Cell, Vol. 107, 419–425, November 16, 2001, Copyright 2001 by Cell PressReviewReconstructing/Deconstructingthe Earliest Eukaryotes:How Comparative Genomics Can Helpplex cellular features and their component parts firstappeared. In the simplest cases, features found in ho-mologous form in all descendants of a common ancestormust, barring lateral transfer, have been present in thatancestor (must be primitive or ancestral features). MoreJoel B. Dacks and W. Ford Doolittle1Program in Evolutionary BiologyCanadian Institute for Advanced ResearchDepartment of Biochemistry and Molecular BiologyDalhousie Universitycomplex cases involve traits found in some descendantsHalifax, Nova Scotia(for instance, as designated “⫹” in species B of FigureCanada1A) but not in others (species A). These could eitherhave been gained (in the lineage leading to B) or lost (inthe lineage leading to A) after their divergence from theirWe could reconstruct the evolution of eukaryote-spe-common ancestor (X). One can tell which by looking atcific molecular and cellular machinery if some living“outgroups” (O). If (and only if) some of these show theeukaryotes retained primitive cellular structures and⫹ trait, parsimony demands that X was ⫹, and that Awe knew which eukaryotes these were. It’s not clearhas lost this feature.that either is the case, but the expanding protist geno-To apply such reasoning, it is essential to know themic database could help us in several ways.phylogeny, and to make interesting and sensible conclu-sions about early eukaryotic cellular evolution, it is es-Introductionsential to have a deeply branched eukaryotic phylogeny.Almost thirty years ago, the Canadian microbiologistToo many eukaryotic features have been assumed toRoger Stanier (Stanier, 1974) asserted that “the differ-be universal (and thus primitive) for all eukaryotes simplyences in structure and function between prokaryoticbecause they are “present from yeast to man.” Thereand eukaryotic cells are many and profound, and noare likely very many early eukaryotic branches (manycontemporary biological group has a cellular organiza-outgroups) below the common ancestor of yeast andtion that can plausibly be interpreted as intermediate.”man, whatever the true structure of the eukaryotic tree.Microbiologists’ understanding now is more nuanced inBefore molecular methods achieved their currentseveral ways. We divide Life into three domains (Bacte-dominance, however, attempts to reconstruct phylog-ria, Archaea, and Eukarya) rather than two (prokaryoteseny were often conflated with attempts to infer whenand eukaryotes). We recognize an astonishing diversitycomplex traits and their components first appeared. Forof form and function within and between the two pro-instance, it seemed reasonable to suggest that the sim-karyotic domains, and the defining positive prokaryoticplest contemporary eukaryotes branched off the maincharacters recognized by Stanier are no longer universaltrunk of the eukaryotic tree very early—because the firstamong them. And increasingly we can confirm the pres-eukaryotic cells obviously must have been simpler thanence in Bacteria or Archaea or both of “primitive” formsmodern ones and simplicity is unlikely to be a second-of key macromolecules and processes previouslyarily derived feature. Among such simple contemporarythought to be eukaryote specific. For instance, bacterialeukaryotes were the diplomonads (including the intesti-MreB, first claimed as a homolog to eukaryotic actinnal parasite Giardia), parabasalids (including the sexu-only on the basis of predicted structural similarity inally transmitted disease-causing agent Trichomonas),ATPase domains (Bork et al., 1992) has recently beenoxymonads, microsporidia (such as the human patho-shown to form “cytoskeletal” actin-like filaments in vivogen Encephalitozoon), pelobionts (Mastigamoeba), en-(Jones et al., 2001) and in vitro (van den Ent et al., 2001).tamoebae, and the heteroloboseid amoebae such asNevertheless, differences at the level of cell structureNaegleria. These protists are variously deficient in suchand function remain “many and profound.” It is still rea-basic eukaryotic cellular components as mitochondria,sonable to speak of the prokaryote-eukaryote transitionperoxisomes, and Golgi dictyosomes.as one of cellular evolution’s greatest leaps. It is stillFor much of the last decade, molecular phylogenysensible to ask how and when the various complex mac-seemed to support an early branching for many of theseromolecular machines (cytoskeletons, endomembranesimple protists. (Note that we should call such eukary-systems, spliceosome, membrane-bounded organelles,otes “early branching” or “deeply diverging” rather thanand many others) that all the best-known eukaryotes“ancestral” or “primitive,” since no contemporary cellhave and all known prokaryotes lack—or possess onlycan be any other’s ancient ancestor, and none is likelyin inchoate form—came into being.to retain primitive states of all important characters.)One can approach this question from the bottom up,Small subunit (SSU) ribosomal RNA gene sequences,looking to diverse prokaryotes for homologs of compo-which have served as the gold standard molecular mea-nents and subassemblies of complex eukaryotic cellularsure for reconstructing phylogenetic relationships, showedmachines (as in the case of actin and MreB), or fromthe amitochondriate diplomonads, microsporidia, andthe top down, searching among eukaryotes for simplerparabasalids at the base of the eukaryotic tree (Figureversions of such machines (as we will describe below).1B). Above them, mitochondriate protist lineages suchEither way, there are principles of logic and parsimonyas Heterolobosea and Euglenozoa emerged, followedthat can help in inferring when in cellular evolution com-by the unresolved cluster of animals, fungi, plants, andother lineages (including some protists) commonlycalled the “eukaryotic crown” (Sogin, 1991).1Correspondence: [email protected] that Cavalier-Smith (1983) called “Archezoa”—comprising, among others, the diplomonads (Giardia),parabasalids (Trichomonas), and microsporidia. Theprevailing belief about mitochondria since Margulis(1970) had been that these eukaryotic organelles arethe degenerate descendants of endosymbiotic bacteriaengulfed by some early amitochondriate eukaryote (thehost). It was logical to propose that the immediate