Multiple Secondary Origins of the Anaerobic Lifestyle in Eukaryotes T

Phil. Trans. R. Soc. B (2006) 361, 1055–1067 doi:10.1098/rstb.2006.1844 Published online 3 May 2006

Multiple secondary origins of the anaerobic lifestyle in eukaryotes T. Martin Embley* Division of Biology, The Devonshire Building, University of Newcastle upon Tyne, NE1 7RU, UK Classical ideas for early eukaryotic evolution often posited a period of anaerobic evolution producing a nucleated phagocytic cell to engulf the mitochondrial endosymbiont, whose presence allowed the host to colonize emerging aerobic environments. This idea was given credence by the existence of contemporary anaerobic eukaryotes that were thought to primitively lack mitochondria, thus providing examples of the type of host cell needed. However, the groups key to this hypothesis have now been shown to contain previously overlooked mitochondrial homologues called hydrogenosomes or mitosomes; organelles that share common ancestry with mitochondria but which do not carry out aerobic respiration. Mapping these data on the unfolding eukaryotic tree reveals that secondary adaptation to anaerobic habitats is a reoccurring theme among eukaryotes. The apparent ubiquity of mitochondrial homologues bears testament to the importance of the mitochondrial endosymbiosis, perhaps as a founding event, in eukaryotic evolution. Comparative study of different mitochondrial homologues is needed to determine their fundamental importance for contemporary eukaryotic cells. Keywords: mitochondria; hydrogenosomes; mitosomes; anaerobic eukaryote evolution

1. INTRODUCTION Despite the plausibility of the above scenario we will Many classical theories for eukaryotic origins suggested probably never know why two disparate ancient cells that eukaryotes originated under anaerobic conditions, came, and then stayed, together. Consequently, and were primitively without mitochondria (e.g. opinions are diverse concerning the ecological context Goksoyr 1967; Cavalier-Smith 1983b). A member of and selective advantages for host and endosymbiont this anaerobic eukaryotic community is posited to have (e.g. John & Whatley 1975; Martin & Mu¨ ller subsequently phagocytosed a eubacterium that could 1998; Cavalier-Smith 2002; Embley & Martin 2006). carry out aerobic respiration and which subsequently What does seem well founded is that the resulting became an endosymbiont ( John & Whatley 1975). The host–endosymbiont consortium forged the common presence of this eubacterial endosymbiont allowed the ancestor of all of the mitochondrion-containing host to colonize aerobic environments. A variety of eukaryotes that are seen today. Thus, a single common mechanisms have been suggested as to why this should origin for all mitochondria is supported by phyloge- be so; the most popular, and one that features in a netic analyses of the genes encoded by mitochondrial variety of different formulations for mitochondrial genomes (Yang et al. 1985; Gray et al. 2004). The origins (Woese 1977; Martin & Mu¨ller 1998; Vellai anaerobic eukaryotes that did not participate in the et al. 1998; Kurland & Andersson 2000), being the mitochondrial endosymbiosis, have been posited either removal of cytosolic oxygen by the endosymbionts’ to have gone extinct, due to an inability to deal respiratory activity. The feasibility of this idea, at least with rising oxygen levels or some other catastrophe as a mechanism, is supported by a modern analogue (Philippe & Adoutte 1998; Vellai et al. 1998), or to have among contemporary ciliate protozoa. Strombidium persisted in anaerobic habitats to the present day purpureum lives in anaerobic marine sands and contains (Cavalier-Smith 1983b; Margulis et al. 2005). facultatively aerobic photosynthetic endosymbiotic The persistence hypothesis is plausible for several bacteria. In the light, Strombidium avoids even traces reasons. Anaerobic habitats have existed throughout of oxygen (less than 1% atmospheric saturation) and Earth’s history and they harbour anaerobic bacteria, the endosymbionts probably use host substrates for providing food for phagotrophic eukaryotes. Extinction anoxygenic photosynthesis. In the dark, the ciliates, in the microbial world may also be rare because of large population sizes, ease of dispersal and the occurrence protected against O2-toxicity by the endosymbionts’ aerobic respiration, accumulate in water with a partial of resistant stages (Fenchel 1993). Even transient anaerobic habitats generally possess protist inhabitants. pressure of O2 of 1–4% atmospheric saturation (Fenchel & Bernard 1993a,b; Bernard & Fenchel As ancient oxygen levels rose, the occupants of 1994). anaerobic habitats on the margins of the aerobic world would actually beneﬁt from the reduced carbon derived from oxygenic photosynthesis. In modern *[email protected] situations, the activities of oxygenic phototrophs One contribution of 14 to a Discussion Meeting Issue ‘Major steps in often provide the reducing power, in the form of cell evolution’. organic material, to maintain anoxic environments;

1055 q 2006 The Royal Society 1056 T. Martin Embley Multiple origins of anaerobic eukaryotes contemporary anaerobic life is most rich in the vicinity for which it was formulated, have been extensively of aerobic life (Fenchel & Finlay 1995). In conclusion, reviewed (Embley et al. 2003b; van der Giezen et al. there are no compelling reasons drawn from con- 2005; Embley & Martin 2006), so only a few points will temporary ecosystems, to suppose that primitively be made here in relation to each type of evidence and to amitochondriate eukaryotes, if they ever existed, controversies, where they exist, surrounding the would have gone extinct simply because they did not interpretation of these data. participate in the mitochondrial endosymbiosis. All of the best-studied archezoans have now been The persistence hypothesis was supported by the shown to contain host–nuclear-encoded genes of discovery of contemporary eukaryotes that apparently putative mitochondrial ancestry (table 1). That is, lacked mitochondria. These eukaryotes, called Arche- they contain genes that encode proteins that in aerobes zoa to indicate that the absence of mitochondria was a typically function in mitochondria and, when the primitive state, not a derived state resulting from proteins are subjected to phylogenetic analysis, they secondary loss, included Entamoeba, Giardia, Tricho- form a monophyletic group with the alpha-proteobac- monas and Microsporidia, and their various close teria. This is the group of bacteria from which the relatives (Cavalier-Smith 1983a,b). As well as not mitochondrial endosymbiont is held to have originated having mitochondria, these species were thought to based upon analyses of genes that are encoded by lack other features common to most eukaryotes, mitochondrial genomes (Yang et al. 1985; Gray et al. including Golgi dictyosomes and peroxisomes, sup- 2001). One criticism of using the genes in table 1 to porting their early emerging status (Cavalier-Smith reject the Archezoa hypothesis is that none of them 1987a; Patterson & Sogin 1992). Archezoa or have ever been found on a mitochondrial genome Hypochondria as they were also known (Patterson & (Gray et al. 2004; Gray 2005). Such a location, which is Sogin 1992), thus became models for studying early found for some of the proteins involved in oxidative eukaryotic evolution and the features of the ﬁrst phosphorylation, the canonical function of aerobic eukaryotic cells. Some suggested that the peculiar mitochondria, would make a direct link between gene features of Archezoa could also be a derived state and organelle. To explain the localization of genes of (Wolters 1991; Siddall et al. 1992), caused, or perhaps mitochondrial ancestry on host nuclear genomes, it is facilitated, by the adaptation of Archezoa to their necessary to posit that they were transferred from the parasitic or anaerobic lifestyles. But that debate was endosymbiont genome to the host nuclear genome unresolved because there was no clear comparative during the process of transforming endosymbiont into evidence that convincingly linked the Archezoa to an organelle ( John & Whatley 1975; Viale & Arakaki eukaryotic groups that possessed mitochondria or the 1994). This process has been called endosymbiotic other debated features. gene transfer and it is still ongoing (Timmis et al. The Archezoa hypothesis was founded in compara- 2004). For example, among plants there are examples tive cytology as a taxonomic hypothesis designed to where particular genes appear on the mitochondrial draw attention to a group of eukaryotes which might genome in some lineages, but in other lineages they are provide a glimpse into a pre-mitochondrial phase of encoded by the host nuclear genome (Adams & Palmer eukaryotic evolution (Cavalier-Smith 1983a,b). It was 2003). Despite the need to invoke such gene transfer, thus enormously useful as a guide to study. It gained the hypothesis that the genes in table 1 came from the considerable support when the ﬁrst phylogenetic trees mitochondrial endosymbiont is probably now the based upon molecular sequence data (Vossbrinck et al. standard interpretation of these data (Boorstein et al. 1987; Sogin et al. 1989; Leipe et al. 1993; Hashimoto 1994; Viale & Arakaki 1994; Clark & Roger 1995). et al. 1997) did indeed place key archezoans before It has also been suggested that archezoans could other eukaryotes with mitochondria. Doubts were have acquired the genes in table 1 from a source other expressed about the veracity of these trees (e.g. Wolters than the mitochondrial endosymbiont. Alternative 1991; Siddall et al. 1992), but the apparent agreement sources that have been posited include alpha-proteo- between molecules and morphology in establishing the bacterial food bacteria (Doolittle 1998) and bacteria timing of the mitochondrial endosymbiosis, one of the related to, but distinct from, the mitochondrial pivotal events in eukaryotic evolution, was compelling endosymbiont (Sogin 1997). Although these ideas and generally seen as a major breakthrough. have been presented as credible alternatives to the standard interpretation discussed above (Dyall & Johnson 2000; Kurland & Andersson 2000; Knight 2. THE DEMISE OF ARCHEZOA 2004; Dyall et al. 2004b; Margulis et al. 2005), there is In an area of research where speculation and conjecture no unambiguous support from phylogenetic analyses abound, the Archezoa hypothesis for early eukaryotic for the separate origin of these Archezoan genes evolution stood out as testable in obvious ways. It could (Horner & Embley 2001; Williams et al. 2002; Hrdy be rejected for individual archezoans if they were shown et al. 2004). to contain genes of mitochondrial ancestry, to branch The strongest evidence that archezoans are not among eukaryotes with mitochondria, i.e. the trees primitively without mitochondria has come from placing them deep were wrong, or to contain previously discoveries that some of the bacterial proteins in overlooked mitochondria. Using these criteria the table 1 are housed in double-membraned organelles. Archezoa hypothesis has now been rejected for all of These organelles are called hydrogenosomes (Mu¨ller the best-studied archezoans. The data and arguments 1993; Bui et al.1996), remnant mitochondria underpinning the conclusion that the Archezoa (Williams et al. 2002) or mitosomes (Tovar et al. hypothesis can now be rejected, for the key species 1999, 2003), with the latter two terms sometimes used

Phil. Trans. R. Soc. B (2006) Multiple origins of anaerobic eukaryotes T. Martin Embley 1057

Table 1. Proteins of mitochondrial ancestry in former Archezoa. species protein references for tree location in cell reference for location

Entamoeba histolytica Mitochondrial 60-kDa Clark & Roger 1995 Mitosome Tovar et al. 1999 chaperonin (Cpn60) Mitochondrial 70-kDa Bakatselou et al. 2000 Not determined heat shock protein (ND) (Hsp70) Giardia intestinalis Cpn60 Roger et al. 1998 Mitosome Regoes et al. 2005 Horner & Embley 2001 Hsp70 Morrison et al. 2001 Mitosome Regoes et al. 2005 Cysteine desulphurase Tachezy et al. 2001 Mitosome Tovar et al. 2003 (IscS) Emelyanov 2003 Trichomonas vaginalis Cpn60 Horner et al. 1996 Hydrogenosome Bui et al. 1996 Bui et al. 1996 Bozner 1997 Roger et al. 1996 Hsp70 Bui et al. 1996 Hydrogenosome Bui et al. 1996 Germot et al. 1996 Bozner 1997 IscS Tachezy et al. 2001 Hydrogenosome Sutak et al. 2004 Emelyanov 2003 Mitochondrial NADH- Hrdy et al. 2004 Hydrogenosome Hrdy et al. 2004 dehydrogenase Dyall et al. 2004a,b Dyall et al. 2004a,b Microsporidia: Nosema locustae Hsp70 Germot et al. 1997 ND Pyruvate dehydrogenase Fast & Keeling 2001 ND Vairimorpha necatrix Hsp70 Hirt et al. 1997 ND Encephatitozoon cuniculi Hsp70 Peyretaillade et al. 1998 ND IscS Emelyanov 2003 ND Encephalitozoon hellem Hsp 70 Peyretaillade et al. 1998 ND Trachipleistophora hominis Hsp70 Williams et al. 2002 Mitosome Williams et al. 2002 interchangeably for the same organelle. There are only Proteins that are destined for the inner mitochondrial two double-membraned organelles, mitochondria and membrane are guided to their destination by internal primary plastids, of undisputed bacterial origin in targeting signals. Others, destined for the mitochon- eukaryotes. Thus the simplest, and hence preferable, drial matrix, are synthesized as pre-proteins carrying a hypothesis to explain the observations that proteins targeting sequence at their amino-terminus that is typically found in mitochondria are present in double- cleaved during import into the organelle. The develop- membrane-bounded Archezoan organelles is that all ment of a system for protein import must have been an such organelles share common ancestry with mito- early and critical step in the evolution of the chondria (Bui et al. 1996; Tovar et al. 1999, 2003; mitochondrial organelle (Cavalier-Smith 1987b). The Williams et al. 2002). Mitochondria, mitosomes and resulting mitochondrial protein import machinery is hydrogenosomes are evolutionary homologues sensu sufficiently complicated, and fidelity of import suffi- Owen and Darwin (Owen 1843; Darwin 1859), and ciently important, that the same pathways are very the differences between them are the products of unlikely to have evolved in different organelles. descent with modification from a common ancestral The hydrogenosomes of Trichomonas vaginalis organelle. The hypothesis that mitochondria, mito- import proteins using both mitochondrial pathways. somes and hydrogenosomes are homologues, predicts Hydrogenosomal ferredoxin carries an N-terminal that, as the organelles are studied more deeply, targeting sequence that guides it into isolated Tricho- additional similarities between them will emerge to monas hydrogenosomes, and the same targeting corroborate the hypothesis. So far this is what has sequence can also sort a marker protein into yeast happened. mitochondria (Plumper et al. 1998). Trichomonas With one notable exception (Boxma et al. 2005), hydrogenosomes also use a member of the mitochon- hydrogenosomes and mitosomes lack a genome, drial carrier family (MCF) to transport ADP and ATP presumably because they no longer require the proteins (Tjaden et al. 2004). Members of the MCF are it encodes for oxidative phosphorylation. Any proteins eukaryotic proteins that are inserted into the inner functioning within hydrogenosomes and mitosomes mitochondrial membrane by internal targeting signals, must therefore be synthesized in the cytosol, and then where they mediate theimportandexportof correctly targeted and imported into the organelle. substrates, including ADP and ATP, required or Although the mitochondrion contains a genome, most produced by the mitochondrion (Kunji 2004). The of its proteins are encoded by the host nuclear genome, insertion of an ADP/ATP carrier into the protomito- so there is a similar requirement for a protein import chondrion membrane was a key step in its transition machinery. Mitochondria have evolved two main into an organelle, because it made symbiont-generated protein import pathways (Pfanner & Geissler 2001). ATP available to the host cell ( John & Whatley 1975).

Phil. Trans. R. Soc. B (2006) 1058 T. Martin Embley Multiple origins of anaerobic eukaryotes

The Trichomonas ADP and ATP carrier is correctly come under increasing scrutiny with the result that they targeted to yeast mitochondria in heterologous trans- are no longer viewed as reliable (Stiller & Hall 1999; fection experiments, suggesting that targeting signals Hirt et al. 1999; Philippe et al. 2000; Inagaki et al. 2004; for import have been conserved between the Trichomo- Thomarat et al. 2004; Roger & Hug 2006). The nas hydrogenosome and yeast mitochondria (Dyall molecular sequences of the former Archezoa often et al. 2000). evolve differently to those of other eukaryotes to which Trichomonas hydrogenosomes have other features in they are being compared. However, most methods of common with mitochondria. They catalyse the enzym- phylogenetic analysis assume a homogeneous process, atic assembly and insertion of Fe–S centres into i.e. that all sequences evolve in the same way, so the apoproteins, using the same enzymes, inherited from aberrant behaviour of Archezoan sequences can make the mitochondrial endosymbiont, as do yeast mito- their phylogenetic position difficult to infer reliably (see chondria (Sutak et al. 2004). Trichomonas hydrogeno- Roger & Hug 2006). The difficulty experienced when somes also contain the NADH dehydrogenase module trying to resolve the origins of the Trichomonas of complex I of the mitochondrial respiratory chain hydrogenosomal NADH dehydrogenase provides just (Hrdy et al. 2004), although the reader should be aware one example of the kind of problems that can be that this last conclusion is controversial (see Gray encountered (Dyall et al. 2004b; Hrdy et al. 2004; Gray 2005), because an alternative origin from another 2005). The case for a rethink is clear-cut for bacterium had previously been suggested for this Microsporidia because most data and analyses now module (Dyall et al. 2004b). While these data are agree in placing them with fungi rather than as deep clearly difficult to analyse using conventional phylogen- branching eukaryotes (Hirt et al. 1999; Keeling et al. etic methods, it is important to appreciate that they 2000; Inagaki et al. 2004; Thomarat et al. 2004). The cannot reject a common origin of Trichomonas and hypothesis that Entamoeba is related to Dictyostelium,an mitochondrial proteins using standard tests (Hrdy et al. aerobic amoeba that contains mitochondria, is also now 2004). Moreover, when corrections are made to well supported by a number of proteins (Horner & mitigate obvious problems with the data, such as Embley 2001; Bapteste et al. 2002). The position of amino acid differences between sequences, the trees Giardia and Trichomonas is uncertain, but there are do support a mitochondrial origin (Hrdy et al. 2004). sufficient rooted trees that argue otherwise, to be wary The discovery of the NADH dehydrogenase activity of making strong claims that they branch before other solves the long-standing puzzle of how the Trichomonas eukaryotes (Horner & Embley 2001; Emelyanov 2003; hydrogenosome regenerates NADC after malate oxi- Arisue et al. 2005). dation (Mu¨ller 2003). Freed from the prejudice that Archezoa must be Entamoeba mitosomes (Tovar et al. 1999)also ‘early branching’ or in some sense ‘primitive’, their contain a mitochondrial carrier protein that transports mitochondrial homologues fit perfectly comfortably ADP and ATP, and in heterologous transfection within the spectrum of ‘non-Archezoan’ eukaryote experiments it too is translocated to the inner biology. mitochondrial membrane of yeast (Chan et al. 2005). Unlike typical mitochondrial ATP/ADP carriers 3. REMNANT MITOCHONDRIA (Klingenberg 1985), the Entamoeba carrier does not IN CRYPTOSPORIDIUM require a positive-outside membrane potential, to Cryptosporidium parvum is an apicomplexan parasite transport ADP and ATP (Chan et al. 2005). This can related to the malaria parasite Plasmodium,and be rationalized by reference to biochemical data together they are part of a broader predominantly (Reeves 1984) and to the recently published Entamoeba aerobic group comprising apicomplexans, ciliates and genome (Loftus et al.2005), which show that dinoflagellates, called the alveolates (Cavalier-Smith Entamoeba lacks the electron transport chain necessary 2002; Adl et al. 2005). Cryptosporidium parvum to make this gradient. contains a double-membraned organelle that has Import pathways are also conserved between been called a relict (Riordan et al. 2003) or degenerate Giardia mitosomes and mitochondria (Dolezal et al. mitochondrion (Abrahamsen et al. 2004), but from its 2005; Regoes et al. 2005). For example, the N-terminal known features it could just as easily be called a presequence from the Giardia mitosomal ferredoxin is mitosome. The Cryptosporidium mitochondrion has lost both necessary and sufficient to guide green fluorescent its genome and the capacity for oxidative phosphoryl- protein into mammalian mitochondria (Regoes et al. ation (Abrahamsen et al. 2004), but it still imports 2005). Study of the microsporidian mitosome has mitochondrial heat shock proteins (Riordan et al. 2003; lagged behind study of other mitochondrial homo- Slapeta & Keithly 2004). There is also circumstantial logues, because Microsporidia are obligate intracellular evidence that it makes Fe–S clusters, but in situ parasites, and there are no homologous transfection localization of the enzymes has not been done (LaGier methods like those established for Entamoeba, Giardia et al. 2003). and Trichomonas. Nevertheless the published genome of the microsporidian Encephalitozoon cuniculi (Katinka et al. 2001) already provides clues to putative shared 4. HYDROGENOSOMES: MITOCHONDRIA functions, e.g. homologous mechanisms of Fe–S THAT MAKE HYDROGEN cluster assembly, between its mitosome and mitochon- Hydrogenosomes were originally discovered (Lindmark dria (Vivares et al. 2002). &Mu¨ller 1973) in the cattle parasite Tritrichomonas Phylogenetic analyses previously taken to support foetus, a close relative of T. vaginalis, but they are also the deep branching positions of archezoans have also found in anaerobic chytrid fungi and diverse lineages

Phil. Trans. R. Soc. B (2006) Multiple origins of anaerobic eukaryotes T. Martin Embley 1059 of ciliate protozoa (Mu¨ller 1993). The evidence for There are biochemical data consistent with the common ancestry of chytrid and ciliate hydrogeno- presence of both enzymes in the hydrogenosomes of somes with mitochondria is now very strong (van der anaerobic ciliates and fungi (Yarlett et al. 1981, 1984, Giezen et al. 2002, 2003; Voncken et al. 2002a; Boxma 1986). By contrast, aerobic eukaryotes use the non- et al. 2005). Among ciliates, mitochondria- and homologous pyruvate dehydrogenase, located in the hydrogenosome-containing groups freely intermingle; mitochondrion, to decarboxylate pyruvate and the there are at least four and probably more ciliate lineages electrons flow via the electron transport chain to that possess hydrogenosomes within this predomin- oxygen. antly aerobic group (Fenchel & Finlay 1995; Embley The presence in hydrogenosomes of PFO and [Fe]- et al. 1995). The transformation of mitochondrion to hydrogenase, enzymes typical of anaerobic bacteria, hydrogenosome can occur over relatively short genetic prompted the early suggestion, recently restated in distances as illustrated by the genus Cyclidium, which modified form, that Trichomonas hydrogenosomes were contains Cyclidium glaucoma with mitochondria and descended from an endosymbiotic anaerobic bacter- Cyclidium porcatum with hydrogenosomes (Esteban ium (Whatley et al. 1979; Dyall et al. 2004a). However, et al. 1993; Embley et al. 1995). Given the ease by neither PFO nor [Fe]-hydrogenase is uniquely associ- which ciliates have evolved hydrogenosomes it was ated with hydrogenosomes, and thus they are not perhaps inevitable (Embley et al.1997) that the reliable indicators of a separate organelle origin. The strongest evidence for the common ancestry of mitochondria of Euglena contain pyruvate:NADP hydrogenosomes and mitochondria would eventually oxidoreductase, a fusion protein containing PFO come from this group. The hydrogenosomes of the domains linked to a C-terminal NADPH-cytochrome anaerobic ciliate Nyctotherus ovalis uniquely retain a P450 reductase domain. The same fusion is found in mitochondrial genome (Boxma et al. 2005), providing C. parvum, but its localization is unknown (Rotte et al. the long-sought direct link between the two organelles 2001). Large fragments of PFO have also been found in (Martin 2005). Saccharomyces cerevisiae where they combine with other Like those of Trichomonas (Clemens & Johnson redox proteins to participate in methionine biosyn- 2000), the hydrogenosomes of the anaerobic fungus thesis (Horner et al. 1999). PFO also occurs in Neocallimastix apparently lack a genome (van der Entamoeba, Giardia and Spironucleus (a close relative Giezen et al. 1997). Neocallimastix also contains a of Giardia), but the location of the protein in these member of the MCF on its genome (van der Giezen species has not been unambiguously demonstrated et al. 2002; Voncken et al. 2002a). This protein has (Reeves et al. 1977; Townson et al. 1996; Brown et al. been shown to import and export ADP and ATP 1998; Rodriguez et al. 1998). in vitro and, crucially, it can restore the function of Hydrogenase genes have now been cloned from mutant yeast mitochondria that lack their own ADP/ T. vaginalis (Bui & Johnson 1996; Horner et al. 2000), ATP carrier (van der Giezen et al. 2002). Thus, fungal the ciliates N. ovalis (Akhmanova et al. 1998)and hydrogenosomes and yeast mitochondria use the same Trimyema sp. (Embley et al. 2003a), and the chytrid pathway for ADP/ATP exchange and they import the fungi Neocallimastix frontalis L2 and Piromyces sp. E2 relevantproteininthesameway.Neocallimastix (Davidson et al. 2002; Voncken et al. 2002b). All of hydrogenosomes also contains mitochondrial Cpn60 them encode iron-only [Fe]-hydrogenases of a type that and Hsp70 and these proteins contain targeting signals is found in eubacteria, but not in archaebacteria. Green that are capable of sorting them, or a green fluorescent algae, such as Chlorella fusca, Chlamydomonas reinhardtii reporter protein, into mammalian mitochondria (van and Scenedesmus obliquus also produce hydrogen, using der Giezen et al. 2003). [Fe]-hydrogenases located in their chloroplasts Anaerobic eukaryotic diversity is poorly sampled for (Horner et al. 2002). The presence of a [Fe]- organelle function so it is unlikely that these groups will hydrogenase in green algal plastids is surprising, since be the final additions to the list of eukaryotes shown to cyanobacteria, the endosymbiotic progenitor of plas- possess hydrogenosomes (Fenchel & Finlay 1995). tids, use the non-homologous [NiFe]-hydrogenase to Transforming mitochondria into hydrogenosomes make hydrogen. The original [NiFe]-hydrogenase appears to be something that diverse eukaryotes have brought by the endosymbiont appears to have been accomplished, raising the question of how it has been replaced by a host-nuclear encoded [Fe]-hydrogenase done. of non-cyanobacterial origin. Genes encoding [Fe]-hydrogenases similar in structure to Trichomonas enzymes, were also recently 5. EVOLUTIONARY ORIGINS OF THE ENZYMES discovered in Giardia, in its relative Spironucleus,and USED TO MAKE HYDROGEN in Entamoeba histolytica (Horner et al. 2000; Nixon et al. Hydrogenosomes are defined by their ability to make 2003). The intracellular location of these enzymes is hydrogen so it is the source of the biochemistry to do unknown at present. Giardia can actually make small this, i.e. key to the transformation of mitochondria into amounts of hydrogen, the elimination of which may hydrogenosomes (Embley et al. 1997). In Trichomonas help to maintain redox balance in this species (Lloyd hydrogenosomes, which are the best studied for their et al. 2002). Given that the structures of the Entamoeba biochemistry, pyruvate:ferredoxin oxidoreductase and Spironucleus genes closely resemble the one in (PFO) carries out the metabolism of pyruvate, and Giardia, it seems likely that these species can also make the electrons generated are transferred via ferredoxin to hydrogen. [Fe]-hydrogenase, producing hydrogen as the reduced Most surprisingly, genes encoding short proteins end product (Mu¨ller 1993; Hrdy & Mu¨ller 1995a,b). related to [Fe]-hydrogenases also occur in the human

Phil. Trans. R. Soc. B (2006) 1060 T. Martin Embley Multiple origins of anaerobic eukaryotes genome (Barton & Worman 1999; Horner et al. 2002). hydrogenase (Embley et al. 2003a), thus helping to These genes have been called NARF-like, after human explain the apparent ease by which diverse ciliates nuclear prelamin A recognition factor (Barton & have made hydrogenosomes (Embley et al. 1995). Worman 1999). In humans, the Narf protein is thought The tree provides no support for a close relationship to be involved in the processing of lamin, a protein between eukaryotic [Fe]-hydrogenases and the only required for maintaining the structural integrity of the sequence from an alpha-proteobacterium, that of nucleus (Barton & Worman 1999). However, detailed Rhodopseudomonas palustris (Davidson et al. 2002). studies on the yeast homologue Nar1p, suggest a rather The Rhodopseudomonas sequence clusters strongly different role for this protein. In yeast, which lacks with one of two different [Fe]-hydrogenases from lamin, Nar1p appears to be an essential protein located Desulfovibrio vulgaris (strain Hildenborough), a in the cytosol where it functions in the synthesis of member of the delta-proteobacteria. As for the PFO extramitochondrial Fe–S proteins (Balk et al. 2004, tree, there is evidence that gene duplications and/or 2005). Database searches have revealed that NARF- potential horizontal gene transfers have influenced the like genes are present in all of the available eukaryotic tree topology for prokaryotic sequences. For example, genomes (Horner et al. 2002; Balk et al.2004), the [Fe]-hydrogenases from Desulfovibrio species do including the smallest eukaryote genome yet not cluster together (figure 2), as their common sequenced, from the intracellular microsporidian classification suggests they should. parasite E. cuniculi (Katinka et al. 2001). Once thought to be exotic exceptions, it seems entirely possible that [Fe]-hydrogenases, in the form of Narf-like proteins, 6. CONCLUSIONS, SPECULATIONS have an important role to play in all eukaryotes. AND OUTLOOK It has been suggested that the genes for PFO and The idea that primitively amitochondriate protists— [Fe]-hydrogenase could have originated from the Archezoa—were alive and well was an attractive one, mitochondrial endosymbiont, as both genes are not least because it lent credibility to theories for common among proteobacteria (Embley et al. 1997; eukaryote origins that required a host of this kind to Martin & Mu¨ller 1998). Phylogenetic analyses have engulph the mitochondrial endosymbiont. Better trees, not provided any support for this hypothesis. Like the discovery of genes of mitochondrial origin and, many eukaryotic metabolic enzymes (Ribeiro & latterly, the discovery of mitochondrial homologues Golding 1998; Rivera et al. 1998; Esser et al. 2004), have allowed the rejection of the Archezoa hypothesis eukaryotic PFOs are more similar in structure to for the key species for which it was formulated. It thus eubacterial enzymes than to those from archaebac- seems possible that all eukaryotes might contain an teria. Moreover, published analyses of PFO (Horner organelle of mitochondrial ancestry, emphasizing the et al. 1999; Rotte et al. 2001; Embley et al. 2003a), pivotal role that the mitochondrial endosymbiosis has recover eukaryotic sequences as a single cluster played in eukaryotic evolution. Although we still need a consistent with an ancient common origin for reliable root for the eukaryotic tree (see Cavalier-Smith eukaryotic enzymes (figure 1). However, the eukary- 2006; Roger & Hug 2006), it is evident that anaerobic otic PFO sequences are not the nearest neighbours of eukaryotes have repeatedly arisen from within ances- sequences from alpha-proteobacteria. Interestingly, trally aerobic groups with mitochondria. Anaerobic the two alpha-proteobacterial sequences do not eukaryotes are neither rare nor primitive as once cluster together in the tree, suggesting that the thought. evolution of eubacterial PFO genes is more complex Most of what is known about mitochondrial than can be explained by vertical inheritance of a function and the importance of mitochondria for the single copy gene (Horner et al. 1999). eukaryotic cell is drawn from the study of yeast, The relationships between eukaryotic [Fe]-hydro- mammal and plant mitochondria. Apart from oxidative genase sequences are not well resolved, to which phosphorylation, other important reactions include the saturation of variable sites is probably a contributing Krebs cycle, haem biosynthesis, beta-oxidation of fatty factor (Horner et al. 2000). Thus, although they do acids, amino acid biosynthesis and the formation and not cluster together in the tree (figure 2), the data export of iron–sulphur (Fe/S) clusters (Reichert & cannot decisively reject the hypothesis of a common Neupert 2004; Lill & Muhlenhoff 2005). It is already and early origin for eukaryotic sequences using evident, from biochemical and genomic data, that standard tests (Horner et al. 2000). There is strong hydrogenosomes and mitosomes can have retained support for the coherence of individual clusters of only a limited subset of these reactions (Katinka et al. eukaryotic sequences, consistent with the hypothesis 2001; Mu¨ller 2003; Abrahamsen et al. 2004; Loftus that the respective ancestors of the sampled green et al. 2005). This raises important questions concern- algae and chytrid fungi already contained a gene for ing the fundamental importance of this compartment [Fe]-hydrogenase. The observation (Embley et al. of endosymbiotic ancestry for the eukaryotic cell, its 2003a) that the enzymes from the two ciliates biochemical flexibility and the limits of organelle Nyctotherus and Trimyema cluster together is particu- reduction. For example, MCF protein diversity closely larly interesting, since these two morphologically mirrors important facets of mitochondrial metabolic distinct species are separated by aerobic mitochon- diversity; non-parasitic aerobic eukaryotes typically dria-containing lineages in the ciliate tree (Embley have between 30 and 60 MCF genes, to transport et al. 1995; Akhmanova et al. 1998). This is consistent different substrates required or produced by the with the hypothesis that the common ancestor they mitochondrion (Kunji 2004). By contrast, E. histolytica shared with aerobic ciliates, already contained a [Fe]- has lost all but a single member of the MCF which

Phil. Trans. R. Soc. B (2006) Multiple origins of anaerobic eukaryotes T. Martin Embley 1061

Figure 1. Phylogenetic tree showing the evolutionary relationships between pyruvate:ferredoxin oxidoreductase (PFO) sequences from eukaryotes and eubacteria. Eukaryotes are shown in bold. The tree is a consensus tree from a Bayesian analysis of aligned protein sequences using custom software (available from [email protected]). Aligned positions (715 sites) were recoded into the six Dayhoff groups: C, STPAG, NDEQ, HRK, MILV and FYW to reduce the effects of mutational saturation (Hrdy et al. 2004). Posterior probabilities for some groups are shown, with a value of 1.0 representing maximum support. The strongly supported groups have also been recovered in previously published analyses using different methods, including maximum-likelihood, and different support measures including bootstrapping (Horner et al. 1999; Rotte et al. 2001). functions in its mitosome to transport ATP and ADP mitochondria are essential to the yeast cell (Lill et al. (Chan et al. 2005; Loftus et al. 2005). The micro- 1999). Key proteins in this pathway; cysteine desul- sporidian Encephalitozoon, an obligate intracellular phurase (IscS) and scaffolding protein (IscU), appear parasite, has gone even further, because its genome to have originated from the mitochondrial endosym- lacks any MCF genes (Katinka et al. 2001). This raises biont (Tachezy et al. 2001; Emelyanov 2003; van der the intriguing question of how the Encephalitozoon Giezen et al. 2004). The maturation and export of Fe/S mitosome acquires ATP. clusters has been suggested as a possible common The maturation of Fe/S clusters, for insertion into function of all mitochondrial homologues (Tachezy the Fe/S proteins that are crucial for all cellular life, is to et al. 2001; Embley et al. 2003a). Key genes for this date the only biosynthetic function for which process are indeed present on all eukaryotic genomes

Phil. Trans. R. Soc. B (2006) 1062 T. Martin Embley Multiple origins of anaerobic eukaryotes

Figure 2. Phylogenetic tree showing the evolutionary relationships between [Fe]-hydrogenase sequences from eukaryotes and eubacteria. Eukaryotes are shown in bold. Details of the analysis on 196 aligned positions are given in the legend to ﬁgure 1. The strongly supported groups have also been recovered in previously published analyses using different methods, including maximum-likelihood, and different support measures including bootstrapping (Horner et al. 2000). examined (Katinka et al. 2001; LaGier et al.2003; Lill & eukaryotes, where they are targeted to the cytosol, Muhlenhoff 2005) but only the Trichomonas hydro- hydrogenosomes, mitochondria, nucleus and plastids. genosome (Sutak et al. 2004) and Giardia mitosome Although hydrogenase and PFO fail to display a unique (Tovar et al. 2003) have actually been demonstrated to afﬁnity for the ‘mitochondrial’ compartment, their contain the pathway. Entamoeba is so far unique among widespread retention among eukaryotes has undoubt- eukaryotes in that it has lost the mitochondrial edly contributed to the facility by which eukaryotes homologues of IscS and IscU (Loftus et al. 2005), have repeatedly evolved hydrogenosomes (Embley et al. and instead it possesses genes for the homologous 1997). The origins of eukaryotic [Fe]-hydrogenase and proteins NifS and NifU, acquired through horizontal PFO are also part of a bigger question concerning the gene transfer from a bacterium related to Campylobacter origins of the eubacterial-like genes that encode much (Ali et al. 2004; van der Giezen et al. 2004). The of eukaryote metabolism (Ribeiro & Golding 1998; location of this pathway is central to testing the Rivera et al. 1998; Esser et al. 2004). There are a hypothesis that Fe/S cluster assembly is the common number of imaginative hypotheses to explain these function of all mitochondrial homologues. genes; as the product of multiple lateral transfers from So far there is no evidence that the genes for [Fe]- different prokaryotes (Doolittle 1998), or the legacy of hydrogenase and PFO, which mediate the conversion different eubacteria that participated in eukaryogenesis of mitochondria to hydrogenosomes, originated from (Martin & Mu¨ller 1998; Moreira & LopezGarcia 1998; the mitochondrial endosymbiont (Embley et al. 1997; Rivera & Lake 2004; Cavalier-Smith 2006). Genomics Martin & Mu¨ ller 1998). However, phylogenetic coupled with more sophisticated phylogenetic analyses, analyses do suggest that early eukaryotes likely should in principle be able to resolve gene origins, or at contained both PFO and [Fe]-hydrogenase, and thus least reveal the limits of resolution that the data can it is possible that they could make hydrogen. The two provide (Penny et al. 2001; Ho & Jermiin 2004; Roger & proteins, in various forms, have been found in diverse Hug 2006). Published work on whole genomes has

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