EEF2 Analysis Challenges the Monophyly of
Archaeplastida and Chromalveolata
Eunsoo Kim¤*, Linda E. Graham
Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
Background: Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and
industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida,
Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and
Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium
(Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and
Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently
Methodology/Principal Findings: This study was designed to test the validity of the Archaeplastida and Chromalveolata
through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein
of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas
truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga
Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2
phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade
composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This
clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from
amino acid replacement before the common origin of these eukaryotic groups.
Conclusions/Significance: Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata,
adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other
morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup.
Citation: Kim E, Graham LE (2008) EEF2 Analysis Challenges the Monophyly of Archaeplastida and Chromalveolata. PLoS ONE 3(7): e2621. doi:10.1371/
Editor: Rosemary Jeanne Redfield, University of British Columbia, Canada
Received March 5, 2008; Accepted June 2, 2008; Published July 9, 2008
Copyright: ? 2008 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant MCB-9977903 from the National Science Foundation. The grant was used to purchase supplies for molecular
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada
Eukaryotes constitute one of the three domains of life,
distinguished from bacteria and archaebacteria by their greater
molecular, cellular, and reproductive complexity. About 1.5 mil-
lion species of eukaryotes have been recognized and named thus
far, with at least several times that number remaining to be
catalogued . Much of eukaryotic diversity occurs among
protists, whose high-level classification remains uncertain in spite
of the need for a reliable, phylogeny-based classification in
ecological, medical, and industrial research.
Eukaryotes can be conservatively classified into about 60 robust
lineages based primarily on ultrastructural features [2,3]. Alterna-
tively, eukaryotes have been grouped into only two major clades—
unikonts and bikonts–based largely on a single gene fusion event,
under the assumption that parallel fusions would be improbable
[4–6]. However, this assumption is refuted by evidence that gene
fusion events do occur independently in different eukaryotic
groups . A fundamental unikont-bikont dichotomy is also
questioned by the phylogenetic position of the bikont Apusozoa
among the ‘‘unikonts’’, as well as other data [8,9]. Other recent
authors have classified eukaryotes into 5 or 6 major supergroups:
Amoebozoa, Opisthokonta, Archaeplastida (or Plantae), Chro-
malveolata, Rhizaria, and Excavata, with the first two grouped as
‘unikonts’ by some authors [10–12]. However, the validity of some
of these supergroups, notably Excavata, Archaeplastida and
Chromalveolata, is controversial [13–21].
The present study was designed to test monophyly of the
Archaeplastida and Chromalveolata, each defined by a single
primary- or secondary-plastid generating endosymbiotic event .
The Archaeplastida is composed of three well characterized
monophyletic groups: the Glaucophyta, Rhodophyta (i.e., red
algae), and Viridiplantae (i.e., green algae plus land plants) . All
members of the Archaeplastida possess double membrane-bound
plastids (i.e., primary plastids), which are believed to have been
derived directly from a cyanobacterial endosymbiont by primary
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endosymbiosis . It should be noted that the rhizarian Paulinella
chromatophora (which does not group with the Archaeplastida)
independently acquired photosynthetic bodies directly from a
cyanobacterium [23,24], although there is debate whether to
Chromalveolata comprises four monophyletic groups—Alveolata,
Cryptophyta (plus Katablepharidae) [8,28], Haptophyta, and
Stramenopiles, each group containing at least some members
harboring plastids thought to be derived from a red alga by
secondary endosymbiosis . Cryptophytes, haptophytes, and
stramenopiles weregrouped as ‘chromists,’ based on shared features
of their plastids. These shared features include the presence of four-
bounding membranes and, with some exceptions , confluence
of the outermost plastid membrane with the nuclear envelope .
It should to be noted, however, that several ‘‘early-diverging’’ clades
within the stramenopiles, and the cryptophyte genus Goniomonas
(plus katablepharid species), do not possess plastids. Alveolates
include ciliates (plastid-less), apicomplexans, and dinoflagellates, the
latter including many plastid-less as well as plastid-bearing members
. Most plastid-bearing dinoflagellates have peridinin as a major
membranes , whereas plastids that are present in the majority of
apicomplexans (i.e., apicoplasts) are exclusively non-photosynthetic
and are bound by 2–4 membranes [33–36]. Recently, a
photosynthetic relative of the apicomplexans has been identified
which harbors plastids with four membranes that are related to
apicoplasts . In contrast to most ‘chromist’ plastids, the
outermostplastid membranesofalveolate plastidsarenotconnected
to the nuclear membrane [32,35].
The major issues surrounding the endosymbiotic origins of
plastid-bearing eukaryotes can be summarized by the following
questions: a) Did the plastids of the Archaeplastida taxa arise from
a single or multiple source(s) of cyanobacteria [38–40]? ; b) Are all
these plastids derived from a single endosymbiotic event [18,41]? ;
c) Can the plastids of the Chromalveolata taxa be traced back to a
single red algal type ? ; d) Were chromalveolate plastids
acquired once or on multiple occasions [43,44]?
As a means of addressing some of these evolutionary concerns,
we carefully targeted molecular phylogenetic markers and taxa to
the specific issue of the monophyly of the Archaeplastida and
Chromalveolata. More focused analyses such as ours can reveal
strong gene-specific evidence for or against phylogenetic relation-
ships, which might be overlooked or unrecognizable in concate-
nation analyses . We chose two conserved, nuclear protein-
coding genes that have been widely used to evaluate eukaryotic
diversification: EEF2 (eukaryotic translation elongation factor 2)
and cytosolic HSP70 (heat-shock protein of 70 kDa) genes. We
generated sequence data from representatives of major eukaryotic
phyla, including cryptophytes, a glaucophyte, a green alga, a
katablepharid, and a rhizarian and analyzed these new sequences
together with existing available database sequences for other
HSP70 is a molecular chaperone which assists in assembly and
folding of proteins and occurs universally in all organisms .
Because of its highly conserved sequence, HSP70 has been
extensively surveyed to address some of the most ancient
evolutionary events such as early bacterial, archaebacterial, and
eukaryotic diversification patterns [46,47]. Like HSP70, EEF2
(and its prokaryotic homolog EF-G) is also highly conserved. EEF2
constitutes an essential component of the translational machinery,
where it is involved in the protein elongation step, specifically in
the translocation of tRNAs and mRNA [48,49]. EEF2 is valued as
a phylogenetic marker because of its large size (,800 amino acids)
and consequently its potential to retain more phylogenetic signal
than smaller proteins. In a previous study, EEF2 phylogeny
strongly suggested a sister relationship between rhodophytes and
Viridiplantae; this observation was argued as nucleocytoplasmic
evidence in support of a single endosymbiotic origin for primary
plastids . Although EEF2 sequences from glaucophytes could
critically test Archaeplastida monophyly, such sequences are so far
In this study, we determined six EEF2 and four cytosolic HSP70
sequences from diverse eukaryotic groups. Importantly, our EEF2
and cytosolic HSP70 phylogenies included, for the first time,
nearly full-length sequences of glaucophytes, katablepharids, or
cryptophytes. The results of our study, specifically the EEF2
analysis, strongly refute the monophyly of the Archaeplastida and
EEF2 and cytosolic HSP70 analyses
EEF2 phylogenetic trees inferred from maximum likelihood
(RAxML, PhyloBayes) and distance (FastME) methods were more
or less similar, although some deep branching patterns that had
low bootstrap support, differed (data not shown). In some cases
posterior probabilities (PhyloBayes) were very high (.0.95) even
when respective bootstrap values (RAxML, FastME) were low
(,70%), although these numbers are not directly comparable as
they have different statistical interpretations. For this reason, we
interpreted a given relationship as being well supported only when
all three supporting values (bootstrap and posterior probability)
were high (.90% or .0.90). In all three analyses, a number of
well-established eukaryotic groups, including rhodophytes, alveo-
lates, opisthokonts, euglenozoans, and Viridiplantae were recov-
ered with .90% bootstrap support and high posterior probabil-
ities (Figure 1). Most interestingly, unlike what is expected from the
Archaeplastida proposal, the glaucophyte C. paradoxa did not
branch with rhodophytes or Viridiplantae in the EEF2 tree.
Instead, EEF2 analysis identified a well-supported clade composed
of cryptophytes, katablepharids, haptophytes, rhodophytes, and
Viridiplantae (Figure 1). Within this large clade, relationships
among the five eukaryotic groups were poorly resolved. Although
Viridiplantae and rhodophytes were each other’s closest sister
group, bootstrap values supporting this relationship were low (49%
for RAxML and 66% for FastME) (Figure 1).
Support for the monophyly of the EEF2 of cryptophytes,
katablepharids, haptophytes, rhodophytes, and Viridiplantae is
further provided in the form of a two amino acid signature
(Figure 2). In these EEF2, two consecutive amino acids, serine (S)
followed by alanine (A), occur at positions 212 and 213 whereas
most other taxa encode the highly conserved amino acid
sequences, glycine (G) and serine (S), at these positions (Figure 2).
This suggests that the SA amino acids arose via amino acid
replacement of the ancestral GS residues.
In contrast to EEF2, major eukaryotic relationships were largely
unresolved in the cytosolic HSP70 phylogeny (Figure S1). Of well-
established eukaryotic groups, only the alveolates, stramenopiles,
euglenozoans, and rhizaria were recovered with .50% bootstrap
support and monophyletic cryptophytes, opisthokonts, rhodo-
phytes, and Viridiplantae were not recovered in the ML tree
(Figure S1). As most nodes in the ML tree were poorly supported,
it is not clear whether some abnormal branching patterns in the
cytosolic HSP70 tree are simply due to the lack of informative sites
or other factors (e.g., incomplete lineage sorting, horizontal gene
transfer, paralogy) that can lead to discordance between the gene
and the species trees. HSP70 phylogeny is also known to be
susceptible to the long branch attraction (LBA) artifact .
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Figure 1. Maximum likelihood tree based on the EEF2 alignment, under the WAG+C+I+F model of protein evolution (RAxML).
Bootstrap support values .50% (RAxML/FastME) and posterior probabilities .0.50 are indicated at the corresponding nodes. Sequences newly
obtained in this study are labeled in bold. Note that the Viridiplantae, Rhodophyta, Haptophyta, Katablepharidae, and Cryptophyta formed a well-
supported clade. NM stands for nucleomorph.
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Concatenated protein phylogeny
As single-gene trees are generally poorly resolved due to the
presence of limited phylogenetic signal , a combined analysis
of six proteins—a-tubulin, b-tubulin, actin, cytosolic HSP70,
cytosolic HSP90, and EEF2—was performed in an attempt to
improve the resolution of the tree by increasing the number of
informative characters. Up to about 40% of missing data for a
particular taxon was permitted for increasing taxonomic sampling,
important for the accuracy of phylogenetic inference [52–54]. The
final alignment included 2,797 amino acids with 278 constant sites
and had in total 5.23% missing data. Well-established groups
including the alveolates, cryptophytes, euglenozoa, haptophytes,
rhodophytes, opisthokonts, stramenopiles, and Viridiplantae were
recovered with strong bootstrap support and .0.95 posterior
probabilities (Figure 3). In addition, higher level-groupings such as
the Opisthokonta-Amoebozoa and the Euglenozoa-Heterolobosea
clades received strong support (Figure 3). Cryptophyta, Katable-
pharidae, and Haptophyta formed a clade with moderate to strong
support, which is consistent with recent multiple-gene phylogenies
that suggested a close relationship between Cryptophyta and
Figure 2. Two amino acid signature within the EEF2. Note that EEF2 of Viridiplantae, Rhodophyta, Haptophyta, Katablepharidae, and
Cryptophyta have amino acids serine and alanine at positions 212 and 213, whereas most other eukaryotes have glycine and serine residues instead.
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Haptophyta (Katablepharidae was not examined in these studies)
[55,56]. As in the EEF2 analysis, a clade comprising the
Cryptophyta, Katablepharidae, Haptophyta, Rhodophyta, and
Viridiplantae was recovered in the combined protein tree.
Although the clade received the highest posterior probability of
1.0 in both analyses, bootstrap support values for the clade
decreased from 98 or 99% in the EEF2 tree to 88 or ,50% in the
combined analysis (compare Figures 1 and 3). The glaucophyte C.
paradoxa did not branch close to rhodophytes or Viridiplantae and
its phylogenetic position to other eukaryotic groups was unre-
solved. Lastly, the rhizarian Thaumatomonas sp. branched with
alveolates and stramenopiles with weak to moderate support
values, consistent with a previous study based upon .100
concatenated protein sequences .
EEF2 phylogeny refutes the monophyly of Archaeplastida
Moreira et al.  showed in their EEF2 tree that rhodophytes
and Viridiplantae were closely related to each other and that a sister
relationship was strongly supported, with a 100% ML bootstrap
Figure 3. Maximum likelihood tree for the concatenated protein data set, under the WAG+C+I model of protein evolution (RAxML)
with the unlinked option. Included proteins are EEF2, actin, cytosolic HSP70, cytosolic HSP90, and a-tubulin, and b-tubulin. Bootstrap support
values .50% (RAxML/FastME) and posterior probabilities .0.50 are shown.
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value. Prior to that, the hypothesis that a single plastid-generating
endosymbiotic event occurred at the origin of glaucophytes,
rhodophytes and Viridiplantae was primarily, if not entirely, based
on plastid-related features, because no nucleocytoplasmic data in
support of the hypothesis were available. Therefore, the Moreira et
al. EEF2 result was regarded as the first strong nucleocytoplasmic
evidence favoring a monophyletic origin of the Archaeplastida
[18,57], although glaucophytes were not examined in their study.
Subsequently, an analysis based on .100 concatenated nucleus-
encoded proteins indicated that glaucophytes branched closely to
rhodophytes and Viridiplantae . Together with plastid-related
evidence (see below for details), these results have convinced many
researchers in the field that the controversy surrounding the origin
of primary plastids was settled (e.g. ). However, both the
Moreira et al.  and Rodriguez-Ezpeleta et al.  studies
suffered from inadequate taxonomic sampling, notably lacking
sequences of cryptophytes, katablepharids, and haptophytes, which
appear to be critical in evaluating the validity of the supergroup
Archaeplastida as well as the Chromalveolata (see discussion below).
In our EEF2 analysis, which included glaucophytes, cryptophytes,
and katablepharids, the monophyly of the Archaeplastida and
Chromalveolata was strongly refuted. In addition, the specific
affiliationofrhodophytes andViridiplantae is nolongersignificantly
supported, although they still form a well-supported clade together
with cryptophytes, katablepharids, and haptophytes (Figure 1).
In recent studies, the strong associations among haptophytes,
rhodophytes, and Viridiplantae in EEF2 phylogenies were
interpreted as evidence for lateral gene transfer from a red algal
endosymbiont to the haptophyte nucleus [56,59]. However, with
the addition of our new cryptophyte and katablepharid EEF2
sequences, it is now clear that the earlier proposal is no longer
tenable (Figure 1). Cryptophytes have a copy of EEF2 gene in the
nucleomorph genome, in addition to one or more nucleus-
encoded copies [60,61]. In our study, as is predicted from its red
algal ancestry, the cryptophyte nucleomorph-encoded EEF2
branched close to the red algal EEF2 (Figure 1). In contrast, the
nucleus-encoded EEF2 of cryptophytes, katablepharids, and
haptophytes did not show specific affiliation to the nucleomorph
or red algal copies (Figure 1). These branching patterns suggest
that the EEF2 gene residing in the nuclei of haptophytes,
cryptophytes, and katablepharids was not obtained through
endosymbiotic gene transfer from the red algal endosymbiont
and probably descended vertically from their ancestors. In
addition, the hypothesis of an endosymbiotic gene transfer of
EEF2 gene requires the a priori assumption that katablepharids and
the cryptophyte genus Goniomonas once possessed plastids, although
there is no molecular or ultrastructural evidence of plastids in these
Concatenated protein analysis
Neither the monophyly of the Archaeplastida nor the Chromal-
veolata were recovered in our concatenated six protein phylogeny
(Figure 3). It should be noted, however, that the clade comprising
cryptophytes, katablepharids, haptophytes, rhodophytes, and Vir-
idiplantae is no longer significantly supported by bootstrap values.
One possible reason might be a long-branch effect of red algal-
derived sequences, especially as their actin and b-tubulin sequences
arerelatively quite divergent(data not shown).On the otherhand,it
cannot be completely ruled out that compared to other molecular
markers, EEF2 has disconcordant phylogenetic signal, although
EEF2 phylogeny does not show any obvious signs of conflict with
the five other protein markers examined in this study (data not
shown). Nevertheless, it is difficult to differentiate between these two
possibilities given the fact that not many other nucleus-encoded
molecular markers have been examined at a similar level of
taxonomic sampling. Finally, it is worth mentioning that in a study
of .100 concatenated nucleus-encoded protein sequences (albeit
with more than 50% of sequence data missing for cryptophytes and
haptophytes), the phylogenetic relationships of cryptophytes &
haptophytes, rhodophytes, or Viridiplantae to other eukaryotic
groups remained unresolved . This suggests that use of markers
selected specifically for their information value may be an effective
alternative to inferring deep phylogenies by the concatenation
approach (or total evidence approach). Given that individual
molecular markers can have differing histories due to lateral gene
transfer, hidden paralogy, and deep coalescence , a concatena-
tion approach can potentially hide strong local phylogenetic signal.
Evaluation of the supergroup Archaeplastida
Over the years, the origin of the plastids in glaucophytes,
rhodophytes and Viridiplantae has drawn considerable attention
[18,19,38,40,65]. These plastids are known as primary plastids as
they are thought to have arisen directly from a cyanobacterial
ancestor that was engulfed by an eukaryotic host. Several molecular
and genomic data support the notion that these primary plastids
arose from a single cyanobacterial endosymbiont. Two particularly
compelling pieces of evidence supporting this hypothesis are the
presence of an inner plastid membrane translocon Tic110 protein
[40,66] and a unique atpA gene cluster . These features are
common to the plastids of the Viridiplantae, glaucophytes, and
rhodophytes, but not found in the cyanobacteria examined thus far.
These features may represent post-endosymbiotic inventions that
occurred prior to the diversification of the three ‘primary’ plastids
[40,67], although it is also possible that they may have been
characteristic of ancestral cyanobacteria of a type so far undiscov-
ered among modern taxa. Triple-helix chlorophyll-binding, light-
harvesting antenna complexes (LHCs) have been suggested as
another case of post-endosymbiotic innovation . Because LHC
homologs have not been identified in glaucophytes , such LHCs
may have evolved after divergence of the glaucophyte plastid.
Plastid genome content and gene phylogenies suggest a single origin
of glaucophyte, rhodophyte and Viridiplantae plastids [58,70],
although such results do not completely rule out alternative
hypotheses . We also note that some other features once
considered specificto plastids, such as inverted repeats in rRNA and
the psbB gene cluster [40,67,71] have been subsequently identified
in cyanobacteria, and thus no longer support (nor refute) a single
origin hypothesis . In summary, although the hypothesis of a
common ancestry for red, green, and glaucophyte plastids is best
supported by current data, additional genomic and molecular data
for cyanobacteria are needed to further test the hypothesis.
In contrast to their plastids, little or no evidence supports an
hypothesis of a common ancestry for the host (i.e., nucleocyto-
plasmic) component of Viridiplantae, glaucophytes, and rhodo-
phytes. These three lineages differ in ultrastructure and biochem-
istry . In addition, nucleus-encoded gene phylogenies have
often been inconclusive [13,19,73,74]. Although in large-scale
phylogenies based on concatenated databases the monophyly of
Viridiplantae, glaucophytes, and rhodophytes was initially recov-
ered with strong support , the addition of cryptophyte or
haptophyte sequences significantly lowered or eliminated support
for monophyly of the three lineages [14,55]. Likewise, mitochon-
drion-encoded gene phylogenies remain largely inconclusive as to
the relationship between Viridiplantae and red algae [75–77]
(mitochondrial genome data for the glaucophyte taxa are not
publicly available for analysis).
Furthermore, although the mechanism of plastid origin by
primary endosymbiosis is widely accepted , this concept is
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primarily based on the presence of two plastid membranes, which
may not be a reliable marker if membranes have been lost over time
. Some dinoflagellates, for example, have plastids with two
membranes that clearly are not of primary origin . Another
example is provided by the transient plastids (i.e., kleptoplastids) of
the sea slug Elysia chlorotica, which have only two membranes,
despite their origin from the stramenopiles Vaucheria litorea. Such
kleptoplastids apparently lost two of the four original plastid
membranes . These observations suggest that loss of plastid
membranes can occur during or after the engulfment of algal
endosymbionts, potentially masking secondary or tertiary origin.
In summary, current data do not provide strong evidence for
monophyly of the host lineage of the Viridiplantae, glaucophytes,
and rhodophytes, whereas our EEF2 data strongly refute the
concept of the Archaeplastida. The observed discrepancy between
the nucleocytoplasmic and the plastid genealogy might be better
explained by postulating multiple acquisitions of plastids in these
eukaryotic lineages. If so, at least one of the ‘primary’ plastids may
actually be of secondary origin.
Evaluation of the supergroup Chromalveolata
The chromalveolate hypothesis, namely that cryptophytes,
haptophytes, stramenopiles, and alveolates arose from a common
ancestor via a secondary endosymbotic event , continues to be
debated [15,17,20,43]. The presence of many, early-diverging
plastid-less taxa within stramenopiles and alveolates [80–82], and
accumulating molecular data, generally conflict with the chro-
malveolate hypothesis or require massive plastid losses, despite the
value of plastids in amino acid, fatty acid and heme biosynthesis, as
well as photosynthesis [13,15]. Lack of any sort of evidence from
nucleus-encoded gene phylogenies casts further doubt on the
chromalveolate hypothesis [8,13]. Although the nucleus-encoded,
plastid targeted glyceraldehyde-3-phosphate
(GAPDH) phylogeny has been presented as evidence for the
chromalveolate hypothesis, cytosolic GAPDH sequences among
‘chromalveolate’ taxa did not form a clade, indicating that
homologs have discordant evolutionary histories [44,83]. In
addition, the plastid-targeted GAPDH tree  is inconsistent
with accepted organismal relationships; the apicomplexan Toxo-
plasma gondii is a sister to haptophytes with strong support, to the
exclusion of peridinin-type dinoflagellates . Overall, the
GAPDH phylogenies seem to be more consistent with multiple
occasions of plastid acquisition among ‘chromalveolate’ taxa.
Plastid-encoded gene phylogenies vary in their level of support for
the chromalveolate hypothesis, depending on taxonomic sampling
and types and number of analyzed genes [42,86–88]. Even when
monophyly of the ‘chromalveolate’ plastids is recovered, it is also
consistent with the ‘‘serial hypothesis’’, which postulates serial
transfer of red algal-derived plastids among ‘chromalveolates’
[88,89]. Finally, recent molecular phylogenies showing that
rhizaria are closely related to stramenopiles and alveolates
[14,56], together with EEF2 evidence presented here appear to
deal a fatal blow to the chromalveolate hypothesis.
Evidence for a new eukaryotic supergroup
Based on EEF2 and some morphological data, we propose an
alternative eukaryotic supergroup that includes cryptophytes,
katablepharids, haptophytes, rhodophytes, and Viridiplantae. We
suggest the name Plastidophila (‘‘friendly to plastid’’) for the
potential clade, because most subclades, except for katablepharid
species and one cryptophyte genus (Goniomonas), are dominated by
plastid-bearing members. Although genomic evidence for Plastido-
phila is yet limited, some morphological features shared among
katablepharids, cryptophytes and Viridiplantae, especially ‘‘early-
diverging’’ prasinophyte green algae, are consistent with this new
concept [62,90–92]. For instance, ejectisomes (i.e., ejectile organ-
elles)ofkatablepharids aresimilartothoseofthe prasinophytegreen
alga Pyramimonas. Although ejectisomes of Pyramimonas form as a
spirally coiled ribbon and those of katablepharids take the shape of
a linear structure . Further, two central flagellar microtubules
that do not penetrate into the flagellar insertion area occur in both
prasinophytes and katablepharids-cryptophytes . In addition,
both the katablepharid Kathablepharis ovalis and the prasinophyte
Pyramimonas possess electron-dense material below the flagellar
terminal plate . The striated root that occurs in katablepharids
has been suggested to be homologous to the system I fibrous roots
found in Viridiplantae . Finally, cell surfaces consisting of a
basal fibrous layer and an upper scaly layer is common to
katablepharids  and scaly green algae such as the ‘‘basal’’
streptophyte green alga Mesostigma  and the prasinophyte
Tetraselmis . Based on comparative morphology, Lee and
Kugrens  and Lee et al.  suggested that katablepharids
represent evolutionary intermediates between cryptophytes and
Viridiplantae. A close relationship between katablepharids and
cryptophytes is supported by SSU and LSU rRNA phylogenies
[8,28].Recent analysesbased on concatenated protein data sets also
suggest a sister relationship between the haptophytes and
cryptophytes (katablepharids were unexamined), although the
phylogenetic position of this clade relative to other eukaryotes
remained unresolved [55,56]. Consistent with these results, our
analyses also suggested that cryptophytes, katablepharids, and
haptophytes are closely related to each other, although a specific
relationship between cryptophytes and katablepharids was not
recovered. If the cryptophyte-katablepharids-haptophyte clade and
the Plastidophila supergroup suggested by EEF2 phylogeny are
indeed correct, it follows that morphological traits common to
katablepharids and ‘‘early-diverging’’ green algae might represent
features that were shared by the common ancestor of Plastidophila.
Hence, katablepharids (plus the cryptophyte Goniomonas) may be
usefulmodels of the heterotrophicflagellate that wasancestralto the
photosynthetic lineage that led to land plants and other algae within
the Plastidophila. Genomic analysis of katablepharids and the
cryptophyte Goniomonas may illuminate nucleocytoplasmic traits of
the plant lineage that existed prior to the massive invasion of genes
from a cyanobacterial precursor to the plastid .
The concepts of Archaeplastida and Chromalveolata do provide
a simple way to explain the distribution of primary and secondary
plastids by minimizing the number of plastid-generating endo-
symbiotic events required. However, our EEF2 data add to a
growing body of evidence that refutes the Archaeplastida and
Chromalveolata. By fostering inaccurate assumptions of relation-
ships, continued use of these supergroup concepts may be
deleterious to progress in studies of ecologically, medically, and
industrially important protists. Given the lack of support for the
monophyly of the Archaeplastida and Chromalveolata, it is
sensible to consider alternative evolutionary models. Based on
EEF2 analysis and some ultrastructural traits, we suggest testing
the concept of a supergroup Plastidophila that links katablephar-
ids-cryptophytes, haptophytes, rhodophytes, and Viridiplantae.
Materials and Methods
Sequencing of EEF2 and cytosolic HSP70 genes
Genomic DNA and/or cDNA were purified from Cyanophora
paradoxa, Goniomonas truncata, Leucocryptos marina (NIES 1335),
PLoS ONE | www.plosone.org7July 2008 | Volume 3 | Issue 7 | e2621
Mesostigma viride, and Thaumatomonas sp. as described in Kim et al.
(2006). EEF2 and cytosolic HSP70 genes, typically ,2.5 Kbp and
,2.0 Kbp in size excluding intron regions, are considered
relatively large for PCR amplification protocols that employ
degenerate primers, so consequently, 2–3 overlapping fragments
were PCR amplified and sequenced to obtain nearly full-length
sequences of each gene or cDNA. Degenerate primers of about
20–30 bp in size were designed to target conserved sequence
regions across diverse eukaryotic taxa within EEF2 and cytosolic
HSP70 genes (Table S1). In most cases, the use of these degenerate
primer pairs enabled the amplification of only partial regions,
hence species-specific primers were subsequently identified from
partial sequencing and used to amplify the adjacent fragment(s)
(Table S2). EST data for C. paradoxa and M. viride were utilized to
identify species-specific primer sites for EEF2 gene amplifications
of these organisms. In many cases, a two-step nested PCR
approach was adapted to obtain larger amounts of PCR fragments
from very little starting DNA material. PCR amplification, PCR
fragment cloning, and sequencing were performed as previously
described . As eukaryotes encode 3 or 4 types of HSP70 (i.e.,
cytosolic, ER, mitochondrial, and plastid forms), each sequenced
HSP70 fragment was carefully examined to verify that it contained
signature sequence sites for the cytosolic form [95,96]. The EEF2
sequence of G. theta was retrieved from the 46genome assembly,
generated by the US Department of Energy Joint Genome
Institute (http://www.jgi.doe.gov/). Newly obtained EEF2 and
cytosolic HSP70 sequences were deposited in GenBank with
accession numbers EU812174–812204 (Table S2).
Molecular sequence analysis
Newly obtained EEF2 and HSP70 sequences were manually
assembled and aligned to sequences downloaded from GenBank
using MacClade ver. 4.08 . Ambiguous regions were excluded.
Phylogenetic analysis was performed based on deduced amino
acid sequences to minimize phylogenetic artifacts caused by codon
usage variations . The final EEF2 and cytosolic HSP70
sequence alignments included 736 and 462 amino acid sites and
had 1.08% and 1.28% missing data, respectively. The two
alignments were analyzed individually and were combined with
a-tubulin, b-tubulin, actin, and cytosolic HSP90 alignments  for
concatenated protein analysis.
Maximum likelihood analysis of amino acid sequence align-
ments was performed using RAxML ver. 7.0.4  and
PhyloBayes ver. 2.3 . For RAxML analysis, ML trees were
inferred with the WAG+C+I+F for the EEF2 data and the
WAG+C+I for the concatenated protein data (4 discrete gamma
rates), and from 100 distinct randomized maximum parsimony
starting trees. The models of protein evolution were selected using
ProtTest ver. 1.4 . For the concatenated data set, the ‘-M’
option was applied so that each protein partition had its own
branch length. Bootstrap analysis was based on 100 re-samplings.
For analysis with PhyloBayes, constant sites were deleted and the
CAT+C model of protein evolution with 4 discrete categories for
gamma distributed rates was applied . Markov chains were
run for 60,000 cycles, the first 5,000 points were discarded as
burn-in, and every 10thtree from the remaining points was
collected to compute the posterior probabilities for individual
nodes. For each analysis, two chains were run in parallel and
compared to check for convergence.
Protein distance analysis was performed using TREE-PUZZLE
ver. 5.2  and FastME . For TREE-PUZZLE analysis,
pairwise maximum likelihood distances were estimated under the
WAG+C+I model with 4 and 8 discrete Gamma distribution rates
for EEF2 and the concatenated data set, respectively. The
resulting distance matrices were then used to construct distance
trees using FastME with the initial tree construction option of the
Greedy Minimum Evolution algorithm and the tree swapping
option of the Balanced Nearest Neighbor Interchanges algorithm.
Bootstrap analysis was based on 100 re-samplings using puzzle-
boot ver. 1.03 (available from www.tree-puzzle.de). Bootstrap
datasets were generated using the SEQBOOT program from the
PHYLIP package ver. 3.66 .
Found at: doi:10.1371/journal.pone.0002621.s001 (0.37 MB EPS)
Found at: doi:10.1371/journal.pone.0002621.s002 (0.27 MB EPS)
Found at: doi:10.1371/journal.pone.0002621.s003 (0.28 MB EPS)
We thank B. Larget and A. J. Roger for access to computation facility for
phylogenetic analysis, A. G. B. Simpson for providing initial sequence
alignments, and A. D. Tsaousis for Greek translation. J. M. Archibald, D.
F. Spencer, and A. J. Roger provided helpful comments on the manuscript.
We also thank J. M. Archibald, M.W. Gray, P. J. Keeling, G. I. McFadden
and C. E. Lane for providing the nucleus-encoded EEF2 sequence from
Guillardia theta, which was obtained from preliminary genome sequence
data produced by the Joint Genome Institute’s Community Sequencing
Conceived and designed the experiments: EK. Performed the experiments:
EK. Analyzed the data: EK. Contributed reagents/materials/analysis
tools: EK LEG. Wrote the paper: EK LEG.
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