Eukaryotic Evolution: Getting to the Root of the Problem

Current Biology, Vol. 12, R691–R693, October 15, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01207-1

Eukaryotic Evolution: Getting to the Root of the Problem Alastair G.B. Simpson and Andrew J. Roger

Comparative analyses of multiple genes suggest most known eukaryotes can be classified into half a dozen ‘super-groups’. A new investigation of the distribution of a fused gene pair amongst these ‘supergroups’ has greatly narrowed the possible positions of the root of the eukaryote tree, clarifying the broad outlines of early eukaryote evolution.

A decade ago, phylogenies based on small subunit ribosomal (r)RNA sequences provided an intuitively appealing evolutionary tree of eukaryotes. Complex eukaryotes, including animals, fungi, plants and most algae, emerged as a broad radiation usually called the ‘eukaryotic crown’ [1]. Below this ‘crown’, more bizarre, and generally simpler, organisms diverged in a ladder-like succession. The small subunit rRNA tree was ‘rooted’ with mitochondrion-lacking unicellular eukaryotes such as diplomonads, parabasalids and microsporidia forming the basal branches (Figure 1a). But several studies now indicate that this rooting was patterned more by methodological artefact than historical signal, temporarily encouraging a view of eukaryote phylogeny as a large unresolved radiation [2]. Recent years have seen tremendous progress in resolving this ‘radiation’. A wealth of single and concatenated gene phylogenies, improved taxon sampling and examination of strong shared-derived characters have revealed several novel eukaryote ‘super-groups’. The hardest question of all — the placement of the root in the eukaryote tree — has now been clarified by Stechmann and Cavalier-Smith’s [3] work on the phylogenetic distribution of a gene fusion in eukaryotes. Most known eukaryotes seem to fall into six ‘supergroups’. The longest recognised super-group is the ‘opisthokonts’, including animals, fungi and a variety of unicellular relatives. As reported by Lang et al. [4] in this issue, the relationships within the opisthokonts — specifically the identification of the protistan sisters of animals — have now been addressed by concatenated mitochondrial protein phylogenies. The ‘plantae’ includes land plants and green algae, as well as red algae and the obscure glaucocystophyte algae. Most other eukaryote algae, and many heterotrophs, belong to the ‘chromalveolate’ assemblage, which unites alveolates (dinoflagellates, ciliates, Apicomplexa), stramenopiles/heterokonts (including brown algae and Canadian Institute for Advanced Research, Program in Evolutionary Biology, Genome Atlantic, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. E-mail: [email protected] and [email protected]

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diatoms), cryptomonads and probably haptophyte algae. A major grouping of flagellates and amoebae, the ‘cercozoa’, emerged from improved taxon sampling of small subunit rRNAs. Recent evidence indicates that the Foraminifera, and perhaps the Radiolaria, may belong with this assemblage [5–7]. Most ‘typical’ amoebae (such as Amoeba and Acanthamoeba), mycetozoan slime moulds, and the mitochondrion-lacking pelobionts and entamoebae form the ‘amoebozoa’. Most controversially, morphological data suggest a grouping, the ‘excavates’, which includes diplomonads and parabasalids (previously thought to be earlydiverging) together with Euglenozoa, Heterolobosea, jakobids and several other protists [8]. But where does the root lie? Philippe et al. [2] noted that land plants (plantae), alveolates (chromalveolates) and some Euglenozoa (excavates) have fused genes encoding a protein with both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities. In animals and fungi, these genes encode separate proteins, as in prokaryotes. Might this gene fusion be a derived feature within eukaryotes? This possibility prompted Stechmann and Cavalier-Smith [3] to examine other groups for evidence of fused DHFR–TS. They determined partial sequences of fused DHFR–TS genes in two chromalveolates (a stramenopile and a ciliate), another euglenozoan, and, most interestingly, a cercozoan plus two unplaced unicellular heterotrophs — an apusomonad, and a centrohelid heliozoan. Apparently, just two of the six super-groups may lack the gene fusion — the opisthokonts and amoebozoa. Under the simplest interpretation, the DHFR–TS fusion character drastically reduces the possible locations for the eukaryote root. Stechmann and Cavalier-Smith [3] argue that just three clusters need consideration: opisthokonts, amoebozoa and the ‘fusion-bearing cluster’ of plantae, chromalveolates, cercozoa (with Foraminifera and Radiolaria) and excavates. Of these, only amoebozoa could ‘include’ the root, because of the putatively derived nature of the DHFR–TS fusion, and the opisthokont-specific EF-1α sequence insertion (see Figure 1 legend). Stechmann and Cavalier-Smith [3] tentatively favour a rooting between amoebozoa plus the fusion cluster on one side, and the opisthokonts alone on the other. This would place animals, fungi and their relatives in the ‘basal’ position in the eukaroytic tree: a humbling reversal for humans when compared to our previous lofty ‘crown’ position under the small subunit rRNAbased model. The DHFR–TS fusion provides the best estimate to date for the placement of the eukaryotic root. The DHFR–TS story could be misleading, however, if fused genes were acquired more than once in eukaryotic evolution, either by multiple fusion events, or by eukaryote-to-eukaryote lateral gene transfers and replacements. Alternatively the fused gene may be ancestral for extant eukaryotes, with the separated

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A

Eukaryote tree, circa 1993 "Eukaryotic crown" Stramenopiles, Alveolates, Red algae, etc.

Animals Fungi Plants

Euglenozoa, Heterolobosea, Entamoebae, Mycetozoa, Various amoebae Parabasalia Diplomonads Microsporidia

Mitochondria Eukaryotic root

B

Eukaryote tree, 2002

Diplomonads, Retortamonads, Carpediemonas, Excavates Parabasalia Euglenozoa, Heterolobosea Malawimonas Oxymonads, Amoebozoa Trimastix "Typical" amoebae Jakobids ? Mycetozoan slime moulds Pelobionts + Entamoebae Animals Choanoflagellates Ichthyosporea Fungi (w/ microsporidia) Nucleariid amoebae

Opisthokonts

Eukaryotes Prokaryotes

Eubacteria

Cercozoa (w/ Foraminifera, w/ Radiolaria??)

Plants + Plantae Green algae Red algae Glaucocystophytes Alveolates Cryptophytes Stramenopiles ? Haptophytes

Chromalveolates

Centrohelid Heliozoa Apusomonads

DHFR-TS fusion Others: Mitochondria

Eukaryotic root

Eukaryotes Prokaryotes

Archaea

"Fusion cluster"

Eubacteria

Archaea

Phalansterium Collodictyonids Spironemids Kathablepharids Telonema Stephanopogon Multicilia etc. Current Biology

Figure 1. Contrasting views of eukaryotic evolution. (A) Eukaryotic evolution, as understood circa 1993 from small subunit ribosomal RNA phylogenies (after [1]). The earliest divergences involve amitochondriate protists, with animals ‘remote’ from the root. (B) Current view of eukaryotic phylogeny, with super-groups as determined primarily by multiple gene phylogenies, and with the deepest structure resolved according to the simplest interpretation of the DHFR–TS fusion, as reported by Stechmann and Cavalier-Smith [3]. Opisthokonts (purple), including animals and Fungi are supported by multiple gene phylogenies, and a large insertion in EF1α (see [13,14]). Relationships with opisthokonts are resolved as in [4]. Amoebozoa (light blue) include mycetozoan slime moulds, which are allied to typical amoebae (Euamoebae) by actin trees and a cox1–cox2 gene fusion [2,9], and to amitochondriate pelobionts and entamoebae in large concatenated gene trees [15]. Plantae, including land plants, are united, somewhat weakly, by concatenated gene phylogenies [16]. Chromalveolates (excluding haptophytes) are weakly grouped by concatenated protein phylogenies [14,15], but share a gene replacement of plastid GAPDH by a nuclear-derived copy, implying a common origin of their secondary plastids, where present [17]. Inclusion of haptophytes is widely expected, but not yet demonstrated [18]. Cercozoa are placed together and ‘adjacent to’ Radiolaria in small subunit ribosomal RNA phylogenies [6,7,18]. Some small subunit rRNA trees weakly place Radiolaria in an exclusive group with cercozoa (A.G.B.S., unpublished data). Actin trees imply a cercozoan-formaniferan relationship [5]. Within excavates, diplomonads plus parabasalids and Euglenozoa plus Heterolobosea groupings are recovered in several gene trees [14,19]. All excavates except parabasalids and Euglenozoa share a suite of cytoskeletal similarities, but almost never form a single group in molecular trees [8,9,14,20]. Apusomonads and centrohelid heliozoa have the DHFR–TS gene fusion [3], but no strong evidence suggests that they fall with any particular super-group. Some other taxa with contemporary identities, but no robust position in tree are listed under ‘others’. Branch lengths are arbitrary, and all multifircations represent uncertainty as to branching order. Branches currently lacking molecular corroboration are indicated with question marks.

genes in opisthokonts being the derived condition. This ‘reversal’ could arise if multiple copies of the fused gene became specialized for different activities, allowing loss of the other half of the gene. Multiple genes could originate through either conventional duplication, or lateral transfer. Stechmann and Cavalier-Smith [3] cite sequence similarities between all eukaryotic forms to refute transfer of either gene from prokaryotes to opisthokonts, but transfer amongst eukaryotes remains possible. Interestingly, ‘eukaryotic type’ DHFR and TS are each present in some viruses. The rooting inference also relies on a reasonably correct underlying ‘super-group’ tree. The DHFR–TS fusion is currently known from only one or a few isolated taxa in each super-group. If the super-group is not a natural group — a ‘clade’ — the inference that all ‘members’ ancestrally had the fusion will be invalid. In the case of excavates, the fusion is known from just one subgroup, Euglenozoa; however, molecular phylogenies generally place Euglenozoa separate from many other excavates, and the latter grouping is not widely accepted. The one analysis where excavates form a natural group, an rRNA tree [7], is in marked

conflict with other examinations of similar datasets [8,9]. Certain excavates, the jakobids, have been implicated in early eukaryotic diversification because of their ancestral bacterial-type mitochondrial RNA polymerases [10]. There are also many eukaryotes that cannot be placed with confidence with any supergroup. In addition to apusomonads and centrohelid heliozoa, which have the fusion, these include Phalansterium, Collodictyonids, Spironemids, Katheblepharids, Stephanopogon, Telonema, Multicilia and many parasitic and/or other amoeboid organisms that may, or may not, be aberrant members of known groups [11]. Any of these might be critical to resolving the exact position of the root. Other aspects of Stechmann and Cavalier-Smith’s [3] view of eukaryote evolution — as illustrated in their Figure 1 — are not directly addressed by the DHFR–TS data and are weakly supported. The largely resolved branching order they depict within the ‘fusion cluster’ is mostly based on evolutionary scenarios for a few contentious morphological characters, and is not supported by current molecular data. They present three arguments to tentatively place amoebozoa on the

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opposite side of the root to opisthokonts. Two are structural similarities — tubular mitochondrial cristae and an anterior flagellum — shared by amoebozoa and the ‘fusion cluster’, which could easily be ancestral features for all extant eukaryotes, rather than sharedderived characters. Their third argument derives from the strong support for the split between opisthokonts and other eukaryotes in many gene trees, but this could equally be interpreted as support for a relatively recent radiation of opisthokonts irrespective of where the root may lie. A more widely accepted depiction of our current state of knowledge is shown in Figure 1b. So where do we go from here? The DHFR–TS fusion gene should be sought in a wider range of eukaryotes, especially a greater diversity of excavates, amoebozoa, and currently unplaced organisms. When more complete DHFR and TS sequences are available, actual phylogenies of these genes might reveal any confounding eukaryote–eukaryote lateral transfers. Most importantly, other strong markers are needed to confidently establish the monophyly of the supergroups and test the possible roots implied by the DHFR–TS fusion. Regardless of the precise position of the root, many other questions regarding early eukaryote evolution persist. Does the difficulty in resolving the highest-level branchings stem from a ‘big bang’ radiation of eukaryote super-groups or does it reflect a ‘saturation’ of phylogenetic signal? How old are eukaryotes anyway? Did they originate in the Archean, as suggested by 2.7 billion year old eukaryotic-like sterane biomarkers [12], or after a ‘snowball Earth’ glaciation only 850 million years ago, as argued by Cavalier-Smith [7]? Are there any living eukaryotes that are simpler in their genetic or cellular makeup than the common ancestor of animals, fungi and plants, or have all ancestrally simple eukaryotes gone extinct [2]? With the wealth of data emerging from comparative protistan genomics efforts and renewed intensive study of the ancient paleontological and geochemical record, we may finally be able to answer these questions.

References 1. Sogin, M.L. (1991). Early evolution and the origin of eukaryotes. Curr. Opin. Genet. Dev. 1, 457–463. 2. Philippe, H., Lopez, P., Brinkmann, H., Budin, K., Germot, A., Laurent, J., Moreira, D., Müller, M. and Le Guyader, H. (2000). Earlybranching or fast-evolving eukaryotes? An answer based on slowly evolving positions. Proc. R. Soc. Lond. B. 267, 1213–1221. 3. Stechmann, A. and Cavalier-Smith, T. (2002). Rooting the eukaryote tree by using a derived gene fusion. Science 297, 89–91. 4. Lang, B.F., O’ Kelly, C.J., Nerad, T.A., Gray, M.W. and Burger, G. (2002). The closest unicellular relatives of animals. Curr. Biol., this issue. 5. Keeling, P.J. (2001). Foraminifera and Cercozoa are related in actin phylogeny: two orphans find a home? Mol. Biol. Evol. 18, 1551–1557. 6. Lopez-Garcia, P., Rodriguez-Valera, F. and Moreira, D. (2002). Toward the monophyly of Haeckel’s Radiolaria: 18S rRNA environmental data support the sisterhood of Polycystinea and Acantharea. Mol. Biol. Evol. 19, 118–121. 7. Cavalier-Smith, T. (2002). The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52, 297–354. 8. Simpson, A.G.B., Roger, A.J., Silberman, J.D., Leipe, D., Edgcomb, V.P., Jermiin, L.S., Patterson, D.J. and Sogin, M.L. (2002). Evolutionary history of ‘early diverging’ eukaryotes: The excavate taxon Carpediemonas is closely related to Giardia. Mol. Biol. Evol. in press.

9. Silberman, J.D., Simpson, A.G.B., Kulda, J., Cepicka, I., Hampl, V., Johnson, P.J. and Roger, A.J. (2002). Retortamonad flagellates are closely related to diplomonads – implications for the history of mitochondrial function in eukaryote evolution. Mol. Biol. Evol. 19, 777–786. 10. Gray, M.W., Burger, G. and Lang, B.F. (1999). Mitochondrial evolution. Science 283, 1476–1481. 11. Patterson, D.J. (1999). The diversity of eukaryotes. Am. Nat. 65, S96–S124. 12. Brocks, J.J., Logan, G.A., Buick, R. and Summons, R.E. (1999). Archean molecular fossils and the early rise of eukaryotes. Science 285, 1033–1036. 13. Baldauf, S.L. and Palmer, J.D. (1993). Animals and fungi are each other’s closest relatives: congruent evidence from multiple proteins. Proc. Natl. Acad. Sci. USA 90, 11558–11562. 14. Baldauf, S.L., Roger, A.J., Wenk-Siefert, I. and Doolittle, W.F. (2000). A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290, 972–977. 15. Bapteste, E., Brinkmann, H., Lee, J.A., Moore, D.V., Sensen, C.W., Gordon, P., Durufle, L., Gaasterland, T., Lopez, P., Müller, M., et al. (2002). The analysis of 100 genes supports the grouping of three highly divergent amoebae: Dictyostelium, Entamoeba, and Mastigamoeba. Proc. Natl. Acad. Sci. USA 99, 1414–1419. 16. Moreira, D., Le Guyader, H. and Philippe, H. (2000). The origin of red algae and the evolution of chloroplasts. Nature 405, 69–72. 17. Fast, N.M., Kissinger, J.C., Roos, D.S. and Keeling, P.J. (2001). Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids. Mol. Biol. Evol. 18, 418–426. 18. Cavalier-Smith, T. (2000). Flagellate megaevolution: the basis for eukaryote diversification. In The flagellates; unity, diversity and evolution B.S.C. Leadbeater and J.C. Green, pp. 361–390. (London: Taylor and Francis), pp. 361–390. 19. Embley, T.M. and Hirt, R.P. (1998). Early branching eukaryotes? Curr. Opin. Genet. Dev. 8, 624–629. 20. Simpson, A.G.B., Radek, R., Dacks, J.B. and O’Kelly, C.J. (2002). How oxymonads lost their groove: An ultrastructural comparison of Monocercomonoides and excavate taxa. J. Eukaryot. Microbiol. 49, 239–248.