LETTER
Evidence Excluding the Root of the Tree of Life from the Actinobacteria
Jacqueline A. Servin,* Craig W. Herbold,* Ryan G. Skophammer, à and James A. Lake* à§1
*Molecular Biology Institute, University of California, Los Angeles; UCLA Astrobiology Institute, University of California, Los
Angeles; àDepartment of Molecular, Cellular, and Developmental Biology, University of California, Los Angeles; and §Department
of Human Genetics, University of California, Los Angeles
The Actinobacteria are found in aquatic and terrestrial habitats throughout the world and are among the most
morphologically varied prokaryotes. They manufacture unusual compounds, utilize novel metabolic pathways, and contain
unique genes. This diversity may suggest that the root of the tree of life could be within the Actinobacteria, although there
is little or no convincing evidence for such a root. Here, using gene insertions and deletions found in the DNA gyrase,
GyrA, and in the paralogous DNA topoisomerase, ParC, we present evidence that the root of life is outside the
Actinobacteria.
Introduction
The Actinobacteria, among the most morphologically
diverse prokaryotes, are widely distributed in both terrestrial and aquatic ecosystems (Embley and Stackebrandt
1994). Actinobacteria employ varied metabolic mechanisms, although no photosynthetic members are known.
They are primarily chemoheterotrophs, which either respire
or ferment. Their oxygen tolerances vary from strictly aerobic, to facultatively anaerobic, to microaerophilic, or to
strictly anaerobic. In addition to utilizing some unique biochemical pathways not found in other prokaryotes, they
also synthesize many macromolecules absent from other
organisms, such as unique cell wall peptidoglycans
(Gokhale et al. 2007). Given their diverse morphological
and biochemical repertoires (Embley and Stackebrandt
1994; Boone and Castenholz 2001; Garrity and Holt
2001), properties that might indicate a deep placement in
the tree of life, we investigate whether the root of life is
contained within the Actinobacteria.
Here, we use top-down rooting to probe the origins of
the Actinobacteria. This method analyzes indels, inserts
and deletions, that are present in ingroup genes but are absent in some or all paralogous outgroup genes. Indel-based
rooting methods are related to traditional methods of
sequence-based rooting (Dayhoff and Schwartz 1980;
Gogarten et al. 1989; Iwabe et al. 1989) but exclude roots
rather than directly reconstructing rooted trees (Rivera
and Lake 1992; Baldauf and Palmer 1993; Lake et al.
2007). We apply the method to all available Actinobacterial, double-membrane prokaryotic, Firmicute, and Archaeal sequences. Together these 4 groups represent all
known prokaryotic life (Boone and Castenholz 2001).
Archaea are primarily extremophiles and include many hyperthermophiles; Firmicutes, formerly named the low-GC
gram positives, contain organisms like clostridia and
bacilli; and double-membrane prokaryotes are a speciose,
1
Present address: 232 Boyer Hall, 611 South Young Drive,
University of California, Los Angeles.
Key words: tree of life, root, indels, cenancestor, Actinobacteria,
prokaryotes.
E-mail: [email protected].
Mol. Biol. Evol. 25(1):1–4. 2008
doi:10.1093/molbev/msm249
Advance Access publication November 13, 2007
Ó The Author 2007. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
possibly primitively photosynthetic taxon exclusively containing all prokaryotes surrounded by double membranes.
Top-down rooting has provided evidence for excluding the root from all but 4 regions of the tree of life
(Skophammer et al. 2006; Lake et al. 2007; Skophammer
et al. 2007). The 4 remaining locations are 1) on the branch
leading to the double-membrane prokaryotes; 2) on the
branch leading to the Actinobacteria; 3) on the branch leading to the clade of the Firmicutes and the Archaea; and 4)
within the Actinobacteria. Applying top-down rooting to
an indel present in the type II DNA topoisomerase (GyrA)
(Gupta 1998) and to the paralogous topoisomerase IV
(ParC) (Champoux 2001), we provide evidence that the root
of the tree of life is excluded from within the Actinobacteria
and, thereby, reduce the number of possible locations for
the cenancestral root.
DNA topoisomerases are essential in eubacteria,
archaea, and eukaryotes. They serve to relieve the topological strains encountered by a cell during replication, transcription, recombination, and chromatin remodeling.
Type II DNA topoisomerases introduce double-strand
breaks and are adenosine triphosphate dependent. Type
II DNA topoisomerases are further subdivided into type
IIA found in all domains of life and type IIB topoisomerases
found only in Archaea. Gyrase and topoIV are welldocumented paralogs in the Topo IIA family and exhibit
extensive sequence similarity (Champoux 2001). The prokaryotic homologs of gyrase and topoIV are heterotetramers. Gyrase contains 4 subunits, 2 each of GyrA and
GyrB. These are homologous to the 2 subunits of topoIV,
ParC, and ParE, respectively. Gyrase genes are ubiquitous,
whereas topoIV genes are present within the Eubacteria but
missing in the Archaea.
Upon comparing alignments of GyrA and ParC sequences, we confirmed that a 4 amino acid GyrA insert
(Gupta 1998) is present in Actinobacterial gyrase sequences, between orthologous Escherchia coli positions 204 and
205 and absent in all other prokaryotic gyrase sequences.
We report that this insert is missing in all eubacterial ParC
sequences (ParC is absent in the Archaea) and analyze this
information using top-down rooting. Representative sequences of these root-informative genes are summarized
in table 1, and complete alignments of nearly 500 GyrA
and ParC sequences are included in the supplementary
2
Servin et al.
Table 1
Summary of the GyrA/ParC Indel
NOTE.—A summary of GyrA and ParC alignments within the NGSSG/GPDFPT region corresponding to Escherichia coli residues 167–217 in the outgroup ParC
sequence. Alignments of individual GyrA and ParC sequences are available in tables S1A and S1B of the supplementary analyses and data (Supplementary Material online).
analyses and data, sections S1 and S2 (Supplementary Material online), respectively.
Our rooting analyses are summarized in figure 1. For 4
taxa, there are 9 possible trees, corresponding to 4 crown
groups, 4 stem groups, and 1 internal branch. The most parsimonious rooted trees for each of the 9 possible rootings are
shown in figure 1. Note that the leaves of the unrooted trees
are divided into 2 separate regions because the groups corresponding to the Actinobacteria (A), the double-membrane
prokaryotes (D), the Firmicutes (F), and the Archaea (R) represent higher level phylogenetic clades rather than single sequences. Thus, the roots within the distal portions of the
leaves (roots 1, 2, 8, and 9) are shown as 2 lines to represent
the branching within these crown groups. The proximal portions of the leaves correspond to roots 3, 4, 5, 6, and 7. As
shown by the large X in figure 1, the least parsimonious
rooted tree—root 2,within the Actinobacteria—requires 3
changes, whereas roots 1 and 3–9 require only 2 changes.
Others have suggested that GyrA has been transferred into
the Archaea (Gadelle et al. 2003). Hence our rooting calculations assume the Archaeal GyrA genes are missing and
uniformly eliminate root 2—the Actinobacterial root (for
analyses, see supplementary sections S3 and S4, Supplementary Material online). Comparisons of indel distributions with GyrA gene trees showed no evidence for indel
homoplasy (supplementary section S5, Supplementary Material online). Analyses of GyrA and ParC indel flanking sequences provide significant statistical support, P , 0.015,
for excluding the root from the Actinobacteria (supplementary section S2, Supplementary Material online). Together
these tests provide strong evidence for excluding an Actinobacterial root.
Previous analyses of directed indels have excluded
roots within the double-membrane prokaryotes, within
the Archaea, on the segment connecting the eukaryotes
to the double-membrane prokaryotes, on the segment connecting the eukaryotes to the Archaea and within the Firmicute–Archaeal–eukaryotic clade (Skophammer et al.
2006; Lake et al. 2007; Skophammer et al. 2007). These
excluded roots, plus the results presented here excluding
FIG. 1.—A top-down rooting analysis of the excluded roots for the
GyrA/ParC indel set. The following taxa are analyzed: the doublemembrane prokaryotes (D or D#); the Actinobacteria (A or A#); the
Firmicutes (F or F#); and the Archaea (R or R#). The character states
corresponding to taxa D, A, F, and R for gene GyrA are , þ, , and , and
the character states corresponding to taxa D#, A#, F#, and R# for gene ParC
are , , , and m, where ‘‘’’, ‘‘þ’’, and ‘‘m’’ correspond to insert absent,
insert present, and gene missing, respectively. Filled rectangles represent
an indel character state change, outlined rectangles represent gene deletions
or insertions, and vertically striped rectangles represent gene duplications.
Indel state changes, gene deletions and insertions, and gene duplications
are weighted equally. The roots are numbered 1–9 as described in the text.
Roots 1 and 3–9 are most parsimonious and correspond to 2 changes. Root
2 is least parsimonious, as indicated by the large X across the tree, and
corresponds to 3 changes. In some cases alternative, but equally
parsimonious, locations for character state changes exist (data not shown).
The Root of the Tree of Life Is Outside the Actinobacteria 3
(Gogarten et al. 1989; Iwabe et al. 1989). The 3 remaining
roots are located on the branch (stem) leading to the doublemembrane prokaryotes, root 1, on the branch leading to the
Actinobacteria, root 2, and on the branch leading to the
Firmicute/Archaeal clade, root 3. We hope that future indels
will facilitate further testing of these roots.
Supplementary Material
Supplementary analyses and data are available at
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
This study is supported by grants from National Science Foundation (NSF) and the University of California,
Los Angeles, National Aeronautics and Space Administration Astrobiology Institute to J.A.L. The authors J.A.S.,
C.W.H., and R.G.S. were supported by a Cell and Molecular Biology Training Grant from National Institutes of
Health (NIH), a Genomic Interpretation and Analysis
Training Grant from NIH, and an Integrative Graduate Education and Research Traineeship training grant from NSF,
respectively.
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FIG. 2.—A summary of the possible locations for the root of the
prokaryotic tree of life, figure 2 top, and for the root of the prokaryotic/
eukaryotic ring of life, figure 2 bottom. The relevant 4 taxa representing
known prokaryotic diversity are the double-membrane eubacteria (D), the
Firmicutes (F), the Actinobacteria (A), and the Archaea (R). The
eukaryotes (K) are present in figure 2, bottom. The 3 possible roots are
numbered 1, 2, and 3. The traditional root is indicated by an ‘‘X’’ and the
root within the double-membrane prokaryotes is indicated by an ‘‘*.’’ The
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Martin Embley, Associate Editor
Accepted November 8, 2007