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Phylogeny and beyond: Scientific, historical, and
conceptual significance of the first tree of life
Norman R. Pace
a,1
, Jan Sapp
b
, and Nigel Goldenfeld
c
a
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309;
b
Biology Department, York
University, Toronto, ON, Canada M3J 1P3; and
c
Institute for Genomic Biology and Department of Physics, University of Illinois, Urbana,
IL 61801
Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved December 20, 2011 (received for review November
29, 2011)
In 1977, Carl Woese and George Fox published a brief paper in PNAS that established, for the first time, that the overall phylogenetic
structure of the living world is tripartite. We describe the way in which this monumental discovery was made, its context within the
historical development of evolutionary thought, and how it has impacted our understanding of the emergence of life and the
characterization of the evolutionary process in its most general form.
Afundamental breakthrough in
biological science occurred in
1977, and most biologists did
not notice. The paper by Woese
and Fox (1) in 1977 was 2.5 pages in length
and contained a single table of numbers
that compared sequence snippets derived
from small subunit rRNAs of different
organisms. The table provided the first
gene sequence-based quantitative assess-
ment of phylogenetic (evolutionary) rela-
tionships between representatives of the
major known kinds of organisms (1). The
paper showed that all cellular life falls into
one of three large relatedness groups:
eukaryotes (our kind of cells, which con-
tain a nuclear envelope), eubacteria
[Woese and Fox (1) termed the group and
this group is where classically studied
bacteria fit], and archaebacteria [an un-
usual group of recently described organ-
isms named by Woese and Fox (1) to
distinguish the group from eubacteria]. In
describing the phylogenetic relationships,
the results also charted the first scientific
view of deep evolutionary history. Both
these fundamental aspects of biology, the
phylogenetic structure of life and the
course of early evolution, previously were
only realms of speculation.
However, the methods and data used in
the work by Woese and Fox (1) were
unfamiliar to most biologists, even mo-
lecular biologists. Traditional biologists,
students of plants and animals, paid little
attention, because the results had little
bearing on their interests. Because of
a joint press release by the National
Aeronautics and Space Administration
and the National Science Foundation that
supported Woese’s research, the paper
was heralded on the front page of The New
York Times for discovery of “a third form
of life”(2). However, the few biologists
who noticed sometimes reacted negatively,
and articles denouncing the claims were
published. Subsequent developments
showed that the methods and conclusions
of the paper were sound.
Several aspects of the paper by Woese
and Fox (1) sparked skepticism. One
was the arcane nature of the molecular
data, which few could appreciate. The re-
liance on a single gene to trace major
trends in evolution was an equally alien
concept. Some quibbled about the name
archaebacteria; others objected to The
New York Times publicity. Most impor-
tantly, however, the conclusions of the
paper flew into the face of the common
wisdom of the time regarding the basic
divisions of biology and the nature of early
evolution. It was generally believed—and
still is taught in our textbooks—that life
is of two kinds: on one hand, eukaryotes
and on the other hand, prokaryotes, which
lack nuclear membranes and as the name
implies, were supposed to have preceded
and evolved into eukaryotes. However,
eubacteria and the newly discovered group
of archaebacteria both lacked nuclear
membranes. Eukaryotes seemed not de-
rived from either bacteria or arch-
aebacteria; all three kinds of organisms
seemed to represent aboriginal lines
of descent.
In this retrospective, we view the 1977
paper by Woese and Fox (1) from three
standpoints. First, we discuss the specific
accomplishments of this landmark paper
and how the program of research initiated
and led by Woese from the late 1960s to
the present day has spawned a revolution
in microbiology and other fields contin-
gent on microbiology, including ecology
and the health sciences. Second, we dis-
cuss the place of that paper in the history
of evolutionary biology, where its un-
precedented use of molecular sequences
associated with rRNA provided the first
window into the deep timeline of life, one
independent of theoretical prejudices that
had flawed earlier efforts to classify life.
Finally, we describe how the under-
standings sparked by the paper are bring-
ing a new face to the study of evolution by
compelling biologists to address founda-
tional issues related to the very concepts of
species and organism and bringing to the
fore the deep limitations of earlier ac-
counts of the evolutionary process.
Lead Up to the Paper
The 1977 paper by Woese and Fox (1) was
an early example of what we would today
call molecular phylogenetics—the com-
parison of macromolecular sequences to
infer genealogical and thereby, evolution-
ary relationships. The notion of comparing
sequences to infer relationships was put
forward in 1958 by Francis Crick (3) and
more formally, by Emil Zuckerkandl and
Linus Pauling in 1965 (4). This was a
time when determination of protein se-
quences had become, to some extent,
tractable with protocols developed by Fred
Sanger (5), who received his first Nobel
Prize for that development and the de-
termination of the amino acid sequence of
insulin in the 1950s (5). Protein bio-
chemists began to develop phylogenetic
relatedness maps, phylogenetic trees,
based on amino acid sequences derived
from various organisms, mainly animals.
Russell Doolittle sketched out vertebrate
evolution using blood-clotting fibrinopep-
tides in the work by Doolittle and Feng
(6); the work by Fitch and Margoliash (7)
used the mitochondrial protein cyto-
chrome C to relate animals and some
fungi. However, not all organisms possess
cytochrome C, and for that reason alone,
its amino acid sequence could not be used
to infer the patterns of relationships
among all of life.
Carl Woese came to the study of evo-
lution from a background in biophysics and
Author contributions: N.R.P., J.S., and N.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Classic Article “Phylogenetic structure of the prokary-
otic domain: The primary kingdoms”on page 5088 in issue
11 of volume 74.
See Classic Profile on page 1019.
1
To whom correspondence should be addressed. E-mail:
Norman.Pace@colorado.edu.
www.pnas.org/cgi/doi/10.1073/pnas.1109716109 PNAS
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PNAS CLASSIC PERSPECTIVE
an interest in the genetic code and its
origins. During the early 1960s, the nature
of protein synthesis and the makeup of the
genetic code were just being worked
out (8). Woese was a contributor to early
thought on the genetic code and had
conducted experimental studies to try to
understand the chemical basis of the ca-
nonical assignments of different amino
acids to particular codons (the DNA or
RNA base triplets that specify the amino
acid sequence of a protein during protein
synthesis) (9). His 1967 book The Genetic
Code: The Molecular Basis for Genetic
Expression focused prescient attention on
the RNA elements of the protein synthe-
sizing machinery (10). Woese, along with
Francis Crick (11) and Leslie Orgel (12)
after him, are considered founding cham-
pions of the idea that nucleic acids played
more than template roles in the origin of
biological systems, thus giving rise to the
notion of a hypothetical prebiotic RNA
world in which nucleic acids served as both
catalytic entities and genetic templates
(13). Woese was concerned that the
emerging paradigm for the mechanism of
protein synthesis was too static and had
no evolutionary dimension. As early as
1969, as articulated in a letter to Francis
Crick, he understood that the only way to
reveal the essence of the process was to
study its conservation and variation in
different organisms—its evolution—in
a phylogenetic framework. He set out to
do that study by comparing rRNA se-
quences. This task was daunting at that
time before the development of rapid se-
quencing protocols, but it was the only way
to quantify evolutionary change.
All cells contain ribosomes, which carry
out protein synthesis and typically are
composed of ∼50% RNA and 50% pro-
tein. The ribosome consists of two sub-
units: a small subunit (SSU), which
contains the 1,500- to 2,000-nt-long SSU
rRNA (also called 16S or 18S rRNA to
denote size), and a large subunit (LSU),
which has two RNA molecules (a 120-nt
5S rRNA and a 3,000- to 5,000-nt LSU
rRNA). [Most eukaryotes contain a fourth
LSU rRNA (5.8S rRNA), but this RNA
corresponds to one end of the bacterial,
archaeal, and some eukaryotic LSU
rRNAs.] Woese began with 5S rRNA,
because its small size rendered it amena-
ble to the sequencing technology of the
time. He studied bacteria because of his
background in working with Bacillus
subtilis and the practical requirement to
prepare highly radioactive RNA for
sequence analysis.
RNA sequencing developed in the mid-
1960s mainly through the efforts of Fred
Sanger and his colleagues using the general
approach used for Sanger’s protein
sequencing protocol (14). A
32
P-labeled
RNA was digested with base-specific
RNases, and the sequences of the resulting
oligonucleotides were determined by di-
gestion with other nucleases. Next, frag-
ments of incomplete digestion of the RNA
were isolated and digested completely,
and the digestion products were analyzed;
eventually, the sequence could be inferred
from the oligonucleotide contents of
overlapping fragments. Mitchell Sogin,
then a graduate student with Woese,
learned the techniques from David H. L.
Bishop, a postdoctoral student from
Fred Sanger’s laboratory who was then
working in the Sol Spiegelman laboratory
at the University of Illinois. Sogin set up
the necessary facility for Woese’s group.
Woese and his students determined sev-
eral bacterial 5S rRNA sequences. They
showed that the rRNA sequences could be
used as phylogenetic markers for bacteria
(15). They also showed that evolutionary
variation in sequences could be used to
determine how the RNAs fold into sec-
ondary structure (so-called phylogenetic
comparative RNA structure analysis) (16).
However, it soon became clear that 5S
rRNA, at only 120 nt in length, was too
small in size and hence, information con-
tent to provide for accurate phylogenetic
assessments.
The SSU rRNA, at 1,500–2,000 nt, was
information-rich, but because of its rela-
tively large size, it was practically impos-
sible to determine the entire sequence
using the Sanger method. Woese posited,
however, that the full sequence of the
RNA was not necessary for phylogenetic
comparisons and that sufficient infor-
mation was available in the collection of
oligonucleotide fragments that result from
specific nuclease digestions of SSU
rRNA. Woese argued that any oligonu-
cleotide six residues or longer had a low
probability of random occurrence in
a molecule the size of the SSU rRNA;
therefore, they could be assumed to be
homologous in different organisms and
have common ancestry, and they could be
used in phylogenetic assessments. He and
his colleagues began to generate catalogs
of sequences of oligonucleotides that re-
sulted from digestion of the RNA with
ribonuclease T1, which cleaves at G resi-
dues and therefore, produces oligonu-
cleotides that are comprised of some
collection of U, C, and A with a single G.
The resulting oligonucleotides were re-
solved by 2D electrophoresis, first on cel-
lulose acetate at pH 3.5 and then on DEAE
cellulose paper at pH 1.9 using Sanger’s
protocols. The autoradiogram shown in
Fig. 1 is an example of such an RNase T1
fingerprint of a
32
P-labeled SSU rRNA.
The positions of the different oligonu-
cleotides in the electropherogram reflect
size, base composition, and sequence of
the particular oligonucleotides. Longer
oligonucleotides were excised from the
paper for secondary and sometimes, ter-
tiary digestions with other ribonucleases to
determine the sequences. Preparation and
analysis of any particular SSU rRNA
were somewhat risky processes. They in-
volved labeling cell cultures with many
millicuries of
32
P-orthophosphate, pro-
cessing the highly labeled RNA, and con-
ducting up to 5- to 8-kV electrophoresis
in 100-L tanks filled with refined kerosene as
a coolant. The analysisof RNA sequences in
these ways probably could not be con-
ducted today because of safety regulations.
Three Kinds of Life
George Fox joined Woese at the University
of Illinois as a postdoctoral fellow. When
the catalogs of SSU sequences proved
informative, Fox worked with William
Balch, then a graduate student in the
laboratory of Ralph Wolfe at the Univer-
sity of Illinois, to prepare
32
P-labeled SSU
rRNAs from methanogens, organisms
that produce methane (natural gas) as
a metabolic product. Although environ-
mentally important, methanogens were
little studied because of their requirement
Fig. 1. Ribonuclease T1 oligonucleotide finger-
print. As outlined in the text, data reported in the
paper by Woese and Fox (1) in 1977 consisted of
catalogs of oligonucleotide sequences derived
from RNase T1 digestion of small subunit (SSU)
rRNAs. The first step of the analysis involved res-
olution of RNase T1 digests of 32P-labeled rRNA
by 2D electrophoresis and locating labeled oligo-
nucleotides on the 80 ×100-cm sheet of electro-
phoresis paper by autoradiography. The result, an
RNase T1 fingerprint of the specific RNA, is shown;
notations were made by Woese during the anal-
ysis of archaeal rRNAs. Larger phylogenetically
informative oligonucleotides are in the upper
one-half of the pattern.
1012
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for extremely anoxic conditions for
growth, and as a result, there was no
classification system of these organisms.
Labeled RNAs went to technician Linda
Magrum for fingerprinting and then to
Woese for work up of oligonucleotide
sequences and compilation of the catalogs.
The SSU catalogs provided the first
phylogeny and classification of those
organisms, but more importantly, the
methanogen oligonucleotide catalogs dif-
fered markedly from catalogs of any bac-
teria that had so far been examined (17).
Today, we would compare DNA or RNA
sequences from different organisms ex-
plicitly in terms of percent identity, an
intuitively meaningful comparison even to
a nonspecialist. Comparison of oligonu-
cleotide catalogs was not so straightfor-
ward, which possibly contributed to the
lack of comprehension and resulting
skepticism that greeted the paper by Woese
and Fox (1). The single table in the work
by Woese and Fox (1) was a matrix
that compared association coefficients,
S
AB
values, of different SSU oligonucleo-
tide catalogs. (S
AB
values for catalogs of
organisms A and B are calculated as
S
AB
=2N
AB
/(N
A
+N
B
), where N
A
and
N
B
are the numbers of nucleotides in se-
quences of hexamers or larger in the
catalogs of organisms A and B and N
AB
is
the number in common to the two
catalogs.) The higher the S
AB
, the more
similar the sequences and the more closely
related must be the organisms repre-
sented by the sequences. The single table
in the paper by Woese and Fox (1), the
matrix of S
AB
numbers, showed clearly
that life—at least SSU rRNA sequences—
fell into three distinct relatedness groups
or urkingdoms as Woese and Fox termed
them. Each urkingdom was further char-
acterized by collections of signature oli-
gonucleotides found only in that group
and not the other groups. In addition to
these idiosyncratic markers, there were
universal signatures, oligonucleotide se-
quences found in all organisms examined.
For the first time, it was actually shown
that all life is related phylogenetically.
This finding was a seminal finding not
previously established but widely and im-
plicitly assumed. However, more strikingly
and wholly unexpectedly was their data
indicating that there were at least three,
not two, primary lineages of life.
The results of the paper showed that
a large-scale map of life’s diversity could
be seen as three branches corresponding
to eukaryotes, eubacteria, and archae-
bacteria. However, where was the root of
the tree (assuming there was one)? Where
was the origin of it all or some place
in the tree that would correspond to a hy-
pothetical last universal common ances-
tor? Were two of the urkingdoms more
closely related to one another than to
the third urkingdom, or did the three
branches spring independently from some
universal ancestor?
These questions could not be answered
from the necessarily limited 1977 data
and indeed, would not be settled for more
than another decade when Iwabe et al. (18)
and Gogarten et al. (19) used paralogous
rooting, a phylogenetic technique de-
veloped by Margaret Dayhoff, a pioneer in
bioinformatics (20), to establish that the
origin was deep on the eubacterial line.
The eukaryotes and archaebacteria
seemed to constitute a sister group to the
exclusion of bacteria. The result came as
no surprise, because by this time (1989),
the phylogenetic work of Woese’s group
had stimulated a large-scale effort, par-
ticularly in Germany, to determine the
molecular biology and biochemistry of
archaebacteria. Many aspects of the fun-
damental molecular biology of eukaryotes
and archaebacteria proved to resemble
each other more than their bacterial
counterparts. For instance, during tran-
scription, eukaryotes and archaebacteria
were both known to use TATA binding
proteins in promoter selection, whereas
bacteria use σ-factors, a different basic
mechanism (21); the DNA replication
enzymologies of eukaryotes and archae-
bacteria resemble one another far more
so than either resembles the eubacterial
version (22). The biochemical and molec-
ular criteria generally supported the root-
ing of the tree and a deep, at least partial
relationship of eukaryotes and archae-
bacteria. The rudiments of a scientifically
grounded universal tree of life were
in place.
In 1990, Woese, with colleagues Otto
Kandler and Mark Wheelis, proposed the
formal designation domains to denote the
three major phylogenetic groups, which
they proposed be named Eucarya,Bacteria,
and Archaea (formerly archaebacteria)
(23). The diagrammatic phylogenetic tree
that they used to support their classifica-
tion is reproduced as Fig. 2. For the first
time, a universal tree of life had been
determined in a scientifically rigorous way.
The field of molecular phylogeny is still
a contentious one, but the large-scale
organization shown in Fig. 2 is generally
accepted.
Revolution in Microbiology
The development of a sequence-based
phylogenetic framework for the identifi-
cation of microbes would revolutionize
microbial ecology (24) and more generally,
bring together evolution and ecological
studies under a single empirically based
framework. It had long been appreciated
that microbial ecology is the critical driver
of the global biosphere. However, progress
to achieving some understanding of envi-
ronmental microbes had been hampered
by the general necessity to culture micro-
organisms, even for identification. This
need was a crippling constraint, because
few environmental microbes, perhaps
much less than 1%, can be cultured using
standard methods (25). Consequently,
the microbial ecologist had little access
to environmental organisms and little
knowledge of even the kinds of microbes
that occur in the environment.
With the phylogenetic framework for
classification in place and the subsequent
development of recombinant DNA and
sequencing technology, rRNA gene
sequences began to be used to sidestep the
requirement for culture to identify envi-
ronmental organisms (26). rRNA genes
could be isolated directly from environ-
mental DNA as recombinant clones or the
products of the PCR and sequenced to
identify environmental organisms phylo-
genetically. Such sequences are incisive
identifiers for microbes, and therefore,
correlations between organisms and envi-
ronments became possible. Moreover,
the rRNA or other gene sequences also
could be used for development of molec-
ular tools, such as in situ hybridization
probes, for the study of environmental
organisms in their native environments.
Surveys of rRNA gene sequences from
different environments continue to expand
knowledge of microbial life in all of the
phylogenetic domains, and it is clear
that, so far, we are only scratching the
surface of a vast reservoir of microbial
Fig. 2. Diagrammatic phylogenetic tree used to illustrate the proposal of three phylogenetic domains in
1990. Modified from Woese et al. (23).
Pace et al. PNAS
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1013
diversity. Currently, about 2 million rRNA
sequences are held by the sequence data-
bases, and more than 99% of these se-
quences are environmental sequences (27).
The 1977 paper (1) and its aftermath
transformed microbiology by introducing
a phylogenetic framework for exploring
life’s diversity and developing a universal
tree of life, a natural system of classification
based on genealogy. This phylogenetic
framework spans most of evolutionary time
on Earth as a microbial world, hitherto
absent from classical evolutionary biology.
Evolution Without Microbes
The neo-Darwinian evolutionary synthesis
that emerged in the 1930s and 1940s was
a conception void of microbial foundation.
It was formed in a world of two kingdoms:
plants and animals. Microbiology largely
remained a world apart from evolutionary
biology until the development of new
concepts and methods for determining the
phylogenetic relationships based on uni-
versal characteristics at the core of the
molecular genetic system of all organisms.
The concept of highly conserved char-
acteristics, far removed from the vicissi-
tudes of life, from which one could
reconstruct the main course of evolution
had been central to evolutionary biology
from its beginnings. In Philosophie
Zoologique, Jean Baptiste de Lamarck
(28) argued that, to classify organisms
correctly, one had to distinguish relatively
trivial characteristics that were modified
through the influence of environmental
conditions from the essential system of
organs. The former adaptive traits repre-
sented the branchings of the tree of life.
The latter represented the course of in-
creasing complexity of organization.
Comparisons of the essential system of
organs, he said, could be made only be-
tween the higher groupings of animals and
not between species or genera; they
were less conspicuous in plants and not
conspicuous at all in the Infusoria (28).
The existence of highly preserved char-
acteristics through which one could trace
the tree of life was equally central to
Charles Darwin’s theorizing in regard to
descent with modification. As Darwin
wrote to his friend Thomas Henry Huxley
in 1857, the “time will come I believe,
though I shall not live to see it, when we
shall have very fairly true genealogical
trees of each great kingdom of nature”
(29). Darwin (30) wrote in On the Origin of
Species that all “true classification is ge-
nealogical; that community of descent is
the hidden bond which naturalists have
been unconsciously seeking, and not some
unknown plan of creation, or ... the mere
putting together and separating objects
more or less alike”(30).
For Darwin no less than for Lamarck, to
have a natural classification that reflected
the course of evolution, one needed to
distinguish trivial characteristics from the
“essential characteristics”or “organs of
high vital or physiological importance”
(30). As he explained in On the Origin
of Species,
[i]t might have been thought ... that those
parts of the structure which determine the
habits of life, and the general place of each
being in the economy of nature, would be
of very high importance in classification.
Nothing can be more false ... It may
even be given as a general rule that the less
any part of the organization is concerned
with special habits, the more important it
becomes for classification (30).
“Embryological characters,”he said,
“are the most valuable of all”(30).
Comparative morphology, anatomy,
embryology, and the fossil record were the
ways to distinguish homology from analogy
and thus, order organisms according to
their evolutionary relatedness. However,
bacteria lacked both morphological com-
plexity and a fossil record. One could not
tell which characteristics were ancient
and of common ancestry, which charac-
teristics were more recent adaptations,
which characteristics were homologous,
and which characteristics were analogous.
Microbiology’s Scandal
In his great work Systema Naturae, Carl
Linnaeus (31) had placed all Infusoria
(microbes) in one species that he pre-
sciently baptized Chaos infusoria. Bacterial
classification remained in chaos for the next
200 y. Bacteria were not put into groups
based on principles of homology and evo-
lution but based on as many characteristics
as possible and principles of utility for in-
dustry and medicine. Bacteria were ar-
ranged into a nested hierarchy of orders,
families, genera, and species based on cell
shape, plane of cell division, ability to form
spores and/or colonies, possession of fla-
gella, whether cells were connected,
whether they were branching, staining re-
actions, relation to temperature and oxy-
gen, pigment production, pathogenicity,
and a broad diversity of biochemical prop-
erties (32).
Although some microbiologists held
out hope for a natural classification, one
based on evolutionary relationships (33,
34), by the middle of the 20th century, they
were forced to admit that their efforts
were futile (35). Admitting defeat, Roger
Stanier, Michael Doudoriff, and Edward
Adelberg (36) declared in the first edition
of their popular text The Microbial World
that “it is a waste of time to attempt
a natural system of classification for bac-
teria ... bacteriologists should concentrate
instead on the more humble practical
task of devising determinative keys to
provide the easiest possible identification
of species and genera”(36).
Even distinguishing different microbes
from one another was troublesome for
microbiologists. For medical researchers,
they were all simply germs as Joseph Lister
called them in 1874; microbe was in-
troduced 2 y later (37). Some thought they
were little animals, and others thought
that they were little plants. Louis Pasteur
spoke sometimes of microscopic plants,
sometimes as animalcules and sometimes
as virus (the Latin word for poison). In
the early 1870s, the term bacteria (Greek
for little staff or rod) also began to be
used for the smallest of germs: “for all
those minute, rounded, ellipsoid, rod sha-
ped, thread-like or spiral forms”(38, 39).
Throughout most of the 20th century,
bacteria were understood to be plants;
their classification was the domain of
botanists who referred to them as the fis-
sion fungi or Schizomycetes as Carl Nägeli
had named them in 1857. This notion
persists in our age today with common
use of microflora to describe microbes.
Breaking out of the plant–animal dualism
would prove to be difficult.
In his speculative phylogenetic tree of
1866, Ernst Haeckel, who coined the
terms ontogeny, phylogeny, and phylum,
placed bacteria and blue-green algae
(now cyanobacteria) in a division that he
called Monera, and he christened another
kingdom the Protista. The Monera were
supposed to lack the fundamental division
of labor of nucleus and cytoplasm exhibited
in true cells and bridge the gap between
the living and the nonliving. Whether the
organization of the bacterium and the
blue-green alga fit Haeckel’sdefinition was
hotly contested throughout the early
20th century (32, 40, 41). A few micro-
biologists proposed that bacteria and the
blue-green algae should be granted their
own kingdom, Monera (34, 42). However,
others doubted that the grouping was
monophyletic. Additionally, it was far
from clear that the blue-green algae
really lacked a nucleus and how smaller
bacteria such as Rickettsia and Chlamydia
could be distinguished from viruses (36).
In a famed paper titled “The Concept of
a Virus,”André Lwoff (43) drew what
he considered to be a unequivocal dis-
tinction between a virus and a bacterium
on the basis of EM and chemistry. The
virus contained only one kind of nucleic
acid, either RNA or DNA, enclosed in
a coat of protein; it possessed few, if any,
enzymes, and it did not reproduce by di-
vision like a cell. In 1962, Stanier and
Van Niel (44) wrote a sister paper, “The
Concept of a Bacterium”, that aimed to
resolve issues about the anatomy of the
bacterium once and for all. “Any good
biologist,”they wrote, “finds it intel-
lectually distressing to devote his life to
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the study of a group that cannot be readily
and satisfactorily defined in biological
terms; and the abiding intellectual scandal
of bacteriology has been the absence of
a clear concept of a bacterium”(44).
Borrowing terms from Lwoff’s former
mentor Edouard Chatton (45, 46), Stanier
and Van Niel (44) distinguished pro-
karyotic cells (Greek for before karyon or
nucleus) from eukaryotic cells (Greek
for true nucleus). The latter divided by
mitosis and possessed a membrane-bound
nucleus, an intricate cytoskeleton, mito-
chondria, and in the case of plants cells,
chloroplasts. Prokaryotic cells were small-
er and lacked those structures. Stanier and
Van Niel (44) wrote that the
principle distinguishing features of the
procaryotic cell are: 1 absence of internal
membranes which separate the resting
nucleus from the cytoplasm, and isolate the
enzymatic machinery of photosynthesis
and of respiration in specific organelles; 2
nuclear division by fission, not by mitosis,
a character possibly related to the presence
of a single structure which carries all the
genetic information of the cell; and 3 the
presence of a cell wall which contains
a specific mucopeptide as its strengthening
element (44).
The prokaryote was, thus, defined
largely negatively. Although they had for-
saken a natural classification for bacteria,
they asserted nonetheless that the pro-
karyote was a monophyletic group of
common origin (44). “All these organisms
share the distinctive structural properties
associated with the procaryotic cell ... and
we can therefore safely infer a common
origin for the whole group in the remote
evolutionary past”(47). “In fact,”they
wrote, “this basic divergence in cellular
structure, which separates the bacteria and
blue-green algae from all other cellular
organisms, probably represents the great-
est single evolutionary discontinuity to be
found in the present-day world”(47).
The kingdom Monera, thus, rose like
a phoenix from the ashes. It was to be one
of five kingdoms, along with animals,
plants, fungi, and protists (microbial eu-
karyotes) (48, 49).
The prokaryote–eukaryote dichotomy
for the description of life’s diversity was
quickly instated among the canons of bi-
ology. Born in a time when the search
for a natural system for microorganisms
had been abandoned, the prokaryote
concept contained just as many untested
assumptions as the old taxonomic con-
jectures: the prokaryote was a mono-
phyletic grouping that preceded and gave
rise to eukaryotes. Indeed, nothing was
known of prokaryote or eukaryote origins;
they could have arisen one or many times.
Clash
The paper by Woese and Fox (1), “Phy-
logenetic Structure of the Prokaryotic
Domain: The Primary Kingdoms,”clash-
ed with the doctrines and methods of
classical microbiology in several ways.
First and foremost was the prokaryote–
eukaryote dualism. Phylogenetically,
Woese and Fox (1) wrote, life is not
“structured in a bipartite way along the
lines of organizationally dissimilar pro-
karyote and eukaryote. Rather it is (at
least) tripartite”(ref. 1, p. 5090). Second,
the eukaryotic cell organelles did not
originate solely in the gradual neo-Dar-
winian manner by gene mutation and se-
lection but rather, saltationally through
symbiosis as long had been suggested.
The SSU rRNA data left “no doubt that
the chloroplast is of specificeubacterial
origin”(1). The case was not yet as cer-
tain for mitochondria. The nature of the
engulfingspecies(thepureurcaryote)
that would have lacked eukaryotic or-
ganelles was unknown.
The manuscript received severe criti-
cisms when it was submitted to PNAS in the
summer of 1977 (1). One reviewer rec-
ommended that it not be published on
methodological grounds that their
claim for a tripartite division of the mi-
crobial world was as unfounded as their
claims in regard to symbiosis and the ori-
gin of eukaryotic organelles. For Woese
and Fox, rRNA was a highly conserved,
nonadaptive structure at the core of all
organisms. Nested deep in the center of
essential cellular functions, the SSU rRNA
was, as Woese (50) later put it, “the ulti-
mate molecular chronometer.”However,
for critics, an rRNA oligonucleotide cata-
log was merely a trait like any other, and
classification on its basis held no more
validity than classifying birds, bats, and
insects on the basis of their possession of
wings. That one molecule or many could
be used to discern phylogenetic relation-
ships was more than many classical mi-
crobiologists could accept; the molecular
approach was pronounced by some as
doomed to failure (51).
The belief that it was impossible to know
the phylogenetic relationships among
microbes because of a lack of fossil record
was well-entrenched in the minds of
microbiologists. As Stanier et al. (52) wrote
in The Microbial World in 1970,
[r]eflection and experience have shown,
however, that the goal of a phylogenetic
classification can seldom be realized. The
course that evolution has actually followed
can be ascertained only from direct his-
torical evidence contained in the fossil
record. This record is at best fragmentary
and becomes almost completely illegible in
Precambrian rocks more than 400 million
years old (52).
However, this publication was well
after Zuckerkandl and Pauling (4) had
written “Molecules as Documents of
Evolutionary History,”and Francis Crick
(3) had written as far back as 1958 that the
amino acid sequences of proteins “are
the most delicate expression possible of
the phenotype of an organism and vast
amounts of evolutionary information may
be hidden away within them”(3). There
was, indeed, a strange disconnect between
microbiology and molecular biology,
one that was finally resolved through the
SSU rRNA sequencing method reported in
the paper by Woese and Fox (1) in 1977.
The SSU rRNA sequencing method
had remarkable predictive success. Most of
the higher taxa above the genus would
have to be reclassified on the basis of SSU
rRNA phylogenies. When that technology
surprised microbiologists by predicting
unexpected relationships, it was corrobo-
rated by other data. Nothing was more
striking than the phenotypically diverse
organisms that came to be included in the
archaebacteria urkingdom by 1980 (do-
main archaea). It comprised methanogens,
found in the guts of rumens, extreme
halophiles known for rotting salted fish,
and extreme thermoacidophiles that live in
conditions that would cook other organ-
isms (53). The group was found to have
many highly conserved characteristics in
common: their walls lacked murein pepti-
doglycan (the only positive characteristic
of the prokaryotes), their tRNAs were
unique, the lipids in their membranes were
ether- and not ester-linked, their tran-
scription enzymes were unlike the enzymes
of the classic bacteria, and their viruses
were unlike the viruses of the classic
bacteria.
The SSU rRNA phylogenies also de-
finitively resolved the venerable question of
whether chloroplasts and mitochondria
originated as symbionts (1, 16, 54). Con-
jectures in regard to the symbiotic origin
of cell organelles had been discussed
throughout the 20th century, but that
question remained far outside the realm of
empirical inquiry (55). As Wilson (56)
commented in his famed book The Cell in
Development and Heredity,“[t]o many,
no doubt, such speculations [symbiotic
origin of organelles] may appear too fan-
tastic for present mention in polite bi-
ological society; nevertheless it is within
the range of possibility that they may
someday call for more serious consider-
ation”(56).
Such speculation did, indeed, call for
more serious consideration in the early
1960s with evidence that mitochondria and
chloroplasts each possessed their own
DNA and translation apparatus. That these
organelles might have originated as sym-
bionts was the conclusion of virtually every
paper, showing that they possessed their
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own DNA (57–59). However, proof of
origin was lacking, and the debates were
sterile. As Woolhouse (60) remarked, “the
time has come to bury this kind of specu-
lation with, by way of an epitaph, a parody
of Wittgenstein’s well-known remark,
‘Whereof one cannot know, thereof one
should not speak’” (60). Stanier (61) de-
creed that the problem of eukaryotic cell
origins would always remain in the realm
of metascience: “[i]t might have happened
thus; but we shall surely never know
with certainty,”Stanier (61) quipped.
Evolutionary speculation constitutes a kind
of metascience, which has the same in-
tellectual fascination for some biologists
that metaphysical speculation possessed
for some medieval scholastics. It can be
considered a relatively harmless habit, like
eating peanuts, unless it assumes the form
of an obsession; then it becomes a vice (61).
SSU rRNA phylogenetics belied that
statement. Molecular evolution revitalized
evolutionary biology and provided a new
basis for empirical investigations of pro-
found new questions concerning the evo-
lution of the cell and its components.
Molecular phylogenetic methods and
concepts constituted a paradigm apart
from classical evolutionary biology. Mo-
lecular methods for classification based on
GC content, DNA–RNA hybridization,
and amino acid sequencing of proteins had
begun to revitalize the aim for a phyloge-
netic classification of bacteria in the 1960s
and 1970s. However, those approaches
could not offer universal and quantitative
methods for a universal tree of life.
Influence on Evolutionary Biology
The work by Woese and Fox (1) and its
continuation within the Woese group have
had an enormous impact (e.g., roughly
one-quarter of the 40,000 total citations to
Woese’s work originate just from refer-
ences) (1, 23, 50, 62), and its methods are
revolutionizing disciplines as varied as
marine science and the study of the human
microbiome for medicine. This success has
perhaps overshadowed the original moti-
vations and may even have stimulated so
much activity in genomics per se that
Woese’s overarching interests in funda-
mental evolutionary questions have been
relatively neglected. We close out this
retrospective with a brief account of how
the work by Woese and Fox (1) began,
the extent to which their work has influ-
enced evolutionary thought, and the way
in which their ideas are still unfolding.
Woese and Fox’s discovery that all living
systems are representatives of one of three
“aboriginal lines of descent”did not
emerge in a vacuum (1). It was a water-
shed event (but not the culmination) in
an iconoclastic program of research that
Woese had been pursuing since the
mid-1960s, one with the goal of under-
standing the evolution of the complex
structure of the modern cell from the
origin of life (32). In this goal, he was
arguably more ambitious than Darwin’s
statement of the problem, which was re-
flectedinthetitleofDarwin’s great work,
On the Origin of Species (30). Indeed,
Woese’s concept of the evolution prob-
lem greatly surpassed prevailing views on
what at that time passed for the deepest
thinking on the question. Woese would
not find it necessary to cite On the Origin
of Species until 1992 (63), reflecting nei-
ther a stubbornness of character nor
a contrived striving for originality. The
fact of the matter is that Woese had
not even read Darwin’s original work
until around 2000, because its relevance
to Woese’sprogramhadseemedremote
until that time.
So what was Woese’s conception of the
evolution problem? First and foremost, it
explicitly drew a connection between the
origin of the genetic code, the translation
mechanism, and the emergence of cells
and their organization. For Woese, speci-
ation was an epiphenomenon of the evo-
lutionary process; the more important
question was that of degree of organiza-
tion. Woese and Fox (1) wrote that “[e]
volution seems to progress in a ‘quantized’
fashion. One level or domain of organi-
zation gives rise ultimately to a higher
(more complex) one ... Ideally one would
like to know whether this is a frequent or
a rare (unique) evolutionary event”(1).
Even earlier, in 1972, Woese (64) had
argued that
there are no such things as ‘special’evo-
lutionary problems. Evolution is not
a mixed bag of various ‘historical acci-
dents’... All evolutionary problems ap-
pear to have important common aspects—
whether they are intracellular problems or
problems on higher levels of biological
organization. Thus in ostensibly addressing
ourselves to evolutions of genetic codes,
translating mechanisms, etc., we are actu-
ally discovering and defining the basic
principles for all evolution ... The nature
of the ‘elements’involved changes from
one example to another, but the patterning
of events in time, the ‘principles of evolu-
tionary construction,’seem to remain in-
variant (64).
Woese understood that the goal of ex-
ploring evolution on the longest possible
time scales was not to elucidate the idio-
syncratic history of genes. “It is too easy to
think of evolution solely in terms of gene
mutations, flow in gene pools, and, of
course, some vague ‘selection’parameters.
Too much emphasis is placed on the
micro-changes at the expense of the
macro-ordering”(64).Hewantedtoun-
derstand universal aspects of the process
of evolution, not the particular sample
path represented by the history of life on
Earth, whose dynamics were imperfectly
captured by the slogan natural selection.
Woese and Fox (1), thus, approached
their work in an open-ended way, one not
constrained by theoretical prejudices
about the particular dynamics of the
evolutionary process. This strategy was
brilliant; not only did they uncover an
unanticipated new domain of life, but
also, they pointed out that there were
many possible modes and tempos of
evolution, of which only one—long
time-scale, core cellular mechanisms—
could be properly probed by molecular
phylogeny.
Woese and Fox’s approach yielded
a major surprise that could be interpreted
directly and plainly without contamination
from theory. The phylogenetic tree im-
plied by table 1 in ref. 1 clearly showed
three fundamental groupings, results that
were subsequently confirmed and elabo-
rated in compelling detail by Woese and
his collaborators (63, 65). At a stroke,
those data demolished the conception of
the prokaryote as a monophyletic group
that preceded and gave rise to the eu-
karyotic cell. The draft of the phylogenetic
tree that became available during the
1990s provided evidence for a last uni-
versal common ancestor, which is sum-
marized above.
Progenote
Woese’s conception of the evolution
question had already led him to surmise by
at least 1971 that life, as we know it today,
descended from an earlier, radically dif-
ferent protocell, which lacked the trans-
lation mechanisms of cells (64). His
rationale for this conclusion is important
to appreciate, because the paper by Woese
and Fox (1) in 1977 bore directly on
the properties of that hypothetical primi-
tive entity.
Woese and Fox (66) had, in parallel to
their experimental work on molecular
phylogeny, published a conceptual paper
that introduced the concept of “aprimi-
tive entity ... called [the] progenote, to
recognize the possibility that it had not
yet completed evolving the link between
genotype and phenotype”(66). The pro-
genotes were hypothetical ancient life
forms in the throes of developing the re-
lationship between nucleic acid and pro-
tein. Woese had suggested the existence
of the progenote from rather detailed
arguments based on his unrivalled
knowledge of the translation apparatus,
whichwasarticulatedinhisbookThe
Genetic Code: The Molecular Basis for
Genetic Expression in 1967 (10); he con-
sidered these arguments to indicate that
a general principle of evolution was that
complexity emerged through (i) a process
of refinement and (ii) a two-step cyclical
1016
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www.pnas.org/cgi/doi/10.1073/pnas.1109716109 Pace et al.
dynamic process that he proposed to
explain the quaternary structure of pro-
teins and that turned out to have re-
markable predictive power (ref. 67, p.
388). These same arguments suggested
a resolution of the old “chicken-and-egg”
paradox of whether translation preceded
the gene or vice versa. The resolution is
that both emerged from what we would
today call a co-evolutionary process of
refinement of translation, in which the
earliest proteins would have been statisti-
cal in character. In other words, early
translation produced not a single protein
but a family of similar proteins, any one of
which was adequate for the task at hand.
Thus, early life was a rickety, free-wheel-
ing construct whose lack of complexity
could tolerate imprecision at the level of
amino acids. The complex modern trans-
lation apparatus would have evolved in
a progenote evolutionary era, with a
tempo and mode based on a high muta-
tion rate of the primitive error-prone
translation system and a pervasive hori-
zontal exchange of genes within the pop-
ulation. During this era, there were no
lineages as such.
Woese and Fox (1) noticed that their
work already implied something funda-
mentally interesting about the progenote
state. Table 1 in ref. 1 established that the
three urkingdoms had to have been at
least 3 billion y old each and therefore,
that the
time available to form each phenotype
(from their common ancestor) is then short
by comparison ... we think that this im-
plies that the common ancestor ... was not
a prokaryote. It was a far simpler entity;
it probably did not evolve at the ‘slow’rate
characteristic of prokaryotes (1).
In short, Woese and Fox’s early data
already suggested to them that life evolved
from at least a two-step process: a late
stage that they characterized as having
a slow rate of evolution, one which could
be measured using the slowly changing
sequence of rRNA, and an earlier stage
refractory to molecular phylogenetic
analysis, where complexity evolved much
more rapidly, leading to the emergence of
genotype and phenotype as separate cel-
lular features along with a fully functional
translation mechanism.
Woese would return to the progenote
speculations in the succeeding years, em-
phasizing especially the characteristics of
high gene mutation and horizontal gene
transfer (HGT) (68), but by 1998 (69), the
context had become genomics and the
ever-increasing evidence for the wide-
spread occurrence of HGT. Its occurrence
was interpreted by some as being anti-
thetical to the entire program of building
a tree of life—all which he had addressed
so many years earlier.
Is There Really a Tree of Life?
HGT is the capability that organisms
possess to transfer genes to a non-
genealogically related recipient (70). Once
thought to be an exclusive aspect of mi-
crobial life, HGT has now been docu-
mented to occur between bacteria and
multicellular eukaryotes (71) as well as
between eukaryotes (72). With genomic
evidence for the widespread occurrence of
lateral gene transfer, the notion of a
universal phylogenetic tree came under
severe scrutiny and criticism (73). How-
ever, among Bacteria and Archaea, the
genes that are horizontally transferred are
typically metabolic genes or genes that
confer such traits as antibiotic resistance
and not the nonadaptive informational
genes that are involved in transcription
and translation.
It is now widely recognized that there
can be wide compositional variations in the
genomes of organisms that are nominally
classified together on the basis of their
SSU rRNA phylogeny. That within-group
variation reflects the presence of cosmo-
politan genes associated more closely with
a particular environment than with any one
particular organism (74, 75). Woese’s
original motivation for constructing a deep
universal phylogenetic tree based on SSU
rRNA sequences to understand the evo-
lution of the molecular genetic system re-
mains largely untouched by HGT. Even
those elements of the translational appa-
ratus that are most susceptible to HGT
(the aminoacyl-tRNA synthetases) show
that the canonical pattern of the rRNA
tree is largely preserved (76). Moreover,
the frequency of HGT is known to depend
on the evolutionary distance between the
lineages concerned, and thus, even if there
is significant HGT, the impact on the
rRNA phylogenetic tree would be ex-
pected to be rather small: the tree can
reflect both the evolutionary history of the
lineage as well as the predominant pat-
terns of gene transfer into and out of the
lineage (77).
Molecular Phylogeny vs. Morphological
Taxonomy
When Woese, Otto Kandler, and Mark
Wheelis (23) formally proposed the three
domains as the natural classification of
taxa in 1990, a direct confrontation with
the status quo ensued. The issues were
well-revealed in Woese’s debates with his
most distinguished opponent, Ernst Mayr
(78, 79). First, there was the question of
the great molecular and biochemical di-
versity of the microbial world compared
with the great morphological diversity ex-
hibited by multicellular eukaryotes. For
Mayr (78, 79), the degree of differences
exhibited within the prokaryotic world
simply could not compare with the mor-
phological diversity so readily observed in
the eukaryotic world, and the measure-
ment of that difference using molecular
sequences was anathema compared with
the more descriptive phenotypic dis-
tinctions that he favored.
A second issue concerned the purpose of
a classification system. Mayr (78, 79)
privileged the facility of information re-
trieval over a system based on evolutionary
relationships. In his view, the classifi-
cation system should enable a biologist to
quickly identify an organism with “a min-
imum of effort and loss of time”(79)
rather than reflect the genealogy of the
organism. In his response, Woese (80)
broadened the discussion to include the
foundational evolutionary questions that
we have presented above and that, by
that time, had occupied his thoughts for
nearly three decades.
Interestingly enough, the Mayr–Woese
debate did not address one of the most
contentious issues arising from the paper
by Woese and Fox (1): the definition of
species in the microbial world. Mayr
(78, 79) had famously defined the Bi-
ological Species Concept in 1942 as groups
of interbreeding natural populations that
are reproductively isolated from each
other, but clearly, this concept did not
apply to Bacteria, Archaea, or even mi-
crobial eukaryotes. With the advent of
SSU rRNA phylogeny, it became common
practice to define operational taxonomic
units by the requirement of close sequence
similarity (typically 97%) (24). However,
as more and more sequencing capability
became available, it became clear that the
spectrum of microbial life was continuous
rather than discrete because of cosmo-
politan genes and HGT. Bacteria seem to
form a radiation, with no specific species
boundaries (27, 81). According to some
estimates, only ∼40% of the genes in
a particular bacterium typically occur in all
of the genomes of that particular named
species; the other ∼60% of genes in the
typical bacterial genome occur only spo-
radically in other representatives of the
species or not at all (81). More than 30 y
after the paper by Woese and Fox (1) was
published, the fundamental biological
concept of species remains unresolved.
Legacy
Woese and Fox set out to determine the
degrees of relatedness between all living
organisms using rRNA sequences as
a marker of cellular evolution on slow
time scales subsequent to the emergence of
the modern lines of descent. They discov-
ered that there are three domains of life,
not two domains as had been previously
believed. Their work also strongly con-
strained the nature of life for times shorter
than about 1 billion y, indicating that
before the emergence of a strong phy-
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1017
logenetic signal of vertical descent, early life
had to evolve rapidly; we now suspect that
its evolution was reticulate in nature.
Modern versions of the techniques used by
Woese and Fox (1) are now routinely used
to sample environments as varied as geo-
thermal hot springs and gastrointestinal
microbiomes, providing unprecedented in-
sight into community structure and dy-
namics. The results challenged the
foundations of classical evolutionary the-
ory, requiring new modes of evolution to be
considered, indicating the presence of an
unexpectedly large microbial pangenome
(“afield of genes”to use Woese’sfavorite
phrase) (1), and forcing us to reconsider
basic concepts such as the nature of species.
Perhaps no other paper in evolutionary
biology has left a richer legacy of accom-
plishments and promise for the future.
ACKNOWLEDGMENTS. We thank Professors Carl
Woese and George Fox for reviewing the manu-
script for accuracy. N.R.P. is supported by the
Alfred P. Sloan Foundation, J.S. is supported by
the Social Sciences and Humanities Research
Council of Canada, and N.G. is supported by
National Science Foundation Grant EF-0526747.
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