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构建基因组树 |
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[编者的话] 作者通过五种不同的方法构建基因组树，以研究物种进化关系，作者发现了对细菌来说，存在着与传统种族树不同的拓扑结构。
Abstract Background The availability of multiple complete genome sequences
from diverse taxa prompts the development of new phylogenetic
approaches, which attempt to incorporate information derived from
comparative analysis of complete gene sets or large subsets thereof.
Such attempts are particularly relevant because of the major role of
horizontal gene transfer and lineage-specific gene loss, at least in the
evolution of prokaryotes. Results Five largely independent approaches were employed to
construct trees for completely sequenced bacterial and archaeal genomes:
i) presence-absence of genomes in clusters of orthologous genes; ii)
conservation of local gene order (gene pairs) among prokaryotic genomes;
iii) parameters of identity distribution for probable orthologs; iv)
analysis of concatenated alignments of ribosomal proteins; v) comparison
of trees constructed for multiple protein families. All constructed
trees support the separation of the two primary prokaryotic domains,
bacteria and archaea, as well as some terminal bifurcations within the
bacterial and archaeal domains. Beyond these obvious groupings, the
trees made with different methods appeared to differ substantially in
terms of the relative contributions of phylogenetic relationships and
similarities in gene repertoires caused by similar life styles and
horizontal gene transfer to the tree topology. The trees based on
presence-absence of genomes in orthologous clusters and the trees based
on conserved gene pairs appear to be strongly affected by gene loss and
horizontal gene transfer. The trees based on identity distributions for
orthologs and particularly the tree made of concatenated ribosomal
protein sequences seemed to carry a stronger phylogenetic signal. The
latter tree supported three potential high-level bacterial clades,: i)
Chlamydia-Spirochetes, ii) Thermotogales-Aquificales (bacterial
hyperthermophiles), and ii) Actinomycetes-Deinococcales-Cyanobacteria.
The latter group also appeared to join the low-GC Gram-positive bacteria
at a deeper tree node. These new groupings of bacteria were supported by
the analysis of alternative topologies in the concatenated ribosomal
protein tree using the Kishino-Hasegawa test and by a census of the
topologies of 132 individual groups of orthologous proteins.
Additionally, the results of this analysis put into question the
sister-group relationship between the two major archaeal groups,
Euryarchaeota and Crenarchaeota, and suggest instead that Euryarchaeota
might be a paraphyletic group with respect to Crenarchaeota. Conclusions We conclude that, the extensive horizontal gene flow
and lineage-specific gene loss notwithstanding, extension of
phylogenetic analysis to the genome scale has the potential of
uncovering deep evolutionary relationships between prokaryotic lineages. Background The determination of multiple, complete genome
sequences of bacteria, archaea and eukaryotes has created the
opportunity for a new level of phylogenetic analysis that is based not
on a phylogenetic tree for selected molecules, for example, rRNAs, as in
traditional molecular phylogenetic studies [1,2],
but (ideally) on the entire body of information contained in the
genomes. The most straightforward version of this type of analysis, to
which we hereinafter refer to as 'genome-tree' building, involves
scaling-up the traditional tree-building approach and analyzing the
phylogenetic trees for multiple gene families (in principle, all
families represented in many genomes), in an attempt to derive a
consensus, 'organismal' phylogeny [3-5].
However, because of the wide spread of horizontal gene transfer and
lineage-specific gene loss, at least in the prokaryotic world,
comparison of trees for different families and consensus derivation may
become highly problematic [6,7].
Probably due to all these problems, a pessimistic conclusion has been
reached that prokaryotic phylogeny might not be reconstructable from
protein sequences, at least with current phylogenetic methods [4]. With the complete genome sequences at hand, it appears
natural to seek for alternatives to traditional, alignment-based
tree-building in the form of integral characteristics of the
evolutionary process. Probably the most obvious of such characteristics
is the presence-absence of representatives of the analyzed species in
orthologous groups of genes, and recently, at least three groups have
employed this approach to build genome trees, primarily for prokaryotes
[8-10]. An
alternative way to construct a genome tree involves using the mean or
median level of similarity among all detectable pairs of orthologs as
the measure of the evolutionary distance between species [11].
Yet another possibility involves building species trees by comparing
gene orders. This approach had been pioneered in the classical work of
Dobzhansky and Sturtevant who used inversions in Drosophila
chromosomes to construct an evolutionary tree [12].
Subsequently, mathematical methods have been developed to calculate
rearrangement distances between genomes, and, using these, phylogenetic
trees have been built for certain small genomes, such as plant
mitochondria and herpesviruses [13,14].
These approaches, however, are applicable only to genomes that show
significant conservation of global gene order, which is manifestly not
the case among prokaryotes [15-17].
Even relatively close species such as, for example, Escherichia coli
and Haemophilus influenzae, two species of the γ-subdivision of Proteobacteria, retain very
little conservation of gene order beyond the operon level (typically,
two-to-four genes in a row), and essentially none is detectable among
evolutionarily distant bacteria and ar chaea [15,16,18].
Very few operons, primarily those coding for physically interacting
subunits of multiprotein complexes such as certain ribosomal proteins or
RNA-polymerase subunits, are conserved across a wide range of
prokaryotic lineages [15,16].
On the other hand, pairwise comparisons of even distantly related
prokaryotic genomes reveal considerable number of shared (predicted)
operons, which creates an opportunity for a meaningful comparative
analysis [19][20,21]. The critical issue with all these approaches to genome
tree building is to what extent each of them reflects phylogeny and to
what extent they are affected by other evolutionary processes, such as
lineage-specific gene loss and horizontal gene transfer. Comparative
analyses have strongly suggested that these phenomena make major
contributions to genome evolution, at least in prokaryotes [7,22-25].
These phenomena have the potential to severely affecting phylogenetic
tree topology, particularly when similar sets of genes are lost
indifferent lineages because of similar environmental pressures, or when
a preferential trend of horizontal gene flow exists between different
lineages. The possibility even has been discussed that the amount of
lateral gene exchange is such that it invalidates the very principle of
representing the evolution of species as a tree; instead, the only
adequate representation of evolutionary history could be a complex
network [6][25].
Genome-trees seem to be the last resort for the species tree concept.
Unless phylogenetic signal can be revealed by at least some approaches
based on genome-wide comparisons, the conclusion seems imminent that
this concept should be abandoned and replaced by a more complex
representation of evolution. Here, we compare the topologies produced with five,
largely independent approaches to genome-tree building: i)
presence-absence of genomes in Clusters of Orthologous Groups of
proteins (COGs); ii) conservation of local gene order (pairs of adjacent
genes) among prokaryotic genomes; iii) distribution of percent identity
between apparent orthologs; iv) sequence conservation in concatenated
alignments of ribosomal proteins; v) comparative analysis of multiple
trees reconstructed for representative protein families. We find that,
while the presence-absence approach is most heavily affected by gene
loss and horizontal transfer, the other four methods reveal stronger
phylogenetic signals. Although the topologies of the trees constructed
with different approaches were only partially compatible, three
previously unnoticed high-level clades among bacteria were revealed with
notable consistency. We suggest that, in spite of all the complexity
brought about by horizontal gene transfer and lineage-specific gene
loss, these groups reflect certain evolutionary reality, i.e. the
trajectory of evolution for a relatively stable gene core. It appears
that this is the only meaningful way to treat the notion of a species
tree: as the history of a relatively large ensemble of genes, not a
comprehensive representation of the history of entire genomes. Results New criteria for genome-tree construction To our knowledge, conserved gene pairs and
distributions of identity level between orthologs have not been used
previously as the basis for phylogenetic tree construction. Therefore we
start by describing the relevant results of prokaryotic genome
comparison in somewhat greater detail. Conserved gene pairs in prokaryotic genomes The results of the present analysis of conserved gene
pairs are consistent with the notion of the fluidity of prokaryotic gene
order caused by extensive recombination. Only 17 invariant genes pairs
were detected, all of which consists of genes for ribosomal proteins and
RNA polymerase subunits. The remaining 4586 gene pairs were missing in
at least one genome. The number of gene pairs represented in three, four
and a greater number of genomes decayed rapidly, with highly conserved
pairs forming the tail of the distribution (Fig. 1).
The 95% quantile of this distribution (excluding the highly conserved
pairs) was found to fit the geometric model with a high statistical
significance (Fig. 1).
This is compatible with random, independent loss of gene pairs during
evolution suggesting that, with the caveat of horizontal transfer, the
number of gene pairs shared by three genomes could reflect the
evolutionary distance between them. The number of conserved gene pairs present in
individual prokaryotic genomes varied from 208 for M. genitalium
to 2314 for P. aeruginosa (Table 1).
Analysis of the co-occurrence of gene pairs among the prokaryotic
genomes shows high values of the Jaquard coefficient, which reflect
partial conservation of gene order (see legend to Table 1),
for closely related species, for example, 0.32 for E. coli and H.
influenzae and 0.35 for M. thermoautotrophicum and M.
jannaschi (Table 1).
The value of this coefficient varied from 0.16 to 0.66, with a mean of
0.26, for archaea, and from 0.04 to 0.87, with a mean of 0.16, for
bacteria. In contrast, for archaeal-bacterial comparisons, the values
varied from 0.04 to 0.18, with the average of 0.08 (Table 1).
These observations appear to indicate that the distribution of conserved
gene pairs among prokaryotic genomes carries a phylogenetic signal. Distributions of identity percentage between probable
orthologs from complete prokaryotic genomes Figure 2
shows a sampling of the distributions of identity percentage between
pairs of apparent orthologs identified as reciprocal best hits from a
range of genome pairs separated by varying phylogenetic distances. Most
of the distributions are clearly unimodal, and the distributions for
pairs of phylogenetically distant genomes, such as those from different
major bacterial lineages or bacteria versus archaea, have their modes
within a relatively narrow range around 33% identity (Figure 2). The use of reciprocal best hits is a conservative way
to identify the set of probable orthologs between pairs of genomes
because some of the orthologs are missed due to complex relationships
between groups of paralogs. Nevertheless, all genome-to-genome
comparisons included at least 100 (for the smallest genomes such as the
mycoplasmas), and typically, a considerably greater number of protein
pairs ([11]
and data not shown). This suggests that parameters of the distributions
of the similarity level between probable orthologs identified in this
fashion could potentially serve as useful measures of the evolutionary
distance between genomes. Genome trees constructed with three different approaches Genome trees were generated using the approaches
described under Material and Methods. All the trees showed a clear
separation of the two major prokaryotic domains, Bacteria and Archaea
(Fig. 3,4,5).
Several terminal bifurcations that reflect clustering of relatively
close species, such as three mycoplasmas (M. genitalium, M.
pneumoniae and U. urealiticum), two spirochetes (B.
burgdorferi and T. pallidum), and H. pylori and C.
jejuni, are also reproduced in all trees (Fig. 3,4,5).
This retention of both the deepest and the terminal branchings shows
that all types of data used for tree construction contained at least a
crude phylogenetic signal. However, beyond these obvious aspects of
topology, and in particular with respect to clustering of distantly
related bacteria and archaea, the trees produced with different
approaches showed significant differences, which appear to reflect the
relative contributions of phenotypic and phylogenetic signals. A
quantitative comparison of the tree topologies using the symmetric
distance method showed that the presence-absence tree was most different
from the trees made by the other methods (Table 2). Presence-absence of genomes in COGs The topology of the parsimony tree built using this
criterion appears to reflect primarily the phenotypes of the respective
organisms (Fig. 3).
This is most clearly manifest in the two major bacterial clusters that
appear in this tree, each with a strong bootstrap support: i) bacteria with large genomes, namely E. coli, B.
subtilis, Synechocystis sp., Deinococcus radiodurans and Mycobacterium
tuberculosis, and free-living bacteria with small genomes, A.
aeolicus and T. maritima ii) parasites with small genomes (mycoplasmas,
spirochetes, chlamydia and rickettsia) Parasites with moderate-sized genomes (H.
influenzae, N. meningitidis, and P. multocida; H. pylori and C.
jejuni) formed two distinct groups. Thus, well-established
phylogenetic relationships between free-living and parasitic bacteria,
such as those within the Proteobacteria (E. coli-H. influenzae-P.
multocida-N. meningitidis) and within low-GC Gram-positive bacteria
(B. subtilis-mycoplasmas), are not reflected accurately in this
tree topology. The two free-living bacteria with small genomes, the
hyperthermophiles A. aeolicus and T. maritima, did not
join either the free-living or the parasitic bacterial cluster, despite
their small number of genes similar to that in bacterial parasites (Fig.
3).
That these bacteria do not group with the parasites despite similar
genome sizes, suggests that it is not the number of genes per se, but
rather the degree of genome degradation and the loss of coherent sets of
genes that affect the topology of the presence-absence tree. The
inclusion of the parasites M. tuberculosis and Pseudomonas
aeruginosa in the cluster of bacteria with large genomes probably
reflects the recent origin of parasitism in these lineages. It is
further notable that, in this tree, the two representative of
Crenarchaeota (A. pernix and S. solfataricus) do not
comprise a sister group of the Euryarchaeota (the remaining archaeal
species), but rather for am branch within the Euryarchaeal cluster (see
discussion below). In previous studies that employed similar approaches
to genome-tree building, phylogenetically reasonable clades were
observed after a simple omission of parasitic species [8,9].
Such an operation could be applied to the tree shown in Fig. 3,
indeed resulting in the correct recovery of the proteobacterial and
Gram-positive bacterial lineages. However, it seems that, because known
natural groups could be reproduced by this approach only after omission
of certain species on the basis of independent prior knowledge, this
method hardly can be useful for delineating new, phylogenetically sound
clades. Conserved gene pairs The topology of the tree based on gene pair
conservation seems to carry a stronger phylogenetic signal than the gene
presence-absence tree because it correctly groups together related
free-living and parasitic bacteria despite major differences in gene
repertoires (Fig. 4).
The bacterial side of this tree consists of three major clades: i)
proteobacterial clade that, in addition to bona fide Proteobacteria,
includes also A. aeolicus, M. tuberculosis, D. radiodurans, and Synechocystis
sp, ii) Gram-positive clade that additionally includes T.
maritima, and iii) an unexpected clade that unites spirochetes and
chlamydia. In the archaeal domain, the two species of the Crenarchaeota
did not form a clade, but instead were present as separate branches
interspersed with euryarchaeal species. To further assess the robustness
of the obtained tree, we varied the parameters of the included conserved
pairs by allowing distances between the genes comprising a pair from 0
to 5 and changing the minimal number of genomes, in which a conserved
gene pair had to be present, from 2 to 4. These changes did not
significantly affect the tree topology (data not shown). The topology of
a neighbor-joining tree constructed by using the number of gene pairs
shared by two genomes to calculate the evolutionary distance between
them was similar to the topology of the maximum parsimony tree (Table 2
and data not shown). At least some unusual aspects of this tree's the
topology could be explained by horizontal transfer of operons between
particular bacterial and archaeal lineages. Specifically, it has been
noticed previously that T. maritima shares a considerable number
of genes and operons with Gram-positive bacteria, to the exclusion of
other bacteria [21];
this seems to be compatible with the position of T. maritima with
the Gram-positive cluster. Similarly, considerable horizontal gene
transfer appear to have occurred between the Sulfolobus and Thermoplasma
lineages, which cluster together in the archaeal part of this tree. The
presence of extra species in the proteobacterial cluster is more
surprising because no obvious trend for operon transfer between these
bacteria and bona fide Proteobacteria has been noticed during systematic
genome comparisons; however, a considerable number of shared gene pairs
was detected during the present analysis (Table 1).
Artifacts of tree construction could also contribute to these
associations. In contrast, the spirochete-chlamydia clade might reflect
a deep phylogenetic relationship (see discussion below). Parameters of percent identity distributions between
orthologs Different characteristics of the distributions of
percent identity between the probable orthologs, such as the mean, the
median, the mode and various quantiles, were used to calculate distances
between genomes and construct phylogenetic trees. Trees built with
different cut-off values for symmetrical best hits, four different
formulas for the evolutionary distance calculation (see Materials and
Methods) and different parameters of the distributions showed
essentially the same topology, with strong bootstrap support for most of
the clades (Fig. 5
and data not shown). The complete proteobacterial and Gram-positive
bacterial clusters were recovered in this tree as well as the unexpected
grouping of chlamydia with spirochete noticed above in the tree based on
conserved gene pairs (Fig. 4,5).
Also similarly to the previous two trees, the Crenarchaea grouped with Thermoplasma
within the archaeal part of the tree. Beyond these groupings, the tree
appeared conservative in the sense that the unassigned bacterial species
formed separate branches near the root of the bacterial subtree. The
closest to the root were the two hyperthermophilic species, A.
aeolicus and T. maritima, which is compatible with the
standard view of their phylogenetic position [1,26]. Alignment-based approaches to the construction of a species
tree The above three approaches involve construction of
genome trees "par excellence", i.e. based on integral
characteristics of genomes (or, more precisely, gene sets) that are not
directly related to more traditional, alignment-based measures, which
are usually employed for calculating evolutionary distances or for
parsimony analysis. These genome tree raise several interesting
phylogenetic questions, for example, do spirochetes and chlamydia indeed
share a common ancestor, and are Euryarchaeota, in fact, a paraphyletic
group with respect to the Crenarchaeota. However, the reliability of the
conclusions drawn from the topology of these trees remains uncertain.
Therefore we decided to complement these genome-oriented approaches with
more traditional ones applied on a large scale. Concatenated alignments of ribosomal proteins The alignments of the 32 ribosomal proteins conserved
in all bacterial and archaeal species were concatenated head-to-tail and
treated as a single alignment containing 4821 columns. The underlying
assumption is that the genes coding for ribosomal proteins that function
as components of a large macromolecular complex are unlikely to undergo
horizontal transfer, which tends to confound comparisons of the tree
topologies for other protein families and would invalidate the
concatenation approach. The resulting maximum-likelihood tree contains
the complete proteobacterial and Gram-positive bacterial clusters as
well as the spirochete-chlamydia cluster noticed in the genome-trees. In
addition to the spirochetes-chlamydia clade, the following non-trivial
affinities were detected with strong bootstrap support: i) a cluster of
the two hyperthemophiles, A. aeolicus and T. maritima, ii) a
cluster including D. radiodurans, Synechocystis, and M. tuberculosis,
which, at a deeper level, joined the Gram-positive bacterial branch
(Fig. 6).
Similar tree topologies were obtained when the ribosomal protein data
were analyzed using the neighbor-joining method and when bacterial
phylogeny was analyzed separately by using a concatenated alignments of
51 ribosomal proteins shared by all bacteria (data not shown). Notably,
in the quantitative comparison of tree topologies, the tree made of
concatenated ribosomal protein alignments showed the closest similarity
to the genome-tree based on the distributions of percent identity
between orthologs (Table 2). The reliability of the observed non-trivial groupings
was further examined by using a maximum likelihood approach (the
Kishino-Hasegawa test). For each clade (usually, species) forming the
group to be tested, trees with alternative topologies were manually
constructed by joining the clade in question to every other major group
in the tree. For example, to assess the support for the
spirochetes-chlamydia grouping, spirochetes were placed, sequentially,
with Thermotoga, Aquifex, the Thermotoga-Aquifex branch, ε-proteobacteria,
the αβγ-proteobacterial
branch, Proteobacteria, the Deinococcus-Synechocystis-Mycobacterium
cluster, the low G+C Gram-positive cluster, the branch that unites the
latter two clusters, and between bacteria and archaea (to the bacterial
root). The same alternatives were tested for chlamydia. Alternative
topologies were compared either directly, using the ProtML program, or
were subjected to local rearrangement first. In cases when the topology
did not revert to the original one, the final, "optimized"
topology was used for the comparison. These tests showed high stability
of the Thermotoga-Aquifex and Deinococcus-Synechocystis-Mycobacterium
groupings (no competing topologies with likelihood within 1 SD unit from
the original; Fig. 7,8,
Table 3,4,5,6).
The affinity of the Deinococcus-Synechocystis-Mycobacterium with
Gram-positive bacteria also was supported, although an alternative
topology, with this cluster joining Proteobacteria could not be ruled
out (Fig. 9,
Table 7).
Assessment of the spirochete-chlamydia grouping revealed two competing
topologies, albeit unusual ones. Specifically, moving ε-proteobacteria from the proteobacterial branch
to the spirochete branch or, alternatively, moving spirochetes with ε-p roteobacteria and
simultaneously moving chlamydia to the bacterial root results in
statistically acceptable topologies (Fig. 10;
Table 8,9).
Also, a minor rearrangement of the topology within the euryarchaeal
branch allowed for a reasonable alternative to the topology in Fig. 8
(euryarchaeal paraphyly), with the Crenarchaea-Euryarchaea radiation at
the archaeal root (Fig. 11,
Table 10). A census of protein families Another approach to the "species tree"
problem involves analysis of phylogenetic trees for as many individual
protein families as possible, in an attempt to identify a prevailing
topology or at least common phylogenetic patterns. A survey of the COG
data set identified 132 COGs, each of which included a large number of
bacterial and archaeal species, but no or few paralogs and thus appeared
to be amenable to a large-scale phylogenetic analysis (Table 11).
Maximum-likelihood trees were constructed for each of these COGs, and a
breakdown of nearest neighbors was derived for species and groups
involved in each of the non-trivial or questionable branchings discussed
above (Crenarchaea, Thermotoga, Aquifex, Deinococcus, Mycobacterium,
Synechocystis, spirochetes, chlamydia, and ε-proteobacteria).
In each case, a wide spread of topologies was observed, but the grouping
that is observed in the concatenated ribosomal proteins tree was
encountered most often, although, for example, for the
spirochete-chlamydia cluster, the lead over other topologies was slim
(Fig. 13,14,15). Discussion and Conclusions The trees constructed with each of the four approaches
employed here reflect both the phylogenetic signal and the phenotypic
(life style) similarities or differences between organisms, but the
relative contributions of these two types of information appear to
differ substantially. The gene presence-absence analysis seemed to be
dominated by the phenotypic signal, primarily that from gene loss. The
tree based on conserved gene pairs appeared to combine phylogenetic
information with major effects of horizontal transfer of operons. In
contrast, the trees based on the distributions of the identity level of
orthologs appear to be more meaningful phylogenetically as indicated by
the recovery of established high-level phylogenetic groups of bacteria,
such as Proteobacteria and Gram-positive bacteria. The ability to
correctly identify these major bacterial subdivisions and the absence of
obviously wrong groupings confer credibility to non-trivial clades
present in these trees, in particular the spirochete-chlamydia clade.
The same logic applied to the tree made of concatenated ribosomal
protein sequences, which included two other non-trivial bacterial
groupings, Aquifex-Thermotoga and Synechocystis-Mycobacterium-Deinococcus,
the latter joining the Gram-positive branch. Furthermore, extensive
testing of alternative topologies using the Kishino-Hasegawa test
largely supported these new bacterial branches. The nature of this
support becomes clearer when one examines the results of the protein
family census. Each of the potential new clades was indeed most common
among the observed topologies, but in no case, was the excess of this
topology overwhelming. Taken together, these results seem to shed light
on the very notion of a "species tree". It appears that, at
best, a species tree can be viewed as a prevailing phylogenetic trend,
which, as far as deep branchings are concerned, may not even apply to a
majority of the genes in a genome. The potential new, deep relationships between
bacterial lineages revealed during this analysis should be considered
preliminary and treated with caution. Nevertheless, an evolutionary
affinity between Cyanobacteria (Synechocystis) and Actinomycetes
(Mycobacterium) appears plausible, particularly given the
presence, in these bacterial groups, of well-developed and partly
similar signal transduction systems [27].
The connection between two hyperthermophilic bacteria, Aquifex
and Thermotoga, also has obvious biological meaning, although, in
this case, particular caution is due, given the possibility of
preferential horizontal gene exchange between these organisms that
inhabit similar environments. However, the strong support for this
grouping obtained in the analysis of concatenated ribosomal proteins
argues against horizontal transfer as the primary cause for the observed
topology. Although recent studies on the phylogeny of ribosomal proteins
suggest some horizontal transfer events, these seem to be largely
restricted to bacteria-specific ribosomal proteins. In the universal set
of ribosomal proteins, only one, S14, showed clear signs of horizontal
transfer [28].
The potential deep phylogenetic connections uncovered during this
analysis call for detailed genome comparisons in search of potential
shared derived characters, such as unique protein domain architectures,
that could support the new clades. The major bacterial lineages are poorly resolved in
rRNA-based trees [2,29]
and those built using alignments of RNA polymerase subunits [30]
and translation elongation factors [29,31].
In the currently accepted taxonomy, which is based primarily (but not
exclusively) on 16S RNA phylogenetic analysis, bacterial lineages that
are suggested by this analysis to form higher-level clusters, tend to
form primary nodes under Bacteria (Chlamydiales, Spirochetales,
Cyanobacteria, the Thermus-Deinococcus group, Aquificales,
Thermotogales). Thus, the genome trees primarily suggest (however
tentatively) new unifications based on deep phylogenetic connections,
rather than split already established clades. A notable exception is the
traditional unification of Actinomycetes, or High G+C gram-positive
bacteria (represented here by Mycobacterium), with low G+C
Gram-positive bacteria (the Bacillus-Clostridium group) under
Firmicutes (Gram-positive bacteria). Such a connection was not supported
by any of the trees analyzed here, and it is also poorly, if at all,
supported by the latest consensus trees for 16S RNA, 23 S RNA and
translation factor EF-Tu [29].
Therefore it seems likely that the Firmicutes clade, at least in its
present composition, does not exist. The new clade that might replace it
consists of low-GC Gram-positive bacteria and the potential
Actinomycetes-Deinococcales-Cyanobacteria group (Fig. 6).
All methods of tree analysis applied here also challenge the traditional
division of the archaeal kingdom into Euryarchaeota and Crenarchaeota,
suggesting instead that Euryarchaeota could be a paraphyletic group with
respect to Crenarchaeota, or in other words, that Crenarchaeota might
have evolved from within the Euryarchaeota. However, the existence of a
statis tically supported alternative topology, with a sister-group
relationship between Euryarchaeota and Crenarchaeota allows for the
possibility that the apparent paraphyly of Euryarchaea is an artifact
caused by rapid evolution in some Euryarchaeal lineages, such as Halobacterium
and Thermoplasma. An independent phylogenetic study of concatenated
ribosomal proteins has been recently published [32].
The main specific conclusion reported in this study was the apparent
association of Synechocystis with Gram-positive bacteria,
although instability of the tree topology dependent on the subset of
sites used for analysis was noticed. Another recent study addressed the
issue of a global tree through phylogenetic analysis of 14 concatenated
sets of orthologous proteins, for which no strong evidence of horizontal
transfer was available [33].
Notably, some of the unexpected groupings within the bacterial domain
reported in this study coincide or overlap with those described here,
namely, a spirochete-chlamydial clade and a Deinococcales-Cyanobacteria
clade. The grouping of the latter clade with Actinomycetes, the
unification of the Deinococcales-Cyanobacteria-Actinomycetes clade with
Gram-positive bacteria and the grouping of the two bacterial
hyperthermophiles were not reproduced in the work of Brown and
co-workers. The differences between the results of the two studies could
owe to the differences between data sets analyzed, the methods used or,
most likely, both. We should note that the present study engaged a
substantially broader data set and more diverse methods for tree
construction. We believe, however, that, in terms of the potential
contribution of genome-wide phylogenetic analysis to phylogenetic
taxonomy, the areas where different methods and independent analyses by
different groups converge might be more important than the areas of
discrepancy. It appears that potential new clades revealed in such
independent studies are strong candidates for new, high-level taxa. The results of the present study suggest that genome
trees based on new, integral criteria do not provide substantial
advantages in phylogenetic reconstruction over more traditional,
alignment-based methods expanded to the genomic scale. In fact, the
latter seem to be more sensitive in detecting potential deep
evolutionary relationships and this is expected to further improve with
the increasing number of completely sequenced genomes becoming available
for analysis. We believe, however, that this conclusion does not
necessarily indicate that genome trees, such as those based on
representation of genomes in orthologous sets or conservation of gene
pairs, are useless. In addition to revealing some new phylogenetic
affinities, they are capable of alerting researchers to other
evolutionary phenomena, such as loss of similar gene sets in different
organisms and preferential horizontal gene exchange between certain
lineages Material and Methods Sequence data The sequences of the proteins encoded in complete
genomes were extracted from the Genome division of the Entrez retrieval
system [34].
The analyzed genomes included those of 30 bacteria: Aquifex aeolicus
(Aquae), Bacillus halodurans (Bacha), Bacillus subtilis
(Bacsu), Borrelia burgdorferi (Borbu), Buchnera sp.
(Bucsp), Campylobacter jejunii (Camje), Caulobacter crescentus
(Caucr), Chlamydia trachomatis (Chltr), Chlamydophila
pneumoniae (Chlpn), Deinococcus radiodurans (Deira), Escherichia
coli (Escco), Haemophilus influenzae (Haein), Helicobacter
pylori (Helpy), Lactococcus lactis (Lacla), Mesorhizobium
loti (Meslo), Mycoplasma genitalium (Mycge), Mycoplasma
pneumoniae (Mycpn), Mycobacterium tuberculosis (Myctu), Neisseria
meningitidis (Neime), Pasteurella multocida (Pasmu), Psudomonas
aeruginosa (Pseae), Rickettsia prowazekii (Ricpr), Staphyloccocus
aureus (Staau), Streptococcus pyogenes (Strpy), Synechocystis
PCC6803 (SynPC), Thermotoga maritima (Thema), Treponema
pallidum (Trepa), Ureaplasma urealyticum (Ureur), Vibrio
cholerae (Vibch), Xylella fastidiosa (Xylfa), and ten
archaea: Aeropyrum pernix (Aerpe), Archaeoglobus fulgidus
(Arcfu), Halobacterium sp. (Halsp), Methanobacterium
thermoautotrophicum (Metth), Methanococcus jannaschii
(Metja), Pyrococcus horikoshii (Pyrho), Pyrococcus abyssi
(Pyrab), Sulfolobus solfataricus (Sulso), Thermoplasma
acidophilum (Theac), Thermoplasma volcanium (Thevo). Phylogenetic tree construction Parsimony trees based on the presence-absence of conserved
gene pairs in prokaryotic genomes The database of Clusters of Orthologous Groups of
proteins (COGs) was used as the source of information on orthologous
genes in prokaryotic genomes [35,36].
Briefly, the COGs were constructed from the results of all-against-all
BLAST [37]
comparison of proteins encoded in complete genomes by detecting
consistent groups of genome-specific best hits (BeTs). The COG
construction procedure does not rely on any preconceived phylogenetic
tree of the included species except that certain obviously related
genomes (for example, two species of mycoplasmas or pyrococci) were
grouped prior to the analysis, to eliminate strong dependence between
BeTs. In order to avoid spurious occurrence of the same gene pair, only
gene pairs conserved in three or more genomes were considered. A pair of
genes from two COGs was considered to be conserved if the respective
genes were adjacent in at least one genome and were separated by no more
than two genes in at least two additional genomes. This relaxed
definition of a conserved gene pair was adopted to take into account the
high level of recombination in prokaryotic genomes. From the data on the
presence-absence of each conserved gene pair in the analyzed genomes
(excluding pairs of closely related species: E. coli-Buchnera sp., H.
influenzae-P. multocida, C. trachomatis-C. pneumoniae, P. horikoshii-P.
abyssi, M. genitalium-M. pneumoniae-U. urealyticum, H. pyroli – C. jejuni, T.
acidophilum-T. volcanium), a 0/1 matrix analogous to the one used
for the presence-absence of individual genes was constructed, and a tree
was built using Dollo parsimony [38].
A parsimony method was chosen for this analysis because the
presence-absence of a conserved gene pair in a genome can be naturally
treated in terms of character states. The Dollo model is based on the
assumption that each derived character state (in this case, the presence
of a gene pair) originates only once, and homoplasies exist only in the
form of reversals to the ancestral condition (absence of a gene pair) [38].
In other words, parallel or convergent gains of the derived condition
are assumed to be highly unlikely. The Dollo parsimony method is not
sensitive to gene loss which is extremely common in evolution of
prokaryotes, but the results can be affected by independent acquisition
of the same gene pair by different genome via horizontal gene transfer.
Phylogenetic analysis was performed by using the PAUP 4.0 program [39],
with 1000 bootstrap replicates performed to assess the reliability of
the tree topology. In addition, the tree topology was analyzed using the
neighbor-joining method [40]. Parsimony trees based on the representation of genomes in
orthologous gene sets The information on orthologous genes in prokaryotic
genomes and the yeast genome was derived from the COGs as in the
previous approach, and the orthology data were similarly represented as
a 0/1 matrix of presence-absence of the analyzed genomes in the COGs. A
Dollo parsimony tree was constructed and the reliability of its topology
was assessed using the bootstrap method as described above. Distance trees based on distributions of identity percentage
between orthologous protein sequences The sequences of all proteins encoded in the analyzed
genomes were compared to each other using the gapped BLASTP program [37].
Reciprocal, genome-specific BeTs were collected at different expectation
(E) value cutoffs (0.01, 0.001, 0.0001, 0.00001). This method for
identification of probable orthologs is, in principle, similar to the
method employed in COG construction, but differs in that there is no
requirement for the formation of triangles of consistent BeTs. The
result of this procedure is a conservative selection of orthologous
pairs because the cases of lineage-specific duplication that result in
non-symmetrical BeTs are excluded and so are orthologous pairs with very
low sequence similarity. However, the limitation of the COG system,
namely the requirement that each orthologous group is represented in at
least three genomes, is avoided. The distributions of identity
percentage among the reciprocal best hits were derived for each pair of
species. The mean, mode, median and different quantiles of the identity
percentage distributions were used for estimating evolutionary
distances. Four distance measures were used, namely: i) P-distances
calculated as the fraction of different residues: d = 1-q,
ii) Poisson distances d = -1nu, iii) geometric distances
calculated using the formula d = 1/u-1, and iv)
logarithmic distances found as a solution of the equation u =
ln(1+2d)/(2d), where d is the evolutionary
distance, q is percent identity, and u = (q-0.05)/0.95
[41,42][43].
Trees were constructed from the distance matrices obtained with the
above distance estimates using the neighbor-joining method [40]
as implemented in the NEIGHBOR program of the PHYLIP package [44].
Bootstrap values were estimated by resampling the set of orthologs
identified for each pair of genomes 1000 times and reconstructing trees
from the distributions of the distances from these resampled sets. Maximum Likelihood trees based on concatenated alignments of
ribosomal proteins Sets of orthologous ribosomal proteins were extracted
from the COG database, and their amino acid sequences were aligned using
the T-Coffee program [45],
with subsequent manual validation and removal of poorly aligned regions.
The alignments are available upon request. Pairwise evolutionary
distances between the sequences in concatenated alignments were
calculated using the Dayhoff PAM model as implemented in the PROTDIST
program of the PHYLIP package [44].
A distance tree was constructed from the resulting distance matrix by
using the least-square [46]
method as implemented in the FITCH program of PHYLIP [44].
The maximum likelihood tree was constructed with the JTT-F model of
amino acid substitutions [47],
as implemented in the ProtML program of the MOLPHY package [48],
by optimizing the least squares tree with local rearrangements.
Alternative topologies were created manually by modifications of the
original tree and directly compared by ProtML. Bootstrap analysis was
performed by using the Resampling of Estimated Log-Likelihoods (RELL)
method as implemented in ProtML [48,49]. Comparative analysis of Maximum Likelihood trees for
individual protein families The representative families were selected from the COG
database according to the following criteria: i) at least 30 species are
represented; ii) no more than two paralogs in any of the species; iii)
no more than 1.2 paralogs per genome on average; iv) at least 100
positions in the alignment containing less than 30% of gaps. This
selection procedure resulted in a set of 132 families (COGs). Alignments
and ML trees were constructed for these families as described above for
the concatenated ribosomal proteins. Quantitative comparison of tree topologies To compare tree topologies quantitatively, the
symmetric distance between trees [50]
was computed using the TREEDIST program of the PHYLIP package (version
3.6a). Briefly, each of the two compared trees is divided by each
internal branch into two partitions. The symmetric distance is the
number of partitions that are found in one tree but not the other. Acknowledgements We thank M. Nei for simulating discussions about the
Dollo parsimony analysis, J. Felsenstein for alerting us of the
inclusion of the TREEDIST program in PHYLIP3.6a and D. Leipe for
discussions on taxonomy. Yuri I Wolf*
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1999-2005 中国科学院上海生命科学研究院生物信息中心 |
