Cell, Vol. 107, 419–425, November 16, 2001, Copyright 2001 by Cell Press
Reconstructing/Deconstructing
the Earliest Eukaryotes:
How Comparative Genomics Can Help
Joel B. Dacks and W. Ford Doolittle1
Program in Evolutionary Biology
Canadian Institute for Advanced Research
Department of Biochemistry and Molecular Biology
Dalhousie University
Halifax, Nova Scotia
Canada
We could reconstruct the evolution of eukaryote-specific molecular and cellular machinery if some living
eukaryotes retained primitive cellular structures and
we knew which eukaryotes these were. It’s not clear
that either is the case, but the expanding protist genomic database could help us in several ways.
Introduction
Almost thirty years ago, the Canadian microbiologist
Roger Stanier (Stanier, 1974) asserted that “the differences in structure and function between prokaryotic
and eukaryotic cells are many and profound, and no
contemporary biological group has a cellular organization that can plausibly be interpreted as intermediate.”
Microbiologists’ understanding now is more nuanced in
several ways. We divide Life into three domains (Bacteria, Archaea, and Eukarya) rather than two (prokaryotes
and eukaryotes). We recognize an astonishing diversity
of form and function within and between the two prokaryotic domains, and the defining positive prokaryotic
characters recognized by Stanier are no longer universal
among them. And increasingly we can confirm the presence in Bacteria or Archaea or both of “primitive” forms
of key macromolecules and processes previously
thought to be eukaryote specific. For instance, bacterial
MreB, first claimed as a homolog to eukaryotic actin
only on the basis of predicted structural similarity in
ATPase domains (Bork et al., 1992) has recently been
shown to form “cytoskeletal” actin-like filaments in vivo
(Jones et al., 2001) and in vitro (van den Ent et al., 2001).
Nevertheless, differences at the level of cell structure
and function remain “many and profound.” It is still reasonable to speak of the prokaryote-eukaryote transition
as one of cellular evolution’s greatest leaps. It is still
sensible to ask how and when the various complex macromolecular machines (cytoskeletons, endomembrane
systems, spliceosome, membrane-bounded organelles,
and many others) that all the best-known eukaryotes
have and all known prokaryotes lack—or possess only
in inchoate form—came into being.
One can approach this question from the bottom up,
looking to diverse prokaryotes for homologs of components and subassemblies of complex eukaryotic cellular
machines (as in the case of actin and MreB), or from
the top down, searching among eukaryotes for simpler
versions of such machines (as we will describe below).
Either way, there are principles of logic and parsimony
that can help in inferring when in cellular evolution com1
Correspondence: [email protected]
Review
plex cellular features and their component parts first
appeared. In the simplest cases, features found in homologous form in all descendants of a common ancestor
must, barring lateral transfer, have been present in that
ancestor (must be primitive or ancestral features). More
complex cases involve traits found in some descendants
(for instance, as designated “⫹” in species B of Figure
1A) but not in others (species A). These could either
have been gained (in the lineage leading to B) or lost (in
the lineage leading to A) after their divergence from their
common ancestor (X). One can tell which by looking at
“outgroups” (O). If (and only if) some of these show the
⫹ trait, parsimony demands that X was ⫹, and that A
has lost this feature.
To apply such reasoning, it is essential to know the
phylogeny, and to make interesting and sensible conclusions about early eukaryotic cellular evolution, it is essential to have a deeply branched eukaryotic phylogeny.
Too many eukaryotic features have been assumed to
be universal (and thus primitive) for all eukaryotes simply
because they are “present from yeast to man.” There
are likely very many early eukaryotic branches (many
outgroups) below the common ancestor of yeast and
man, whatever the true structure of the eukaryotic tree.
Before molecular methods achieved their current
dominance, however, attempts to reconstruct phylogeny were often conflated with attempts to infer when
complex traits and their components first appeared. For
instance, it seemed reasonable to suggest that the simplest contemporary eukaryotes branched off the main
trunk of the eukaryotic tree very early—because the first
eukaryotic cells obviously must have been simpler than
modern ones and simplicity is unlikely to be a secondarily derived feature. Among such simple contemporary
eukaryotes were the diplomonads (including the intestinal parasite Giardia), parabasalids (including the sexually transmitted disease-causing agent Trichomonas),
oxymonads, microsporidia (such as the human pathogen Encephalitozoon), pelobionts (Mastigamoeba), entamoebae, and the heteroloboseid amoebae such as
Naegleria. These protists are variously deficient in such
basic eukaryotic cellular components as mitochondria,
peroxisomes, and Golgi dictyosomes.
For much of the last decade, molecular phylogeny
seemed to support an early branching for many of these
simple protists. (Note that we should call such eukaryotes “early branching” or “deeply diverging” rather than
“ancestral” or “primitive,” since no contemporary cell
can be any other’s ancient ancestor, and none is likely
to retain primitive states of all important characters.)
Small subunit (SSU) ribosomal RNA gene sequences,
which have served as the gold standard molecular measure for reconstructing phylogenetic relationships, showed
the amitochondriate diplomonads, microsporidia, and
parabasalids at the base of the eukaryotic tree (Figure
1B). Above them, mitochondriate protist lineages such
as Heterolobosea and Euglenozoa emerged, followed
by the unresolved cluster of animals, fungi, plants, and
other lineages (including some protists) commonly
called the “eukaryotic crown” (Sogin, 1991).
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420
Figure 1. Parsimony Diagram and Representative Tree of Eukaryotes
(A) demonstrates the principles of parsimony in evolutionary deduction (see text). (B) is a redrawn version of the tree of eukaryotes
based on SSU rDNA from Sogin (1991). This tree, and the early
and sequential divergence of several protist groups it implies, have
provided an invaluable stimulus and guide to much evolutionary
research in the last two decades. However, the agnostic representation in Figure 2 may be more consistent with the bulk of available
reliable data.
Now however, things are much less certain. In some
cases, the simplicity of supposed deeply diverging protist lineages is obviously due to secondary loss of complex cell structures, likely a consequence of adopting a
parasitic lifestyle. Although surely the first eukaryotes
were simpler in structure than modern cells, there is no
compelling evidence that the last common ancestor of
all surviving eukaryotes was. Nor is there any agreed
upon and robust deep eukaryotic phylogeny. While the
lack of resolution may be largely a reflection of our methods of phylogenetic analysis, some feel that eukaryotes
diversified so rapidly (in a “Big Bang” radiation) that
the order of the early branchings will never be known
(Philippe et al., 2000a). Here, we summarize the problems, and consider whether comparative genomics
might hold the answers. Our take on these issues is not
unlike (and was influenced by) that presented earlier by
Embley and Hirt (1998) and by Roger (1999), and of
necessity leaves out much: for instance, the complex
supportive arguments for early mitochondria based on
hydrogenosomes (Tachezy et al., 2001).
Primitive Simplicity versus Secondary
Simplification
The deepest of the deeply diverging eukaryotes were
until recently thought to be those mitochondrion-lacking
protists that Cavalier-Smith (1983) called “Archezoa”—
comprising, among others, the diplomonads (Giardia),
parabasalids (Trichomonas), and microsporidia. The
prevailing belief about mitochondria since Margulis
(1970) had been that these eukaryotic organelles are
the degenerate descendants of endosymbiotic bacteria
engulfed by some early amitochondriate eukaryote (the
host). It was logical to propose that the immediate ancestor of this eukaryote left other surviving descendants
that never harbored such endosymbionts or never converted them to mitochondria. Those descendants would
have been the direct ancestors of modern archezoa.
This appealing “Archezoa Hypothesis” now seems likely
to be false, based on two types of evidence: First, several gene sequences (Hirt et al., 1999; Keeling and Doolittle, 1996; Van de Peer et al., 2000) disagree with SSU
rDNA on the deep branching of microsporidia (placing
them instead among or as sister to fungi), while the deep
divergences of diplomonads and parabasalids obtained
with several molecular datasets are now seen as “long
branch artifacts” (see below). Second, all three lineages
harbor, in their nuclear genomes, at least one gene that
obviously derived from mitochondrial genomes. Initial
evidence for this came in 1996, when four groups simultaneously described a mitochondrial-type cpn60 gene
in nuclear DNA of Trichomonas vaginalis (Bui et al., 1996;
Germot et al., 1996; Horner et al., 1996; Roger et al.,
1996). The same gene was since found in the diplomonads Giardia lamblia (Roger et al., 1998) and Spironucleus barkhanus (Horner and Embley, 2001), and thorough phylogenetic analyses place all such “archezoal”
cpn60s with a mitochondrial cluster (Horner and Embley,
2001). Of course, their functions cannot be “mitochondrial”: current thinking is that, at least in parabasalids,
cpn60 serves the hydrogenosome—a hydrogen-generating organelle that likely shares a common origin with
mitochondria. Several other amitochondriate lineages
have been shown to have nuclear genes of mitochondrial origin (Clark and Roger, 1995; Germot et al., 1997) or
to be embedded in groups that have likely mitochondrial
homologs (Dacks et al., 2001), and thus (by the logic of
Figure 1A) likely also to be secondarily deficient in these
organelles. At the moment, it is unclear that there are
any living direct descendants of the premitochondriate
“host,” if such ever existed. Thus, parsimony says that
the last common ancestor of all currently known eukaryotes had mitochondria.
Deep protists should also, according to prevailing theory, lack introns. Not only would this be consistent with
the general observation that more complex organisms
generally have more introns (Logsdon, 1998), it would
jibe with the popular theory that spliceosomal introns
derive from group II introns were first introduced into
eukaryotes by the premitochondrial symbiont (CavalierSmith, 1991). But if all known eukaryotes once had mitochondria, did they also all once have (indeed do they
all still have) spliceosomal introns? Microsporidia surely
do: introns have now been described in some of their
genes (Biderre et al., 1998) and U2 and U6 spliceosomal
RNAs are present (Fast et al., 1998). Although no introncontaining genes have yet been described in diplomonads or parabasalids, a gene for the essential and highly
conserved splicing component PRP8 has been found in
Trichomonas (Fast and Doolittle, 1999) and a PRP8 gene
and several other splicing factors are listed at the Giardia
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421
Table 1. Presence of Endomembrane Genes in Diverse Eukaryotic Genome Projects
Higher taxon
organism
Syntaxin
Snap25
Rab
ARFGap
COPI
Sec1
Ykt6p
Fungi
Land Plants
Animal
Diplomonad
Kinetoplastid
Apicomplexa
Apicomplexa
Slime molds
Entamoebae
Red Algae
Stramenopiles
Green algae
Saccharomyces
Arabidopsis
Human
Giardia
Trypanosoma
Plasmodium
Theileria
Dictyostelium
Entamoeba
Porphyra
Phytophthora
Chlamydomonas
****
****
****
***
*
***
*
****
*
**
**
**
****
****
****
NI
NI
NI
NI
NI
NI
NI
NI
NI
****
****
****
***
****
***
****
****
****
**
*
****
****
***
****
**
*
***
*
NI
*
NI
**
****
****
****
****
*
***
***-a
*
NI
*
**
*
**
****
****
****
**
*
***
*
**
*
NI
**
**
****
****
****
**
*
*
*
**
*
**
**
**
This summarizes searches as of Oct 2001. **** indicates a published reference, *** means that a homolog is listed in GenBank, ** denotes an
identified BLAST hit listed on the relevant genomic project web page, * indicates that our search identified a putative homolog by BLAST
with a score less than p ⫽ 0.05. NI ⫽ Not Identified: a homolog could not be reliably identified by any of the above criteria. a ⫽ there is a
COPI homolog identified in GenBank for the apicomplexan Toxoplasma.
This data was obtained by searching, using keywords/BLASTing either GenBank or the following genomics projects, which we thank for their
contributions:
Giardia ⫽ http://hermes.mbl.edu/baypaul/Giardia-HTML/index2.html,
Chlamydomonas and Porphyra ⫽ http://www.kazusa.or.jp/en/plant/database.html,
Dictyostelium ⫽ http://www.csm.bio1.tsukuba.ac.jp/cDNAproject.html,
Phytophthora ⫽ http://www.ncgr.org/pgc/indes.html and
Theileria ⫽ http://www.tigr.org/tdb/e2k1/tpa1/.
While preliminary lists of this type can allow general statements about evolutionary events, to make robust and detailed conclusions using
genomic data, it is also important to obtain near complete open reading frames (ORF) of putative proteins identified by BLAST. Potentially
misleading BLAST results of partial sequences aside, the full ORF can be used for phylogentic analysis, or to identify those rare evolutionary
events discussed below. The verified sequence will also allow for comparative analysis of functionally characterized residues.
genome project web site. The few gene sequences available for other putatively early eukaryotes such as Reclinomonas (Archibald et al., 2001; Edgcomb et al., 2001)
and Mastigamoeba (Stiller et al., 1998) contain introns,
sometimes at high density. Although one awaits proof
of spliceable introns in genes of Giardia and Trichomonas, the odds are that all known eukaryotes have—or
once had—these elements in their genes, and the
wherewithal to remove them.
A number of putatively deeply diverging lineages including Heterolobosea, diplomonads, Entamoeba, Mastigamoeba, and oxymonads lack recognizable stacked
Golgi bodies, and some were once thought to have diverged prior to this key eukaryotic innovation (CavalierSmith, 1983). In the case of Giardia (Lujan et al., 1995)
and Entamoeba (Ghosh et al., 1999), however, there is
evidence that a Golgi-like structure can be induced, and
that some Golgi enzymes are present in the cell. In the
case of such a complex and variable system, mapping
components and subassemblies to eukaryote phylogeny might provide a picture of progressive complexification. The diversity of Rab proteins (GTPase proteins
involved in vesicular transport) has been examined, with
homologs identified in animals, fungi (Armstrong, 2000),
and plants (Borg and Poulsen, 1994) as well as diverse
protists (Bush et al., 1993; Field and Boothroyd, 1995;
Janoo et al., 1999; Rodriguez et al., 2000). However,
the majority of proteins involved in the endomembrane
system have not been examined through comparative
genomics. Table 1 summarizes our recent preliminary
searches of completed and in-progress genomes for
several crucial genes involved in the endomembrane
system. Although Snap-25 homologs were not found
outside animals, plants, and fungi, most proteins
searched did have at least one homolog present in di-
verse taxa. The last common ancestor of all extant eukaryotes likely had complicated vesicular transport machinery.
These are but three examples of systems once
thought to be primitively absent in deeply diverging lineages that seem now more likely to have been secondarily lost in such cells, or cryptically present in all eukaryotes. Others, including the protein folding apparatus
(Archibald et al., 2000) and translation elongation release
factors (Inagaki and Doolittle, 2000), are also well documented. The last common eukaryote ancestor would
probably not be thought primitive, were it available for
examination today. Either (1) the “many and profound”
differences between eukaryotes and prokaryotes appeared very quickly in evolution, (2) many transitional
stages have left no survivors, or (3) those survivors have
yet to be identified.
Problems with Phylogeny
Another barrier to reconstructing the prokaryoteeukaryote transition is uncertainty about the phylogenetic relationships of the lineages that are known. Many
protein sequences from eukaryotes, developed to be
reliable markers of eukaryotic organismal evolution, do
not reproduce the small subunit rDNA tree. Similar disharmony among prokaryotic phylogenies based on different genes may frequently be due to lateral gene transfer (Doolittle, 1999), and eukaryotes are surely not
immune to transfer. Nevertheless, such incongruence
in eukaryotes has most often been attributed to failures
in methods of phylogenetic reconstruction. For computational reasons, early models of phylogenetic analysis
involved radical and unrealistic simplifications. These
included the assumption that all sites in a protein vary
equally, and that all changes between nucleotides or
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amino acids occur with the same frequency (Swofford
et al., 1996). Most importantly, however, was the faulty
assumption that the same gene in different organisms
evolves at a constant rate. This led to an artifact called
long branch attraction (LBA) (Felsenstein, 1978). In phylogenetic reconstruction, sequences that evolve at
higher rates are artificially attracted to each other and
(for the same reasons) to sequences that are very different because they diverged very long ago. For eukaryotic
trees rooted with bacterial or archaeal outgroup sequences (which present long branches because they are
indeed anciently diverged), the result will be the artifactual
placement of rapidly evolving sequences at the root of
the tree.
The recognition of this artifact, and attempts to compensate for it with more sophisticated computer algorithms and biologically accurate models of sequence
evolution, have thrown into doubt the ancient nature
of many of the organisms once thought to be deeply
diverging. Microsporidia were the first to go. Phylogenies of tubulins and RNA polymerase as well as reanalyses of other markers have shown that the microsporidia
are not ancient eukaryotes, but either degenerate members or very close relatives of the fungi (Hirt et al., 1999;
Keeling and Doolittle, 1996; Van de Peer et al., 2000).
The ancient nature of the parabasalids and diplomonads
has also come into question: these organisms do present long branches in many phylogenetic reconstructions
(Hirt et al., 1999; Stiller and Hall, 1999). However, no
alternative placement has been suggested for these taxa
and so their status as deeply diverging lineages is only
in doubt, not disproved.
In doubt as well, though, is the very structure of the
eukaryotic tree. Philippe et. al. (2000b) have argued that
long branch attraction can provide false support for the
ladder-like structure of sequentially emerging taxa in
the eukaryotic phylogenetic tree, seen in Figure 1B. They
show that correction for this artifact produces a multifurcated tree, whose deep branching order is unresolved
(Philippe et al., 2000a). This, they propose, is the result
of a real biological phenomenon, not methodological
failure. Their “Big Bang” hypothesis suggests that many
of the extant eukaryotic lineages evolved very rapidly
from one another: the branching pattern is unresolved
because, for most markers, not enough mutations occurred between branchings to allow reconstruction of
the order of events (Philippe et al., 2000a). Such a view
is of course consistent with (although not required by)
the conclusion drawn above that extant characterized
eukaryotes diverged from an already complex eukaryotic ancestor. However, a rapid radiation does not necessarily mean that the phylogeny of eukaryotes is an insurmountable or unresolvable question.
How Genomics Might Help with Phylogeny
Scientists almost always assert that what is needed is
“more data.” Here, we need more data of at least three
sorts.
First, one can look harder for surviving direct descendants of the primitively simple eukaryotes that must
once have thrived in ancient anaerobic habitats, according to all currently fashionable theories of cell evolution (Lopez-Garcia and Moreira, 1999; Margulis, 1970;
Figure 2. Eukaryotic Relationships Based on Consensus Data
This scheme takes into account (Baldauf and Palmer, 1993; Baldauf
et al., 2000; Cavalier-Smith and Chao, 1996; Dacks et al., 2001; Fast
et al., 2001; Moreira et al., 2000; Simpson and Patterson, 2001),
among others. Taxa with full genome sequencing or GSS projects
underway are shown in Blue, while those with EST projects only are
shown in Red. Taxa with both EST and full genomic initiatives are
shown in purple.
Martin and Muller, 1998). Searching by microscopic
methods in benthic environments have uncovered interesting new organisms such as Reclinomonas (Flavin and
Nerad, 1993), Trimastix (Brugerolle and Patterson, 1997),
Malawimonas (O’Kelly and Nerad, 1999), and Carpediemonas (Simpson and Patterson, 1999). For prokaryotes,
it is estimated that we only know 1% of the actual biodiversity that exists globally (Pace, 1997). For eukaryotes
the fraction might be higher than that since diversity
can be more easily seen, but by how much? Recent
environmental PCR studies of ocean water have revealed
(through their SSU rDNA sequences) novel eukaryotic
groups (Lopez-Garcia et al., 2001). When environmental
genomic sampling methods (such as characterizing BAC
clones from whole communities or ecosystems [Beja et
al., 2000]) become widely applied, our appreciation of
eukaryotic diversity, not only phylogenetic but genomic,
physiological, and structural diversity, will expand enormously.
Second, the multiplicity of new individual gene sequences that emerge from genome projects and genome surveys (such as single-pass and EST analyses)
will allow different and more compelling sequencebased phylogenetic reconstructions. As we have seen
in the past few years, single strong pieces of evidence
that are consistent with previous inconclusive data can
help us lock down phylogenetic positions. Sequences
from different genes will be useful for reconstructing
relationships between different organisms. The debate
about whether red and green algae share a recent common ancestor seemed played to a stalemate, until sequences of the EF2 gene strongly placed them together
(Moreira et al., 2000). Similarly adding an additional organism can help to tack down a taxon that had gone
phylogenetically adrift, as in the case of the oxymonad
and Trimastix relationship (Dacks et al., 2001). More
genome sequences will help us find more insertion/deletions such as the EF1␣ insertion that unites animals and
Genome Review
423
fungi (Baldauf and Palmer, 1993). These can give great
confidence because they rely on positive evidence for
extremely unlikely events, in addition to whatever phylogenetic signal their sequences provide. Increasing gene
sequence from genomic projects also allows us to combine many genes into concatenated multigene datasets.
This approach has been used successfully a number of
times in the past few years to yield robust evidence for
relationships that were previously contested (Martin et
al., 1998; Baldauf et al., 2000). Some of these new or
newly affirmed relationships are illustrated in Figure 2,
which summarizes what we feel to be the most wellsupported relationships among eukaryotes, based on
many sorts of data.
Third, data from EST and partial and complete genome projects for many protists will allow comparative
analyses of their (partial or complete) gene complements, or “proteomes,” in popular parlance. There are
several ways in which information on gene content could
augment sequence-based phylogenetic analyses of individual or concatenated genes, by revealing unique
rare gene duplication, fusion, or transfer events. These
can convincingly link existing groups to single inferred
ancestors, just as insertions and deletions within genes
can do. For instance, Philippe and collaborators (2000a,
2000b) recently identified a fusion of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) genes
that unites plants, alveolates, and Euglenozoa. In a more
complex case, Fast et al. (2001) propose that the plastidtargeted GAPDH (glyceraldehyde-3-phosphate dehydrogenase) genes common to apicomplexa, dinoflagellates, heterokonts, and perhaps cryptomonads derive
from a unique duplication and retargetting of cytosolic
GAPDH at the base of this group, which also includes
the secondarily aplastidic ciliates.
Unambiguous cases of such unique genetic events
may not only serve to unite groups, but also to root the
eukaryotic tree. Rooting requires events that unite all
eukaryotic taxa save one; that one, provided it convincingly shares the pre-event character state with prokaryotic outgroups, can then be taken as the deepest
branch. Keeling and Palmer (2000) recently found two
deletions in enolase shared by all known eukaryotes
save parabasalids (such as Trichomonas). Parabasalids
are themselves like archaea in enolase structure, and
on this basis these authors nominated parabasalids as
the deepest eukaryotes. Any single such one-of-a kind
event can in principle establish relationships between
the unconnected groups of Figure 2, but molecular evolutionists are as often wrong as right in specific cases.
Thus, it is comforting that genomic data will likely provide scores of such examples.
Protist genomics is in its infancy, about where prokaryotic genomics was six years ago when the first complete sequences began to appear. What evolutionary
microbiologists will soon have are phylogenetic trees
for tens of thousands of genes and partial or complete
gene contents (through EST, genome surveys and complete sequences) for dozens of protists about many of
whose evolutionary relationships we can currently only
guess. As these data accumulate, comparative analyses
of the “parts lists” of eukaryotic molecular machines
and cellular organelles much more detailed and complete than Table 1 will be assembled. Our conclusion
that that last common eukaryotic ancestor was a complex cell notwithstanding, there will surely be taxonspecific differences in parts lists that support more inclusive groupings of the taxa in Figure 2. Conversely,
as such groupings (and the eukaryotic root) become
established in multiple overlapping analyses, one can
undertake a detailed reconstruction of the evolutionary
history of specific eukaryotic cellular structures. If we do
find new eukaryotic lineages that are primitively simple,
then the “many and profound” differences that distinguish eukaryotes from prokaryotes can be tracked to
their origins. If we don’t, we can content ourselves with
detailing the many elaborations wrought in such structures over one to two billion years of eukaryotic cellular
radiation.
From a practical standpoint, until we have a wellresolved phylogeny, it is necessary to sample as many
eukaryotes as possible. EST projects provide evidence
for protein presence and expression levels. Full genome
sequences are going to be harder to come by, but,
importantly, provide information about protein absence,
crucial when ascribing simplified protein complements
to potentially early-diverging lineages. Although genomics projects are currently clustered in the better-known
taxa, as a wider diversity is covered, we will be able
to glean more and more information about eukaryotic
biology. By looking at the detailed phylogenies and paralog complements from diverse eukaryotes, we may be
able to reconstruct, on a molecular level, aspects of our
earliest eukaryotic ancestors’ cell biology.
Acknowledgments
We would like to thank A.J. Roger, J.M. Logsdon, A.G.B. Simpson,
and R.A. Singer for helpful discussion, and the latter two also for
critical reading of the manuscript. This work was supported by Canadian Institutes of Health Research (C.I.H.R.) grant MT4467 to W.F.D.
J.B.D. is supported by a C.I.H.R. Doctoral Research Award and
W.F.D. by a Canada Research Chair.
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