Probing early eukaryotic evolution using phylognetic methods

1. Probing early eukaryotic evolution using phylognetic methods

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

3. The SSU Ribosomal RNA Tree for Eukaryotes Mitochondria? Prokaryotic outgroup Archezoa

4. 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

5. 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

6. Chaperonin60/GroEL phylogeny pyruvate ferredoxin oxidoreductase PFO Pyruvate Acetyl CoA Fe-hydrogenase 2H+ H2 Pi + ADP ATP Is Trichomonas an Archezoan?

7. Trichomonas chaperonin 60 shares common ancestry with mitochondrial chaperonins Mitochondria alpha - proteobacteria

8. 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

9. Chaperonin 60 Protein Maximum Likelihood Tree(PROTML, Roger et al. 1998, PNAS 95: 229) Note 100% Bootstrap support A case of Eukaryote Eukaryote HGT?

10. 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

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

12. 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

13. Cpn-60 Protein ML tree (PROTML) from variable sites with outgroups removed

14. 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?

15. 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

16. 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?

17. 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

18. 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

19. 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%

20. 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)

21. 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)

22. 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)

23. 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?

24. 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

25. 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

26. 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

27. 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

28. Hydrogenosomes • Strange anaerobic eukaryotic organelles which make hydrogen

29. 12um Methanocorpusculum (Uses H2 and CO2) Metopus endosymbiont

31. Anaerobic eukaryotes from different phylogenetic groups make hydrogen - how?

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

33. 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

34. Spironucleus

36. DNA ML tree for Fe hydrogenases

37. 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)

38. 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

39. 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

41. Iron hydrogenase ML tree Eukaryotic compartment Trichomonas Hydrogenosome Cytosolic? Plastid Ciliate Hydrogenosome

42. 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

43. 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

44. The mitosome, a novel organelle related to mitochondria in Entamoeba histolytica Tovar et al., 1999. Slide shows epitope tagged recombinant cpn60 localised to mitosome

45. 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)