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].
D. Moreira, H. Philippe / Res. Microbiol. 152 (2001) 771–780
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,
D. Moreira, H. Philippe / Res. Microbiol. 152 (2001) 771–780
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
D. Moreira, H. Philippe / Res. Microbiol. 152 (2001) 771–780
[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
D. Moreira, H. Philippe / Res. Microbiol. 152 (2001) 771–780
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
sporozoan plastids origin and the eukaryote family tree, J.
Euk. Microbiol. 46 (1999) 347–366.
Cavalier-Smith T., Membrane heredity and early chloroplast
evolution, Trends Plant. Sci. 5 (2000) 174–182.
Cavalier-Smith T., Allsopp M.T., Chao E.E., Chimeric conundra: are nucleomorphs and chromists monophyletic or
polyphyletic?, Proc. Natl. Acad. Sci. USA 91 (1994) 11368–
11372.
Chan R.L., Keller M., Canaday J., Weil J.H., Imbault P.,
Eight small subunits of Euglena ribulose 1-5 bisphosphate
carboxylase/oxygenase are translated from a large mRNA
as a polyprotein, EMBO J. 9 (1990) 333–338.
Delwiche C.F., Tracing the thread of plastid diversity through
the tapestry of life, Am. Nat. 154 (1999) S164–S177.
Delwiche C.F., Palmer J.D., The origin of plastids and
their spread via secondary endosymbiosis, Plant System.
Evol. 11 (1997) 139–148.
Douglas S., Zauner S., Fraunholz M., Beatin M., Penny S.,
Lang-Tuo D., Wu X., Reith M., Cavalier-Smith T., Maier U.G.,
The highly reduced genome of an enslaved algal nucleus,
Nature 410 (2001) 1091–1096.
Douglas S.E., Murphy C.A., Spencer D.F., Gray M.W.,
Cryptomonad algae are evolutionary chimaeras of two
phylogenetically distinct unicellular eukaryotes, Nature 350
(1991) 148–151.
Edlind T.D., Li J., Visvesvara G.S., Vodkin M.H., McLaughlin
G.L., Katiyar S.K., Phylogenetic analysis of beta-tubulin
sequences from amitochondrial protozoa, Mol. Phylogenet.
Evol. 5 (1996) 359–367.
Eschbach S., Hofmann C.J., Maier U.G., Sitte P., Hansmann
P., A eukaryotic genome of 660 kb: electrophoretic karyotype
of nucleomorph and cell nucleus of the cryptomonad alga,
Pyrenomonas salina, Nucleic Acids Res. 19 (1991) 1779–
1781.
Fast N.M., Kissinger J.C., Roos D.S., Keeling P.J., Nuclearencoded, plastid-targeted genes suggest a single common
origin for apicomplexan and dinoflagellate plastids, Mol. Biol.
Evol. 18 (2001) 418–426.
Felsenstein J., Cases in which parsimony or compatibility
methods will be positively misleading, Syst. Zool. 27 (1978)
401–410.
Fraunholz M.J., Moerschel E., Maier U.G., The chloroplast
division protein FtsZ is encoded by a nucleomorph gene in
cryptomonads, Mol. Gen. Genet. 260 (1998) 207–211.
Gajadhar A.A., Marquardt W.C., Hall R., Gunderson J.,
Ariztia-Carmona E.V., Sogin M.L., Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and
Crypthecodinium cohnii reveal evolutionary relationships
among apicomplexans, dinoflagellates, and ciliates, Mol.
Biochem. Parasitol. 45 (1991) 147–154.
Germot A., Philippe H., Le Guyader H., Evidence for loss
of mitochondria in Microsporidia from a mitochondrial-type
HSP70 in Nosema locustae, Mol. Biochem. Parasitol. 87
(1997) 159–168.
Gibbs S.P., Nuclear envelope-chloroplast relationships in
algae, J. Cell Biol. 14 (1962) 433–444.
Gibbs S.P., The route of entry of cytoplasmically synthesized
proteins into chloroplasts of algae possessing chloroplast
ER, J. Cell Sci. 35 (1979) 253–266.
779
[26] Gibbs S.P., The chloroplasts of some algal groups may
have evolved from endosymbiotic eukaryotic algae, Ann. NY
Acad. Sci. 361 (1981) 193–208.
[27] Gilson P.R., McFadden G.I., The miniaturized nuclear
genome of eukaryotic endosymbiont contains genes that
overlap, genes that are cotranscribed, and the smallest
known spliceosomal introns, Proc. Natl. Acad. Sci. USA 93
(1996) 7737–7742.
[28] Gray M.W., Evolution of organellar genomes, Curr. Opin.
Genet. Dev. 9 (1999) 678–687.
[29] Gustafson D.E. Jr., Stoecker D.K., Johnson M.D., Van
Heukelem W.F., Sneider K., Cryptophyte algae are robbed
of their organelles by the marine ciliate Mesodinium rubrum,
Nature 405 (2000) 1049–1052.
[30] Hibberd D.J., Norris R.E., Cytology and ultrastructure of
Chlorarachnion reptans (Chlorarachniophyta Divisio nova,
Chlorarachniophyceae, Classis nova), J. Phycol. 20 (1984)
310–330.
[31] Hirt R.P., Healy B., Vossbrinck C.R., Canning E.U., Embley T.M., A mitochondrial Hsp70 orthologue in Vairimorpha
necatrix : molecular evidence that microsporidia once contained mitochondria, Curr. Biol. 7 (1997) 995–998.
[32] Hirt R.P., Logsdon J.M. Jr., Healy B., Dorey M.W., Doolittle
W.F., Embley T.M., Microsporidia are related to fungi: evidence from the largest subunit of RNA polymerase II and
other proteins, Proc. Natl. Acad. Sci. USA 96 (1999) 580–
585.
[33] Hülsmann N., Hausmann K., Towards a new perspective in
protozoan evolution, Europ. J. Protistol. 30 (1994) 365–371.
[34] Jeffrey S.W., Chloroplast pigment patterns in dinoflagellates,
J. Phycol. 111 (1975) 374–384.
[35] Keeling P.J., Doolittle W.F., Alpha-tubulin from earlydiverging eukaryotic lineages and the evolution of the tubulin
family, Mol. Biol. Evol. 13 (1996) 1297–1305.
[36] Keeling P.J., Luker M.A., Palmer J.D., Evidence from betatubulin phylogeny that microsporidia evolved from within the
fungi, Mol. Biol. Evol. 17 (2000) 23–31.
[37] Kohler S., Delwiche C.F., Denny P.W., Tilney L.G., Webster
P., Wilson R.J., Palmer J.D., Roos D.S., A plastid of probable
green algal origin in Apicomplexan parasites, Science 275
(1997) 1485–1489.
[38] Lewitus A.J., Glasgow H.B., Burkholder J.M., Kleptoplastidy
in the toxic dinoflagellate Pfiesteria piscicida (Dinophyceae),
J. Phycol. 35 (1999) 303–312.
[39] Maier U.G., Douglas S.E., Cavalier-Smith T., The nucleomorph genomes of cryptophytes and chlorarachniophytes,
Protist 151 (2000) 103–109.
[40] Margulis L., Origin of eukaryotic cells, Yale University Press,
New Haven, CT, 1970.
[41] Martin W., Kowallik K.V., Annotated English translation of
Mereschkowsky’s 1905 paper ’Über Natur und Ursprung
der Chromatophoren im Pflanzenreiche’, Eur. J. Phycol. 34
(1999) 287–295.
[42] Martin W., Stoebe B., Goremykin V., Hansmann S.,
Hasegawa M., Kowallik K.V., Gene transfer to the nucleus
and the evolution of chloroplasts, Nature 393 (1998) 162–
165.
[43] McFadden G.I., Gilson P.R., Hofmann C.J., Adcock G.J.,
Maier U.G., Evidence that an amoeba acquired a chloroplast
780
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
D. Moreira, H. Philippe / Res. Microbiol. 152 (2001) 771–780
by retaining part of an engulfed eukaryotic alga, Proc. Natl.
Acad. Sci. USA 91 (1994) 3690–3694.
Mereschkowsky C., Über natur und usprung der chromatophoren im pflanzenreiche, Biologisches Centralblatt. 25
(1905) 593–604.
Moreira D., Le Guyader H., Philippe H., Unusually high
evolutionary rate of the elongation factor 1 alpha genes
from the Ciliophora and its impact on the phylogeny of
eukaryotes, Mol. Biol. Evol. 16 (1999) 234–245.
Moreira D., Le Guyader H., Phillippe H., The origin of red
algae and the evolution of chloroplasts, Nature 405 (2000)
69–72.
Müller M., What are the Microsporidia?, Parasitol. Today 13
(1997) 455–456.
Palmer J.D., Delwiche C.F., Second-hand chloroplasts and
the case of the disappearing nucleus, Proc. Natl. Acad. Sci.
USA 93 (1996) 7432–7435.
Palmer J.D., Delwiche C.F., in: Soltis P.S., Soltis D.E.,
Doyle J.J. (Eds.), Molecular systematics of plants. II. DNA
sequencing, Kluwer Academic, Boston, 1998.
Philippe H., Adoutte A., in: Coombs G., Vickerman K., Sleigh
M., Warren A. (Eds.), Evolutionary relationships among
Protozoa, Chapman & Hall, London, 1998, pp. 25–56.
Philippe H., Germot A., Phylogeny of eukaryotes based
on ribosomal RNA: long-branch attraction and models of
sequence evolution, Mol. Biol. Evol. 17 (2000) 830–834.
Philippe H., Germot A., Moreira D., The new phylogeny of
eukaryotes, Curr. Op. Genet. Develop. 10 (2000) 596–601.
Philippe H., Laurent J., How good are deep phylogenetic
trees?, Curr. Opin. Genet. Dev. 8 (1998) 616–623.
Philippe H., Lopez P., Brinkmann H., Budin K., Germot A.,
Laurent J., Moreira D., Muller M., Le Guyader H., Earlybranching or fast-evolving eukaryotes? An answer based
on slowly evolving positions, Proc. R. Soc. Lond. B. Biol.
Sci. 267 (2000) 1213–1221.
Ragan M., Gutell R., Are red algae plants?, Bot. J. Linn.
Soc. 118 (1995) 81–105.
Sogin M.L., Early evolution and the origin of eukaryotes,
Curr. Opin. Genet. Dev. 1 (1991) 457–463.
Sogin M.L., Elwood H.J., Gunderson J.H., Evolutionary
diversity of eukaryotic small-subunit rRNA genes, Proc. Natl.
Acad. Sci. USA 83 (1986) 1383–1387.
Sogin M.L., Gunderson J.H., Structural diversity of eukaryotic small subunit ribosomal RNAs. Evolutionary implications, Ann. NY Acad. Sci. 503 (1987) 125–139.
Stiller J.W., Hall B.D., The origin of red algae: implications
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
for plastid evolution, Proc. Natl. Acad. Sci. USA 94 (1997)
4520–4525.
Tengs T., Dahlberg O.J., Shalchian-Tabrizi K., Klaveness D.,
Rudi K., Delwiche C.F., Jakobsen K.S., Phylogenetic analyses indicate that the 19 Hexanoyloxy-fucoxanthin-containing
dinoflagellates have tertiary plastids of haptophyte origin,
Mol. Biol. Evol. 17 (2000) 718–729.
Turmel M., Otis C., Lemieux C., The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast
genomes, Proc. Natl. Acad. Sci. USA 96 (1999) 10248–
10253.
Van de Peer Y., Ben Ali A., Meyer A., Microsporidia: accumulating molecular evidence that a group of amitochondriate
and suspectedly primitive eukaryotes are just curious fungi,
Gene 246 (2000) 1–8.
Van de Peer Y., De Wachter R., Evolutionary relationships
among the eukaryotic crown taxa taking into account siteto-site rate variation in 18S rRNA, J. Mol. Evol. 45 (1997)
619–630.
Van de Peer Y., Rensing S.A., Maier U.G., De Wachter R.,
Substitution rate calibration of small subunit ribosomal RNA
identifies chlorarachniophyte endosymbionts as remnants of
green algae, Proc. Natl. Acad. Sci. USA 93 (1996) 7732–
7736.
Vossbrinck C.R., Maddox J.V., Friedman S., DebrunnerVossbrinck B.A., Woese C.R., Ribosomal RNA sequence
suggests microsporidia are extremely ancient eukaryotes,
Nature 326 (1987) 411–414.
Waller R.F., Keeling P.J., Donald R.G., Striepen B., Handman E., Lang-Unnasch N., Cowman A.F., Besra G.S., Roos
D.S., McFadden G.I., Nuclear-encoded proteins target to the
plastid in Toxoplasma gondii and Plasmodium falciparum,
Proc. Natl. Acad. Sci. USA 95 (1998) 12352–12357.
Zauner S., Fraunholz M., Wastl J., Penny S., Beaton M.,
Cavalier-Smith T., Maier U.G., Douglas S., Chloroplast
protein and centrosomal genes, a tRNA intron, and odd
telomeres in an unusually compact eukaryotic genome, the
cryptomonad nucleomorph, Proc. Natl. Acad. Sci. USA 97
(2000) 200–205.
Zhang Z., Green B.R., Cavalier-Smith T., Single gene circles
in dinoflagellate chloroplast genomes, Nature 400 (1999)
155–159.
Zhang Z., Green B.R., Cavalier-Smith T., Phylogeny of ultrarapidly evolving dinoflagellate chloroplast genes: A possible
common origin for sporozoan and dinoflagellate plastids, J.
Mol. Evol. 51 (2000) 26–40.