-hydrogenase

Trichomonas chaperonin 60 shares commonancestry with mitochondrial chaperonins

alpha - proteobacteria

Mitochondria

.10

Homo sapiensRatus norvegicusMus musculus

Heliothis virescensTrichomonas vaginalis

Ehrlichia chaffeensisRickettsia tsutsugumushi

Dictyostelium discoidiumTrypanosoma brucei

Zea maysArabidopsis thaliana

Naegleria fowleri

Saccharomyces cerevisiaeAjellomyces capsulatum

Schizosaccharomyces pombeNeocallimastix frontalis

Mitochondrial genes in Archezoa

Giardia /Spironucleus

Trichomonas

Microsporidia

Heat shock 70, Chaperonin 60

Heat shock 70, Chaperonin 60

Heat shock 70

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

Proteins of mitochondrial origin*Archezoa

Chaperonin 60 Protein Maximum Likelihood Tree(PROTML, Roger et al. 1998, PNAS 95: 229)

A case ofEukaryote Eukaryote

HGT?

Note 100% Bootstrap support

Long branches may cause problems forphylogenetic analysis

• Felsenstein (1978) made a simple model phylogeny includingfour taxa and a mixture of short and long branches

• Methods which assume all sites change at the same rate(e.g. PROTML) may be particularly sensitive to thisproblem

A

B

C

D

TRUE TREE WRONG TREE

A B

C D

ppq

qq p > q

Chaperonin 60 Protein Maximum Likelihood Tree(PROTML, Roger et al. 1998, PNAS 95: 229)

Longestbranches

• Does the Cpn60 tree topology change:

– If we remove long-branch outgroups

– If we remove sites where every specieshas the same amino acid

A simple experiment:

Cpn-60 Protein ML tree (PROTML) from variablesites with outgroups removed

Giardia

Entamoeba

Dictyostelium

30

31

Plants

Apicomplexa

Euglena & Trypanosoma

Trichomonas

Animals & Fungi

Competing Hypotheses for Microsporidia

AnimalsFungi

Ciliates + ApicomplexaStramenopiles

Euglenozoa

GiardiaTrichomonas

Plants / green algae

Red algae

Entamoebae

Choanozoa

Dictyostelium

Physarum

Microsporidia

Percolozoa“Microsporidia Early”SSU rRNA, EF-1 alphaEF-2

Microsporidia + FungiTubulin, mitHSP70

• HGT from Fungi to Microsporidia? (Sogin, 1998)• Another artefact of the method of analysis?

• 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

Microsporidia have a numberof unusual features

• Retention of ancestral features ofthe eukaryote cell at an early stageof evolution?

Or are they• Adaptations to an obligate

intracellular lifestyle?

Alternative explanations ofMicrosporidia unusual features

Elongation Factor 2 protein ML tree (PROTML)(Hashimoto et al. 1997 Arch. Protist. 148:287)

Plant

Trichomonas

Giardia

Sulfolobus

HalobacteriumMethanococcus

Trypanosoma

Cryptosporidium

Animals + Fungi

EntamoebaDictyostelium

Microsporidia

Archaebacteria outgroups

Eukaryote root

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

75

88

Shared nucleotide or amino acid composition biasescan also cause problems for phylogenetic analysis

Truetree

Wrongtree

Aquifex Thermus

Bacillus Deinococcus

Aquifex (73%)

Thermus (72%)

Bacillus (50%)

Deinococcus(52% G+C)

16S rRNA

The correct tree can be obtained if amodel is used which allows base/aacomposition to vary betweensequences -LogDet/ParalinearDistancesHeterogeneous Maximum Likelihood

Thermus

Deinococcus

Aquifex

Bacillus

LogDet/Paralinear distances for EF-2 DNAvariable sites codon positions 1+2

Animals

ChlorellaTrypanosoma

Trichomonas

Giardia

DictyosteliumEntamoebaSacharomyces

MicrosporidiaCryptosporidium

SulfolobusMethanococcus 44%

Halobacterium 58%

60

25

76

70

Archaebacteriaoutgroups

Note thatroot haschanged

45% G+C

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

0 20 40 60 80 100

Methanococcus outgroup(low G+C)

0

20

40

60

80

100

Halobacterium outgroupHigher G+C

0

20

40

60

80

100

0 20 40 60 80 100

(Microsporidia, outgroup)

Fraction of constant sites removed

LogDetBootstrap

values

MLestimate

0 20 40 60 80 1000

20

40

60

80

100

0

20

40

60

80

100

0 20 40 60 80 100

(Microsporidia, outgroup)(Microsporidia, Fungi)

Fraction of constant sites removed

Bootstrapvalues

A combination of factors (outgroup GC content & siterate heterogeneity) influence the EF-2 DNA tree

Methanococcus outgroup(low G+C)

Halobacterium outgroupHigher G+C

A combination of factors (outgroup GC content & siterate heterogeneity) influence the EF-2 DNA tree

0 20 40 60 80 1000

20

40

60

80

100

0

20

40

60

80

100

0 20 40 60 80 100

(Giardia, Trichomonas, outgroup)Fraction of constant sites removed

Bootstrapvalues

Methanococcus outgroup(low G+C)

Halobacterium outgroupHigher G+C

Competing hypotheses for Microsporidia

AnimalsFungi

Ciliates + ApicomplexaStramenopiles

Euglenozoa

GiardiaTrichomonas

Plants / green algae

Red algae

Entamoebae

Choanozoa

Dictyostelium

Physarum

Microsporidia

Percolozoa

“Microsporidia Early”SSU rRNA

Microsporidia + FungiTubulin, RNA polymerase,LSU rRNA, HSP70, TATA binding protein,EF-2, EF-1 alpha

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

Summary I

• Making trees is not easy:– Among-site rate heterogeneity, “fastclock” species, shared nucleotide or aminoacid composition biases

– Different data sets may be affected byindividual phenomena to different degrees

– Biases need not be large if phylogeneticsignal is weak

Summary II

• Are Archezoa ancient offshoots?– Microsporidia are related to fungi– Evidence for Giardia and Trichomonas branching

deeper than other eukaryotes is based on treesmade 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 unrootedtree for eukaryotes?

• Combining different genes in a single analysismay provide a more robust eukaryotic tree

• One argument is that phylogenetic signalshould be additive whereas gene-specific“noise” will pull in different directions

DNA ML tree found using a model which allows bothbase composition and site rates to vary across the tree

Plasmodium

Cryptosporidium

Halteria /Stylonichia

Tetrahymena Green algae

Arabidopsis

Chondrus

Trypanosoma

EuglenaTrichomonas

Giardia

Sacharomyces

Schizosaccharomyces HumanDrosophila

Dictyostelium

Entamoeba

another red algaActin+tubulin+EF-2

Animals + fungi + slimemoulds

Ciliates plusapicomplexa

Red and greenalgae/plants

Giardia andTrichomonas

Hydrogenosomes• Strange anaerobic eukaryotic organelles

which make hydrogen

Methanocorpusculum(Uses H2 and CO2)

Metopus endosymbiont

12um

Anaerobic eukaryotes from differentphylogenetic groups make hydrogen - how?

Origin(s) of Hydrogenosomes

• Is a 2 part problem– The organelle (the bag)– The biochemistry to produce hydrogen

particularly hydrogenase

MalatePyruvateME

NAD(P)+NAD(P)H

2Fd-2Fd

PFOAcetyl-CoA Acetate

ASCT

Succinyl-CoASuccinate

ADP + PiATP

AAC

STK

[Fe]HydNAD(P)-FO

2H+

H2

Doublemembrane

hsp70

cpn60

Schematic Map of Hydrogenosomes(after Muller 1993)

CO2

CO2

ADPATP

Transitpeptides

Proteinimport

N

Enzyme found also in mitochondriaAlpha-proteobacterial ancestry

Unknown ancestry

CoASH

Fungi and Trichomonas

Spironucleus

DNA ML tree for Fe hydrogenases

0.1 substitutions per site

Clostridium acetobutylicum Clostridium pasteurianum

Clostridium perfringensClostridium acetobutylicum

Thermotoga maritima

Clostridium thermocellum

Thermotoga maritima Entamoeba histolytica

Spironucleus barkhanus

Trichomonas vaginalis

Trichomonas vaginalis

Nyctotherus ovalis

Desulfovibrio vulgaris

Desulfovibrio fructosovorans

Clostridium difficile

48

45

72 100

100 Desulfovibrio spp.

A likelihood ratio test of monophyly(Huelsenbeck, Hillis & Nielson 1996)

The Test Statistic (δδδδ) = lnL1 - lnL0

• Where lnL1 is the likelihood of the best treeand lnL0 is the likelihood of the bestmonophyly tree

• The null (eukaryote monophyly) distribution ofδδδδ is generated by simulation under anappropriate model (parametric bootstrapping)

Parametric Bootstrapping to estimate atest distribution

Estimate ML modelparameters using original

data

Simulate 1000 newsequence data sets usingthis model over the best

monophyly tree

For each new data setestimate L0 and L1 using

ML, with model re-optimised each time

Plot d for each of the1000 data sets to give thetest distribution and the95%confidence interval

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

Calculate d for original data andcompare to distribution - if it fallsoutside of the 95% interval it isbigger than expected by chance andmonophyly can be rejected

The likelihood ratio test rejects the hypothesisthat eukaryotic hydrogenases are monophyletic

9.6495%

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

δ= lnL1 - lnL0

δ for original dataδδδδ Original data95%

Eukaryotic compartment

Trichomonas Hydrogenosome

Cytosolic?

Ciliate Hydrogenosome

Plastid

Iron hydrogenase ML treeClostridium acetobutylicum p262Clostridium perfringens

Clostridium pasteurianumClostridium acetobutylicum ATCC824

Shewanella putrefaciens

Dehalococcoides ethanogenes

Desulfovibrio vulgaris (Oxamicus)

Desulfovibrio vulgaris (Hildenborough)Desulfovibrio fructosovorans

Megasphaera elsdenii

Treponema denticola

Thermotoga maritimaDesulfovibrio vulgaris Hyd-g

Nyctotherus ovalisClostridium thermocellum.

Clostridium difficileDesulfovibrio fructosovorans (Hildenborough)

Trichomonas vaginalis (long form)Trichomonas vaginalis (short form)

Spironucleus barkhanusEntamoeba histolytica

Chlamydomonas reinhardtiiScenedesmus obliquus

Conclusions I• Hydrogenosomes share common ancestry with

mitochondria• Hydrogenase has been acquired at least twice

and can be targeted to different cellcompartments in different eukaryotes– Humans, plants and fungi also contain remnants of

iron hydrogenases

• There is no evidence from phylogenetic analysisthat the “bag” and hydrogenase share a commonorigin from the mitochondrion endosymbiont

Conclusions II• Phylogeny is hard, there are lots of potential problems

with data, so we need to be careful in ourinterpretations of what trees mean - includesinferences of HGT

• Better methods hold promise of more reliable trees(allowing re-analysis of SSUrRNA data)

• Archezoa contain genes which originated withmitochondrion endosymbiont and the jury is still out onwhether former archezoa have lost the mitochondrialbag

• We don’t know which eukaryotes are early branching -for this we need a rooted tree

The mitosome, a novel organelle relatedto mitochondria in Entamoeba histolytica

Tovar et al., 1999.

Slide shows epitopetagged recombinantcpn60 localised tomitosome

Are there still organelles of common ancestrywith mitochondria in Giardia and Microsporidia?

• Giardia:– “What are the ovoid pellicular bodies (in Giardia)? The

study made suggests that they might be nothing butchanged mitochondria with a few crysts or tubules”

– “The ultrastructure of mitochondria may be related withthe oxygen deficiency in Lamblia environment”

(Cheissen, 1965)

• Microsporidia:– “There are reports of mitochondria-like structures in

several microsporidia” (Vavra, 1976)