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cytokinesis and are enhanced by Rho 18. Yamada, T., Hikida, M., and Kurosaki, T. (2006). and RGA-4 in the germ line and in the early and suppressed by Rac. J. Cell Biol. 166, Regulation of cytokinesis by mgcRacGAP in B embryo of C. elegans. Development 134, 61–71. lymphocytes is independent of GAP activity. 3495–3505. 16. Severson, A.F., Baillie, D.L., and Bowerman, B. Exp. Cell Res. 312, 3517–3525. (2002). A formin homology protein and a 19. Schonegg, S., Constantinescu, A.T., Hoege, C., profilin are required for cytokinesis and and Hyman, A.A. (2007). The Rho GTPase- Department of Molecular Genetics and Cell Arp2/3-independent assembly of cortical activating proteins RGA-3 and RGA-4 are Biology, University of Chicago, Chicago, microfilaments in C. elegans. Curr. Biol. 12, required to set the initial size of PAR domains IL 60637, USA. 2066–2075. in Caenorhabditis elegans one-cell E-mail: [email protected] 17. Zhang, W., and Robinson, D.N. (2005). Balance embryos. Proc. Natl. Acad. Sci. USA of actively generated contractile and resistive 104, 14976–14981. forces controls cytokinesis dynamics. Proc. 20. Schmutz, C., Stevens, J., and Spang, A. (2007). Natl. Acad. Sci. USA 102, 7186–7191. Functions of the novel RhoGAP proteins RGA-3 DOI: 10.1016/j.cub.2008.12.028

Evolution: Revisiting the Root of the been controversial since they were first proposed [6]. Now, with the Tree rapid accumulation of genome-scale data for diverse species, a flurry of phylogenomic analyses A recent phylogenomic investigation shows that the enigmatic flagellate [7–9] are putting these hypotheses is a distinct anaerobic lineage within the eukaryote super-group to the test. and challenges the unikont– rooting of the tree of A recent paper by Minge et al. [7] . reports phylogenomic analyses of the enigmatic protist Breviata anathema, Andrew J. Roger1,2 ‘’ (all other super-groups), a small -like cell with an and Alastair G.B. Simpson1,3 as shown in Figure 1. Both the six anterior flagellum. Breviata is super-groups model and the interesting for two major reasons: it In the 1980s and 1990s, prevailing unikont–bikont root hypothesis have lives in low oxygen conditions and views of the eukaryote tree of life were strongly influenced by phylogenies of small subunit ribosomal RNA (rRNA) [1]. Although these analyses ‘Unikonts’ ‘Bikonts’ placed many eukaryotes into major groups, it became clear that the relationships amongst these groups Apusomonads (= Plantae) could not be determined because + Amoebozoa Breviata of the limited information available Slime moulds in a single , as well as Stramenopiles methodological artifacts [2]. More 0 1 2+ recently, a ‘six super-groups’ Alveolata Multicilia hypothesis for deep eukaryote 1 1/2 2 2 2 1 2 phylogeny emerged as a synthesis of Lobose 2 analyses of sequence data for rRNAs, amoebae 0 2 concatenated sets of conserved 2 proteins and organellar genomes, 2* and some detailed ultrastructural inc. comparisons [3]. The six super-groups and Fungi proposed are the opisthokonts, Eukaryotes Amoebozoa, Archaeplastida, Prokaryotes chromalveolates, Rhizaria and Excavata. In the absence of outgroup sequences that are sufficiently Eubacteria Archaebacteria Current Biology closely related to allow reliable rooting of eukaryotes in molecular Figure 1. The placement of Breviata anathema in the eukaryote tree of life. phylogenies, Cavalier-Smith and The relationships amongst the six super-groups of eukaryotes are shown as recovered by Minge colleagues proposed that the et al. [7] and other recent phylogenomic analyses [8,9]. The hypothesized super-groups are presence or absence of colour-coded as follows: opisthokonts (purple), Amoebozoa (light blue), Archaeplastida (green), a dihidrofolate reductase-thymidylate chromalveolates (orange), Rhizaria (dark blue) and Excavata (brown). Note that recent evidence synthase (DHFR-TS) gene fusion [4] suggests that Rhizaria are specifically related to some chromalveolates [8,9]. The tree is shown and specific myosin gene families [5] as rooted according to the unikont–bikont hypothesis [5,14]. Anaerobic/microaerophilic in diverse eukaryotes could be used protistan lineages that lack classical mitochondria are shown in red. The numbers in ellipses show the inferred ancestral number of basal bodies per kinetid (flagellar unit) in the various to infer that the eukaryote root falls eukaryote lineages. The plus (+) indicates that Breviata may contain more basal bodies than between so-called ‘unikonts’ the number cited whereas the asterisk (*) indicates that one basal body is non-flagellated. (opisthokonts and Amoebozoa) and Dashed lineages indicate uncertainty in the location of that branch on the tree (see text). Current Biology Vol 19 No 4 R166

cytoskeleton and other . sequence features in the small Suddenly Breviata became of key subunit rRNA genes from Breviata. interest for early eukaryote evolution: On the other hand, divergent lineages it was not assigned to any major are often placed too deep in eukaryote group, and it was just phylogenetic analyses as a result of possible that Breviata was the only long-branch attraction artifacts [2], primitively amitochondriate eukaryote and removal of rapidly-evolving lineage still alive today. ‘noisy’ sites is sometimes an Minge et al. [7] soundly refute this effective strategy for combating possibility. They conducted an this artefact. Although Minge expressed-sequence tag (EST) et al.’s [7] preference is reasonable, survey of Breviata anathema and, it will need to be tested by future from these data, constructed a data analyses with better sampling of set of 75 proteins for phylogenetic amoebozoan species. investigations. Sophisticated Even more interesting, the placement analyses indicate that Breviata is of Breviata as a basal amoebozoan most closely related to Amoebozoa, calls into question the nature of the the super-group to which the common ancestor of extant eukaryotes Archamoebae also belong. Provided implied by the unikont–bikont root the root of the eukaryotic tree falls hypothesis. Cavalier-Smith [14] Figure 2. Transmission electron micrograph elsewhere, this sister-group proposed the names ‘unikonts’ and showing the kinetid of Breviata. relationship between Breviata and the ‘bikonts’ based on a scenario for the The two arrows indicate the two basal bodies, ancestrally -containing evolution of the flagellar apparatus. In one of which gives rise to the flagellum (F). Amoebozoa demonstrates that most eukaryotic cells the flagellar Image kindly provided by Giselle Walker (University of Cambridge) and Aaron Heiss Breviata too descends from apparatus is the centre of organization (Dalhousie University). a mitochondrion-containing ancestor. for the cytoskeleton. Its core is usually Even more convincingly, the a single ‘kinetid’ consisting of one or authors found within their EST data more basal bodies, which may either lacks classical mitochondria; and its sequences encoding mitochondrial give rise to flagella, or be non- flagellar apparatus has at least one marker proteins such as chaperonin flagellated. Cavalier-Smith argued that additional non-flagellated basal body 60 and tim17. These findings, ancestral eukaryotes had a simple (Figure 2). Breviata was originally combined with electron kinetid with one basal body anchoring assumed to be a member of the microscopy evidence of a double one flagellum. He further suggested Archamoebae, a group of membrane-bounded [13], that the ‘unikonts’ had retained this mitochondrion-lacking amoebozoans suggest that Breviata does ancestral organization, whereas the that includes organisms such as the contain some sort of relict ‘bikonts’ descended from a common human amebic dysentery parasite mitochondrion. ancestor that had evolved a kinetid with Entamoeba histolytica and the giant Anaerobic and microaerophilic two flagella, one anterior and one multinucleate amoeba with mitochondrion-derived posterior. palustris. Archamoebae gained organelles are scattered across the In ‘bikonts’ studied to date, notoriety in the 1980s because they eukaryotic tree (Figure 1), and a vibrant a characteristic ontogenetic flagellar were widely believed to be primitive sub-field of evolutionary cell biology is transformation process occurs during eukaryotes that had diverged prior devoted to understanding how these cell division. New basal bodies always to the endosymbiotic origin of organelles evolved to function in low become the anterior units, while mitochondria [10]. The subsequent oxygen conditions [10]. Does Breviata existing basal bodies become the finding of mitochondrial represent another independent lineage posterior units in the daughter cells. marker proteins and relict of eukaryotes with modified This means that the anterior flagellum mitochondrion-derived organelles mitochondria? In Minge et al.’s [7] of the parent transforms into of unknown function in several main analysis, Breviata branches a posterior flagellum in one of the Archamoebae [11,12] proved this robustly as the sister to all other daughters. Cavalier-Smith [14] hypothesis wrong. Indeed, evidence Amoebozoa, as shown in Figure 1, suggested that the bikont kinetid for the retention of relict mitochondria implying that they are distinct from and associated flagellar transformation in many other anaerobic protists other taxa with modified mitochondria. are important shared derived (Figure 1) suggests that no Intriguingly, however, analyses that characteristics of the ‘bikonts’. eukaryote lineages persist from exclude the rapidly evolving sites in These derived characters combined a pre-mitochondrial phase of their protein alignments [7] place with the the DHFR-TS gene fusion eukaryote evolution [10]. Breviata inside Amoebozoa as the character exclude the possibility Several molecular studies, sister group of Archamoebae, that the root of eukaryotes falls within however, suggested that Breviata suggesting they might share the bikonts. was not related to the other a common anaerobic ancestor. One problem for this hypothesis was Archamoebae, and a recent Which phylogenetic position is the fact that many ‘unikonts’ actually ultrastructural investigation [13] correct? Minge et al. [7] prefer the have more than one flagellum or basal showed that Breviata has a completely deep-branching position, citing the body in their kinetids. Opisthokonts different organization of its lack of conserved Amoebozoa-specific with flagella characteristically have two Dispatch R167 basal bodies — one bears a flagellum, ‘bikonts’ should really refer to the 4. Stechmann, A., and Cavalier-Smith, T. (2002). Rooting the eukaryote tree by using a derived while the second does not. Meanwhile, group containing all living eukaryotes gene fusion. Science 297, 89–91. the flagellated cells of some and would cease to be useful. 5. Richards, T.A., and Cavalier-Smith, T. (2005). amoebozoan slime moulds are But what about the placement of the Myosin evolution and the primary divergence of eukaryotes. Nature 436, biflagellated, or at least have a second root in the eukaryote tree? So far, 1113–1118. basal body in the kinetid [15,16]. phylogenomic analyses support 6. Parfrey, L.W., Barbero, E., Lasser, E., Dunthorn, M., Bhattacharya, D., Cavalier-Smith [14] argued that the a bipartition between so-called Patterson, D.J., and Katz, L.A. (2006). ‘unikonts’ with a second flagellum or ‘unikonts’ and ‘bikonts’, but they can Evaluating support for the current basal body resulted from convergent say nothing about the location of the classification of eukaryotic diversity. PLoS Genet. 2, e220. evolution, rather than common root. Although the arguments 7. Minge, M.A., Silberman, J.D., Orr, R.J.S., ancestry with ‘bikonts’. To support the regarding the myosin gene family data Cavalier-Smith, T., Shalchian-Tabrizi, K., Burki, F., Skjæveland, A˚ ., and Jakobsen, K.S. independent evolution argument, he and the DHFR-TS gene fusion that (2008). Evolutionary position of breviate pointed to an early study of the support a ‘unikont’–‘bikont’ root have amoebae and the primary eukaryote myxogastrid slime mould Physarum yet to be refuted, they were based on divergence. Proc. R. Soc. Lond. B, in press. 8. Rodrı´guez-Ezpeleta, N., Brinkmann, H., polycephalum, where the flagellar a very narrow sampling of eukaryotic Burger, G., Roger, A.J., Gray, M.W., transformation pattern was interpreted genomes then available, many of which Philippe, H., and Lang, B.F. (2007). Toward resolving the eukaryotic tree: the phylogenetic to be the reverse of that found in completely lacked recognizable positions of and cercozoans. Curr. ‘bikonts’ [17]. Consistent with the myosin, DHFR or TS genes. Biol. 17, 1420–1425. independent evolution argument, Furthermore, at least one group, the 9. Burki, F., Shalchian-Tabrizi, K., and Pawlowski, J. (2008). reveals biflagellated slime moulds seemed to apusomonads, has the bikont-type a new ‘megagroup’ including most branch inside Amoebozoa, which DHFR-TS fusion character [4], but photosynthetic eukaryotes. Biol. Lett. 4, 366–369. otherwise have one basal body per shows phylogenetic affinity with 10. Embley, T.M., and Martin, W. (2006). Eukaryotic kinetid, or none at all. (and could fall within) ‘unikonts’ [19]. evolution, changes and challenges. Nature 440, This is where Breviata becomes key The position of apusomonads is in 623–630. 11. Aguilera, P., Barry, T., and Tovar, J. (2008). to the discussion, as an additional, urgent need of clarification. As Entamoeba histolytica : organelles deep-branching Amoeobozoan lineage genomic data become available in search of a function. Exp. Parasitol. 118, 10–16. with at least two basal bodies. If we from many more protists, it will be 12. Gill, E.E., Diaz-Trivin˜ o, S., Barbera` , M.J., map the number of basal bodies per important to watch for the presence Silberman, J.D., Stechmann, A., Gaston, D., kinetid onto a likely eukaryote of gene families and gene fusions Tamas, I., and Roger, A.J. (2007). Novel mitochondrion-related organelles in the phylogeny including Breviata (Figure 1), that are discordant with the anaerobic amoeba balamuthi. the idea the ‘unikonts’ were ancestrally original unikont/bikont root Mol. Microbiol. 66, 1306–1320. 13. Walker, G., Dacks, J.B., and Martin Embley, T. unikont looks increasingly untenable. If hypothesis. (2006). Ultrastructural description of Breviata the basal position of Breviata within All of this suggests that, with the anathema, n. gen., n. sp., the organism Amoebozoa is correct, it is at least as current pace of change in our previously studied as ‘‘Mastigamoeba invertens’’. J. Eukaryot. Microbiol. 53, 65–78. parsimonious that the last common understanding of the eukaryote tree of 14. Cavalier-Smith, T. (2002). The phagotrophic ancestor of Amoebozoa had two basal life, we should proceed with caution. origin of eukaryotes and phylogenetic classification of . Int. J. Syst. Evol. bodies rather than one, if outgroups are There are several important taxa whose Microbiol. 52, 297–354. not considered. But as every other phylogenetic affinities to the major 15. Spiegel, F.W. (1990). Phylum plasmodial slime major group of eukaryotes can be super-groups remain controversial molds class Protostelida. In Handbook of Protoctista, L. Margulis, J.O. Corliss, inferred to have originally had two (the apusomonads, for example) or M. Melkonian, and D.J. Chapman, eds. basal bodies (Figure 1), the most unknown (the collodictyonids, for (Boston: Jones and Bartlett Publishers), pp. 484–497. parsimonious interpretation is clearly example) and whose cellular properties 16. Walker, G., Simpson, A.G.B., Edgcomb, V., that the last common ancestor of the once clarified and placed in a robust Sogin, M.L., and Patterson, D.J. (2001). Opisthokonts and Amoebozoa was phylogenetic context could radically Ultrastructural identities of Mastigamoeba punctachora, Mastigamoeba simplex and ‘bikont’, as was the most recent alter our view of early eukaryote Mastigella commutans, and assessment of common ancestor of all extant evolution. Resolution of the remaining hypotheses of relatedness of the pelobionts (Protista). Eur. J. Protistol 37, 25–49. eukaryotes! questions regarding the eukaryote tree 17. Wright, M., Moisand, A., and Mir, L. (1980). What about the supposedly of life and the nature of the last Centriole maturation in the amoebae of backwards flagellar transformation in common ancestral eukaryote almost Physarum polycephalum. Protoplasma 105, 149–160. the biflagellated unikont Physarum?An certainly depends on both genomic 18. Gely, C., and Wright, M. (1986). The centriole apparently overlooked re-examination and ultrastructural studies of a wider cycle in the amoebae of the myxomycete Physarum polycephalum. Protoplasma 132, of flagellar ontogeny in Physarum [18] array of protistan species that better 23–31. indicates that original interpretation represent the true diversity of 19. Kim, E., Simpson, A.G., and Graham, L.E. [17] was incorrect, and that its flagellar eukaryotes. (2006). Evolutionary relationships of apusomonads inferred from taxon-rich transformation process resembles analyses of 6 nuclear encoded genes. that of bikonts after all. Regardless References Mol. Biol. Evol. 23, 2455–2466. of whether ‘unikonts’ are truly 1. Sogin, M.L. (1991). Early evolution and the origin of eukaryotes. Curr. Opin. Genet. Dev. 1, 1 a super-clade of eukaryotes, the name 457–463. Centre for Comparative Genomics and 2 ‘unikonts’ is probably a misleading 2. Roger, A.J., and Hug, L.A. (2006). 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