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
PLoS ONE | www.plosone.org1 July 2008 | Volume 3 | Issue 7 | e2621
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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