Sure facts and open questions about the origin and evolution of photosynthetic plastids

Res. Microbiol. 152 (2001) 771–780  2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923-2508(01)01260-8/REV

Mini-review

Sure facts and open questions about the origin and evolution of photosynthetic plastids David Moreira∗ , Hervé Philippe Equipe Phylogénie, Bioinformatique et Génome, UMR CNRS 7622, Bâtiment B, 6eme étage, Université Pierre et Marie Curie, 9 quai Saint Bernard, 75252 Paris cedex 05, France Received 28 December 2000; accepted 4 May 2001

Abstract – Some eukaryotic groups carry out photosynthesis thanks to plastids, which are endosymbiotic organelles derived from cyanobacteria. Increasing evidence suggests that the plastids from green plants, red algae, and glaucophytes arose directly from a single common primary symbiotic event between a cyanobacterium and a phagotrophic eukaryotic host. They are therefore known as primary plastids. All other lineages of photosynthetic eukaryotes seem to have acquired their plastids by secondary or tertiary endosymbioses, which are established between eukaryotic algae, already containing plastids, and other eukaryotic hosts. Both primary and secondary symbioses have been followed by extensive plastid genome reduction through gene loss and gene transfer to the host nucleus. All this makes the reconstruction of the evolutionary history of plastids a very complex task, indissoluble from the resolution of the general phylogeny of eukaryotes.  2001 Éditions scientifiques et médicales Elsevier SAS plastids / chloroplasts / evolution / phylogeny / protists / algae

1. Introduction The plethora of photosynthetic eukaryotes that thrive today on our planet depends for its function on a very particular type of organelle, the plastids. The history of research on the origin of these organelles goes far back in time. In 1905, Mereschkowsky brilliantly suggested that photosynthetic plastids derive from endosymbiotic photosynthetic bacteria (in particular cyanobacteria) [44] (for a recent translation of Mereschkowsky’s work see [41]). This idea was revived several decades later by Margulis in her formulation of the serial endosymbiotic theory [40] and, after an initial resolute opposition, an endosymbiotic origin of plastids (and also mitochondria) is now widely accepted. In fact, a wealth of data strongly suggests that all known photosynthetic plastids are monophyletic and that they have a cyanobacterial origin (reviewed in [13]). This common ancestry could appear difficult to reconcile with the outstanding diversity displayed by eukaryotic plastids in terms of morphology, ultrastructure, pigmentation, and gene

∗ Correspondence and reprints.

E-mail address: [email protected] (D. Moreira).

content. However, an important part of this diversity arises from the particular evolutionary pathways followed by the different eukaryotic photosynthetic lineages. In fact, plastids may derive from two types of symbiotic events: primary endosymbiosis, which is established directly between cyanobacteria and the eukaryotic host, and secondary and tertiary endosymbioses, which are established between a eukaryotic alga already equipped with plastids and a second eukaryotic host. Plastids may therefore be primary, secondary, or tertiary (figure 1). Some secondary (and tertiary) plastids can be recognised because distinctive characteristics of the eukaryotic cell that hosted them remain. The most dramatic case concerns chlorarachniophytes and cryptophytes, whose plastids (which arose by endosymbiosis of green and red algae, respectively) have retained several membranes and, more impressively, the nucleus of these eukaryotic endosymbionts that is called the nucleomorph [11, 16, 27, 64]. However, in other cases secondary plastids may have lost these distinctive characteristics by reductive evolution, making it very difficult to trace back their origins. This uncertainty gives rise to important questions that still remain partially or completely open: Which lineages have primary or secondary plastids? How many primary en-

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Figure 1. The complex evolutionary pathway of photosynthetic eukaryotes and their plastids. Three groups (green algae, red algae, and glaucophytes) directly derive from the primary endosymbiosis between a cyanobacterium and a phagotrophic eukaryote. Thereafter, green algae were probably acquired secondarily by euglenozoans and chlorarachniophytes, and red algae were probably acquired secondarily by alveolates (dinoflagellates and apicomplexans), heterokonts, cryptophytes and haptophytes, to give rise to secondary plastids. In the case of some dinoflagellates, their red algal-type plastids were replaced by green algal- and haptophyte-type plastids through secondary replacement and tertiary symbiosis processes. Note that some secondary plastids preserve their eukaryotic nuclei (the nucleomorphs), and that the number and topology of plastid membranes is diverse. The contents of this figure have been largely inspired by work from Charles Delwiche [13] and Tom Cavalier-Smith [10].

dosymbioses occurred? How many secondary ones? This article deals with these questions presenting an overview of our current knowledge on plastid evolution, with special attention to the fact that the answers

are intimately related to the general phylogeny of eukaryotes, since photosynthetic lineages are believed to be widely intermixed with nonphotosynthetic lineages [56].

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2. Three lineages of primary plastids, but how many primary endosymbioses? Most authors accept that structural characteristics (in particular the presence of a double membrane) and phylogenetic analyses point to the existence of primary plastids only in three eukaryotic lineages: green algae + plants (a group that we will refer to as the green plants), red algae, and glaucophytes [13, 14, 42] (figure 1). Despite being considered of primary origin, these three plastid lineages display important differences. For instance, green plant plastids contain chlorophyll a and b and stacked thylakoids, while red algae and glaucophytes only contain chlorophyll a and unstacked thylakoids. On the contrary, these two lineages show phycobilisomes (granules containing light-harvesting pigments closely associated with the photosynthetic apparatus), which are absent in green plants. These differences raised doubts on the hypothesis of a unique origin of the three lineages, suggesting that they could have been the result of three independent primary endosymbioses with three different cyanobacterial lineages. This was initially supported by the first phylogenetic analyses of the corresponding eukaryotic hosts carried out using the small subunit rRNA (SSU rRNA) as a phylogenetic marker [2, 56 – 58]. These analyses showed that these three eukaryotic groups, albeit located in the apical part of the tree known as the crown, do not form a monophyletic clade. These results thus supported an independent origin of the hosts and hence of their plastids. This possibility was in clear contradiction with the phylogenies based on plastid genes, initially on rRNA sequences, which strongly supported the monophyly of all plastids (reviewed in [13]). However, recent studies using nucleus-encoded protein sequences as phylogenetic markers, in particular the RNA polymerase RPB1, seem to sustain several independent origins, especially for green plants and red algae [59]. Nevertheless, other nuclear markers, such as the actin and α-tubulin, support, albeit weakly, the sisterhood of both lineages [55]. Nuclear markers therefore give an equivocal answer to this problem. The determination of mitochondrial genome sequences added new valuable information coming from a third cellular compartment. In contrast to nuclear rRNA and some protein coding genes but in agreement with plastid markers, mitochondrial sequences strongly supported the sisterhood of green plants and red algae [4]. Moreover, preliminary data from the mitochon-

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drial sequence of the glaucophyte Cyanophora paradoxa lend support to its close proximity to the green plants + red algae group (F. Lang, personal communication). As we will discuss in other sections of this review, the congruence between the data from the different cellular compartments, especially the nucleus and chloroplasts, is essential in order to accurately count the number of endosymbiotic events. The disparity in the results obtained using different nucleusencoded markers made it impossible to determine the precise number of primary endosymbioses. This disparity most likely results from the scarcity of available information (few phylogenetic markers and poor taxonomic sampling) and of tree reconstruction artefacts that unequally affect the different genes. One of the best-known artefacts is the long branch attraction (LBA), which provokes the artificial grouping of fast evolving sequences in the reconstructed trees [20], and their misplacement towards the basal region of the trees when distant outgroup sequences are used [50, 52 – 54]. 3. A single birth of all primary plastids: nuclear markers embrace chloroplast and mitochondrial data We decided to address this problem by analysing an alternative nuclear marker, elongation factor 2 (EF2), since preliminary analyses suggested that it possesses satisfying characteristics for phylogenetic reconstruction, in particular a rather homogeneous evolutionary rate among the different lineages (more homogeneous than other common markers such as the SSU rRNA or elongation factor 1α [32]). To enlarge the taxonomic sampling for this marker, we determined its sequence for two red algae (Gelidium canariensis and Chondrus crispus) and several other protists. The subsequent analysis of the new EF2 data set provided, for the first time, strong support from a nuclear marker for the green plants + red algae sisterhood [46]. Moreover, the nonmonophyly of this group was statistically rejected by this marker. This result openly contradicted the results based on the RPB1 sequences [59]. We therefore reanalysed this marker including more sequences, and found that the monophyly of green plants + red algae was still not retrieved, but it was no longer statistically rejected. In fact, the emergence of red algae far from green

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plants could represent a tree reconstruction artefact, due to the apparent high evolutionary rate of the red algae RPB1 sequences. The combined analysis of a large fusion of the 13 nuclear markers for which an adequate taxonomic sampling is available (including EF2 and RPB1) once again produced a strong and statistically significant support for the green plants + red algae sisterhood. A shorter fusion (6 genes) including C. paradoxa showed, with modest support, that glaucophytes are the closest relatives to the green plants + red algae clade [46]. These findings, together with the evidence provided by chloroplasts and mitochondria, strongly argue for a unique primary endosymbiosis, which predated the divergence of glaucophytes, green plants, and red algae. A clade comprising these three groups was already proposed by Cavalier-Smith as the kingdom Plantae [6] but its complete recognition lacked the valuable phylogenetic congruence explained above. Glaucophytes, which have retained the classical bacterial peptidoglycan cell wall in their chloroplasts, are still poorly studied, but their thorough characterisation will be of key importance to understand the evolution of primary plastids. 4. Secondary endosymbioses: the nucleomorphs The three eukaryotic lineages derived from the primary endosymbiosis only account for a modest fraction of the diversity of plastid-containing photosynthetic eukaryotes. Where is the origin of those other plastids then? Almost three decades ago, Gibbs provided the first undeniable evidence that plastids can also originate from secondary endosymbiosis when she studied the ultrastructure of cryptomonads, unicellular algae with red pigmentation. The most outstanding characteristic of these organisms is the presence of a highly reduced eukaryotic nucleus, known as the nucleomorph, in addition to the principal nucleus [24]. This convincingly supported the hypothesis that the chloroplasts of cryptomonads did not derive directly from endosymbiotic cyanobacteria, but from engulfed eukaryotic (red) algae through secondary endosymbiosis. Such a process generates a complex cell that has been called by some authors a “meta-alga” [10]. Within this cell, the secondary endosymbiont is bound by a characteristic double membrane, called the chloroplast endoplasmic reticulum, with the outer plastid membrane continuous with

the nuclear envelope [26] (figure 1). The true plastid itself is surrounded by the classical double membrane found in primary plastids. Therefore, its cytoplasm is separated from that of the host by four membranes. Chlorarachniophytes, amoeboid organisms with green plastids, represent a similar case. Like cryptophytes, these protists show a nucleomorph and a chloroplast endoplasmic reticulum [30] (figure 1). Molecular phylogenetic studies of nucleomorph genes supported the hypothesis that the secondary endosymbionts of this group were of green algal origin, as suggested by their green pigmentation [43]. The plastids found in the primary endosymbiotic lineages (glaucophytes, green plants, and red algae) have very reduced genomes, which do not encode all the macromolecules and activities required for plastid maintenance and function [28]. This indicates that plastids have undergone a process of genome reduction, which occurs through loss of genes unnecessary for the endosymbiont in its new cellular environment but, more significantly, also through horizontal transfer towards the host nucleus and through recruitment of pre-existing nuclear genes [28]. The latter probably took place after the establishment of an efficient system of import between the host and the plastid [10]. Mitochondria underwent a similar process that explains their tiny current genomes [28]. Transferred genes were integrated within the host genome, where they generally acquired signal peptides that allowed redirecting the gene products back into the plastid (although some of them are recruited by the host and function in its cytoplasm and even in the nucleus). Nucleomorphs, which contain the smallest known eukaryotic genomes, represent a similar process of genome miniaturisation, but starting from a eukaryotic genome. The nucleomorph of the cryptophyte alga Pyrenomonas salina contains three chromosomes with a total genome size of 660 kb [18], while the nucleomorph of a chlorarachniophyte species also contains three chromosomes, but its total genome size is of only 380 kb [27]. The sequencing of nucleomorph genomes revealed some clues about the mechanisms of genome compaction. First analyses, carried out on the chlorarachniophyte nucleomorph, showed that introns, albeit numerous, were reduced to the minimal size known so far (18–20 base pairs) and, more surprisingly, overlapping and cotranscribed genes were found [27]. Interestingly, similar results were recently found from the complete sequence of the nucleomorph of the cryptophyte Guillardia theta,

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which shows very few but larger (although still small) introns (42–55 base pairs), and overlapping genes [15]. A true cotranscription of genes is not found in cryptophyte nucleomorphs, which instead show an inaccurate 3 end-processing of mRNAs, so that mRNAs often include parts of downstream located genes [21, 67]. Nevertheless, the similarity of the streamlining phenomena in chlorarachniophyte and cryptophyte nucleomorphs is remarkable, and most likely an excellent example of convergent evolution. Convergence is in fact suggested by a similar final result (genome compaction), but with slightly different mechanisms to produce it (number and size of introns, cotranscription dynamics) in the nucleomorphs from both groups of algae. 5. Other secondary (and tertiary) endosymbioses Nucleomorphs reveal that secondary endosymbiosis may be followed by drastic genome reduction and therefore the possibility of complete extinction of the nucleomorph genome can even be envisaged. In fact, they are maintained in several groups because they encode chloroplast proteins. However, complete nucleomorph genome sequences show that few such genes are present [15, 39]. They could therefore be rather easily transferred to the host nucleus and then the useless nucleomorph may disappear, as this seems to have been the case in several algal lineages. In these organisms the secondary nature of their plastids can be recognised by means of the number of membranes (more than two) surrounding them. Three or more membranes are present because the phagosomal vacuole that the host used to engulf the secondary endosymbiont remains stabilised as a perialgal membrane, unlike the case for primary endosymbiosis. An example can be found in heterokonts (a vast assemblage of organisms including, among others, brown algae and diatoms), which exhibit a characteristic four-membrane complex enclosing their plastids that is very similar to the chloroplast endoplasmic reticulum of cryptomonads [26] (figure 1). Phylogenetic analyses show that the heterokont plastids are indeed close relatives of those of red algae [16] while the respective host cells are only distantly related [63]. This disagreement between data from the two cellular compartments suggests that heterokonts acquired a photosynthetic red alga that finally lost its own nucleus. The result of this process was a cell with

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red algal-type chloroplasts lacking a nucleomorph. A similar situation is found in haptophytes (also known as coccolithophorids), whose secondary plastids are also of red algal origin [25] (figure 1). The three-membrane plastids found in dinoflagellates are also suspected to be of secondary origin (figure 1). Typical (but not all, see below) dinoflagellate plastids are characterised by their pigmentation with chlorophylls a and c2 , and peridinin [34]. Recent phylogenetic analyses suggest that dinoflagellate peridinin-containing plastids have a secondary origin analogous to that of heterokont plastids, but have lost one of their four membranes [10, 48, 68, 69]. Dinoflagellates, some of which are well known to be causative agents of red tides, form, together with apicomplexans and ciliates, a vast taxonomic assemblage called the alveolates [22]. Interestingly, plastid-like structures were also found in the parasitic apicomplexan genera Eimeria, Plasmodium, and Toxoplasma [37]. These plastids, known as the apicoplasts, are unpigmented four-membrane organelles (figure 1), which contain 35-kb circular chromosomes. Phylogenetic analyses of the elongation factor tu gene present in these plastids grouped them with cyanobacteria and other plastids, in particular with green algal plastids. This indicates that the apicomplexans acquired a plastid by secondary endosymbiosis, apparently from a green alga [37]. However, apicomplexan plastid genes are characterised by very high evolutionary rates, so that their precise location in phylogenetic trees is difficult. In fact, phylogenetic analyses of 16S and 23S rRNA, and psbA sequences support the notion that apicomplexan and dinoflagellate plastids have a common origin [3, 69]. This suggests that the common ancestor of both groups, and perhaps of all alveolates, already possessed a secondary plastid. If this is the case, apicomplexan plastids would have a red algal origin [69], in agreement with the analysis of rRNA operon sequences [3] (figure 1). Recent analyses of nuclear-encoded, plastidtargeted GAPDH genes have provided further evidence for this hypothesis, showing strong support for the sisterhood of apicomplexan and dinoflagellate plastid sequences [19]. Regardless of the origin of these apicomplexan plastids, they represent a very interesting example of preservation of an organelle after the loss of its major ability, in this case photosynthesis. Most apicomplexan parasites could have retained relict plastids because they are the only cellular source of fatty acid synthesis enzymes, since these

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species have lost their eukaryotic pathway after the acquisition of the plastid [66]. Euglenozoans also possess three-membrane plastids [26] (figure 1), and the nuclear-encoded proteins that have to be transported into them show characteristic transit peptides which are similar to those found in chlorarachniophytes and apicomplexans [12, 66]. Given these characteristics, together with the green algal-type pigmentation of their plastids and the apparent disconnection of euglenoids and green algae in phylogenetic trees based on nuclear markers, it has been proposed that euglenoid plastids arose by secondary endosymbiosis with a green alga [26]. This hypothesis has been strengthened by the results of the phylogenetic analysis of complete chloroplast genome sequences, which placed the euglenoids within the green algae [61]. Some atypical dinoflagellate plastids provide examples of an additional level of endosymbiosis complexity. In fact, several species do not host the typical peridinin-containing dinoflagellate plastids. Instead, these species may contain plastids with pigmentation closely resembling that of green algae or of haptophytes [14, 60] (figure 1). Since the species carrying these atypical plastids emerge in phylogenetic trees clearly intermixed with those containing the secondary peridinin-containing plastids, it has been suggested that their plastids have been incorporated through tertiary endosymbiosis, replacing the peridinin-containing plastids already present in the hosts (namely, a process of tertiary plastid replacement) (Cavalier-Smith, 2000). However, organisms carrying secondary plastids have not been thoroughly studied, especially from a molecular perspective. Plastid loss is a phenomenon that also remains only partially analysed. However, it may be a frequent process that, in turn, may favour secondary plastid replacement and the acquisition of plastids by tertiary endosymbiosis, since it avoids possible conflicts between different plastids coexisting within the same cell. It may also favour the acquisition of what have been called the kleptoplasts (i.e., “stolen” plastids). These are transient plastids derived from ingested algae that are not completely digested. They may remain active within the host cytoplasm for some time, but they are finally lost. These kleptoplasts are difficult to distinguish from secondary or tertiary plastids, although the latter are stable. They have been found, for instance, in ciliates [29] and di-

noflagellates, which complicates the already puzzling distribution of plastids within this latter phylum [38]. 6. The number of secondary endosymbioses We have seen that very diverse groups of eukaryotic algae carry secondary plastids, which suggests several instances of endosymbiosis. As stated above for the case of primary endosymbioses, the determination of the exact number of symbiotic events requires a precise knowledge of the phylogenetic relationships both of the plastids and their hosts. Thus, using the information based upon the analysis of the SSU rRNA as a phylogenetic framework, a relatively large number of secondary endosymbioses has been suggested [13, 49]. In fact, nuclear SSU rRNA shows, although with low statistical support, that chlorarachniophytes, euglenozoans, dinoflagellates, cryptophytes, heterokonts, and haptophytes emerge as independent lineages. This would imply at least six independent secondary endosymbioses. Moreover, dinoflagellates with green algaltype plastids and apicomplexans may represent additional secondary endosymbiosis events, increasing the total number up to seven or eight [13, 49]. This relatively large number of secondary endosymbioses, compared with a unique primary endosymbiosis [46], would indicate that the establishment of this initial association between two very different organisms, a cyanobacterium and a eukaryotic host, may have been much more arduous than the subsequent secondary symbioses between already integrated algae and different eukaryotic hosts. However, recent evidence is challenging this view. As discussed above, dinoflagellates and apicomplexans possibly share a photosynthetic ancestor with red algal-type plastids and, consequently, green algaland haptophyte-type dinoflagellate plastids would be of tertiary origin [69]. In addition, the phylogenetic analysis of protein sequences is providing increasing support for the sisterhood of alveolates (containing dinoflagellates and apicomplexans, as well as ciliates) and heterokonts [1, 19]. This would imply that both groups shared a photosynthetic common ancestor, namely, a single secondary endosymbiosis at their origin [10, 19]. Moreover, radical revisions of the phylogeny of photosynthetic eukaryotes have proposed the assembling of cryptophytes, haptophytes and heterokonts within a kingdom Chromista

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[8], which would form together with alveolates a huge supergroup of photosynthetic origin, the chromalveolates [9]. On the other hand, on the basis of peculiarities of protein import into the plastids, a common origin of euglenoids and chlorarachniophytes has been also posited [9]. If these proposals turn out to be correct, they will reduce the number of secondary endosymbioses to only two (figure 1). The possibility of introducing this kind of intensive phylogenetic reorganisation reflects the current state of uncertainty that concerns our knowledge of the relationships between eukaryotic phyla (for review see [52]). As shown above, some parts of the eukaryotic tree based on SSU rRNA sequences, traditionally granted as having a robust phylogenetic framework, have been seriously challenged when information from other phylogenetic markers has become available [50]. In particular, the classical view that the SSU rRNA phylogeny was a faithful reflection of a progressive evolution of the eukaryotic cell from simpler protists to complex multicellular organisms [33, 56], was revealed to have important failures. The best example concerns a group of very simple parasitic protists, the microsporidia, which lack mitochondria and other typical eukaryotic features. In addition to their extreme simplicity, they emerge at the very base of the eukaryotic SSU rRNA tree, so that they were proposed to be “living fossils” from early times of the eukaryotic evolution [7, 65]. However, the discovery of genes of likely mitochondrial origin in the nuclear genome of these organisms demonstrated that their ancestor probably possessed mitochondria, which were lost secondarily [23, 31]. In addition, protein-based phylogenetic analyses strongly supported the notion that microsporidia were close relatives to fungi [17, 32, 35], and even that they actually emerge from within the fungi [36]. Several phenotypic characters, such as the presence of chitin in the cellular walls, confirmed this relationship (reviewed in [47]). The high evolutionary rate of the microsporidian SSU rRNA genes provokes the basal emergence of this group in the tree because of an LBA artefact [51, 62]. This provides an eloquent case of how the results based on a single phylogenetic marker can be entirely misleading for some parts of the tree. This problem affects, in particular, the basal regions of the phylogenetic trees. We have recently proposed that these regions are actually artefactual, being the result of LBA problems. This explains why the discrepancies among

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the trees constructed with different markers are concentrated in these regions, since for each marker the fast evolving species (those affected by LBA and misplaced to the base of the tree) may be different. Considering that all basal branches may be indeed misplaced by LBA, the actual phylogeny of eukaryotes would be closer to a vast radiation of all extant eukaryotic phyla, which has been proposed as the “big bang” hypothesis [45, 50, 52 – 54]. The subsequent acceleration of the evolutionary rate of some phyla makes them emerge artificially as basal groups (e.g., the microsporidia). The true distances between the different groups would be greatly reduced, so that individual genes may not contain enough information to solve the relationships between them. This “big bang” in eukaryotic evolution makes conceivable the new phylogenetic groupings proposed above (two supergroups with only two secondary endosymbioses at their origin), given that all groups are indeed phylogenetically close. 7. Concluding remarks and perspectives The study of the evolution of plastids, as well as the complete research field of eukaryotic evolution, is undergoing a period of exciting discoveries and fertile discussion. Some concepts traditionally accepted as solid interpretations of nature are now under critical scrutiny, while more recent ones are gaining important consensus. An important advance is the plausible confirmation of the single origin of primary plastids, now supported (with differing degrees of confidence) by three independent lines of evidence: plastids, mitochondria, and nuclear markers [46]. Most open questions hence apply to secondary plastids, in particular to the number of secondary endosymbioses. Since photosynthetic lineages could be widely scattered upon the eukaryotic tree, the answer to these questions necessarily implies the resolution of the general phylogeny of eukaryotes. For this aim, the use of information derived from different phylogenetic markers, analysed individually or combined into large fusions, will be of great value [1, 46]. The proliferation of complete genome sequencing projects will provide the raw material for these analyses, so it is possible that in the near future many open questions of eukaryotic phylogeny will be unveiled. With the exception of euglenoids, whose early branching in SSU rRNA trees is most likely due

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to an LBA artefact, all photosynthetic eukaryotic groups (both with primary and secondary plastids) appear to have diverged within a short time span. This means that the secondary symbioses probably occurred shortly after the origin of the primary plastids, when the two primary partners (the cyanobacterium and the eukaryotic host) were still not fully integrated. The fact that no stable, more recent secondary endosymbioses have been described suggests that secondary endosymbiosis was somehow facilitated by that still-on-the-way status of primary symbiosis. Two possible explanations for this fact concern the transfer of genes from the plastid to the nucleus and the complex architecture of membranes enclosing the plastids. After the primary endosymbiosis, the plastid genome began its reduction process through gene loss and gene transfer to the nucleus of its eukaryotic host. An early secondary endosymbiosis would allow the transfer of essential plastid genes directly to the nucleus of the secondary eukaryotic host, which thus took over control on the plastid and stabilised the symbiosis, without the necessity of a nucleomorph. Cases where the nucleomorph persists seem to be due to the presence of plastid housekeeping genes in the nucleomorph genome [34, 64]. In these cases, only gene transfer from the nucleomorph to the secondary host nucleus would allow the complete disappearance of the nucleomorph genome. Stabilisation of the membrane complexes enclosing plastids and the development of systems to import proteins from the host cytosol into the plastid could also have been important factors in the establishment of the secondary endosymbioses [9, 10]. In relation to the previous discussion on transferred genes, soon after the primary endosymbiosis and when the first plastid genes were transferred to the host nucleus, a system for the relocation of the corresponding gene products back into the plastid became necessary. Secondary symbiosis would have been easier if it had occurred soon after primary endosymbiosis, when the transport system between the primary host and the primary endosymbiont are not completely established. This would have facilitated the direct transport from the secondary host to the photosynthetic endosymbiont, avoiding the coexistence of various overlapping transport systems (i.e., between the primary host and the endosymbiont and between the secondary host and the endosymbiont) [10]. To conclude, an important question remains open: When did the origin of plastids occur? Available in-

formation does not provide a clear answer, so that only suggestions can be advanced. Cavalier-Smith hypothesises that the divergence of photosynthetic eukaryotes could have followed the warming of the Earth that occurred 600 million years ago, in the late Proterozoic [10]. Philippe and coworkers advance an earlier but similar date for the diversification of all (not only photosynthetic) eukaryotic phyla (between 700 million and 1 billion years ago), coupled to the rise of atmospheric oxygen and, perhaps, to the acquisition of mitochondria [54]. However, the recent discovery of putative red algal fossils of 1.2 billion years weakens these hypotheses [5]. An accurate evaluation of the new paleogeological, paleontological, and phylogenetic data will probably provide new answers. Acknowledgements We thank the Editorial Board of Research in Microbiology for the invitation to contribute this mini-review, and Purificación López-García, Philippe Lopez, and an anonymous referee for critical reading of the manuscript. References [1] Baldauf S.L., Roger A.J., Wenk-Siefert I., Doolittle W.F., A kingdom-level phylogeny of eukaryotes based on combined protein data, Science 290 (2000) 972–977. [2] Bhattacharya D., Helmchen T., Bibeau C., Melkonian M., Comparisons of nuclear-encoded small-subunit ribosomal RNAs reveal the evolutionary position of the Glaucocystophyta, Mol. Biol. Evol. 12 (1995) 415–420. [3] Blanchard J.L., Hicks J.S., The non-photosynthetic plastid in malarial parasites and other apicomplexans is derived from outside the green plastid lineage, J. Eukaryot. Microbiol. 46 (1999) 367–375. [4] Burger G., Saint-Louis D., Gray M.W., Lang B.F., Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae, Plant Cell 11 (1999) 1675–1694. [5] Butterfield N.J., Bangiomorpha pubescens n. gen., n. sp.: implications for the evolution of sex, multicellularity, and the Mesoproterozoic/Neoproteorozoic radiation of eukaryotes, Paleobiol. 26 (2000) 386–404. [6] Cavalier-Smith T., Eukaryote kingdoms: seven or nine?, Biosystems 14 (1981) 461–481. [7] Cavalier-Smith T., Kingdom protozoa and its 18 phyla, Microbiol. Rev. 57 (1993) 953–994. [8] Cavalier-Smith T., A revised six-kingdom system of life, Biol. Rev. Camb. Philos. Soc. 73 (1998) 203–266. [9] Cavalier-Smith T., Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and

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