624 Early branching eukaryotes? T Martin Embley* and Robert P Hirt Recent phylogenetic analyses suggest that Giardia, Trichomonas and Microsporidia contain genes of mitochondrial origin and are thus unlikely to be primitively amitochondriate as previously thought. Furthermore, phylogenetic analyses of multiple data sets suggest that Microsporidia are related to Fungi rather than being deep branching as depicted in trees based upon SSUrRNA analyses. There is also room for doubt, on the basis of a lack of consistent support from analyses of other genes, whether Giardia or Trichomonas branch before other eukaryotes. So, at present, we cannot be sure which eukaryotes are descendants of the earliest-branching organisms in the eukaryote tree. Future resolution of the order of emergence of eukaryotes will depend upon a more critical phylogenetic analysis of new and existing data than hitherto. Hypotheses of branching order should preferably be based upon congruence between independent data sets, rather than on single gene trees. Addresses Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK *e-mail: tme@nhm.ac.uk Correspondence: T Martin Embley +, mitochondrial homologue detected; –, no mitochondrial homologue detected; P, likely α−proteobacterial origin (i.e. consistent with a mitochondrial origin); ?, no published data. mHsp70, mitochondrial Hsp 70. Current Opinion in Genetics & Development 1998, 8:624–629 http://biomednet.com/elecref/0959437X00800624 © Current Biology Ltd ISSN 0959-437X Introduction Establishing a phylogeny for eukaryotes is central to our attempts to understand contemporary eukaryote diversity: for example, patterns of character change can be mapped over a phylogeny to fuel hypotheses of common ancestral states prior to lineage splitting. Hypotheses of character evolution can be made without recourse to phylogenetic analysis but phylogenies help to identify independent evolutionary events and to distinguish cause from effect in comparative analysis [1]. If, through phylogenetic analysis, we were able to identify descendants of the first eukaryotic branches, it might even be possible to infer something about the early stages of eukaryote evolution. As such, there has been considerable effort spent in trying to identify early branching eukaryotes and the purpose of this review is to discuss prevailing ideas pertaining to this topic. The Archezoa hypothesis for early branching eukaryotes In the premolecular era, the absence of functional mitochondria in protists such as Giardia, Trichomonas and Microsporidia was interpreted [2] as resulting from their early separation from other eukaryotes (i.e. prior to the mitochondrion symbiosis) which is thought to have occurred once [3]. The hypothesis that Giardia, Trichomonas and Microsporidia were relicts of a pre-mitochondrial phase of eukaryote evolution was formalised by calling them ‘Archezoa’ to denote a Table 1 List of nuclear encoded genes found in Giardia, Trichomonas and Microsporidia that probably originated from the endosymbiont that gave rise to mitochondria. Trichomonas vaginalis Giardia lamblia Microsporidia Hsp10 + [9] ? ? cpn 60 + [8–10] + [17••] ? mHsp70 + [9,11] ? + [14••–16••] Valyl-tRNA synthetase Triose-phosphate isomerase Adenylate kinase + [18••] + [18••] ? ? P [13] ? + [12] – [12] ? primitively amitochondriate condition [2,4•]. The Archezoa hypothesis was apparently supported for these taxa at least, when phylogenetic trees based upon small subunit ribosomal RNA [5] and translation elongation factors EF-1α and EF-2 [6], showed Giardia, Trichomonas and Microsporidia branching before eukaryotes which contain mitochondria (see Figure 1a). The Archezoa hypothesis stimulated widespread interest in Giardia, Trichomonas and Microsporidia because their biology was expected to reveal stages in the acquisition of eukaryote cellular features; however, recent data now suggest that Giardia, Microsporidia and Trichomonas contain genes which are most likely of mitochondrial origin. Mitochondrial genes in early branching eukaryotes It is accepted that mitochondrial genes were transferred to the host nucleus during the evolution of the symbiosis because phylogenetic analysis of host nuclear genes betrays their mitochondrial origin (e.g. [7]). Genes for which phylogenetic analysis indicates (or is consistent with) a mitochondrial origin have now been reported in Giardia, Trichomonas and Microsporidia (Table 1). They include adenylate kinase, 10, 60 and 70 kDa heat shock proteins (Hsp10, cpn60, Hsp70), triose phosphate isomerase and valyl tRNA-synthetase [8–13,14••–18••]. Alternative explanations, other than one-time possession of mitochondria, have been proposed to account for the presence of these genes in Giardia, Trichomonas or Microsporidia [19•,20•] but these seem unnecessary to explain the strongly supported positions of cpn60s and/or Hsp70s within clades otherwise defined by mitochondrial homologues and Early branching eukaryotes? Embley and Hirt 625 Figure 1 (a) 'Crown' taxa Animals Fungi Choanozoa Plants/green algae Alveolates Stramenopiles Rhodophytes Dictyostelium Entamoebae Euglenozoa Physarum Percolozoa H Microsporidia Trichomonas H Giardia Acquisition of mitochondria? Origin of eukaryotes? (b) H H ? Animals Fungi i Microsporidia Choanozoa ii Dictyostelium Physarum Entamoebae Plants/green algae Rhodophytes Stramenopiles ? Alveolates H Percolozoa H Euglenozoa vii Trichomonas H Giardia H Unidentified early branching eukaryote? Archaea Putative [pre-1996 in (a)] primitively amitochondriate taxa Secondary loss of functional mitochondria in some or all taxa Highlighted taxa are those branching before the 'Crown' (boxed) in most published SSUrRNA trees H Hydrogenosomes in some or all taxa Acquisition of mitochondria? iii v iv vi Origin of eukaryotes? (2) (1) Archaea Unidentified early branching primitively amitochondriate eukaryote? Current Opinion in Genetics & Development Alternative phylogenetic hypotheses for eukaryotes. (a) Relationships among eukaryotes as interpreted on a schematic SSUrDNA tree suggest the acquisition of mitochondria after the divergence of Giardia, Trichomonas and Microsporidia (all Archezoa sensu [2]) whose relative order of branching at the base of eukaryotes is uncertain. The placement of Archaea as the sister-group to eukaryotes is discussed in the text. (b) Composite phylogeny (i.e. no single gene tree supports all branches) depicting some alternative hypotheses for eukaryote relationships. The diagram was constructed by considering published trees from different protein datasets and some of these relationships are more speculative than others. All relationships should be viewed simply as more or less well-founded hypotheses which can be supported or refuted through more data and further analyses. The data considered to support particular hypotheses are: branch i, (see Table 2); ii, EF-1α [50], actin [5] and β-tubulin (e.g. [35•]); iii–vii, actin and β-tubulin (e.g. [5,35•]) and valyl-tRNA synthetase [18••] (and see Table 2). Branch lengths are meaningless in both diagrams and polytomies indicate lack of resolution not support for explosive radiation. Alternative hypotheses for the origin of the eukaryotic cell relative to the acquisition of mitochondria are indicated in (b): (1) The eukaryotic cell originated before the acquisition of the mitochondrion endosymbiont, thus there may be primitively amitochondriate eukaryotes remaining to be discovered. (2) The Hydrogen hypothesis [43••] posits that eukaryotes originated through symbiotic association between an Archaea (nominally the ‘host’) and the proteobacterium (the ‘symbiont’) which subsequently gave rise to mitochondria and hydrogenosomes. In this sense the pre-‘mitochondrial’ branches in ‘host’ trees will be Archaea. The documented distribution of hydrogenosomes among eukaryotes [51•] is shown on both trees, as are hypotheses of primitive or secondary absence of functional mitochondria. The possibility that Stramenopiles may lack functional mitochondria while retaining the organelle is discussed in [52]. Note that the perceived distribution of hydrogenosomes may well be conservative as anaerobic habitats are in general poorly described for the eukaryotes they contain or for the organelles that such eukaryotes may posses [38]. they are not founded upon phylogenetic analyses which support alternative origins for these or other genes (Table 1). We interpret the presence of genes in Archezoa which cluster with mitochondrial orthologues, as support for the hypothesis that Giardia and Microsporidia once had mitochondria, and for the hypothesis that Trichomonas has converted its mitochondria to hydrogenosomes [21,22]. hypothesis that a eukaryote may lose its mitochondria but still retain mitochondrial genes is now supported by recent phylogenetic analyses indicating a relationship between Microsporidia and Fungi (see below). Alternative phylogenetic relationships for early branching eukaryotes Phylogenetic analyses of hydrogenosome-containing ciliates [23] and hydrogenosome-containing fungi [24] suggest that they are derived from aerobic ancestors with mitochondria. For ciliates, fungi and Trichomonas, there is also other data consistent with a mitochondrial origin for the hydrogenosome organelle (reviewed in [25]). The The finding of mitochondrial genes in Giardia, Trichomonas and Microsporidia does not preclude these taxa from branching early in eukaryote evolution but there are alternative, and in some cases now better supported, hypotheses of relationships for these taxa. The deep position of Microsporidia was first challenged by tubulin gene trees which depicted a relationship between Microsporidia and 626 Genomes and evolution Table 2 Alternative hypotheses of relationship for Giardia, Trichomonas and Microsporidia from two or more proteins. α-tubulin β-tubulin RPB1 EF-1α EF-2 mHsp70 cpn60 Valyl-tRNA synthetase AK GDH Microsporidia + fungi +[27] +[26] +[29••] – –/+[29••] –/+[14••,15••] ? ? ? ? Giardia + Trichomonas – +[35•] – +[6] –/+[29••] ? +[17••] +[18••] – ? Giardia +/or Trichomonas + plants – – – – – – – +[18••] +[12] +[36] +, support for relationship; –/+, support for relationship from some analyses; ?, data unavailable. —, no apparent support for this relationship. RPB1, largest subunit of RNA polymerase II; mHsp70, mitochondrial Hsp 70; AK, adenylate kinase; GDH, glutamate dehydrogenase. Fungi [26,27]. This phylogenetic position for Microsporidia, surrounded by aerobic mitochondrial taxa, consistent with a mitochondria-loss hypothesis for Microsporidia discussed above. Judged against the apparently consistent story from SSUrRNA, EF-2 and EF-1α, that Microsporidia branch deep, however, the tubulin trees were treated with scepticism. Methodological artefact caused by a high substitution rate for fungal tubulins [28] or lateral transfer of tubulin genes from Fungi to Microsporidia [19•] being proposed as explanations of the aberrant tubulin relationships. Recent analyses of sequences from the largest subunit of RNA polymerase II (RPB1) also provide strong support for a relationship between Microsporidia and Fungi [29••] and analyses of mitochondrial Hsp70 support it weakly [14••,15••]. Furthermore, re-analysis of the EF-1α and EF-2 datasets show that they do not convincingly support the hypothesis that Microsporidia branch before other eukaryotes. When known sources of artefact affecting phylogenetic inference were accounted for, EF-2 was shown to support Microsporidia plus Fungi [29••]. The gene for EF-1α in the microsporidian Glugea plecoglossi is potentially saturated [29••], making it unreliable for tree construction. It does, however, contain an insertion encoding 11 amino-acids which appears otherwise diagnostic for the EF-1α genes of Fungi and Metazoa [30], which form a clade on the basis of phylogenetic analyses of different molecular datasets [5,30]. Thus, only SSUrRNA analyses appear to provide strong support for the deep divergence of Microsporidia [5] but even here there is some dissent; Kumar and Rzhetsky [31] concluded that the position of Microsporidia was difficult to resolve based on SSUrRNA. Furthermore, a recent analysis of large subunit rRNA, although not supporting a specific relationship with fungi, does not support an early divergence for the microsporidian Encephalitizoon [32•]. Are Giardia and Trichomonas really early branching? The history of systematics suggests that it is unreasonable to expect any single gene (such as SSUrRNA) or another small sample of characters, to resolve all relationships equally and some strongly supported patterns depicted in published gene trees may not reflect phylogeny [33,34]. It is has been suggested already (e.g. [35•]) that the deep positions of Giardia and Trichomonas (and other protists) in SSUrRNA trees may represent examples of long branch attraction, whereby long branches in trees cluster together irrespective of phylogenetic relationships. Although we agree that this is plausible, the patterns in the data supporting this potential criticism of the SSUrRNA topology have not yet been identified analytically. Interestingly, neither RPB1 or reanalysis of EF-2 [29••] and EF-1α [6,29••] provide compelling support for Giardia or Trichomonas diverging before other eukaryotes as is depicted in SSUrRNA trees. There are also alternative hypotheses of relationships for Giardia or Trichomonas (Figure 1b; Table 2) and for the protist lineages which branch before the ‘crown’ in SSUrRNA trees (Figure 1a). Some of these hypotheses are perhaps not as initially compelling as the signal from tubulin for Microsporidia plus Fungi but they cannot be dismissed on present analyses. For example, there is some support (Table 2, Figure 1b) for a sister group relationship between Giardia and Trichomonas. Much more speculatively — because limited data, uneven and limited taxon sampling, hinder tree comparison — there is a hint of a relationship to plants (Table 2) but not necessarily as a sister group. A relationship between Giardia and Trichomonas would support, if the Trichomonas hydrogenosome is indeed a modified mitochondrion, the hypothesis of secondary loss of mitochondria by Giardia. A relationship to plants might explain, through hypothesised plastid loss in Giardia, the relationship between Giardia adenylate kinase and glutamate dehydrogenase and the same proteins located, respectively, in maize or Chlorella plastids [12,36] (but see also [37] for a discussion of when organelle localisation might not reflect symbiotic origins). Where now in the search for the first branches in the eukaryote tree? If we believe that an ancestral anaerobic or microaerophilic eukaryote gained mitochondria by engulfment of an Early branching eukaryotes? Embley and Hirt α-proteobacterium, this event need not necessarily exclude the descendants of other eukaryotes (which did not participate in this symbiosis) from persisting in anaerobic or microaerophilic habitats (branch [1] in Figure 1b). Anaerobic or microaerophilic habitats often support large communities of microbial eukaryotes but relatively few of these been studied in any detail [38]. As well as trying to isolate the eukaryotes in such samples, it might be of value to undertake a ‘fishing expedition’ for eukaryotic genes from anaerobic or microaerophilic samples, using the environmental gene library approach used to discover novel Archaea [39]. Which gene(s) might be most informative in such a fishing trip, which aims to discover early branching eukaryotes, is difficult to predict without doing the experiments but it would be sensible to use more than just SSUrRNA. Furthermore, even if Giardia, Microsporidia and Trichomonas are probably not primitively amitochondriate, it does not mean that all Archezoa which lack mitochondria once contained the organelle. For example, the phylogenetic positions of the amitochondriate oxymonads and retortomonads are still uncertain [40,41]. Lastly, we should not forget that the trees upon which existing phylogenetic hypotheses are based are poorly sampled and only a small number of anaerobic or aerobic protist lineages have been explored in any detail, or for more than a single gene; for example, Patterson recently listed his ‘residua’ — several hundred genera of unknown phylogenetic or ultrastructural affinity [42]. Interestingly, the ‘hydrogen hypothesis’ [43••] posits that eukaryotes originated through symbiotic association between an Archaea (nominally the ‘host’) and the proteobacterium (the ‘symbiont’) which subsequently gave rise to mitochondria and hydrogenosomes (branch [2] in Figure 1b). In this sense the pre-‘mitochondrial’ branches in ‘host’ trees will be Archaea. Interestingly, this prediction is consistent (ignoring contemporary phenotypes — see [43••]) with phylogenetic analyses of translation elongation factors [44–46] which suggest that some Archaea may be more closely related to eukaryotes than others. Was there a ‘big bang’ in eukaryote evolution? Most of the preceding discussion assumes that phylogenetic analyses will ultimately permit us to identify the early branches of the eukaryote tree, given more critical attention to methods and more data. It has been suggested recently, however, that the major eukaryote groups might have diversified so quickly that resolution of their order of emergence, early or late, may be either difficult or impossible. This hypothesis of a rapid cladogenesis or explosive radiation giving rise to most or all major groups of eukaryotes has been called the ‘big bang’ hypothesis [35•]. 627 published. If it depends on the rapid emergence of the morphological or cellular features which are used to define taxonomic groups, then the characters and taxa supporting the hypothesis have not been described. And if the hypothesis uses both types of data, it has not been demonstrated that each can be analysed separately to provide independent support for the hypothesis. Without this information it is impossible to see what relationships or character distributions might critically test the big bang hypothesis. If, for example, tubulin trees (or Figure 1b) were to subsequently prove accurate for some of the relationships they display, would these relationships and character distributions either support or refute the big bang hypothesis? Our suggestion that the big bang hypothesis needs to be stated unambiguously is motivated by its potential implications for our ability to resolve branching order, but also by a desire to avoid the confusion which has surrounded other explosion hypotheses including the most famous, the ‘Cambrian explosion’. Here, the fossil data have been interpreted to support the hypothesis that the major metazoan phyla appeared during less than 20 million years in the early Cambrian [35•]. In contrast, Gould has written that “The Cambrian explosion embodies a claim for a rapid spurt of anatomical innovation within the animal kingdom, not a statement about times of genealogical divergence” [47]. Interestingly, a cladistic analysis of morphology [48], new fossils [47], and, most recently, new methods for analysing molecular data [49], are more consistent with a gradual preCambrian diversification of metazoan phyla rather than an explosive appearance of new clades. Conclusions The results of phylogenetic analyses to date, have demonstrated that organelle absence, perceived cytological simplicity, or particular phenotypes are uncertain guides for identifying early branching eukaryotes prior to phylogenetic analysis. Thus Giardia and Trichomonas are probably not primitively amitochondriate and the strong support for them being deep-branching appears to come mainly from the SSUrRNA dataset. Microsporidia are related to Fungi on the basis of several datasets. Indeed, if early branching eukaryotes can be identified through new data, or more rigorous analysis can resolve the order of emergence supported by multiple data sets, their features may yet surprise us. Acknowledgements RPH is supported by a Wellcome Trust Career Development Fellowship (number 045702/Z/95/Z). The authors thank W Martin, D Seibert, A Roger and H Philippe for critical comments on parts of this manuscript. References and recommended reading At present, this interesting idea has not been formulated sufficiently precisely for us to see what data actually supports it. For example, if it is based upon rates of lineage splitting as revealed by rigorously evaluated branching diagrams for different genes, then this data has not been Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Harvey PH, Leigh Brown AJ, Maynard Smith J, Nee S: New uses for new phylogenies. Oxford, UK: Oxford University Press; 1996. 628 Genomes and evolution 2. Cavalier-Smith T: A 6 kingdom classification and a unified phylogeny. In Endocytobiology II. Edited by Schwemmler W, Schenk HEA. Berlin: De Gruyter; 1983:1027-1034. 3. Lang BF, Burger G, O’Kelly CJ, Cedergreen R, Golding GB, Lemieux C, Sankoff D, Turmel M, Gray MW: An ancestral mitochondrial DNA resembling a eubacterial genome in miniature. Nature 1997, 387:493-497. 4. Keeling PJ: A kingdom’s progress: Archezoa and the origin of • eukaryotes. Bioessays 1998, 20:87-95. An interesting review of the history of the Archezoa concept, including some taxa not featured in our own review. It summarises features and the biology of the main groups and current ideas about their relationships. It includes a discussion of the tubulin trees and non-tree data consistent with a relationship between Microsporidia and Fungi. 5. Sogin ML, Silberman JD, Hinkle G, Morrison HG: Problems with molecular diversity in the eukarya. In Evolution of Microbial Life. Edited by Roberts DM, Sharp P, Alderson G, Collins M. Cambridge, UK: Cambridge University Press; 1996:167-184. 6. Hashimoto T, Nakamura Y, Kamaishi T, Hasegawa M: Early evolution of eukaryotes inferred from the amino acid sequences of elongation factors 1a and 2. Arch Protistenkd 1997, 148:287-295. 7. Viale AM, Arakaki AK: The chaperone connection to the origins of the eukaryotic organelles. FEBS Lett 1994, 341:146-151. 8. 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