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CorrespondenCe
Yet viruses cannot be included in the
tree of life
Purificación López-García and David Moreira
We welcome the comments on our Review
article (Ten reasons to exclude viruses from
the tree of life. Nature Rev. Microbiol. 7,
306–311 (2009))1 by Bayry and colleagues
(Reasons to include viruses in the tree of life.
Nature Rev. Microbiol. 29 June 2009 (doi:
10.1038/nrmicro2108-c1))2; Navas-Castillo
(Six comments on the ten reasons for the
demotion of viruses. Nature Rev. Microbiol.
29 June 2009 (doi: 10.1038/nrmicro22108-c2))3; Claverie and Ogata (Ten good
reasons not to exclude giruses from the evolutionary picture. Nature Rev. Microbiol. 29
June 2009 (doi: 10.1038/nrmicro2108-c3))4;
Ludmir and Enquist (Viral genomes are
part of the phylogenetic tree of life. Nature
Rev. Microbiol. 29 June 2009 (doi: 10.1038/
nrmicro2108-c4))5; Koonin and colleagues
(Compelling reasons why viruses are
relevant for the origin of cells. Nature Rev.
Microbiol. 29 June 2009 (doi: 10.1038/
nrmicro2108-c5))6; and Raoult (There is
no such thing as a tree of life (and of course
viruses are out!) Nature Rev. Microbiol. 29
June 2009 (doi: 10.1038/nrmicro2108-c6))7.
They offer us an opportunity to clarify
our ideas further and to dissipate some
confusion owing to the mixing of very different concepts and levels of interpretation
regarding the actual possibility of including
viruses in a tree of life (TOL). We realize that
much of this confusion comes from the fact
that many virologists and other biologists
are not familiar with the theory and practice
of molecular phylogeny. Their reaction is
therefore more sentimental than rational, as
if denying the possibility of including viruses
in the TOL would imply dismissing their
importance in evolution. First, we emphasize
that we agree that viruses have been, and still
are, important for the evolution of cellular
organisms. They are vehicles of horizontal
gene transfer (HGT) between cells, contribute to the acceleration of gene evolutionary
rates and exert fundamental selection
pressures on their host populations, thereby
generating and regulating biodiversity (for
example, through ‘kill-the-winner’ strategies8). However, their importance in biological evolution does not necessarily authorize
their inclusion in the TOL. Let us make clear
the essential points of our opinion article
in a more systematic way. We stated that
viruses do not belong in the TOL based on
two types of arguments: epistemological
arguments, which refer to what ‘should not
be done’, and methodological arguments,
which refer to what ‘cannot be done’ and
are more practically conclusive.
Our first argument relates to the definition of life and whether viruses are alive. It is
epistemological in nature and deals with the
way in which humans conceptualize their
surrounding natural world. It is neither metaphysical, as Koonin et al.6 state, nor religious
(even much less so) as unfoundedly claimed7.
As biological scientists, we deal exclusively
with empirical data from the material world,
from which we extract information that
allow hypotheses to be constructed and
tested through the hypothetico–deductive
methodology that is the common practice
in our discipline. Any form of spirituality,
including religion7 or intelligent design4, is
out of the scope of our scientific activities.
We disagree that defining life should be left
to philosophers alone6, as it is biologists who
actually study life. Defining life, as defining
species, is a problematic issue owing to the
difficulty of delimiting barriers to some kind
of natural continuum. However, once a definition is established it should be applied logically. When some biologists say that viruses
are alive, they are accepting some kind of
definition. A logical syllogism would ensue:
if viruses were alive they ‘should be’ placed
in the TOL (epistemologically, an ideal construction in which all living entities would
have a place) whereas if viruses were not alive
they ‘should not’. The problem is that current
definitions of life based on self-replication
and evolution do not accommodate viruses
because viruses cannot self-replicate. This is
not because of the occurrence or absence of
ribosomes, as is often thought5,9, but because
of the fact that viruses, unlike cells (including
cellular parasites), are unable to transform
energy and matter (that is, to actively generate order from disorder). In this sense, we
agree with van Regensmortel10 that viruses,
as genes or transposons, are biological elements but not living entities. By contrast,
a self-replicating molecule, for example a
self-replicating ribozyme, would be alive
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according to the above definition. However,
viruses are not self-replicating ribozymes
or DNAzymes and consequently cannot be
qualified as living. Nevertheless, one can
avoid defining life or use a more inclusive
definition to accommodate viruses. Let us
accept for the benefit of this discussion that
anything that can be replicated (that is, does
not necessarily self-replicate) and evolves
is alive. Viruses and, by the same token,
languages, popular legends, cooking recipes
and human technology, would be alive. We
contend that it would still be impossible to
place viruses in the TOL because of purely
methodological reasons (nine reasons out
of the ten that we proposed1).
Regarding the methodological arguments
to exclude viruses from the TOL, we must
differentiate between two types of hypotheses
and we must also explain what a TOL is in
this context: a molecular phylogenetic tree
based on universally conserved (core) genes
that are supposed to reflect organismal evolution. Whether this molecular phylogenetic
tree truly reflects the most important lines of
organismal evolution is a matter of discussion but, in practice, it is helpful and the basis
of natural systematics (for an example, see
the Tree of Life Web Project). Paradoxically,
those that propose that a TOL does not exist
because it is blurred by HGT use molecular
phylogenies to show HGT11 and recognize
the existence of archaea, primarily defined
as an independent domain of life by their
segregation in a conserved gene-based TOL.
The first type of hypotheses, sustained
by Koonin et al.12 and others before them13,
propose that viruses pre-date cells in biological evolution. Koonin et al.6,12 recognize
that “viruses do not have universal genes”
but claim that there is a core of ancient
(precellular) virus hallmark genes that have
persisted to date, dismissing convergence and
HGT as explanations for their widespread
distribution. We acknowledge that many
viral genes are shared within viral families,
but we still claim that dating them back to
a common precellular origin is impossible
because viruses are polyphyletic and because
structural convergence and HGT are wellattested phenomena that can account for that
distribution. Although simple structures are
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CorrespondenCe
1
0.4
Aquifex aeolicus (NP_214275)
Thermotoga maritima (NP_228495)
Petrotoga mobilis (YP_001568440)
Psychroflexus torquis (ZP_01251649)
1
.62
Bacteroides fragilis (YP_100510)
Bacillus subtilis (ZP_03589675)
.82
1
Streptococcus agalactiae (ZP_00789310)
Dehalococcoides ethenogenes (YP_181327)
.97
Gloeobacter violaceus (NP_924555)
.63
Nostoc sp. PCC7120 (NP_488972)
1
1
Synechococcus sp. BL107 (ZP_01469011)
Treponema denticola (NP_97318)
.37
Streptomyces coelicolor (NP_628248)
1
1
Bifidobacterium longum (ZP_00121363)
Geobacter metallireducens (YP_386359)
.62
1
Myxococcus xanthus (YP_630176)
Escherichia coli (YP_001457315)
.50
1
Neisseria gonorrhoeae (YP_207872)
Magnetococcus sp. (YP_865652)
.63
1
Rhodobacter sphaeroides (YP_353787)
.57
Rickettsia rickettsii (YP_001650684)
Sulfolobus solfataricus (NP_342275)
Thermofilum pendens (YP_919680)
1 1
Pyrobaculum aerophilum (NP_558807)
Ignicoccus hospitalis (YP_0014358)
1
Staphylothermus marinus (YP_001041227)
1 .74
Pyrococcus horikoshii (NP_142122)
Methanococcus voltae (ZP_02193668)
1
Thermoplasma volcanium (BAB60660)
1
.54
Picrophilus torridus (YP_023365)
Natrialba
magadii (ZP_0369220)
1
1
Halobacterium sp. (NP_280914)
.57
Methanosaeta thermophila (YP_842699)
Archaeoglobus fulgidus (NP_070884)
.66
Mimivirus R395 (YP_142749)
.83
Entamoeba histolytica (XP_651156)
1
Entamoeba invadens (AANW02000208)
.94
Hemiselmis andersenii (XP_001712450)
.92
Cyanidioschyzon merolae (CMF110C)
Paramecium
tetraurelia
(XP_00146169)
.45
Trichomonas vaginalis (XP_001301493)
1
.74
1
E. siliculosus virus (NP_077572)
Toxoplasma gondii (EEB04101)
.75
.68
Babesia bovis (XP_001609386)
.84
Plasmodium falciparum (XP_001350805)
Trypanosoma brucei (XP_829019)
.78
.37
Naegleria gruberi (JGI_68675)
Phaeodactylum tricornutum (XP_002184059)
1 1 Thalassiosira pseudonana (XP_002296611)
.68
Aureococcus anophagefferens (JGI_59087)
.91
Phytophthora sojae (JGI_108506)
Chlorella vulgaris (JGI_00341)
.72 .84
Micromonas sp. (ACO68741)
1 Physcomitrella patens (XP_001752392)
Aspergillus fumigatus (XP_747574)
.97
1
Candida glabrata (XP_447581)
.95
Pan
troglodytes
(XP_001149874)
.81
1
Hydra magnipapillata (XP_002160048)
.55
Dictyostelium discoideum (XP_629875)
.95
Physarum polycephalum (Contig4271)
Babesia bovis (XP_001611428)
1
Plasmodium falciparum (XP_001349685)
.84
Paramecium tetraurelia (XP_001424767)
1
Tetrahymena thermophila (PEP_TTL00001239)
Dictyostelium discoideum (XP_637900)
.89
Mimivirus R510 (YP_142864)
Entamoeba invadens (AANW02000319)
.73 .86
Cyanidioschyzon merolae (CML307C)
.81
Drosophila persimilis (XP_002022783)
.93
Monodelphis domestica (XP_001370165)
.72
.88
Trichoplax adhaerens (XP_002111821)
.91
Aspergillus fumigatus (XP_754784)
.61
Debaryomyces hansenii (XP_461055)
.65
E. siliculosus virus (NP_077667)
.89
.83
Phytophthora sojae (JGI_134191)
.79
Trichomonas vaginalis (XP_001323818)
Physcomitrella patens (XP_001767691)
.77
Micromonas pusilla (EEH58311)
.88
.83
Ostreococcus lucimarinus (XP_001419724)
Entamoeba invadens (AANW02001003)
Entamoeba
hystolytica (XP_651283)
1
.91 Entamoeba moshkovskii (mosh123d08)
Mimivirus L499 (YP_142853)
Naegleria gruberi (JGI_29498)
.79
Trypanosoma cruzi (XP_808715)
.93
Trichomonas vaginalis (XP_001329029)
.69
Cryptosporidium hominis (XP_667086)
Plasmodium yoelii (XP_72452)
.93
.84
.76
E. siliculosus virus (NP_077709)
.86
Tetrahymena thermophila (PEP_TTL00008276)
.77
.99
Paramecium tetraurelia (XP_001435167)
Physcomitrella patens (XP_001771105)
.89
Chlorella vulgaris (JGI_120255)
.82
Ostreococcus lucimarinus (XP_001416450)
.79
.69
Phytophthora sojae (JGI_109673)
Aureococcus anophagefferens (JGI_71065)
1
Phaeodactylum tricornutum (XP_002179670)
.73
1
Thalassiosira pseudonana (XP_002293495)
Dictyostelium discoideum (XP_640301)
1
Dictyostelium
purpureum (GID1_0043829)
.76
Laccaria bicolor (XP_001875137)
1
Saccharomyces cerevisiae (EDN6253)
.73
Monosiga brevicollis (XP_001751040)
Pan troglodytes (XP_001156346)
1
Drosophila virilis (XP_002051157)
1
.85
Nematostella vectensis (XP_001633193)
.94
Bacteria
Archaea
Eukaryotes
Paralogue 1
Eukaryotes
Paralogue 2
Eukaryotes
Paralogue 3
Figure 1 | Taxon-rich phylogenetic tree of clamp
loader proteins. This 106-taxa tree was constructed on 174 conserved sites with the Bayesian
approach implemented in PhyloBayes, using a mixture model (CAT) that was less sensitive to compositional bias and evolutionary rate heterogeneity
between species18. Note that, in contrast to Claverie
and Ogata’s 20-taxa tree4, there is not a single
eukaryotic group but three distinct paralogues and
that, as a consequence of the richer taxonomic
sampling, viral sequences emerge within (and not
at the base of) the eukaryotic groups, suggesting
that they were acquired by horizontal gene transfer
from their eukaryotic hosts. Numbers at nodes are
posterior probabilities. Sequence accession
numbers are given in parentheses.
more prone to convergence, structures that
are more complex can also be affected by it
(for example, the presence of complex eyes in
some unicellular dinoflagellates is an amazing
case of convergence with metazoan eyes14).
Koonin et al. maintain that a “virus-like stage
of precellular evolution appears inevitable”.
There is some ambiguity about what ‘viruslike’ implies (not all genetic elements are
viruses; the distinction is important in this
debate). If virus-like implies self-replicating
elements, we might agree, but if it implies
genetic elements that are dependent for replication on other systems (what viruses actually
are), we disagree. It would seem more logical
that life derives from those systems that
were already able to replicate than from their
molecular parasites (these might, however,
foster the evolution of such self-replicating
systems, just as viruses contribute to cell
evolution today). Nonetheless, the problem is
that this hypothesis cannot be tested. It cannot be proved or disproved by phylogenetic
analysis with contemporary empirical data.
Consequently, hypotheses proposing that
viruses antedate cells, however unlikely they
might be (it is difficult to imagine that parasites pre-date their hosts), cannot be falsified
and remain valid. However, proponents of
such models would agree with us that viruses
cannot be placed in a phylogenetic TOL. This
is methodologically impossible as they share
no universally conserved genes with cells.
If such genes ever existed, their sequences
have evolved beyond homology recognition,
losing all phylogenetic signal from putative
pre-cellular times.
The second type of hypotheses on virus–
cell relationships, including those of Raoult
et al.15 and Claverie and Ogata4, claim that
viruses (particularly large DNA viruses such
as the Mimivirus) can indeed be placed in
a TOL on the basis of phylogenetic analysis
of their conserved cell-like genes (those that
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CorrespondenCe
are used to construct the cellular, however
imperfect, TOL by molecular phylogeny).
On the basis of these analyses, viruses would
define a fourth domain of life4,15. Ironically,
Raoult, who was the first to propose that the
“Mimivirus appears to define a new branch
distinct from the three other domains”
(Ref. 15), partly motivating our article1, now
contradicts himself saying that there is no
TOL7. Claverie and Ogata, on the contrary,
maintain that position4 and present an
additional tree of the clamp loader protein
from Mimivirus (MIMI_R395) and from
Ectocarpus siliculosus virus-1 (ESV-1) with
their cellular homologues. The viruses
appear at the base of eukaryotes, which is
taken as “evidence of deep Mimivirus gene
ancestry” (Ref. 4). This kind of assertion
can be tested. using proper phylogenetic
analyses, the vast majority of genes shared
by Mimivirus and cells can be shown to have
been acquired by recent (by contrast to precellular or pre-domain diversification) HGT
from their cellular hosts (and associated bacteria) and not the other way around16. The
apparent basal positions of Mimivirus genes
are easily explained both by long-branch
attraction artefacts owing to the higher
evolutionary rate of these genes in viruses
and by poor taxon sampling. The case of the
clamp loader is illustrative, as Claverie and
Ogata’s tree includes a poor representation
of taxa4. Homologues from the lineages to
which Mimivirus and ESV-1 hosts belong
(amoebae and stramenopiles) are excluded.
A taxon-rich, detailed phylogenetic analysis
shows a more complicated history for
this gene, which has three paralogues in
eukaryotes. Interestingly, Mimivirus also has
three copies of the gene. However, instead
of being at the base of the three eukaryotic
branches (which would be expected if
the Mimivirus genes were ancestral), each
Mimivirus gene appears nested within its
respective paralogue group close to amoebae
genes (fIG. 1). Similarly, ESV-1 appears nested
within the eukaryotic groups (the slowest
evolving ESV-1 gene — paralogue 2 — even
branches with a stramenopile sequence),
far from any of the Mimivirus copies.
This demonstrates that the viral clamp
loader genes were recently, and independently, acquired by HGT from eukaryotic
hosts, and highlights the importance
of adequate taxonomic sampling 17. By
showing their tree of the clamp loader,
Claverie and Ogata provide a further
example of what ‘cannot be done’ from
a molecular phylogenetic point of view:
concluding that large DNA viruses form
an independent domain in the TOL from
a poor phylogenetic analysis using genes
that can be shown to have been acquired
by HGT from cells. This and previous
analyses argue for massive cell-to-virus
HGT1,16. Consequently, these hypotheses
proposing that viruses define a fourth
domain of life that can be placed in a TOL
can be, and in our opinion have been,
experimentally refuted.
In conclusion, even if viruses were
considered to be alive and pre-date primitive cells, viruses could not be placed in a
universal TOL by purely methodological
reasons owing to the absence of shared
genes and/or the loss of phylogenetic signal
over billions of years of evolution. The claim
that viruses can be placed in a TOL using
cell-like genes is based on artefactual results
and can be shown to be wrong.
NATuRE REVIEWS | Microbiology
Purificación López-García and David Moreira are at
the Unité d’Ecologie, Systématique et Evolution, CNRS
UMR 8,079, Université Paris-Sud 11, bâtiment 360,
91405, Orsay Cedex.
e-mail: [email protected], [email protected]
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