The prokaryotic tree of life: past, present... and future?

The prokaryotic tree of life: past,
present. . . and future?
James O. McInerney1, James A. Cotton2 and Davide Pisani1
1Department of Biology, National University of Ireland Maynooth, Maynooth, County Kildare, Ireland
2School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, UK
No accepted phylogenetic scheme for prokaryotes
emerged until the late 1970s. Prior to that, it was
assumed that there was a phylogenetic tree uniting all
prokaryotes, but no suitable data were available for its
construction. For 20 years, through the 1980s and 1990s,
rRNA phylogenies were the gold standard. However,
beginning in the last decade, findings from genomic
data have challenged this new consensus. Gene trees
can conflict greatly, and strains of the same species can
differ enormously in genome content. Horizontal gene
transfer is now known to be a significant influence on
genome evolution. The next decade is likely to resolve
whether or not we retain the centuries-old metaphor of
the tree for all of life.
Where does the tree come from?
The use of trees to model evolutionary relationships can be
traced back to the work of Jean-Baptiste Lamark. How-
ever, it was Haeckel who was mainly responsible for
popularising the idea [1]. Indeed, the tree metaphor was
so effective that the search for a unique tree, representing
the relationships among all cellular organisms, has con-
tinued to this day [2–9].
From the very beginning, plant and animal phylogenies
could be based on embryological and morphological char-
acters. However, there was no such luxury for the prokar-
yotes, as these organisms lack complex intracellular
structures and have extremely simple external
morphologies [1]. In the first half of the last century, the
lack of phylogenetically informative prokaryotic characters
resulted in a great debate centered on whether or not it
would ever be possible to recover a sensible phylogenetic
classification for this group. Many were convinced that a
simple, ‘phenetic’ classification of the prokaryotes was
more useful than a phylogenetic one and, from its inception
in 1923 and for some time afterward, Bergey’s Manual of
Determinative Bacteriology disregarded any attempt to
classify prokaryotes according to phylogenetic principles.
The feeling among the editors of Bergey’s Manual was
summarised by Breed [10] in 1939 when he distinguished
between ‘realistic workers’ and ‘idealists,’ stating that
idealists had introduced ‘‘unjustified speculations regard-
ing relationships between the various groups of Bacteria.’’
Stanier and van Niel [11] rejected this viewpoint
and opined in 1941 that when it came to classifying
prokaryotes, ‘‘there is a good reason to prefer an
admittedly imperfect natural system to a purely empirical
one.’’ Interestingly, in hindsight, Stanier and van Niel
queried why the absence of sexual reproduction was not
included by the editors of Bergey’s Manual as a defining
feature of prokaryotes. At that time it was generally
accepted that prokaryotic reproduction was clonal and that
all genetic material was vertically inherited.
Review
Glossary
Archaebacteria: Single-celled organisms lacking a nucleus (prokar-
yotes), with ether-linked lipids in their membranes and lacking
murein in their cell walls.
Compositional bias: Not all DNA and protein sequences in all organ-
isms have the same nucleotide or amino acid composition. Deviation
from an equal composition of all nucleotides or amino acids is typical
and, unless these compositional differences are adequately
accounted for in evolutionary analyses, the resulting phylogenetic
tree can often appear to be more like a classification of sequences
according to their composition rather than their evolutionary history.
Eubacteria: Single-celled organisms lacking a nucleus (prokaryotes),
with ester-linked lipids in their membranes and murein in their cell
walls.
Eukaryotes: All single-celled and multicellular organisms with a
membrane-bound nucleus.
Long-branch attraction: In a situationwhere different lineages evolve
at different rates, rapidly evolving sequences can converge on the
same character state. Some phylogenetic methods, most notably
maximum parsimony, can interpret these homoplastic characters as
synapomorphies and place the long-branch lineages as sister taxa.
Methods of avoiding long-branch attraction include the use of more
appropriate evolutionary models and the breaking of long branches
by the addition of appropriate taxa.
Orthology: Two genes that are members of the same gene family are
said to be orthologs if they trace their most recent common ancestor
to a speciation event.
Paralogy: Two genes that are members of the same gene family are
said to be paralogs if they trace their most recent common ancestor
to a gene duplication event.
Prokaryotes: Unicellular organisms without a membrane-bound
nucleus (see Box 3).
Quartet-based tree construction: It is often desirable to break up an
evolutionary analysis into a series of smaller analyses. The smallest
nontrivial phylogenetic tree is composed of four sequences. The
analysis of all or a large number of the possible quartets that can
be made from a data set can provide useful information for evaluat-
ing congruent phylogenetic signals, for making phylogenetic trees
and for making phylogenetic supertrees.
SSU rRNA: The RNA component of the small subunit of the ribo-
some.
Supertree: A supertree is a phylogenetic tree that is generated by
amalgamating several phylogenetic trees into a single tree. If the leaf
sets on the input trees are not identical, then the resulting tree is a
supertree. If the leaf sets are identical on all the input trees, the result
is a type of consensus tree.
Corresponding author: McInerney, J.O. (james.o.mcinerney@nuim.ie).
276 0169-5347/$ – see front matter ! 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tree.2008.01.008 Available online 25 March 2008

Five years later, Lederberg and Tatum [12] presented
evidence for ‘‘the existence of a sexual stage’’ inEscherichia
coli, showing for the first time that genetic recombination
existed in prokaryotes. A decade later, Stanier and col-
leagues wrote about van Niel that he had completely
revised his position and ‘‘has expressed the opinion that
it is a waste of time to attempt a natural classification for
bacteria’’ [13]. This skepticism pervaded the microbiology
community for almost another 20 years, during which
bacteriology had to proceedwithout an evolutionary frame-
work.
The rise of the small-subunit rRNA-based tree of life
The advent of molecular phylogenetics heralded another
sea-change in the perceived usefulness of a prokaryotic
phylogeny. In the 1960s, Zuckerkandl and Pauling [14]
defined the new research area of molecular evolution.
Within a decade, Woese and colleagues [2,3], using indirect
methods of oligonucleotide cataloguing from small-subunit
rRNA (SSU rRNA), identified one particularly important
split within the prokaryotes: that separating the Archae-
bacteria from the Eubacteria. The identification of this
split [2,3] meant that theremight be a thirdmajor category
of living things on the planet, as divergent fromEubacteria
and Eukaryota as each is from the other. Confirmation of
themonophyly of these three potential groups seemed to be
found in 1989, when the SSU rRNA tree was rooted using
anciently duplicated genes [15,16].
These early studies showed that there was substantial
phylogenetic structure within the prokaryotes, and
prompted microbiologists to look at their diversity from
a new evolutionary point of view. From then on, phyloge-
netic studies of prokaryotic evolution took off. By 1980,
SSU rRNA had been characterised from around 170 pro-
karyotic species, giving a broad outline of the major eubac-
terial and archaebacterial groups [17]. By 1987, when Carl
Woese published his authoritative 50-page review ‘‘Bac-
terial Evolution’’ summarising his earlier work [18],
around 500 Eubacteria had already been characterised
by oligonucleotide cataloguing, and complete SSU rRNA
sequences were coming onstream. This led to the view of
the SSU rRNA as ‘‘the ultimate molecular chronometer’’
[18]. Optimism concerning the existence of a single unify-
ing tree of life was at its peak; the SSU rRNA tree was
crowned as the ‘‘universal tree of life,’’ and was used to
devise a new (three-domain-based) phylogenetic classifi-
cation of life [19]. The legacy of this work is that today there
are more than 400 000 SSU rRNA sequences in the public
sequence repositories, the vastmajority of which have been
sequenced for phylogenetic purposes.
Genomics and the rise of the network of life
hypothesis
During the 1990s, gene sequences started to accumulate at
an ever-increasing rate, and by 1995 the first complete
eubacterial genome was publicly available [20]. As more
genetic markers became available, the universal tree of
life was put to the test ever more frequently. Interestingly,
phylogenies inferred from alternative markers were often
found to be incongruent with the topology of the SSU rRNA
tree [21]. Some of these incongruent phylogenies were
based on genes whose biological function was as essential
as that of the SSU rRNA (e.g. the RNA polymerase gene
[22]). Accordingly, it became less clear why the SSU rRNA
should have been preferred over other markers to derive
the universal tree of life [23]. The impression that the SSU
rRNA tree might be inadequate was further exacerbated
by the realization that trees derived from markers that
initially seemed to support the SSU rRNA tree (e.g. ATP
synthase and the elongation factors) were unreliable either
because they were affected by horizontal gene transfer
(HGT [24]) or because of phylogenetic artifacts (e.g. long-
branch attraction [25,26]). Finally, the presumed super-
iority of the SSU rRNA gene as a phylogenetic marker was
revealed to be untenable when it was discovered that the
placement of many groups (e.g. the Microsporidia [22] – a
group of fungi) in the SSU rRNA tree was the result of
systematic errors (e.g. long-branch attraction).
These findings caused a shift in thinking. Among the
first to state that at least for some genes and organisms
there was a network rather than a tree of life were Hilario
and Gogarten [24] and Martin and colleagues [27]. This
viewpoint became increasingly popular as the first com-
parative genomic studies showed that the genomes of
organisms that were closely related in the universal tree
of life had significantly different gene content. In particu-
lar, Lawrence and Ochman claimed that the genome of E.
coli acquired 18% of its genes via HGT after its divergence
from its closest relative (Salmonella enterica) [28]. Because
E. coli and S. enterica diverged!100million years ago, this
might have meant that the vertical phylogenetic signal in
the E. coli genome could be completely erased every 500
million years, assuming the rate of HGT per gene was
homogeneous through time. Even vertebrates have a fossil
history that is longer [29], whereas cellular life is likely to
have existed on Earth for more than 3.8 billion years [30].
However, rates of HGT are gene specific, and vertically
inherited genes might persist in bacterial genomes for
more (or less) than 500 million years. On the other hand,
even if a gene can persist in a genome for more than 500
million years, it seems unlikely that it might persist in a
genome for billions of years. In fact, it has recently been
shown that every gene family can be transferred intoE. coli
[31], and that (at the least in the laboratory) barriers to
HGT are very low. We do not know whether this result will
hold for prokaryotes in general. Nonetheless, it implies
that at most prokaryotes can swap their genes with E. coli
very easily, and that a gene that could be considered a
universal chronometer that has kept track of the clonal
history of cellular life since its origin is thus unlikely to
exist [32]. Indeed, if the HGT rates observed inE. coliwere
found to apply to all prokaryotes, their evolutionary history
would be essentially horizontal. That is, prokaryotes would
effectively share a common gene pool and, from an evol-
utionary point of view, they would behave in a way that is
not unlike that of the populations of a single species.
The new uncertainty at the end of the last century was
different from that of half a century before. Prior to the
molecular era, the absence of data caused uncertainty.
With the arrival of complete genomes, uncertainty was
not anymore caused by lack of data. Instead, now the very
existence of the universal tree of life was on trial.
Review Trends in Ecology and Evolution Vol.23 No.5
277

The sound of ideologies clashing
Toward the end of the 1990s, asmore andmore prokaryotic
genomes were being sequenced, it became customary to
report the proportion of their genes that were of foreign
origin. Methods used to identify horizontally transferred
genes were based on the identification of compositional
biases (either codon usage biases or nucleotide composition
biases; e.g. Ref. [28]) and BLAST score analysis (reviewed
in Ref. [33]). These methods (see Box 1) have their limita-
tions, and their results were relatively inaccurate, and
often incongruent [21]. However, they consistently showed
that HGTwas anything but rare, and in some cases foreign
genes seemed to have been transferred between organisms
that were very distantly related (according to the SSU
rRNA tree). For example, it was reported that 24% of
the genome of the bacterium Thermatoga maritima [34]
was acquired from archaebacterial donors, whereas 30% of
the genes in the genome of the Archaebacteria Methano-
sarcinamazei andM. acetivoranswere of eubacterial origin
[35,36]. Obviously,many researchers remained skeptical of
HGT [37–39], and in particular Kurland went as far as
stating that ‘‘global LGT’’ (i.e. the view that HGT was a
major evolutionary force; LGT = HGT) was ‘‘an ideology
that is begging for deconstruction’’ [38]. Nevertheless, the
view that HGT was a ‘‘rampant’’ phenomenon gained
credibility, and was eventually formalised by Doolittle,
who concluded ‘‘the history of life cannot properly be
represented as a tree’’ [23]. According to Doolittle, HGT
was such a powerful force that the evolutionary history of
the prokaryotes was better represented using a network in
which edges represent HGTs (see Figure 1).
The last word was still far from said. Studies attempting
to estimate global rates of HGT could not agree (see above),
and methods to identify these events (see Box 1) were
questioned [40–43]. This cast doubt on the real frequency
of HGT, and on the network of life hypothesis [38,39]. Yet,
when the genomic sequences of three ecologically distinct
E. coli strains were compared, it was shown that they
Figure 1. A network of life. (a) Eukaryotes are known to be chimeric, with chloroplast and mitochondrial genes having a different origin from nuclear genes, and originating
from different groups of Eubacteria. There is more debate over whether lateral gene transfer between Eubacteria, Archaebacteria and Eukaryota and between major groups
of prokaryotes is common or rare. (b) Lateral gene transfer is known to be so common between members of some groups of bacteria that they are effectively panmictic,
whereas (c) it can be completely absent from other groups.
Box 1. Identification of HGT
A variety of approaches have been developed to identify genes that
have been laterally transferred. These include BLAST-based
approaches and methods that compare base composition or codon
usage biases of different genes in a set of genomes. Discovering
HGT is best carried out by identifying significant disagreement
between phylogenetic trees inferred from different genes. This
approach assumes that there exists an underlying species phylo-
geny with which alternative gene trees can be compared, and with
which they should be congruent in the absence of HGT, paralogy
and systematic biases (like those caused by long-branch attraction
and amino acid composition bias). Any phylogenetic tree is a
statistical inference from the sequence data and, like any inference,
is subject to error. Careful analysis is thus needed to identify
significant incongruence which cannot be explained as being a
result of estimation error or paralogy, and so is potentially indicative
of HGT. The tree-based approach to identifying HGT has the
disadvantage that one has to assume a reference tree with which
the individual gene trees are compared. Ideally, this reference tree
should be the organismal phylogeny of the considered taxa.
However, as we pointed out (see main text), this tree might not
exist. This is not necessarily a problem, as any nonrandom tree, for
example, a genomic tree (see Box 2), which might not represent a
species tree (see text), might be used as a reference to infer HGTs. In
fact, because in the absence of HGT every tree derived from a set of
orthologs should have the same topology, even a single gene tree,
such as the SSU rRNA [68], can be used as a reference against which
the congruence of other gene trees might be compared to infer
potential HGT events. However, if the source of incongruence
between a gene tree and a reference tree is phylogenetic inaccuracy
rather than HGT, tree-based approaches will overestimate HGT.
Review Trends in Ecology and Evolution Vol.23 No.5
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shared only 40% of their genes [44]. This patchy
distribution of genes could be explained by assuming that
the genome of the common ancestor of the three E. coli
strains included all the genes found in the genomes of its
descendants. The partially overlapping distribution of
genes would then be the result of lineage-specific gene
deletions. However, this explanation would entail that
the last common ancestor of these strains had an unrea-
listically large genome. A more reasonable explanation
would be that most of the genes that are not shared among
the three strains (!60% of the total) have been indepen-
dently acquired by HGT. Similarly, the analysis of the
genomes of five Cyanobacteria showed that also the
chromosomes of these organisms were littered with hori-
zontally transferred genes [45].
Evidence accumulating during the 1990s and the ear-
liest years of the new millennium seemed to suggest that
there was no tree of life, but counterarguments were also
being proposed. Phylogenetic model misspecification and
misidentification of orthologs are confounding influences
that might cause the overestimation of HGT (see Box 1).
Additionally, even if HGT were rampant, it seemed that
not all genes were equally likely to be transferred [46,47].
This observation was formalised as the complexity hypoth-
esis by Lake and coworkers [46], who suggested that
operational genes (those involved in the day-to-day pro-
cesses of cell maintenance) are more likely to be trans-
ferred than informational genes (those involved in DNA
replication, transcription and translation), and that a core
set of nontransferable or rarely transferable genes might
exist.
In August 2003, Daubin and his collaborators [48], using
a variety of interspecies and intraspecies data sets and a
quartet-based method, attempted to test whether trees
inferred from protein-coding genes significantly disagreed
with the SSU rRNA tree. Using this approach, they con-
cluded that ‘‘orthologs available for phylogenetic recon-
struction are compatible with a single tree.’’ The same
month, Kurland and his coworkers [39] published a tren-
chant criticism of the notion that interspecies gene transfer
had obliterated the signal of vertical descent. This criticism
focused on the methods that were being used to infer
HGTs, which did not seem particularly robust (see above
and Box 1). Additionally, they also suggested that although
HGT might result in a gene finding its way into a genome,
this did not necessarily mean that it would stay for an
appreciable length of time; its presence might only be
transient.
Kurland and his coworkers forcefully claimed that a
prokaryotic tree existed even in the presence of HGT, and
provided some sensible arguments to support their claim
[39]. However, even if a universal tree of life exists, it does
not mean that this tree is also recoverable. We have used
supertree approaches (see Box 2) to investigate congruence
across large sets of ortholog trees. These studies concluded
that for relatively recent groups of prokaryotes such as the
g- and the a-Proteobacteria, congruence is high. Conver-
sely, at the deepest levels of prokaryotic history, congru-
ence fades significantly [7,9]. Even if a prokaryotic tree
exists [37–39,49,50], its most basal nodes might not be
recoverable because of phylogenetic signal erosion [51],
HGT, ortholog misidentification or a combination of all
three.
Future prospects for the tree of life
In 1998, Woese proposed his genetic annealing model for
the earliest stages of prokaryotic life [52]. In this model,
HGT was initially the dominant mode of evolution within
single-celled communities. Later on, HGT became less
frequent and vertical inheritance became dominant (as
in the species seen today). However, more recently, Gold-
enfeld and Woese [53] stated that HGT seems to have been
so pervasive that it must be one of the most significant
parts of any discussion about species or phylogenies. The
minimal assumption for the existence of a prokaryotic tree
is the existence of a core set of nontransferable (i.e. verti-
cally inherited) genes, but evidence suggests that such a
core set of genes cannot exist. Informational genes, in-
cluding rRNA, can and have been horizontally transferred
[21,54–58]. Some of these genes have been extensively
swapped between domains. Probably the most extreme
case is that of the aminoacyl tRNA synthetases [55], as
none of the genes in this family agree with the SSU rRNA
tree. Additionally, Miller and colleagues found a hybrid
SSU rRNA gene in a chlorophyll d-producing cyanobacter-
ium [59]. Finally, in E. coli [31], barriers to HGT are very
low, and no single gene family is completely untransferable
(by laboratory approaches) into this species [32].
As currently defined, the core represents less than 1% of
the average prokaryotic genome [60] and its existence is
Box 2. Inferring phylogenies from multigene data sets
Three main approaches can be used to infer phylogenies from large
collections of genes
The gene concatenation approach
The sequences of different genes are concatenated into a single data
set, which is then analysed using one of the available phylogenetic
methods (e.g. maximum likelihood, parsimony, Bayesian analysis
and so on; see e.g. Refs [8,69]). The obvious advantage of this
approach is that it can reduce (potentially even eliminate [69])
stochastic error. It also allows the combination of weak phylogenetic
subsignals in different genes [51], which might result in the
discovery of new clades that do not obtain significant support from
single gene analyses. This approach ignores HGT, as it assumes that
all loci have the same history. Gene concatenation is the method
generally used when recovering core gene-based phylogenies [8].
The supertree approach
In the supertree approach, individual gene trees are inferred using
standard phylogenetic methods. These trees are then combined
using one of the available supertree methods to derive a consensus
phylogeny; see Ref. [9] for an example. The advantage of the
supertree approach is that the gene sequences are not combined
before the phylogenetic inference. This can avoid the combination
of genes with incompatible phylogenetic histories (i.e. genes vexed
by HGT). A second advantage of the supertree approach is that it can
be used to derive phylogenies based on extremely large numbers of
genes (on the order of thousands [9]).
Gene content-based methods
These approaches use the presence or absence of specific genomic
features (e.g. protein families), rather than sequence alignments, as
discrete characters to be used in phylogenetic analyses; see Ref. [6]
for an example. Matrices construed using such characters are
analysed using standard phylogenetic methods.
Review Trends in Ecology and Evolution Vol.23 No.5
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difficult to defend [32]: as more genomes are being
sequenced, more genes are found to have been horizontally
transferred and are thus removed from the core [57].
Indeed, 44 putative core genes have been identified for
Eubacteria [61] and 45 putative core genes have been
identified for the Archaebacteria [62], but only 31 core
genes could be identified when Archaebacteria, Eubacteria
and Eukaryota were considered [8].
Ernst Mayr defined species as ‘‘groups of interbreeding
natural populations that are reproductively isolated from
other such groups’’ [63]. Mayr was a zoologist and his
biological species concept is one that essentially fits
animals. Microbiologists might surely modify Mayr’s defi-
nition to accommodate some level of HGT. However, when
HGT seems to be able to move virtually every gene in the
genome [32], the meaning of a tree derived from a tiny
minority of putatively vertically inherited core genes
become fuzzy [58]. This caused much disagreement on
what is the real meaning of the core gene-based trees
[49,50,60,64–66] (see Box 2). Indeed, in agreement with
others [58,60,66], we think that the congruence of these
core genes is unlikely to have any special significance. A
core gene tree could only recapitulate clonal history if the
core genes truly were vertically inherited. Unfortunately,
the only approach to identifying vertically inherited genes
is the congruence of gene trees with a species phylogeny.
These putatively vertically inherited genes are mostly
coding for proteins that are part of, or tightly associated
with, the ribosomalmachinery (e.g. ribosomal proteins [8]).
Phylogenetic congruence among these genes might there-
fore reflect coevolution, not clonal history. In any case (see
also Ref. [66]), the phylogeny derived from these genes
cannot represent a species tree, as prokaryotic lineages are
not reproductively isolated in any meaningful sense and
thus are not species (sensu Mayr [63]).
Trees inferred from complete genomes [6,9], are also
unlikely to represent species phylogenies. However, we
conjecture that these trees might be more useful than
those based on core genes because they represent
some sort of central tendency and can be used as general
frameworks in the study of prokaryotic evolution (e.g. Ref.
[67]), irrespective of the existenceof aprokaryotic tree of life.
Indeed, the same genomic tree can either be considered a
representation of the prokaryotic tree of life, if such a tree
can be shown to exist, or a phenetic dendrogram represent-
ing the proximity (under a given distance measure) of the
prokaryotic lineages (if the prokaryotic tree of life does not
exist). By contrast, a core gene-based tree will loose its
predictive power in the absence of a tree of life.
Eukaryotes are outside the scope of this review. How-
ever, it is important to point out that the current genomic
evidence suggests that eukaryotes emerged from the
‘fusion’ of an archaebacterium and a eubacterium [6,9].
If these results are confirmed then there is no tree of life,
but a ring-like network of life [32]. In the next 10 years, as
an ever-increasing number of genomes will become avail-
able, we might see either the rebirth of the tree of life
hypothesis or its ultimate downfall. It is still too early to
say in which direction the evidence will swing. Nonethe-
less, it is not overly presumptive to state that 70 years after
Breed (1939) made the suggestion, it is possible that we
might have to revert to a realistic approach to prokaryotic
classifications, eventually divorcing ourselves from the
idea that a natural classification of the prokaryotes can
be defined.
Acknowledgements
The authors would like to thank Robert Beiko and four anonymous
reviewers for their comments and suggestions. This work was partially
supported by a Science Foundation Ireland Research Frontiers
Programme grant to J.O.McI.
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Box 3. Should we be using the term Prokaryota?
Some authors assign evolutionary significance to the group
Prokaryota, that is, they assume prokaryotes to be monophyletic
(e.g. Refs [25,49]). However, in the traditionally rooted [15,16] SSU-
based universal tree of life, the prokaryotes do not form a
monophyletic group: Archaebacteria are recovered as the sister
group of the Eukaryota, instead of grouping with Eubacteria.
Because the traditionally rooted SSU rRNA tree does not support
the monophyly of the Prokaryota, Pace [50] recently started to
campaign for the dismissal of the word ‘‘prokaryotes.’’ To
corroborate his view, this author claimed that no positive definition
has ever been given for this group, that is, they have always been
clustered because they lack some feature (e.g. a membrane-bound
nucleus), rather than because they share a defining phenotypic
character. As a reaction to Pace [50], a positive definition of the term
‘‘prokaryotes’’ was provided by Martin and Koonin [70], who
pointed out that the prokaryotes can be defined as the organisms
that co-transcriptionally translate their main chromosomes. Because
of the current uncertainties about the tree of life, in this article we
have not assumed the monophyly of any prokaryotic group.
However, we think that abandoning the word prokaryotes is
unnecessary, as it unambiguously identifies an organizational
grade: that of the single-celled organism without a membrane-
bound nucleus, all of whom perform co-translational transcription
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