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Eukaryotic evolution, changes and challenges
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"� 2006 Nature Publishing Group Eukaryotic evolution, changes and challenges T. Martin Embley 1 & William Martin 2 The idea that some eukaryotes primitively lacked mitochondria and were true intermediates in the prokaryote-to- eukaryote transition was an exciting prospect. It spawned major advances in understanding anaerobic and parasitic eukaryotes and those with previously overlooked mitochondria. But the evolutionary gap between prokaryotes and eukaryotes is now deeper, and the nature of the host that acquired the mitochondrion more obscure, than ever before. N ew findings have profoundly changed the ways in which we view early eukaryotic evolution, the composition of major groups, and the relationships among them. The changes havebeendrivenbyafloodofsequencedatacombinedwith improved?butbynomeansconsummate?computational methods of phylogenetic inference. Various lineages of oxygen-shunning or parasitic eukaryotes were once thought to lack mitochondria and to have diverged before the mitochondrial endosymbiotic event. Such key lineages, which are salient to traditional concepts about eukaryoteevolution, includethediplomonads(forexample, Giardia), trichomonads (for example, Trichomonas) and microsporidia (for example, Vairimorpha). From today?s perspective, many key groups have been regrouped in unexpected ways, and aerobic and anaerobic eukaryotesinterminglethroughouttheunfoldingtree.Mitochondria in previously unknown biochemical manifestations seem to be universal among eukaryotes, modifying our views about the nature of the earliest eukaryotic cells and testifying to the importance of endosymbiosis in eukaryotic evolution. These advances have freed the field to consider new hypotheses foreukaryogenesis and toweigh these, and earlier theories, against the molecular record preserved in genomes. Newer findings even call into question the very notion of a ?tree? as an adequate metaphor to describe the relationships among genomes. Placing eukaryotic evolution within a time frame and ancient ecological context is still problematic owing to the vagaries ofthemolecularclock and thepaucityof Proterozoicfossil eukaryotes that can be clearly assigned to contemporary groups. Although the broader contours of the eukaryote phylogenetic tree are emerging fromgenomicstudies,thedetailsofitsdeepestbranches,anditsroot, remain uncertain. The universal tree and early-branching eukaryotic lineages The universal tree based on small-subunit (SSU) ribosomal RNA 1 provided a first overarching view of the relationships between the different types of cellular life. The relationships among eukaryotes recovered from rRNA 2 , backed up by trees of translation elongation factor (EF) proteins 3 , provided what seemed to be a consistent, and hence compelling, picture (Fig. 1). The three protozoa at the base of these trees (Giardia, Trichomonas and Vairimorpha), along with Entamoeba and its relatives, were seen as members of an ultrastruc- turallysimple,paraphyleticgroupofeukaryotescalledtheArchezoa 4 . Archezoawerethoughttoprimitivelylackmitochondria,havingsplit from the main trunk of the eukaryotic tree before the mitochondrial endosymbiosis: all other eukaryotes contain mitochondria because theydiverged after this singularsymbioticevent 5 . Therefore,Archezoa were interpreted as contemporary descendants of a phagotrophic, nucleated, amitochondriate celllineage thatincludedthehost for the mitochondrial endosymbiont 6 . The apparent agreement between molecules and morphology depicted the relative timing of the mitochondrial endosymbiosis (Fig. 1) as a crucial, but not ancestral, event in eukaryote phylogeny. Chinks in the consensus Mitochondrial genomes studied so far encode less than 70 of the proteins that mitochondria need to function 5 ; most mitochondrial proteins are encoded by the nuclear genome and are targeted to REVIEWS Figure 1 | The general outline of eukaryote evolution provided by rooted rRNA trees. The tree has been redrawn and modified from ref. 92. Until recently, lineages branching near the root were thought to primitively lack mitochondria and were termed Archezoa 4 . Exactly which archezoans branched first is not clearly resolved by rRNA data 2 , hence the polytomy (more than two branches from the same node) involving diplomonads, parabasalids and microsporidia at the root. Plastid-bearing lineages are indicated in colours approximating their respective pigmentation. Lineages furthest away from the root, including those with multicellularity, were thought to be the latest-branching forms and were sometimes misleadingly (see ref. 60) called the ?crown? groups. 1 School of Biology, The Devonshire Building, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK. 2 Institute of Botany III, University of Du�sseldorf, D-40225 Du�sseldorf, Germany. Vol 440|30 March 2006|doi:10.1038/nature04546 623 � 2006 Nature Publishing Group mitochondria using a protein import machinery that is specific to this organelle 7 . The mitochondrial endosymbiont is thought to have belonged to the a-proteobacteria, because some genes and proteins still encoded by the mitochondrial genome branch in molecular trees among homologues from this group 5,8 . Some mitochondrial proteins, such as the 60- and 70-kDa heat shock proteins (Hsp60, Hsp70), also branch among a-proteobacterial homologues, but the genes are encoded by the host nuclear genome. This is readily explained by a corollary to endosymbiotic theory called endosym- biotic gene transfer 9 : during the course of mitochondrial genome reduction, genes were transferred from the endosymbiont?s genome to the host?s chromosomes, but the encoded proteins were re- imported into the organelle where they originally functioned. With the caveat that gene origin and protein localization do not always correspond 9 , any nuclear-encoded protein that functions in mito- chondria and clusters with a-proteobacterial homologues is most simply explained as originating from the mitochondrion in this manner. By that reasoning 10 , the discovery of mitochondrial Hsp60 in E. histolytica was taken as evidence that its ancestors harboured mitochondria. A flood of similar reports on mitochondrial Hsp60 and Hsp70 from all key groups of Archezoa ensued 11 , suggesting that their common ancestor also contained mitochondria. At face value, those findings falsified the central prediction of the archezoan concept. However, suggestions were offered that lateral gene transfer (LGT) in a context not involving mitochondria could also account for the data. But that explanation, apart from being convoluted, now seems unnecessary: the organisms once named Archezoa for lack of mitochondria not only have mitochondrial-derived proteins, they have the corresponding double-membrane-bounded organelles as well. Mitochondria in multiple guises Theformerarchezoansaremostlyanaerobes,avoidingallbutatraceof oxygen, and like many anaerobes, including various ciliates and fungi that were never grouped within the Archezoa, they are now known to harbour derived mitochondrial organelles?hydrogenosomes and mitosomes. These organelles all share one or more traits in common with mitochondria (Fig. 2), but no traits common to them all, apart from the double membrane and conserved mechanisms of protein import, have been identified so far. Mitochondria typically?but not always (the Cryptosporidium mitochondrion lacks DNA 12 )? possess a genome that encodes components involved in oxidative phosphorylation 5 .Withonenotableexception 13 ,allhydrogenosomes Figure 2 | Enzymes and pathways found in various manifestations of mitochondria. Proteins sharing more sequence similarity to eubacterial than to archaebacterial homologues are shaded blue; those with converse similarity pattern are shaded red; those whose presence is based only on biochemical evidence are shaded grey; those lacking clearly homologous counterparts in prokaryotes are shaded green. a, Schematic summary of salientbiochemical functionsinmitochondria 5,88 ,includingsomeanaerobic forms 16,17 . b, Schematic summary of salient biochemical functions in hydrogenosomes 14,19 . c, Schematic summary of available findings for mitosomes and ?remnant? mitochondria 32?34,93 . The asterisk next to the Trachipleistophora and Cryptosporidium mitosomes denotes that these organisms are not anaerobes in the sense that they do not inhabit O 2 -poor niches, but that their ATP supply is apparently O 2 -independent. UQ, ubiquinone; CI, mitochondrial complex I (and II, III and IV, respectively); NAD, nicotinamide adenine dinucleotide; MCF, mitochondrial carrier family protein transporting ADP and ATP; STK, succinate thiokinase; PFO, pyruvate:ferredoxin oxidoreductase; PDH, pyruvate dehydrogenase; CoA, coenzyme A; Fd, ferredoxin; HDR, iron-only hydrogenase; PFL, pyruvate:formate lyase; ASC, acetate-succinate CoA transferase; ADHE, bi-functional alcohol acetaldehyde dehydrogenase; FRD, fumarate reductase; RQ, rhodoquinone; Hsp, heat shock protein; IscU, iron?sulphur cluster assembly scaffold protein; IscS; cysteine desulphurase; ACS (ADP), acetyl-CoA synthase (ADP-forming). REVIEWS NATURE|Vol 440|30 March 2006 624 � 2006 Nature Publishing Group andmitosomesstudiedsofarlackagenome.Theorganismsinwhich they have been studied generate ATP by fermentations involving substrate-levelphosphorylations, rather than through chemiosmosis involving an F 1 /F 0 -type ATPase 12,14,15 . Entamoeba, Giardia and Trichomonas live in habitats too oxygen-poor to support aerobic respiration 14 , while others, like Cryptosporidium and microsporidia havedrastically reducedtheirmetaboliccapacities during adaptation to their lifestyles as intracellular parasites 12,15 . Between aerobic mitochondria, which use oxygen as the terminal electron acceptor of ATP-producing oxidations, and Nyctotherus hydrogenosomes, which (while retaining a mitochondrial genome) use protons instead of oxygen 13 , there are a variety of other anaero- bically functioning mitochondria. They occur in protists such as Euglena, but also in multicellular animals such as Fasciola and Ascaris, which typically excrete acetate, propionate or succinate, instead of H 2 OorH 2 , as their major metabolic end-products 16,17 . Hence, mitochondria, hydrogenosomes and mitosomes are viewed most simply as variations on a single theme, one that fits neatly within the framework provided by classical evolutionary theory 18 . They are evolutionary homologues that share similarities because of common ancestry, but?like forelimbs in vertebrates?differ sub- stantially in form and function across lineages owing to descent with modification. Hydrogen-producing mitochondria Hydrogenosomes oxidize pyruvate to H 2 ,CO 2 and acetate, making ATP by substrate-level phosphorylation 19 that they export to the cytosol using a mitochondrial-type ADP/ATP carrier 20,21 . They have been identified in trichomonads, chytridiomycetes and ciliates 13,22 ; their hydrogen excretion helps to maintain redox balance 14 in these organisms. Important similarities between Trichomonas hydrogenosomes and mitochondria include the use of common protein import pathways 23 , conserved mechanisms of iron?sulphur- cluster assembly 24 , conserved mechanisms of NAD � regeneration 25 , and conservation of a canonical ATP-producing enzyme of the mitochondrial Krebs cycle?succinate thiokinase 26 . On the basis of electron microscopy and ecology, additional, and diverse, eukaryotic lineages are currently suspected to contain hydrogenosomes 27,28 , but hydrogen production?the defining characteristic of hydrogeno- somes 19 ?by those organelles has not yet been shown. In contrast to most mitochondria, hydrogenosomes typically contain pyruvate:ferredoxin oxidoreductase (PFO) and iron [Fe] hydrogenase. Common among anaerobic bacteria, these enzymes prompted the early suggestion that trichomonad hydrogenosomes arose from a Clostridium-like endosymbiont 29 . In a recent rekindling of that idea 30,31 , trichomonad hydrogenosomes were suggested to be hybrid organelles, derived from an endosymbiotic anaerobic bacter- ium (the source of PFO and hydrogenase genes), a failed mitochon- drial endosymbiosis (the source of nuclear genes for mitochondrial Hsp60 and Hsp70), plus LGT from a mitochondrially related (but non-mitochondrial) donor (the source of NADH dehydrogenase). However, independent work suggested a mitochondrial, rather than hybrid, origin of the Trichomonas NADH dehydrogenase 25 . Further- more, the hybrid hypothesis fails to account for the presence of [Fe]- hydrogenase homologues in algal chloroplasts, PFO homologues in Euglena mitochondria, or the presence of either enzyme and hydro- genosomes in other eukaryotic lineages 25 ; hence, a single common ancestry of mitochondria and hydrogenosomes sufficiently accounts for current observations. Mitochondria reduced to bare bones Mitosomes were discovered in Entamoeba 32 as mitochondrion- derivedorganellesthathaveundergonemoreevolutionary reduction than hydrogenosomes. They are also found in Giardia 33 and micro- sporidia 34 . Mitosomes seem to have no direct role in ATP synthesis because, so far, they have been found only among eukaryotes whose core ATP synthesis occurs in the cytosol 14 or among energy parasites 15 . Mitosomes import proteins in a mitochondrial-like manner 35?37 , and Giardia mitosomes contain two mitochondrial proteins of Fe?S cluster assembly?cysteine desulphurase (IscS) and iron-binding protein (IscU) 33 . Fe?S clusters are essential for life: they are cofactors of electron transfer, catalysis, redox sensing and ribosome biogenesis in eukaryotes 38 . Fe?S cluster assembly is an essential function of yeast mitochondria 38 and it has been widely touted as a potential common function for mitochondrial homologues 15,22 .ItistheonlyknownfunctionofGiardiamitosomes, which, like Trichomonas hydrogenosomes 24,37 , promote assembly of [2Fe?2S] clusters into apoferredoxin in vitro 33 . By contrast, and (so far) uniquely among eukaryotes, Entamoeba uses two proteins of non-mitochondrial ancestry for Fe?S cluster assembly 39 ; the location of this pathway in Entamoeba is currently unknown. Branch migrations and evolutionary models ThediscoveryofmitochondrialhomologuesinGiardia,Trichomonas and microsporidians, which had been the best candidates for eukaryotes that primitively lacked mitochondria, has pinned the timing of the mitochondrial origin to the ancestor of all eukaryotes studied so far. But that does not mean thatthe basal position of these groups in the SSU rRNA tree (Fig. 1) and EF trees 3 is necessarily incorrect. That issue hinges on efforts to construct reliable rooted phylogenetic trees depicting ancient eukaryotic relationships: a developing area of research that is fraught with difficulties. The tempo and mode of sequenceevolution is farmorecomplicated than is assumed by current mathematical models that are used to make phylogenetic trees 40 . In computer simulations, where the true tree is known, model mis-specification can produce the wrong tree with strong support 41 . Differentsites inmolecular sequencesevolveatdifferentrates, and failure to accommodate this rate variation, something early methods failed to do, can lead to strongly supported but incorrect trees owing to a common problem called ?long-branch-attraction? 42 . This occurs whenbranches thatarelong or?fast evolving?,relativeto others inthe tree, cluster together irrespective of evolutionary relationships. The molecular sequences of Giardia, Trichomonas and microsporidia often form long branches in trees and thus are particularly prone to this problem 25,43,44 . The traditional models that placed microspor- idia deep within trees 2,3 assumed that all sequence sites evolved at the same rate, even though they clearly do not. In these trees, the long-branch microsporidia are next to the long branches of the prokaryotic outgroups. More data and better models have produced trees that agree in placing microsporidia with fungi 45,46 , suggesting that the deep position of microsporidia in early trees was indeed an artefact. The position of Giardia and Trichomonas sequences at the base of eukaryotic molecular trees is also suspect, given that they also form long branches in the trees that place them in this way, and because other treesandmodels placethem together asaninternal branchofa rooted eukaryotic tree 47 . Resolving which position is correct is particularly important, because Giardia and Trichomonas are still commonly referred to as ?early-branching? eukaryotes. Given the evident uncertainties of such phylogenies, and the importance of the problem, the onus is on those who would persist in calling these species?earlybranching?toshowthattreesplacingthemdeepexplain the data significantly better than trees that do not. The root of the eukaryotic tree The usual way to root a phylogenetic tree is by reference to an outgroup; the rRNA and EF trees used prokaryotic sequences to root eukaryotes on either the Giardia, Trichomonas or microsporidia branch (Fig. 1), but these rootings have not proved robust 43?45 . The sequencesof outgroups areoften highlydivergentcomparedto those of the ingroup, making it difficult to avoid model mis-specification and long-branch-attraction 44,48 . Analternativemethod ofrootinganexisting tree istolookforrare NATURE|Vol 440|30 March 2006 REVIEWS 625 � 2006 Nature Publishing Group changes in a complex molecular character where the ancestral state can be inferred. This method was used 49 to infer that the root of the eukaryotic tree lies between the animals, fungi and amoebozoa (together called unikonts) on the one side, and plants, algae and mostprotozoa(bikonts)ontheother.Infungiandanimals,thegenes for dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are separate 44 , astheyareinprokaryote outgroups; but theyarefused in the bikonts sampled so far. Assuming that the fusion occurred only once and that its subsequent fission did not occur at all, the DHFR?TS fusion would be a derived feature uniting bikonts, suggesting that the eukaryote root lies outside this group 49 . The coherence of animals, fungi and various unicellular eukaryotes (together called opisthokonts) is supported by phylogenetic trees and other characters 50 . The presence of a type II myosin in opistho- konts and amoebozoa unites them to form the unikonts 51 . If both unikonts and bikonts are monophyletic groups, and together they encompass extant eukaryotic diversity, then the root of eukaryotes would lie between them. Placing the eukaryote root between unikonts and bikonts would help to bring order to chaos, if it is correct. However, it assumes that the underlyingtree?over whichthe rooting character ismapped?is known, when in fact the relationships?especially for bikonts and many enigmatic protistan lineages 52 ?remain uncertain. The rooting also depends upon a single character of unknown stability sampled from only a few species. An additional caveat is that Giardia and Trichomonas lack both DHFR and TS?parasites relinquish genes of various biosynthetic pathways, stealing the pathway products from their hosts instead. Hence, the missing fusion character does not address their position in the tree. New hypotheses of eukaryotic relationships New data and analyses from many laboratories have been used to formulate a number of hypotheses of eukaryotic relationships (Fig.3)thatfundamentallydiffer fromthosein theSSUrRNAtree.It isapparentthathydrogenosomes andmitosomes appearondifferent branches; the absence of traditional mitochondria and presence of a specialized anaerobic phenotype are neither rare nor ?primitive?, as oncethought.Mitochondriawithagenomeencodingelementsofthe respiratory pathway also appear on both sides of the tree (Fig. 3), suggesting that this pathway has been retained since earliest times; although,asmodernexamplesattest 16,17 ,itneednothavealwaysused oxygen as the sole terminal electron acceptor. On the basis of the unfolding tree, it would seem entirely possible?if not likely?that aerobic and anaerobic eukaryotes, harbouring mitochondrial homologues of various sorts, have co-existed throughout eukaryote history. The relationships between major groups of eukaryotes are uncer- tain because of the lack of agreement between different proteins and different analyses; this uncertainty is depicted as a series of polytomies in Fig. 3. Most groups are still poorly sampled for species andmolecularsequences?factorsthatimpederobustresolution 53 .It has been suggested 54 that the lack of resolution in deeper parts of the eukaryotic tree stems from an evolutionary ?big bang? or rapid radiation for eukaryotes, perhaps driven by the mitochondrial endosymbiosis 54 . However, both theory and computer simu- lations 40,41 suggest that a lack of resolution at deeper levels is to be expected given sparse data, our assumptions about sequence evolu- tion, and the limitations of current phylogenetic methods. Thus, loss ofhistorical signal provides asimple nullhypothesisfor theobserved lack of resolution in deeper parts of the eukaryotic tree. More good theories for eukaryotic origins than good data Eukaryotic cell organization is more complex than prokaryotic, boasting, inter alia, a nucleus with its contiguous endoplasmic reticulum, Golgi, flagellawith a?9�2? patternofmicrotubule arrange- ment, and organelles surrounded by double membranes. There are no obvious precursor structures known among prokaryotes fromwhich such attributes could be derived, and no intermediate cell types known that would guide a gradual evolutionary inference between the prokaryotic and eukaryotic state. Accordingly, thoughts on the topicarediverse,andnewsuggestionsappearfasterthanoldonescan be tested. Biologists have traditionally derived the complex eukaryotic state from the simpler prokaryotic one. In recent years, even that has been Figure 3 | Schematic tree of newer hypotheses for phylogenetic relationships among major groups of eukaryotes. The composite tree is based on work from many different laboratories and is summarized elswhere 52 ; no single data set supports all branches. Polytomies indicate uncertainty in the branching order between major groups. The naming of groupsfollowscurrentpopularusage 52,60 .Thecurrentdebatethattherootof the tree may split eukaryotes into bikonts and unikonts is discussed in the text. Lineages containing species with comparatively well-studied hydrogenosomes (H) or mitosomes (M) are labelled. The depicted distribution of hydrogenosomes and mitosomes is almost certainly conservative, as relatively few anaerobic or parasitic microbial eukaryotes have been studied in sufficient detail to characterize their organelles. The strict coevolution of host nuclear and algal nuclear plus plastid genomes within the confines of a single cell in the wake of secondary endosymbiosis (28), irrespective of whether or not the secondary nucleus or plastid has persisted as a separate compartment, is indicated by doubled branches. Diversity of pigmentation among photosynthetic eukaryote lineages is symbolized by different coloured branches. REVIEWS NATURE|Vol 440|30 March 2006 626 � 2006 Nature Publishing Group called into question, as some phylogenies have suggested that prokaryotes might be derived from eukaryotes 55 . However, the ubiquityofmitochondrialhomologuesrepresentsastrongargument thatclearlypolarizestheprokaryote-to-eukaryotetransition:because the common ancestor of contemporary eukaryotes contained a mitochondrial endosymbiont that originated from within the proteobacterial lineage, we can confidently infer that prokaryotes arose and diversified before contemporary eukaryotes?the only ones whose origin requires explanation?did. This view is consistent with microfossil and biogeochemical evidence 56 . Current ideas on the origin of eukaryotes fall into two general classes: those that derive a nucleus-bearing but amitochondriate cell first, followed by the origin of mitochondria in a eukaryotic host 57?61 (Fig. 4a?d), and those that derive the origin of mitochondria in a prokaryotic host, followed by the origin of eukaryotic-specific features 62?64 (Fig. 4e?g). Models that derive a nucleated but amito- chondriate cell as an intermediate (Fig. 4a?d) have suffered a substantial blow with the demise of Archezoa. Models that do not entail amitochondriate intermediates have in common that the host assumed to have acquired the mitochondrion was an archaebacter- ium not a eukaryote; hence, the steep organizational grade between prokaryotes and eukaryotes follows in the wake of radical chimaer- isminvolvingmitochondrialorigins(Fig.4e?g).Acriticismfacingall ?archaebacterial host? models is that phagotrophy (the ability to engulf bacteria as food particles) was once seen as an absolute prerequisite for mitochondrial origins 60 . This argument has lost some of its strength with the discovery of symbioses where one prokaryote lives inside another, non-phagocytotic prokaryote 65 . The elusive informational ancestor With the exception of the neomuran hypothesis, which views both eukaryotes and archaebacteria as descendants of Gram-positive eubacteria 60,61 (Fig. 4d), most current theories for eukaryotic origins (Fig. 4) posit the involvement of an archaebacterium in that process. The archaebacterial link to eukaryote origins was first inferred from shared immunological and biochemical similarities of their DNA-dependent RNA polymerases 66 . Tree-based studies of entire genomes 67,68 extended this observation: most eukaryotic genes for replication, transcription and translation (informational genes) are related to archaebacterial homologues, while those encoding biosyn- thetic and metabolism functions (operational genes) are usually related to eubacterial homologues 8,67,68 . The rooted SSUrRNA tree 1 depicts eukaryotes and archaebacteria as sister groups, as in the neomuran (Fig. 4d) hypothesis 60,61 .By Figure 4 | Models for eukaryote origins that are, in principle, testable with genome data. a?d, Models that propose the origin of a nucleus-bearing but amitochondriate cell first, followed by the acquisition of mitochondria inaeukaryotichost.e?g,Modelsthatproposetheoriginofmitochondriain a prokaryotic host, followed by the acquisition of eukaryotic-specific features. Panels a?g are redrawn from refs 57 (a), 58 (b), 59 (c), 60 and 61 (d), 62 (e), 63 (f) and 64 (g). The relevant microbial players in each model are labelled. Archaebacterial and eubacterial lipid membranes are indicated in red and blue, respectively. NATURE|Vol 440|30 March 2006 REVIEWS 627 � 2006 Nature Publishing Group contrast, the eocyte (Fig. 4c) hypothesis 69,70 proposes that eukaryotic informational genes originate from a specific lineage of archae- bacteria called the eocytes, a group synonymous with the Crenarch- aeota 1 . In the eocyte tree, the eukaryotic genetic machinery is descended from within the archaebacteria. Although the rooted rRNA tree is vastly more visible to non-specialists, published data are equivocal: for every analysis of a eukaryotic informational gene that recoversthe neomuran topology, a different analysis of the same molecule(s) has recovered the eocyte tree 70?74 , with the latter being favoured by more sophisticated phylogenetic analyses 69,73,74 and by a shared amino-acid insertion in eocyte and eukaryotic elongation factor 1-a 70 . More recently, genome trees based on shared gene content have been reported. These methods are still new, and?just like gene trees?give different answers from the same data, recovering for informational genes either eukaryote?archaebacterial sisterhood 75 , the eocyte tree 76 or a euryarchaeote ancestry 77 . The dichotomy of archaebacteria into euryarchaeotes and eocytes/crenarchaeotes 1 remains unchallenged. The issue, so far unresolved, is the relationship of eukaryotic informational genes to archaebacterial homologues: inheritance from a common progenitor (as in the neomuran hypothesis) or a direct descendant; and if by direct descent, from eocytes/crenarchaeotes like Sulfolobus 76 , or euryarchaeotes such as Thermoplasma 64,78 , Pyrococcus 77 or methanogens 58,62 . The problems associated with the phylogenetic relationships discussed above are exacerbated at such deep levels, and there is currently neither consensus on this issue nor unambiguous evidence that would clarify it. The vexing operational majority Of those eukaryotic genes that have detectable prokaryotic homo- logues, the majority 67 , perhaps as much as 75% 8 , are eubacterial and correspond to the operational class. Here arises an interesting point. Although individual analyses of informational genes arrive at fundamentally different interpretations 76,77 , no one has yet suggested that more than one archaebacterium participated in eukaryote origins. The situation is quite different with operational genes, where differing phylogenies for individual genes are freely inter- preted as evidence for the participation of more than one eubacterial partner. The contribution of gene transfers from the ancestral mitochondrion to nuclear chromosomes has been estimated as anywhere from 136?157 (ref. 77) to ,630 genes 79 , depending on the method of analysis. An issue that still requires clarification concerns the origin of thousands of eukaryotic operational genes that are clearly eubacterial, but not specifically a-proteobacterial, in origin 8 (disregarding here the cyanobacterial genes in plants 80 ). There are currently four main theories that attempt to account for those genes. (1) In the neomuran hypothesis (Fig. 4d), they are explained through a direct inheritance from the Gram-positive ancestor 60,61 ; however, few eukaryote genes branch with Gram- positive homologues. (2) In hypotheses entailing more than one eubacterial partner at eukaryote origins (Fig. 4a?c), they are explained as descending from the non-mitochondrial eubacterium; however,thesegenesbranchallovertheeubacterialtree,notwithany particular lineage. (3) In models favouring widespread LGT from prokaryotestoeukaryotes,theyareexplainedasseparateacquisitions fromindividual donors 81 ; although someLGT clearly has occurred 82 , the jury is still out on its extent because of a lack of detailed large- scale analyses of individual genes using reliable methods. (4) In single-eubacteriummodels(Fig.4e?g),theyareeithernotaddressed, orexplainedasacquisitionsfromthemitochondrialsymbiont,witha twofold corollary 8 of LGT among free-living prokaryotes since the origin of mitochondria, and phylogenetic artefact. LGT among prokaryotes 83 figures into the origin of eukaryotic operational genes in a fundamental manner that is often overlooked. Most claims of outright LGT to ancestral eukaryotes (that is, from donors distinct from the mitochondrion) implicitly assume a static chromosome model in which prokaryotes do not exchange genes among themselves; finding a eukaryotic gene that branches with a group other than a-proteobacteria is taken as evidence for an origin from that group (the vagaries of deep branches notwithstanding). But if we embrace a fluid chromosome model for prokaryotes, as some interpretations of the data suggest we should 84 , then the expected phylogeny for a gene acquired from the mitochondrion would be common ancestry for all eukaryotes, but not necessarily tracing to a-proteobacteria, because the ancestor of mitochondria possessed an as yet unknown collection of genes. The timing and ecological context of eukaryote origins Diversified unicellular microfossils ofuncertain phylogenetic affinity (acritarchs), but widely accepted as eukaryotes, appear in strata of ,1.45billion years (Gyr) of age 85 , providing a minimum age for the group. Bangiomorpha, a fossilized multicellular organism virtually indistinguishable in morphology from modern bangiophyte red algae, has been found in strata of ,1.2Gyr of age 86 , placing a lower bound on the age of the plant kingdom. A wide range of molecular clockestimatesofeukaryoteagehavebeenreported,butthesearestill uncertain, being contingent both on the use of younger calibration pointsandonthephylogeneticmodelandassumedtree 87 .Atpresent, a minimum age of eukaryotes at ,1.45Gyr and a minimum age of the plant kingdom at,1.2Gyr seem to be criteria that the molecular clock must meet. The classical view of early eukaryote evolution posits two main ecological stages: (1) the early emergence and diversification of anaerobic, amitochondriate lineages, followed by (2) the acquisition ofan oxygen-respiringmitochondrial ancestorin onelineage thereof and the subsequent diversification of aerobic eukaryotic lineages 78 . Concordant with that view, mitochondrial origins have traditionally been causally linked to the global rise in atmospheric oxygen levelsat ,2Gyragoand anassumed ?environmental disaster?forcellslacking the mitochondrial endosymbiont 63,88 , providing a selective force (oxygendetoxification)for theacquisitionofthemitochondrion 63,88 . Two observations challenge this model. First, it is now clear that the contemporary anaerobic eukaryotes did not branch off before the origin of mitochondria. Second, new isotope studies indicate that anaerobic environments persisted locally and globally over the past 2Gyr. That oxygen first appeared in the atmosphere at ,2Gyr ago is still generally accepted, but it is now thought that, up until about 600Myr ago, the oceans existed in an intermediate oxidation state, with oxygenated surface water (wherephotosynthesiswasoccurring), andsulphide-rich(sulphidic) and oxygen-lacking (anoxic) subsurface water 89,90 . Hence, the ?oxy- gen event? in the atmosphere should be logically decoupled from anoxic marine environments, where anaerobic eukaryotes living on the margins of an oxic world could have flourished, as they still do today 27 . Outlook In the past, phylogenetic trees have produced a particular view of early eukaryote history that was appealing, but turned out to be wrong in salient aspects. Simply testing whether a model used to make a tree actually fits the data 40 would do much to restore confidence in the merits of deep phylogenetic analyses. The fact that monophyly of plants can be recovered using molecular sequences 91 , an event that should predate 1.2Gyr, suggests that ancient signal can be extracted, but how far back we might expect to be able to go is uncertain. The persistence of mitochondrially derived organelles in all eukaryotes, and plastids in some lineages, provides phylogeny-independent evidence for the occurrence of those sym- bioticevents.Butindependentevidencefortheparticipationofother prokaryotic endosymbionts is lacking. Analysis of mitochondria in their various guises has revealed that their unifying trait is neither respiration nor ATP synthesis; the common essential function, if any, for contemporary eukaryotes remains to be pinpointed by REVIEWS NATURE|Vol 440|30 March 2006 628 � 2006 Nature Publishing Group comparativestudy.Itmaystillbethataeukaryoteislurkingoutthere that never possessed a mitochondrion?a bona fide archezoan?in which case prokaryote-host models (Fig. 4e?g) for eukaryogenesis can be abandoned. However, morphological studies and environ- mental sequencing efforts performed so far from the best candidate habitats to harbour such relics?anaerobic marine sediments?have not uncovered new, unknown and more-deeply branching lineages; rather, they have uncovered a greater diversity of lineages with affinities to known mitochondriate groups 28,61 . 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S., Langreth, S. G., Buttle, K. F. & Mannella, C. A. Electron tomographic and ultrastructural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles. J. Eukaryot. Microbiol. 52, 132?-140 (2005). Acknowledgements We thank M. Mu�ller, J. Archibald, R. Hirt, K. Henze and L. Tielens, and members of our laboratories, for discussions. Author Information Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests. Correspondence should be addressed to T.M.E. (martin.embley@ncl.ac.uk) or W.M. (w.martin@uni-duesseldorf.de). REVIEWS NATURE|Vol 440|30 March 2006 630 "
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