Determining which branches of the tree of life have derived features narrows down the possible location of the root. Currently the polarization of indels done by Lake _et al_.^1-5^ and the polarizing transitions of Cavalier-Smith^6^ arrive at contradictory positions for the root of the tree.  We have analyzed the sequence based indel arguments using protein structure wherever possible. Structure strongly supports some of the polarizations, but in other indels it argues for a different conclusion. We conclude that there is no contradiction between Lake _et al_. and Cavalier-Smith; the root of the tree of life must be near the Chloroflexi.&#xd;&#xa

Philip E. Bourne

Ruben E. Valas

English

Ruben Valas

Philip Bourne

Crossref

Structural Analysis of Polarizing Indels Argues the Root of the Tree of Life is Near the Chloroflexi

Nature Precedings

An insert in EF-2 excludes the root from 
Eukaryotes and Archaea
EF-2 is the Archaeal and Eukaryotic homolog of EF-G. EF-2 contains an insert that is not 
present in EF-G or EF-Tu which implies the Archaea and Eukaryotes are derived. How-
ever, the authors’ sequence alignment has been called into question. We performed a 
structural alignment EF-G (2BV3 colored blue), EF-Tu (1EFC colored cyan), EF-2 (1N0U 
colored red) to investigate whether their conclusion is valid. Our analysis is compli-
cated by a disordered region in the structures that precedes the region of interest. The 
well conserved RGIT motif does align correctly when using structure alone. This is 
highlighted by showing the difference in position of the conserved Glycine on the C 
end of this region (colored green) as well the conserved Aspartic acid in the N end 
(colored purple). The 4 positions highlighted in red are aligned in the original align-
ment, which is why the initial result is controversial. This is further complicated by an 
additional insert in Eukaryotes relative to the Archaea, which is boxed in black. Neither 
the sequence alignment, nor structural alignment is strictly correct. However, when 
one combines both pieces of data it becomes clear that there must a derived insertion 
in the Archaea, which implies the root cannot be in the Archaea or the Eukaryotes.
Structural Analysis of Polarizing Indels Argues 
the Root of the Tree of Life is Near the Chloroflexi
Ruben Valas, Philip Bourne      Contact: rvalas@sdsc.edu
Bioinformatics Program, University of California, San Diego
Abstract
Determining which branches of the tree of life have derived features narrows down the 
possible location of the root. Currently the polarization of indels done by Lake et al.[1-5] 
and the polarizing transitions of Cavalier-Smith[6] arrive at contradictory positions for 
the root of the tree.  We have analyzed the sequence based indel arguments using pro-
tein structure wherever possible. Structure strongly supports some of the polarizations, 
but in other indels it argues for a different conclusion. We conclude that there is no con-
tradiction between Lake et al.  and Cavalier-Smith; the root of the tree of life must be 
near the Chloroflexi.
Introduction
Darwin’s theory implies that all life arose from a common ancestor. The first split in the 
branches of the tree corresponds to that Last Universal Common Ancestor (LUCA). The 
search for LUCA has been composed of arguments of primacy as well as exclusion. How-
ever, arguments about which extant species are the most primitive rely on assumptions 
about what primitive life was like, which leads to circular reasoning when searching for 
the root. Exclusive methods do not rely on such assumptions.
B
A
E
A’
B’
E’
Orthologs
Paralogs
Derived Deletion
A  B  E A’  B’  E’ B  A  E  B’ A’  E’
E  B  A  E’  B’  A’
+    +   +  +   +    -+    +   +  +   +    -
+   +    +    -    +    +
E  B  A  E’  B’  A’
+   +    +    -    +    +
B  A  E B’ A’  E’
+    +   +  +  +     -
A  B  E A’  B’  E’
 +   +    +  +   +    -
The Mreb Hsp70 
indel is inconclusive
The first indel that apparently disagrees with Cavalier-Smith’s root is 
in Hsp70. Hsp70 has a large insert in the Gram-negatives relative to 
the Gram-positives. Lake et al. claim this region is conserved between 
the Gram-positives and the paralogous MreB. This would imply that 
the Gram-negative must be derived. However, a structural alignment 
of representatives of these 3 proteins (MreB 1JCF:A in blue, Hsp70- 
2V7Y:A in red, and Hsp70+ 1DKG:D in orange) reveals that Hsp70 from 
Gram-negatives has a significant insert relative to MreB. This makes 
the ancestral state of the paralogs ambiguous, so the 2 forms of 
Hsp70 cannot be polarized.  This would be insignificant since there 
are 2 other indels that apparently exclude the root from the Gram-
negatives, but we will argue below that neither of these robustly ex-
cludes the root from the Eobacteria. 
The quaternary structure of PyrD excludes 
the root from the Firmicutes and Archaea 
The small indel in PyrD has been polarized using several different outgroups to reach 
contradictory conclusions that are not mutually compatible[2, 5].  The best outgroup is 
probably HisA, which implies the Gram-negatives and Actinobacteria are derived. 
However the different forms of the indel correspond to different quaternary structures 
in this case. PyrD 2 is a monomer. PyrD 1A is a homodimer (1JUB colored cyan to the 
left), and PyrD 1B is a heterotetramer. The homodimer interface at the center of PyrD 
1B (1EP3 colored blue to the left) is similar to the interface in PyrD 1A. PyrD 1B has an 
additional subunit PyrK (colored red to the left). This implies that PyrD 1B is derived 
from 1A. All the sequences that have the apparently ancestral deletion are in the PyrD 
1B family. We argue that in this case the structural polarization trumps the sequence 
based argument as it a smaller evolutionary event to lose two amino acids than it is to 
gain an entire protein-protein interface. PyrD 1B is present across the Firmicutes and 
Archaea, so we can exclude the root from both of these clades.
The HisA HisF indel does not exclude the 
root from the Eobacteria
2 of the indel polarizations that exclude the root from the Gram-negatives are flawed.  
This would be a moot point if the HisA HisF indel robustly excluded the root from all 
Gram-negatives. HisA and HisF are ideal paralogs for indel polarization because they 
are widely distributed and highly conserved at the sequence level. That reduces the 
chance of a conclusions being based on an alignment artifact. Our analysis of this 
data is mostly in agreement with the authors. There is a derived insertion in many 
HisA sequences that can be used to exclude the root. However, we noticed that this 
insert does not appear to be present in any Eobacteria. A sequence alignment be-
tween the Eobacteria with a representative sequence from Actinobacteria (that have 
the insert), other Gram-negatives (that have the insert), and from Firmicutes (that lack 
the insert) shows that all the Eobacterial sequences have the same form of the indel 
as the Firmicutes. The polarization of this indel excludes the root from all Actinobac-
teria and Gram-negatives except the Eobacteria.
Conclusion
Exclusive rooting methods offer great promise in determining the nature of LUCA. 
However, any single argument could be wrong due to the difficulty of correctly po-
larizing a transition. The insert in EF-2 excludes the root from the Archaea and Eu-
karyotes. The insert in GyrA robustly excludes the root from the Actinobacteria. Our 
analysis of the indel data finds no evidence that excludes the root from all Gram-
negatives. If any of these 3 arguments held up it would prove Cavalier-Smith’s root-
ing wrong. We take the fact that none of these arguments can robustly exclude the 
root from Eobacteria as evidence supporting Cavalier-Smith’s rooting. When the 
indel data is combined with other the polarizations a consensus mapping of the 
major evolutionary events emerges: 1) the root of the tree of life is in the Eobacteria, 
2) the Gram-positives are derived from the Gram-negatives, and 3) the Archaea are 
derived from the Gram-positives. At this point we will not argue whether the ances-
tor of the Archaea is more like a Firmicute or an Actinobacteria so that line is drawn 
ambiguously.
Archaea     
Firmicutes 
  
Actinobacteria  
     
Other Gram-negatives 
Eobacteria 
EF-2
PyrD
GyrA
HisA
References
1. Lake, J.A., et al., Rooting the tree of life using nonubiquitous genes. Mol Biol Evol, 2007. 24(1): p. 130-6.
2. Lake, J.A., et al., Evidence for a new root of the tree of life. Syst Biol, 2008. 57(6): p. 835-43.
3. Servin, J.A., et al., Evidence excluding the root of the tree of life from the actinobacteria. Mol Biol Evol, 
2008. 25(1): p. 1-4.
4. Skophammer, R.G., et al., Evidence that the root of the tree of life is not within the Archaea. Mol Biol Evol, 
2006. 23(9): p. 1648-51.
5. Skophammer, R.G., et al., Evidence for a Gram-positive, eubacterial root of the tree of life. Mol Biol Evol, 
2007. 24(8): p. 1761-8.
6. Cavalier-Smith, T., Rooting the tree of life by transition analyses. Biol Direct, 2006. 1: p. 19.
EF-2           EF-G          EF-Tu
PyrD 1A
PyrD 1B      PyrK
Hsp70+      Hsp70-       MreB
One exclusive method is indel polarization. As an example, consider the hypothetical set 
of 2 paralogous genes above. For simplicity lets assume these genes are universally dis-
tributed and the duplication that resulted in the paralogs occurred before LUCA.  All the 
Eukaryotes have an indel in E’. It would normally be ambiguous whether this is the result 
of an insertion or deletion.  However, we know all the paralogous and some of the or-
thologous sequences have that region, which implies the ancestor of both genes had it. 
So E’ must be a derived form of the gene. There are 6 ways of rooting a tree with 3 taxa. 
The most parsimonious scenario for explaining the deletion of that region for each of 
these trees is presented above. A rooting within the Eukaryotes requires at least 2 losses, 
so it less parsimonious than the other 5 trees, and we can exclude the root from the Eu-
karyotes in this example.
Reality is trickier; horizontal transfer and gene loss muddy the waters. Lake et al. have de-
veloped a method to polarize indels despite these factors called top-down rooting[1]. 
They have presented 8 polarized indels that root the tree of life between two clades, 
shown below. The first is the Actinobacteria (A) and Gram-negatives (D). The second is 
the Firmicutes (F) and Archaea (R). Cavalier-Smith has presented 13 polarizing transitions 
that place the root within the Gram-negative bacteria, near the Chloroflexi (marked with 
an * below). It is important to note that both of these methods agree that the Archaea 
are derived from Gram-positive Bacteria. However, the major disagreement on the place-
ment within the Bacteria must be resolved. At least one of the polarizations in these 
methods must be wrong.
        
                  
               Reproduced from[2]
A quality alignment is the prerequisite for properly identifying and polarizing an indel. 
Paralogs that duplicated before LUCA are required to exclude the root from a portion of 
the tree. That means that polarized indel arguments require alignments between para-
logs that diverged over 3.5 Gya. The Achilles’ heel of indel polarization is the alignment 
step. We have used structural alignments and information about quaternary structure to 
analyze Lake et al.’s conclusions. Their exclusion of the root from Actinobacteria based on 
an insert in GyrA appears incredibly robust based on sequence alone. Our data supports 
the controversial indel in EF-2 that excludes the root from Archaea and Eukaryotes. How-
ever, we find that none of the 3 arguments the authors present can actually exclude the 
root from all Gram-negatives as they claim. Instead we find they actually exclude the 
root from all Gram-negatives except the Eobacteria (Deinococcus-Thermus and Chloro-
flexi) which is completely consistent with Cavalier-Smith’s rooting near the Chloroflexi.
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Evidence excluding the root of the tree of life from the actinobacteria. Mol Biol Evol,

Evidence for a Gram-positive, eubacterial root of the tree of life. Mol Biol Evol,

Evidence that the root of the tree of life is not within the Archaea. Mol Biol Evol,

Rooting the tree of life by transition analyses. Biol Direct,

Rooting the tree of life using nonubiquitous genes. Mol Biol Evol,

http://precedings.nature.com/documents/3290/version/1

Structural Analysis of Polarizing Indels Argues the Root of the Tree of Life is Near the Chloroflexi

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