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Prokaryote Systematics: The Evolution of a Science

CARL R. WOESE

Introduction
The editors of The Prokaryotes are taking the bold and scientifically proper step of organizing this
second edition along phylogenetic lines. The idea of classifying bacteria in this way, according to
their “natural” relationships, is by no means new; it is, in fact, as old as bacteriology itself. What is
new is our capacity to do so. Before the advent of molecular sequencing—particularly of nucleic
acids—the microbiologist simply did not have the tools with which to generate the types of data
required to construct a reliable bacterial phylogeny. Now the microbiologist does. This edition of
The Prokaryotes thus marks a major turning point in the history of bacteriology; the familiar order
of the old determinative classification (with its misleading phylogenetic overtones) is giving way to
the meaningful order of a phylogenetic system. Replacing the old determinative classification with
a phylogenetically valid one may not at first sight appear to be a radical or even important
departure for the field of microbiology. Yet it is; and hopefully this will become apparent as the
present chapter unfolds and as the reader derives insights and other benefits from the succeeding
chapters in this phylogenetically organized edition of The Prokaryotes.

Microbiology—the Science Without a Past

Organisms are in essence historical documents; their structure at all levels reflects (is determined
by and records) their evolutionary history. For this reason a knowledge of evolution and of
evolutionary relationships is an integral part of explanation and understanding in almost all aspects
of biology. Unlike zoology and botany, microbiology developed without this historical dimension,
without knowledge of the phylogenetic relationships among the organisms it studied. It is
impossible to say exactly what effect this has had on the science of microbiology. However, there
can be little doubt that knowing the phylogeny of bacteria would have profoundly altered the
course not only of microbiology, but of biology as a whole.

Consider as an example the role phylogenetic classification plays in zoology: A new beetle is
discovered. It is found to represent a novel genus within the Coleoptera. The biologist, therefore,
knows many things about this discovery that would not otherwise be known, and has explanations
for many of the insect's features that come only from knowing the evolutionary history, the
comparative anatomy, of beetles. This knowledge strongly influences the biologist's scientific
orientation, the interpretation of experimental results, etc.

Contrast this to the microbiologist's classification of a new bacterial isolate. Placing the bacterium
in a determinative system is helpful when it provides precise identification, as when the isolate is
found to be identical (or closely related) to some known species. In this case preexisting
knowledge can be invoked in studying or otherwise dealing with a new isolate. However, if the
isolate is sufficiently unusual that it can be placed only within one of the higher taxa in the
deterministic order, the organism's classification can actually work against understanding the new
species, unless that taxon chances to have phylogenetic validity. The assumption that this familiar
order is actually a meaningful order leads to confusion, not enlightenment. The determinative
system presented in the 8th edition of Bergey's Manual (1974) is full of higher level groupings of
this nature—all polyphyletic scramblings, familiar orders conveying the semblance of understanding
without its substance. Examples are the gliding bacteria, the sheathed bacteria, the appendaged
bacteria, the spiral and curved bacteria, the rickettsiae, Flavobacterium, and, of course, the classic
Pseudomonas. We often read statements about some property of the “pseudomonads,” in which
the author fails to identify the particular species of Pseudomonas possessing that property, on the

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mistaken assumption that the genus Pseudomonas (which means “false unit”!) is a truly
monophyletic grouping, of which this particular strain must, therefore, be representative.

The microbiology that developed in the absence of an evolutionary framework is a science strong
on the applied side; it has also been a rich mine for the biochemist (who treats organisms merely
as bags of enzymes), and one bacterium in particular, Escherichia coli, has proven a most
convenient and productive source of genetic and molecular understanding. However, bacteria hold
a special place in the global ecology: they are the bases of food chains and/or the main mechanism
for recycling organic material. They are agents of mineral deposition and the source of our oxygen
atmosphere. (The role of plants here is not being overlooked; their photosynthetic capacity is of
bacterial origin, however.) Bacteria have ex-isted for at least three-quarters of the earth's history,
nearly 10 times as long as the metazoans, upon which the biologist bases the concept of evolution.
Yet the microbiology that could properly conceptualize and develop these aspects of bacteria never
came into existence. Only now that a phylogenetically oriented microbiology exists can we begin to
relate the history of life on this planet to concomitant geologic changes (i.e., relate biochemistry to
geochemistry), to develop a deep understanding of bacterial ecology and to advance the frontiers
of evolution.

Early Attempts to Determine the Natural Relationships Among Bacteria

The 19th century witnessed the transformation of the Linnean classification system into a
phylogenetic systematics (albeit somewhat imperfect) for the metazoa. The reason this was
possible and the reason for the system's success lay in the morphological complexity of animals
and plants, the ontogenetic recapitulation of the phylogenetic sequence, and the existing
knowledge of the fossil record. Early microbiologists recognized the benefits of similarly classifying
the bacteria:
… the only truly scientific foundation of classification is to be found in appreciation of the available facts from
a phylogenetic point of view. Only in this way can the natural interrelationships of the various bacteria be
properly understood … it cannot be denied that the studies in comparative morphology made by botanists
and zoologists have made phylogeny a reality. Under these circumstances it seems appropriate to accept the
phylogenetic principle also in bacteriological classification (Kluyver and van Niel, 1936).

However, the facts that bacteria are morphologically very simple, that they do not have meaningful
developmental stages, and that bacterial fossils (which are phylogenetically uninformative in any
case) had yet to be discovered, meant that attempts to develop a natural classification system for
the bacteria were doomed from the start, and would even adversely affect the development of the
science of microbiology.

During the lifetime of Louis Pasteur and Ferdinand Cohn (mid- to late 19th century) a natural
system of bacterial classification was not very important. Bacteriologists then were wrestling with
the basic question of the nature of a bacterium, with problems such as the apparently pleomorphic
nature of bacteria (the result of working with impure cultures), the difficulty of their isolation and
cultivation, and the search for a sufficient number of characters by which to make useful
distinctions among them. Thus, it was relatively unimportant whether bacteria were related to
plants (as Cohn believed) or were the Monera of Haeckel's new third (protist) kingdom; or whether
or not bacteria and blue-green algae were truly related. Cohn's original “form-genera” classification
of bacteria, a simple system based upon shape and behavior, gave the microbiologist a useful start
on the problem.

By the turn of the century, however, the evolution of bacteria (and so a natural system of
classification) had become an important consideration, as witnessed by the above quote from
Kluyver and van Niel. Bacteriologists debated the proper criteria upon which to base a natural
system, as well as the appropriate assumptions to make regarding bacterial evolution. Emulating
metazoan taxonomists, most early microbiologists assumed the morphological characters were the
fundamental ones: “It is self-evident that the shape of the cells is of outstanding importance for
determining the place of a bacterium in any phylogenetic system” (Kluyver and van Niel, 1936).
And in defining the higher taxa there could be “no doubt that in this respect morphology remains
the first and most reliable guide.” (Kluyver and van Niel, 1936). Physiological characters were
suspect “because of the wide range of adaptation which is manifested in bacteria” (Kluyver and van
Niel, 1936) and such characters were thus definitely secondary. Prévot (1933), among others, felt
that physiological characters were reliable only in making the lowest level, interspecies distinctions.
Their use as primary (high-level taxonomic) characters was so at variance with conventional
wisdom in the first part of this century that Kluyver and van Niel (1936) judged “Orla-Jensen's

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contribution to systematics—the prominent place assigned to rightly evaluated physiological

characters— … a milestone in the development of bacterial taxonomy.” Still, van Niel a decade later
cautioned that “… we cannot yet use physiological or biochemical characters as a sound guide for
the development of a ‘natural system’ of classification for bacteria” (van Niel, 1946).

By the 1940s, a number of “natural,” albeit procrustean, bacterial classifications had been devised
—e.g., by Migula (1907), Pringsheim (1923), Buchanan (1925) (the beginnings of Bergey's
Manual), Kluyver and van Niel (1936) (later emended by Stanier and van Niel [1941]), and Prévot
(1940)—all based primarily on morphology, and so all basically similar.

The original Kluyver and van Niel (1936) system serves as a good example of these efforts and the
assumptions underlying them: the primary assumption—given that morphology reigned—was that
the simplest shape, the sphere, was the original shape. In keeping with Oparin's (1938) primitive
ocean theory, bacterial metabolism was seen as initially heterotrophic . More implicity embodied
in the scheme was the notion—taken again from metazoan experience—of an evolutionary
development; evolution proceeded toward increasingly intricate forms, toward more complex life
cycles, “in the direction from unicellularity to multicellularity” (van Niel, 1946). The postulated
ancestral coccoid eubacterium was seen as developing along four lines: in the family
Micrococcaceae, the cell shape remained spherical, but a progression from single cells, through
tetrads, to cells grouped in packets (sarcinae) occurred, culminating in “the highest developmental
stage … the cocci able to form endospores” (Kluyver and van Niel, 1936). The fact that “motility
occurs … sporadically among the cocci … indicates that also the flagellated rods find their origin in
this primitive group, the more so since the two typical modes of flagellation are already
encountered” (Kluyver and van Niel, 1936). Here then is the origin of the Pseudomonadaceae,
“cephalotrichous (and related immotile) rod-shaped bacteria,” spiral, curved or straight, but largely
Gram-negative; and the Bacteriaceae, “peritrichous (and related immotile) rod-shaped bacteria,”
either sporeforming or not, but in the main, Gram-positive. The final family, the Mycobacteriaceae,
arises from a lineage that “leads via the streptoccoci to the short Gram-positive rods in the group
of lactic acid bacteria and corynebacteria. The further development of these universally immotile
bacteria can have given rise to the mycobacteria which apparently form the connecting link with
the simpler actinomycetes” (Kuyver and van Niel, 1936). Although (purple and green)
photosynthetic bacteria were enfolded into the various categories of this scheme (their
morphologies were typical and their physiologies incidental) the emphasis on morphological
characters forced the exclusion of the spirochetes, myxobacteria, and blue-green algae.

Stainer and van Niel (1941) later completed (and emended) the scheme, using the Kingdom
Monera to include all the prokaryotes within two divisions, the Myxophyta (blue-green algae) and
the Schizomycetae (bacteria). The division of schizomycetes contained three classes “of
polyphyletic origin” (Stainer and van Niel, 1941) Eubacteriae, Myxobacteriae, and Spirochaetae. At
this time the photosynthetic bacteria were also separated from their nonphotosynthetic
counterparts in a separate order Rhodobacteriales; showing that the microbiologist had now
developed greater confidence in physiological characters.

The Dream Ends

These early bacteriologists cannot be faulted for their efforts at phylogenetic classification. They
followed scientific principles and were generally temperate and tentative in their conclusions and
pronouncements. Their goal was to create a framework that would generate understanding and
successfully guide bacteriology into the future. However, they dealt with a vast array of facts—
shapes, sizes, arrangement and behavior of cells, flagellar patterns, spore characteristics, Gram
stain, fixation and staining artifacts, pigmentation, colony characteristics, nutritional requirements,
catabolic properties, and so on—that were ill-defined; and the relationships among these facts
were confusing. Moreover, the majority of their facts represented relatively superficial properties of
bacteria. This hazy, unsortable continuum from useful to meaningless facts and relationships did
not become the basis for building a useful natural classification of bacteria, but rather served as a
convenient and powerful device by which prejudices could be laundered into scientifically
respectable pronouncements. Bacteriologists of the day readily justified their view that blue-green
algae are not really bacteria; “… pigmentation does not afford any indication of relationship
between Bacteria and Myxophyceae …” (Pringsheim, 1949). They just as readily put certain
bacteria, e.g., Beggiatoa and Thiothrix, together with the blue-green algae, because
“representatives of … [these] genera show such unmistakable morphological affinities with the
Cyanophyceae that in our opinion they must be considered as colourless derivatives of the genera

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Oscillatoria, Phormidium and Schizothrix. …” (Kluyver and van Niel, 1936).

Eventually, most microbiologyists came to realize the futility of these attempts to construct a
phylogenetic systematics for the bacteria. Van Niel put his rejection of the whole program (the end
of his dream of a natural system for bacteria) this way in 1955:

What made Winogradsky (1952) grant that the systematics of plants and animals on the basis of the Linnean
system is defensible, while contending that a similar classification of bacteria is out of the question? The
answer must be obvious to those who recognize in the former an increasingly successful attempt at
reconstructing a phylogenetic history of the higher plants and animals, based upon comparative-anatomical,
embryological, distributional, ecological, and paleontological studies and who feel that comparable efforts in
the realm of the bacteria (and bluegreen algae) are doomed to failure because it does not appear likely that
criteria of truly phylogenetic significance can be devised for these organisms (van Niel, 1955).

This became microbiology's new catechism:

For [most] major biological groups [including the bacteria], the general course of evolution will probably
never be known, and there is simply not enough objective evidence to base their classification on
phylogenetic grounds. For these and other reasons, most modern taxonomists have explicitly abandoned the
phylogenetic approach. … (Stanier et al. 1970).

It was one thing to recognize the impossibility of classifying bacteria phylogenetically by the
existing criteria. It was quite another to conclude that they could never be so classified. Yet, this
indeed is what the field of microbiology had done—clinching this scientific exorcism by adopting the
attitude that, determinable or not, knowing the phylogenetic relationships among bacteria was not
important in any case—an attitude that seems to have been held from the start by the “realists”
who had “… been impatient with idealists … [for] unjustified speculations regarding relationships
between … bacteria” (Breed, 1939), and who evinced little cognizance of evolution. Where were the
“idealists” now, who understood that “… the mere fact that a particular phylogenetic scheme has
been shown to be unsound by later work is not a valid reason for total rejection of the phylogenetic
approach” (Stanier and van Niel, 1941)?

Henceforth, speculation concerning bacterial evolution became solely an extracurricular activity,

best done over a glass of port; classification would be solely determinative; and criticism of the
system would center merely upon its organization, which “… based on the Linnean approach, not
only is unjustifiably pretentious, but also impedes the best utilization of established characteristics
because they are employed for the construction of mutually exclusive combinations” (van Niel,
1955).

The outcome of all this was an atavistic replacement of the concern with natural relationships by
the still-unsettled problem of what a bacterium (a prokaryote) actually is:
… the biological nature and relationships of the bacteria have been subjects of perennial discussion. Why
have these questions obsessed some members of each succeeding generation of microbiologists? There can
be no doubt about the principal reason. Any good biologist finds it intellectually distressing to devote his life
to 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. … Our first joint
attempt to deal with this problem … 20 years ago … was framed in an elaborate taxonomic proposal, which
neither of us cares any longer to defend. But even though we have become sceptical about the value of
developing formal taxonomic systems for bacteria …, the problem of defining these organisms … in terms of
their biological organization is clearly still of great importance … [italics added] (Stanier and van Niel, 1962).

While great progress would be made in characterizing and defining the prokaryotic cell, a
fundamental answer to this new/old question of bacterial organization nevertheless rested upon
having a natural classification for bacteria; to fully interpret that historical document the cell,
comparative analysis in the context of a phylogenetic framework is essential.

The Molecular Revolution

The historical irony here is that as the interest in determining bacterial phylogenies waned, as the
enterprise became increasingly distrusted, discredited, and disregarded, the tools for generating a
valid phylogenetic classification for bacteria were being developed by molecular characterizations of
the cell. The reason why this would make possible what seemed impossible is obvious: on the
molecular level, prokaryotes are just as complex, just as stable, and just as easily and clearly
defined, as eukaryotes are.

In their seminal article “Molecules as documents of evolutionary history,” Zuckerkandl and Pauling
(1965) emphasized the considerable historical information contained in macromolecular sequences.

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What makes molecular sequences so informative is that, in addition to being essentially linear (and
so relatively simple to interpret), they are perfectly well defined and generally comprise hundreds
to thousands of more-or-less-independent characters (amino acids or nucleotides). The number of
different possible sequences is enormous. Thus (extensive) similarity always signifies evolutionary
relatedness; convergence ceases to be a consideration. Or, as Zuckerkandl and Pauling (1965) put
it: “… a recognition of many differences between two [sequences] does not preclude the
recognition of their similarity.”

From the extent (and nature) of the differences among sequences that correspond in different
organisms to the same function, one can reconstruct molecular genealogies, build evolutionary
trees for the organisms (Fitch and Margoliash, 1967; Fitch 1971; Felsenstein, 1982; Olsen, 1988).

The initial molecular approaches used to characterize bacteria gave less precise, less reliable, and
more restricted phylogenetic information than the underlying full sequences later would—however,
these were a far cry from the ill-defined, ambiguous, and misleading phenotypic characterizations
previously available. Genetic studies, nucleic acid hybridizations, and immunological cross-
reactivities all helped to provide a phylogenetic basis for a variety of bacterial genera. DNA base
ratios provided a widely accepted and useful (but generally overvalued) phylogenetic measure as
well. Molecular analyses of the bacterial cell wall brought meaningful order to the “high GC” Gram-
positive bacteria (Schleifer and Kandler, 1972). And full sequences were even determined for a few
cytochromes (Ambler et al., 1979a and b) and ferredoxins (see George et al., 1985). It was not
until the late 1970s, however, after the partial sequencing method known as oligonucleotide
cataloging had been extensively applied to bacterial ribosomal RNAs, that the full panoply of
bacterial phylogeny was finally glimpsed (Fox et al., 1980).

The Nature of Molecular Chronometers

The “evolutionary clock” (Kimura, 1983) is one of the great discoveries of the 20th century: The
fact that in different organisms different (but clearly related) molecular sequences correspond to
what appears to be the very same molecular function implies that most of the (net) changes that
become fixed over time in any given molecular sequence are selectively neutral; they are of no
phenotypic consequence (Kimura, 1983). Such changes must happen more or less randomly in
time, and so can be used to measure time in a relative sense. In other words, on the genotypic
level a more-or-less steady pace of evolutionary change occurs that is quasi-independent of the
sporadic “real” evolutionary changes happening in the overlying phenotype. This independent
“evolutionary clock” embedded in the genotype gives the biologist the capacity to infer
evolutionary histories and relationships (Woese, 1987); and it has also freed the bacteriologist
from the phenotypic quagmire of ill-defined, confusing, or conflicting, and generally
phylogenetically uninterpretable morphological and physiological characters.

Not all molecular chronometers are equally useful for reconstructing genealogies, however (Woese,
1987): Some are more accurate than others. Some are restricted to particular taxonomic levels
and/or particular bacterial groups. Others give distorted answers because they have undergone
(subtle) functional, i.e., selected, changes. The most reliable molecular chronometers might seem
to be those free of direct connections with the overlying phenotype, such as parts of introns,
pseudogenes, “junk DNA.” Changes in their sequence would necessarily happen randomly, and so
would be interpretable in a straightforward way. However, changes become fixed so rapidly in these
sequences that the range of organisms over which they provide phylogenetically useful information
is extremely narrow (Kimura, 1983; Woese, 1987).

The most useful molecular chronometers actually are molecules like the ribosomal RNAs—
molecules with universal, constant, and highly constrained functions that were established at early
stages in evolution, functions that are not affected by changes in the organism's environment
(except for changes in basic physical parameters such as intracellular pH and temperature). (A
strong indicator of the constancy of rRNA function is the near constancy of the molecule's
secondary structure within each of the primary kingdoms, and the approximate constancy that
holds even between kingdoms [Gutell et al., 1985]). Because rRNAs are large molecules, they
contain considerable information; their size also makes them less erratic chronometers than
smaller molecules (Woese, 1987). Moreover, rRNAs are easy to isolate in relatively large quantities;
they seem not to be subject to lateral gene transfer; and they can be sequenced directly (without
resort to gene cloning).

The fact that a precisely functioning molecule such as RNA is under strict functional constraints is

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both an advantage and a disadvantage. The advantage lies in the constancy of the constraints on
the various positions in the sequence and the fact that some positions change far more slowly than
others; this last makes the molecule like a clock that includes both a second hand and a calendar
—i.e., it can measure a wide range of time intervals. The disadvantage is that the stringent
constraints lead to more (local) sequence convergence than otherwise, and the vast differential in
rates at which positions change makes analysis more problematic than it would be for a uniform
rate situation. (Another of its advantages would appear to be that RNA is a “nonlinear”
chronometer and so is potentially capable of distinguishing rapidly vs. slowly evolving lineages,
and, therefore, localizing the root of a tree without the need to resort to the use of outgroup
sequences [Woese, 1987]).

Construction of Phylogenetic Trees from Sequence Data

The initial step in extracting phylogenetic information from molecular sequences is to align them.
An ideal alignment procedure places all the homologous positions in a set of sequences in
correspondence. We generally equate statistically significant sequence similarity with homology,
the retention of ancestral attributes, and so align sequences by maximizing their similarity.
However, sequence similarity is a somewhat inaccurate indicator of homology (especially the
“maximized” similarities generated by some of the computer algorithms now in use). The issue of
what constitutes sequence homology is not as straightforward as it initially seems, but it is
inappropriate to discuss this in detail here. Suffice it to say that homologous sequence positions
are often difficult to identify (as sequence similarities) in the absence of knowledge of higher order
structural (and functional) homology—especially when sequences span the vast evolutionary
distances they do in the microbial realm. (Recall that genes under no phenotypic constraint change
composition very rapidly on an evolutionary time scale [Kimura, 1983]), Thus, significant higher
order structural similarity is generally taken to mean homology, and in turn is used together with
sequence similarity in attempts to construct sequence alignments that maximize homology (Woese
et al., 1983).

Two main types of analysis are used today to infer phylogenetic trees from sequence alignments,
evolutionary distance analysis (Fitch and Margoliash, 1967; Felsenstein, 1982) and maximum
parsimony analysis (Fitch, 1971; Felsenstein, 1982). The former takes into account only the
number of positions that differ in composition between pairs of sequences—this pair-wise
difference being formally a distance. The latter looks at the quality of the difference between
sequences, i.e., at which positions have different compositions in different sequences and the
nature of these differences. Evolutionary distance analysis uses less of the information in an
alignment than does maximum parsimony analysis. A crude analogy that to some extent captures
the distinction between the two is that of a person arriving at a city from some far-away place,
having traveled by some arbitrarily chosen circuitous route. A distance measurement would
concern itself only with how far the person has traveled, not the route, the travel time or where the
traveler began. A parsimony analysis attempts to reconstruct the actual route taken.

With the evolutionary distance method, the observed number of differences between two
sequences is an underestimate of the actual number of differences, because more than one change
will have taken place at some positions; and in some instances these will appear to be no change.
The greater the actual distance between two sequences, the greater their observed difference
underestimates it. Consequently, sequence distance measurements make distantly related lineages
seem relatively closer than they actually are—often making them appear specifically related, when
indeed they are not (Felsenstein, 1982; Olsen, 1988). Fortunately, this underestimate of distances
can be corrected statistically, at least to some extent (Jukes and Cantor, 1969; Olsen, 1988).

The maximum parsimony method assumes that evolution proceeds by a “least action” principle,
i.e., that in generating sequence B from sequence A, the evolutionary process tends to make the
minimum number of changes required to do so. Thus the “correct” tree (evolutionary branching
order) is assumed to be the one that allows the minimum number of changes in going from some
(assumed) ancestral sequence to the collection of its descendant sequences (being considered). As
with the (uncorrected) evolutionary distance method, maximum parsimony analysis fails to take
multiple changes at a given position in an alignment into account, and so shows a strong tendency
to cluster distant lineages, whether or not they are specifically related to one another (Felsenstein,
1982).

All methods of phylogenetic tree inference are imperfect, due to the effects just mentioned, as well
as others (Felsenstein, 1982; Olsen, 1988; Jin & Nei, 1990). They perform particularly badly when

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dealing with lineages that evolve at different rates and with sequence alignments wherein the
different positions change composition at vastly different rates. Unfortunately, these are the real-
world conditions. If nothing else, it is important to confine analyses of a sequence alignment to
those positions that are the most “phylogenetically informative”, i.e., those that have not changed
composition frequently during the time span covered by the alignment (Felsenstein, 1982; Olsen,
1988; Achenbach-Richter et al., 1988).

The construction of phylogenetic trees has become an arcane art, and most biologists are unable to
assess the validity of such trees. A major part of the problem here lies with the practitioners of the
art, who feel no responsibility to present their methods and conclusions to the broader scientific
community in intelligible forms; in some cases it is even impossible to reconstruct a particular
analysis from its published account. To make matters worse, different experts using different
methods usually disagree about the correct branching orders. However, it appears (based largely
upon the consistency of the rRNA trees derived under different conditions, from alignments of
varying compositions, etc.) that most phylogenetic trees now being inferred from bacterial
sequence data by the standard methods are at least first approximations to the correct trees.

Since bacterial relationships so far are based almost exclusively upon a single molecule, the 16S
rRNA (or the functionally related but far-smaller 5S rRNA), it remains an assumption (yet a
reasonable one) that the phylogenies so inferred represent the organism as a whole. It would
seem, therefore, that the scientifically proper stance for the microbiologist to take at this juncture
would be to treat these phylogenies as hypotheses, and test them using other molecules,
phenotypic characteristics of the organisms, and so on. When the same or very similar
relationships are given by different molecular systems or when new phenotypic similarities
consistent with the projected phylogeny turn up, then that (general) phylogeny can be confidently
accepted. A few such tests have already been done, and the results have almost always confirmes
the major taxa inferred from ribosomal RNA sequence analysis. For example, a large structural
idiosyncrasy in an ATPase sequence common only to the (true) flavobacteria and the bacteroides
(Amann et al., 1988) confirms a relationship that was suggested solely on the basis of the analysis
of 16S rRNA.

Prokaryotes and Eukaryotes

So long as their phylogeny remained an unresolved, confused matter, and their nature was so little
understood, the relationship of bacteria to eukaryotes was not a fruitful subject for discussion.
Although there was little doubt that eukaryotes were a monophyletic group (because it seemed
unlikely that such a complex cell would arise twice), the same could not be said of bacteria.
Microbiologists disagreed as to whether the bacteria were distant relatives of plants (Cohn's view),
whether some (the blue-green algae) were plant-like and others not, whether they belonged to
Haeckel's kingdom of protists, and so on; but there was general agreement that bacteria were not
necessarily all related to one another. This could be seen in the strong distinction made between
blue-green algae and bacteria, and in the reluctance to include myxobacteria and spirochetes, for
example, in the usual bacterial taxonomic schemes (Pringsheim, 1949; Stainer and van Niel,
1941). There was just too much morphological and physiological diversity among them for the
(premolecular) bacteriologists to feel comfortable with the concept that bacteria were a
monophyletic group.

In the 1930s, Chatton (1937) attempted to bring order into this confusion by placing all living
systems into one of two categories, defined on the basis of fundamental cytological distinctions:
“Eukaryotes” were those organisms whose cells contained a membrane-bounded nucleus and
certain organelles; “prokaryotes” lacked these features.

Bacteriologists were not initially attracted to Chatton's suggestion, because it defined prokaryotes
in a completely negative way and thus did nothing to address the bacteriologists' concerns about
the nature of bacteria and their relationships.

The entirely negative characteristics upon which this group is based should be noted, and the possibility of …
convergent evolution.… be seriously considered (Pringsheim, 1949).

… the bacteria and blue-green algae encompass a number of distinct major groups, which do not now appear
to be closely related to one another; their only common character is that they are procaryotic. It thus
appears that the procaryotic cell has provided a structural framework for the evolutionary development of a
wide variety of microorganisms. (Stanier and van Niel, 1962)

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The matter was further confused by suggestions (based upon artifacts and misinterpretations) that
some bacteria might actually have cytological structures similar to those that were considered
unique to eukaryotes (DeLamater, 1952; Tulasne, 1947).

However, with the advent of the electron microscope and other molecular tools, the prokaryote (as
well as the eukaryote) began to take on a well-defined submicroscopic and molecular character of
its own. It was now possible to define and distinguish prokaryote from eukaryote in comparable
terms, e.g., the differences in ribosome structure (Woese, 1970; Woese and Fox, 1977a). These
detailed studies strongly reinforced the notion that “prokaryote” was a monophyletic unit. Yet, the
ready acceptance of this conclusion was strange, for despite the above-cited reservations of some
bacteriologists and the warnings of others:

… there are remarkably few comparative studies. The result is that the application of the newer adjuncts of
morphology for taxonomic purposes entails generalization from limited cases (Murray, 1962),

the “characteristic” features of prokaryotes were derived from very few examples thereof.
Escherichia coli was the only prokaryote to be extensively studied. This can only mean that the
monophyletic nature of prokaryotes was assumed a priori by those interested in their molecular
characterization.

By the late 1960s the prokaryote-eukaryote dichotomy had become dogma; and the critics had
become converts:

There is little doubt … that biologists can accept the division of cellular life … into two groupings at the
highest level expressing the encompassing characters of procaryotic and eucaryotic cellular organization.
(Murray, 1974)

All organism except viruses can be assigned to one of two primary groups, readily distinguishable by
differences in cellular organization.… The definition of bacteria and blue-green algae in terms of positive
rather than negative characters had to await the revolution in our knowledge of cell structure which followed
the introduction of the electron microscope as a tool in biological research.… These differences [between
prokaryotes and eukaryotes] are now so widely recognized that descriptions of them can be found in the
better textbooks of general biology …, a sure indication that they have acquired the status of truisms
(Stanier, 1970).

The strength of the eukaryote-prokaryote dogma was convincingly demonstrated by the incredulity
expressed by many, perhaps the majority, of biologists at the discovery of the archaea
(archaebacteria), almost all of it unpublished, unfortunately. Biologists could no longer even
imagine that a class of prokaryotes could exist that were no more related to other prokaryotes
than they were to eukaryotes!

The prokaryote-eukaryote distinction did much to focus and refine the biologist's concept of the
primary phylogenetic relationships. However, in an important sense it was wrong and so, wrongly
shaped the biologist's thinking, with the result that it impeded as much as it fostered scientific
progress. The seductive symmetry of this simplistic dichotomy is almost certainly the source of the
problem: monophyletic eukaryotes (the one side of the equation) imply monophyletic prokaryotes
(the other). (One wonders how long the discovery of the archaea was delayed by this all-too-
simple catechism).

The concept has an even more pernicious aspect, however. The prefix pro- in prokaryote connotes
three interrelated things: 1) prokaryotes are older than eukaryotes; 2) prokaryotes are simpler and
more primitive than eukaryotes; and 3) prokaryotes are an ancestral stage in the evolution of
eukaryotes. A clear example of these assumptions can be seen in the works of L. Margulis (e.g.,
1988). A constant theme, readily apprehended in her diagrams, is the aboriginal cell that gives rise
to an ancestral bacterium, that in turn spawns a cascade of sublineages, certain ones of which go
on to form various aspects of the ancestral chimeric eukaryotic cell (through endosymbiotic
interactions). The mycoplasmas (“aphragmabacteria” in her terminology) provide the body of the
eukaryotic cell (the “host” component), whose mitochondria are derived from the “omnibacteria” (a
group vaguely reminiscent of the purple bacteria); cyanobacteria become the ancestral
chloroplasts, spirochetes the ancestral flagella, and so on.

The microbial phylogenies based upon molecular sequence comparisons fail to support this picture
and its underlying assumptions (Fox et al., 1980; Woese, 1987). The eukaryotes seem as ancient a
group as the bacteria; the basic eukaryotic cell (its organelles aside) does not appear to have
arisen from either prokaryotic group; and the notion that the eukaryotic cell was a prokaryote that
evolved to become more “advanced” is no more than anthropocentric bias. (In any case, the fact
that we now know two disparate types of prokaryotic organization exist casts any presumed
prokaryotic stage in the evolution of the eukaryotic [host] cell in a whole new light).

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Prokaryotic Evolution

How Valid Were the Old Phylogenies?

The bacterial phylogeny that emerges from molecular sequence comparisons has little in common
with our preconceptions. The old microbiologists' worst fears concerning the phylogenetic validity
of the phenotypic characters that they had to deal with have been fully realized. Morphology is not
the guiding principle they once took it to be; most, if not all bacterial morphological characters are
just too simple not to have evolved independently numerous times. Cocci generally arise as
degenerate variants of less simple shapes, as in the cases of Micrococcus, Staphylococcus,
Leuconostoc, Paracoccus, and other genera the reader will encounter throughout this volume. It is
doubtful that patterns of cell aggregation are useful phylogenetic characters even for making
species distinctions. Bacterial appendages, particular modes of motility or division, and flagellar
patterns all show a scattered, polyphyletic distribution (Fox et al., 1980; Woese, 1987). A few of
the more complex morphological traits, such as endospore formation, have some phylogenetic
validity; but what is a phylogenetically meaningful morphological character cannot be assessed a
priori.

The traditional physiological characters fare no better, though far less had been expected of them.
Autotrophy and heterotrophy, and oxidation and reduction of the same compounds, can be found
within different species in the same genus or family, and occasionally even in the same organism
(Fox et al., 1980; Woese, 1987). Phylogenetic sense has yet to be made of the catabolic patterns in
groups such as the clostridia. The most phylogenetically consistent physiological trait is
photosynthesis (Woese, 1987): each of the five known variations of chlorophyll-based
photosynthesis represents a different bacterial phylum (division).

Phenotypic characters, both morphological and physiological, tend to define incomplete

(paraphyletic) groupings; close relatives lacking the trait in question almost always occur (Woese,
1987). For instance, nonsporeforming relatives of spore formers are common, and “colorless”
relatives often outnumber the photosynthetic bacteria in a number of taxa. (Ironically, however,
none of the bacteria that the early bacteriologist felt certain were colorless relatives of the blue-
green algae [see above] turned out to be so.

It is clear that the microbiologists who would now construct a new (phylogenetic) taxonomy for
bacteria must take as open minded and creative an approach to developing the new system as
possible. It will not do unthinkingly to apply the old Linnean formula, to continue merely to use
only the old criteria for defining and naming taxa, or to make the new system a chimera of old and
new concepts and categories (Stackebrandt et al., 1988).

The Scope of Prokaryote Phylogeny

The ability to determine bacterial phylogenetic relationships, which culminated in the discovery of
the archaea (Woese and Fox, 1977b), has had three important consequences: 1) it has permitted
the construction of a comprehensive phylogenetic tree that relates all living systems (see Fig. 1);
2) it provides the basis for a proper understanding of the relationship between prokaryotes and
eukaryotes; and 3) it gives us a framework within which to begin developing a meaningful concept
of the universal ancestor of all life on this planet.

Fig. 1. An unrooted universal phylogenetic tree. The tree was produced from an evolutionary
distance matrix derived from an alignment of small subunit rRNA sequences. Only those positions
in the alignment judged to be homologous among all three domains were used in the calculation.
Detailed descriptions of these procedures can be found in Woese (1987) or references cited
therein.

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In dealing with prokaryotic evolution, one has to think on an entirely different time scale than
when dealing with classical (metazoan) evolution: the plant/animal/fungal branchings in Fig. 1 are
relatively superficial by comparison to some of the prokaryotic branchings (or, for that matter, to
the protist branchings on the eukaryotic lineage itself). Yet in defining microbial taxa we err in the
opposite direction, grossly underclassifying them relative to the metazoa: the human and the frog,
two different classes of animals, are separated by less evolutionary distance—about 5% in rRNA
sequence terms—than separates most species of the genus Bacillus. This taxonomic “double
standard” undoubtedly contributes to our mistaken (or at least exaggerated) impression that
microbial evolution has a far more protean quality than metazoan evolution.

The Two Prokaryotic Kingdoms

In general, the (eu)bacteria and archaea present rather different evolutionary pictures. The
archaea are a small but diverse collection of phenotypes—the methanogens, the extreme
halophiles, the extreme thermophiles, and the sulfate-reducing species (Balch et al., 1979; Jones
et al., 1987; Stetter et al., 1987). They differ in this respect from the bacteria, whose greater
phenotypic variety appears at the same time more connected, more interrelated. The tree shown in
Fig. 1 can be interpreted to suggest that the archaea in general evolve more slowly than bacteria
do. While the majority of the bacterial phyla arise in such a tight radiation that their exact order of

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branching has yet to be resolved, branching order among the major archaeal groups is readily
discernible. Photosynthesis is distributed among the bacteria in such a way as to suggest that it is
a fundamental bacterial characteristic, either being present in the ancestor common to all bacteria,
or arising very early in bacterial history (Woese, 1987). Archaeal photosynthesis, on the other
hand, which is based upon an entirely different mechanism than is bacterial photosynthesis
(involving bacterial rhodopsin [Stoeckenius, 1985]), appears to have arisen in, and is confined to,
one relatively superficial subline (Woese and Olsen, 1986). Aerobic phenotypes are plentiful among
the bacteria, rare among archaea (Woese, 1987). Thermophilic species, especially extreme
thermophiles, are so numerous among archaea that they are considered characteristic of the
group; the majority of bacteria are mesophilic (Woese, 1987).

The differences between archaea and bacteria run deep. Each has its own characteristic version of
the ribosomal RNAs, almost as different from one another as either is from eukaryotic rRNAs. And
in their ribosomal proteins the archaeal and bacterial versions tend to resemble one another less
than the former do the corresponding eukaryotic versions. Indeed, some archaeal ribosomal
proteins do not even have homologs among the bacteria; yet they do among the eukaryotes.
Archaeal DNA-dependent RNA polymerase finds its closest relative in one of the three eukaryotic
polymerases (pol II), rather than in the bacterial RNA polymerase, which is only distantly related to
these two. This same story repeats itself in most molecular systems studied in the archaea—in the
ATPases, where archaeal and eukaryotic versions are definitely more alike than either is like the
bacterial version, in certain key factors involved in the translation process, and in some DNA-
associated proteins. To all this can be added the ways that archaea are unique, such as their ether-
linked lipids, methanogenic metabolism and the modifications they make to certain bases in their
tRNAs.

By comparing sequences of genes that appear to have been duplicated (and functionally diverged
somewhat) in the universal ancestral state, i.e., before any of the three major lineages emerged, it
has been possible to locate the root of the universal tree (Iwabe et al., 1989). The root occurs on
the bacterial branch of the tree (below the bacteria proper) shown in Fig. 1. If true, this means
that archaea are specific relatives of (and possibly resemble ancestral forms of) the eukaryotes!

By now, the reader is well aware that the unfamiliar term “archaea” is being used in lieu of the
customary term “archaebacteria” (and that “bacteria” is used instead of “eubacteria”). The term
archaebacteria incorrectly connotes a relationship to (eu)bacteria. Both are prokaryotes, but, as we
have seen, this reflects only cytological resemblance, not kinship. To view archaea as “just
bacteria” in any sense is not to appreciate their uniqueness. Therefore, specifically to overcome the
misleading connotation of the term “archaebacteria” and generally to bring formal taxonomy into
line with the natural system emerging from molecular data, a new formal system of organisms has
been proposed (Woese et al., 1990), in which at the highest taxonomic level all life is divided into
three “domains”—the Bacteria, the Archaea, and the Eucarya. Within each domain, there are two
or more kingdoms (for example, the Animalia and Plantae within the Eucarya).

A detailed phylogenetic tree for the Archaea is shown in Fig. 2 (Woese and Olsen, 1986; Woese,
1987; Achenbach-Richter et al., 1987b). The root of the tree divides the Archaea into two main
lineages. One, the kingdom Crenarchaeota (Woese et al., 1990) is a phenotypically pure collection
of extreme thermophiles. The other, the kingdom Euryarchaeota (Woese et al., 1990) is a diverse
collection of lineages that collectively embrace all four of the major archaeal phenotypes
(Achenbach-Richter et al., 1988). On this latter, cosmopolitan branch of the tree a group of typical
extreme thermophiles, members of the genus Thermococcus, provides the deepest branching,
which is succeeded by the branching of the sulfate reducers (the genus Archaeoglobus). The three
methanogen branchings then follow, the Methanococcales being the deepest branching among
them. This branching pattern suggests that methanogenic metabolism somehow arose from the
sulfur-reducing thermophilic metabolism of Thermococcus-like entities (perhaps through the
intermediary of a sulfate-reducing metabolism) and that the biochemistry of the extreme
halophiles originated in methanogenic biochemistry.

Fig. 2. Phylogenetic tree for the Archaea. The tree, produced as described in the caption to Fig. 1,
was rooted using outgroup sequences from the other two domains. The positions in the alignment
used for the analysis met two conditions: 1) they were represented by a known nucleotide in all
archaeal sequences used; and 2) at least 50% of the sequences showed the same composition at a
given position. The three major methanogen groups are indicated by roman numerals. Generic
abbreviations are: Mc., Methanococcus; Mt., Methanothermus; M., Methanobacterium; Ms.,

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Methanospirillum; Mp., Methanoplanus; Msa., Methanosarcina; Mth., Methanothrix; H.,

Halobacterium; T., Thermoplasma; A., Archaeoglobus; Tc., Thermococcus; Tp., Thermoproteus; Tf.,
Thermofilum; P., Pyrodictium; S., Sulfolobus; and D., Desulfurococcus.

Fig. 3 provides a comparably detailed look at the bacteria (Woese, 1987). The lowest branching in
this case involves the Thermotogales lineage, a little-studied but very interesting group of
thermophiles. While one or two other branchings from the common stem can be discerned above
this, the bulk of the major bacterial lineages arise in an unresolved radiation. If one accepts that
the most ancient stromatolites (3.5 billion years old [Walter, 1983]) were produced by
photosynthetic bacteria (as stromatolites are today), then it appears that at least the earliest
photosynthetic bacteria (the green nonsulfur bacterium, Chloroflexus, being an example) had
evolved by that time.

Fig. 3. Phylogenetic tree for the Bacteria. The tree was produced as described in the captions of
Figs. 1 and 2. The alignment used comprised one to three representative sequences from each of
the 11 recognized bacterial phyla.

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A reasonable argument can be made to the effect that both prokaryotic domains arose from
thermophilic ancestors (Achenbach-Richter et al., 1987a; Woese, 1987). The case is stronger for
the archaea: 1) No mesophilic lineages have been found on the crenarchaeal side of the archaeal
tree although they have been looked for extensively—strongly suggesting that this branch of the
Crenarchaeota has a thermophilic origin; 2) only the extreme thermophile phenotype is found on
both main branches of the archaeal tree; 3) both the Methanococcales and the Methanobacteriales
contain species that grow at very high temperatures; these define the deepest branchings in each
group and so presumably represent the group's ancestral phenotype; and 4) the shortest lineages
in the tree are all associated with thermophilic species of one kind or another. (A short lineage
implies that the phenotype is relatively little changed from some common ancestral phenotype.)
The analogous argument for the bacteria is based upon the fact that on one side of that tree, only
thermophilic species are known (the Thermotogales group), and their lineages are shorter than
those of other bacteria, while on the other side, thermophilic lineages are at least common,
particularly among the deeper branching lineages, e.g., the green nonsulfur bacteria and the
deinococcus-thermus group (Achenbach-Richter et al., 1987a; Woese, 1987). A thermophilic origin
for all prokaryotes is in keeping with the geologist's picture of early earth, a planet whose
biosphere was far warmer than it is today (Walker et al., 1983; Worsley et al., 1986).

Mechanism of Prokaryotic Evolution: Its Relationship to Geologic

Change
In that prokaryotic evolution spans almost the entire history of this planet, and that prokaryotes
have such an intimate connection with certain types of geologic processes, one of the more
interesting classes of questions the microbiologist faces concerns the relationship between
prokaryotic evolution and geologic change—what geologic events influenced prokaryotic evolution
and how they did so; how prokaryotic evolution in turn effects changes in the biosphere; etc.
These are certainly questions for the future, but the beginnings of understanding seem to be visible
in the little we have discovered so far about prokaryotic evolution.

The strongest correlation emerging from molecular phylogenetic measurements of prokaryotes is

that the rate of evolution measured at the genotypic (i.e., sequence) level is in some way

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connected with the types of change that occur at the overlying phenotypic level; The more rapid
the rate of evolution at the genotypic level (one manifestation of which is a lineage on a
phylogenetic tree that is abnormally long), the more unusual and atypical are the resulting
phenotypic changes. Such a “tempo-mode” relationship has been observed, extensively
documented, and discussed on the metazoan level in paleontological terms for many years (Mayr,
1942; Simpson, 1944). It is only recently that what appears to be its counterpart on the cellular
and molecular level has been encountered in the prokaryotic world (Woese et al. 1985; Woese,
1987).

It is by no means a straightforward matter to identify an “unusual” or “atypical” prokaryotic

phenotype. Different organisms tend to be characterized in different noncomparable ways; and the
judgement “atypical” is subjective in any case. A useful direction to develop here may be the
comparison of genotypic classification of species (based upon some chronometer such as rRNA) to
their numerical taxonomic classification (where the latter does not include molecular sequence
data). Those organisms whose phenotypes are “unusual” compared to their relatives should tend to
show a discrepancy between their genotypic and phenotypic (numerical taxonomic) classifications.
On a cruder level this can already be seen when species that actually belong to one genus are
placed instead in some new genus.

The clearest examples of the tempo-mode relationship so far encountered are the mycoplasmas.
Phenotypically, mycoplasmas constitute a separate bacterial class; their uniqueness includes not
only lack of a cell wall, but also the small size of their genomes and certain biochemical and
cytological idiosyncrasies (Razin, 1978). Some biologists have gone so far as to declare them to be
primitive forms of life, unrelated specifically to bacteria (Wallace and Morowitz, 1973). By contrast,
mycoplasmas genotypically i.e., by rRNA sequence measure) are quite ordinary bacteria. The
mycoplasma group stems from the bacillus-lactobacillus-streptococcus lineage, which is one of a
number of lineages within the Gram-positive phylum; in other words, the mycoplasmas represent a
relatively superficial branching within the bacterial tree (Woese et al., 1980; Woese, 1987). What is
unusual about the mycoplasmas by this genotypic measure is that their individual lineages tend to
be relatively long; they appear to be evolving more rapidly than normal bacteria (Woese et al.,
1980; Woese, 1987). Another unusual characteristic of their rRNAs, which also appears to reflect
their rapid evolution, is that the compositions of the “highly conserved” positions in the molecule
tend to be less conserved in this group (Woese et al., 1980, 1985). Indeed all bacterial lineages
that appear to be rapidly evolving show this same tendency to alter the composition of the more
conserved positions (Woese, 1987).

While this relationship between rapid rate and unusual phenotype is most clearly demonstrated by
the mycoplasmas, it can be seen in more typical bacteria as well—where its converse can also be
recognized; i.e., slow rates, as judged by shorter lineages and a higher than average tendency to
conserve composition of the “highly conserved” sequence positions, correlate with phenotypes that
would be described as typical or ancestral in a group. A number of examples of both types can be
seen among the Gram-positive bacteria and the purple bacteria (groups for which extensive
sequence collections now exist). Among the Gram-positive bacteria, examples of rapid evolution
are Leuconostoc oenos, Fusobacterium, Haloanaerobium, and the general group of “high GC”
Gram-positive bacteria. (The members of this last group have evolved an impressive variety of
unusual and complex morphological traits, as their taxonomic treatment by early bacteriologists
amply demonstrates [Kluyver and van Niel, 1936; Stanier and van Niel, 1941]). Typical or
presumed ancestral Gram-positive phenotypes would include organisms such as Clostridium, (e.g.,
C. pasteurianum), Lactobacillus (e.g., L. casei), Bacillus, and Heliobacterium (its photosynthesis
being taken as ancestral), all of which are characterized on the genotypic level by relatively short
lineages and relative high retention of composition for the highly conserved sequence positions in
16S rRNA (Woese, 1987). Among the purple bacteria, one sees Chromatium, whose phenotype can
be judged ancestral and whose evolutionary tempo gives evidence of being slow; as opposed, for
example, to Haemophilus, an atypical “enteric” bacterium that shows a decidedly rapid
evolutionary tempo.

If it is true that drastic evolutionary change among prokaryotes invariably accompanies rapid
evolutionary rate, the question then becomes what brings about such rate increases. This
discussion is too long and too tenuous for the present context. Let me simply leave the reader with
a few brief comments: Increased evolutionary tempo among prokaryotes seems to reflect an
increase in the (optimum) mutation rate of a lineage. (Mycoplasmas are a special case because
they have very small genomes and so do not require mutation rates as low as those found among
normal bacteria [Woese, 1987].) This increase may be brought about by environmental factors,
such as by being in unsuitable niches, in which merely survival, rather than survival-of-the-fittest,
is the criterion, and in which the selection constraints in a sense are “internal,” rather than external

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(Woese, 1987). Under these conditions, an increased optimum mutation rate may not only be
acceptable, but even advantageous. Such evolutionary situations then produce evolutionary
saltations, those major changes that bring into existence new high-level taxa. Many, if not most,
such evolutionary situations will probably involve global environmental shifts—major changes in
the physical state of the planet that result in prolonged periods of relative instability. (This would
explain the occurrence of major evolutionary radiations.) Thus, it is conceivable that biologists in
the future will find a close connection between major evolutionary changes among prokaryotes and
major changes in the state of the planet: the history of prokaryotes that is written in their genes
may also include a history of the planet itself.

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