4 The Tree of Life An Overview

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S. L. Baldauf

D. Bhattacharya

J. Cockrill

P. Hugenholtz

43

Most of life, for most of life’s history, is about single-celled

organisms, which come in one of two types, eukaryotic and

prokaryotic. Most of life is probably prokaryotic, in terms of

numbers of cells, numbers of species, and time on Earth. Two

of the three domains of life are prokaryotic, the Archaea and

the Bacteria, and theirs are the oldest fossils, found in the oldest

unmetamorphosed rock [3.5 Byr (billion years) old; Schopf

et al. 2002; but see Van Zuillen et al. 2002]. Therefore, the last

universal common ancestor of all life (LUCA) was probably

prokaryotic, that is, a small cell (1–5 mm diameter), with a small

genome [~1–10 megabases (million bases)], few or no internal

membrane-bound structures, and able to meet all its living

requirements using simple compounds (autotrophic).

Eukaryotes were almost certainly derived from prokaryotes

(but see Philippe, ch. 7 in this vol.). The oldest even

arguably eukaryotic fossils are only ~1.8 Byr old (Brocks et al.

1999). All well-studied eukaryotes have cells that are at least

an order of magnitude larger than those of prokaryotes with

genomes (100–10,000 megabases). However, we now know

that bacterial-sized eukaryotes, probably with nearly bacterialsized

genomes (picoeukaryotes; described below), are common

(Moon-van der Staay et al. 2001, Lopйz-Garcia et al.

2001), but even these are clearly distinct from prokaryotic

cells. Thus, eukaryotic cells are more structurally complex than

those of prokaryotes, having various internal membranebound

organelles, such as a nucleus, and are, for the most

part, energetically dependent on endosymbiotic bacteria, that

is, mitochondria and chloroplasts.

Until the 1980s, universal trees of life were based on a

combination of structural and biochemical data characters,

but these generally have either too much or too little variation

to reflect reliably ancient evolutionary relationships.

Therefore, before the advent of molecular biology, constructing

an evolutionarily meaningful tree of life was a dubious

undertaking, at best. It was the discovery of the conservative,

ubiquitous nature of ribosomal RNAs that changed this.

All living cells make protein and in pretty much the same

way using ribosomes that consist of a large and small subunit

(LSU and SSU). The catalytic core of each ribosomal

subunit is an RNA molecule, the ribosomal RNAs (rRNAs).

LSU and SSU rRNAs are large molecules, highly conserved

across all life, and extremely abundant. It is these characteristics

that make them such excellent “molecular phylogenetic

markers,” particularly SSU rRNA (also known by its sedimentation

coefficient of 12S for mitochondrial or 16S–18S for

nuclear SSU or rRNA; Green and Noller 1997).

The highly conserved nature of SSU rRNAs allows these

sequences to be obtained relatively easily from most living

organisms and meaningfully compared with each other.

Thus, SSU rRNA data provided, for the first time, large numbers

of clearly homologous characters across all life and led

to the first universal evolutionary trees derived by objective,

quantitative criteria. The most startling early discovery was

that prokaryotic cells are actually two fundamentally different

groups of organisms, archaebacteria (Archaea) and true

bacteria (Eubacteria or simply “Bacteria”), as different from

J. Pawlowski

A. G. B. Simpson

44 The Origin and Radiation of Life on Earth

each other as either is from eukaryotes (Eucarya; Woese and

Fox 1977).

There are now more than 40,000 SSU rRNA sequences

in the public domain (Benson et al. 2004). These clearly identify

many (but likely not all) major taxonomic groups, some

previously only guessed at or entirely unknown. Parts of the

molecule are so highly conserved that they can be used as

primers to determine SSU rRNA sequences from even trace

amounts of DNA using polymerase chain reaction (PCR).

This technology has recently been adapted to allow sequencing

of SSU rRNA from uncultured organisms or even from

mixed pools of total environmental DNA, an approach called

environmental or culture-independent PCR (ciPCR; Amann

et al. 1995, Moreira and Lopйz-Garcia 2001, Hugenholtz

et al. 1998; see also Pace, ch. 5 in this vol.). This has revealed

a tremendous diversity of previously unknown organisms at

all taxonomic levels.

SSU rRNA data first defined the universal Tree of Life and

remain the cornerstone of molecular systematics. Although

protein genes trees have revealed important discrepancies in

the SSU rRNA tree, each protein gene tree seems to have its

own, unique inaccuracies as well. Nonetheless, on the whole,

there is a general consensus on most branches among most

molecules, although no single gene seems able to accurately

reconstruct them all (Baldauf et al. 2000). Individual genes

also seem to lack sufficient information to resolve the deepest

branches in the tree. For this reason, most studies of deep

phylogeny now employ multigene “concatenated” data sets

(CDSs). However, even this may not work for bacteria and

archaeans because of frequent trading of genes among even

very distantly related taxa [lateral gene transfer (LGT); see

Doolittle, ch. 6 in this vol.).

The following is a summary of the major groups of life

as we currently see them, and our best guesses as to how they

are related to each other. We have tried to provide a brief

description of each of the major groups, a summary of their

likely higher order relationships, and the nature of the supporting

data, both molecular and nonmolecular. The reader

should keep in mind that the deepest divergences in these

trees require large CDSs to test them, and only a few of these

are yet available. Furthermore, most habitats remain unsampled

by ciPCR studies, and the identities of these new

“ciPCR taxa” need to be confirmed with other data. Therefore,

the following is very much a summation of a work in

progress, but, with a little luck, one we can continue to build

on for a while.

Overview of the Tree

Figure 4.1 summarizes our current best guess as to the composition

of and relationships among the major groups of

living organisms based on a large number of independent,

partially overlapping studies. Emphasis is placed on SSU

rRNA trees, because these are the most comprehensive, and

on CDS trees, because these are the most accurate. The integrity

of the three domains of life, Archaea, Bacteria, and

Eucarya, is now confirmed by a tremendous body of data,

including nearly 100 completely sequenced genomes. The

identities of most of the major groups within these domains

are also confirmed by many different data, both molecular

and nonmolecular. Some of the relationships among the

major groups (“deep branches”) are also well resolved by

substantial bodies of data, but the majority of these deepest

branches are still only tenuously supported (shaded bars

on figure 4.1).

Arguably the single most outstanding question in the Tree

of Life is the position of the root. This can theoretically be

tested using ancient gene duplications that occurred before

the origin of the last common ancestor of all life. A number

of these duplications are known, and all seem to tell the same

story, that the root of the universal tree lies within Bacteria,

making Archaea and Eucarya sister taxa (Gogarten et al.

1989, Iwabe et al. 1989). This agrees with the striking similarities

between Archaea and Eucarya in nearly all aspects of

cellular information processing. Nonetheless, these are still

only a handful of genes each with only a small number of

universally alignable positions. These limitations, together

with the immense evolutionary distance involved (2–4 Byr),

make this an extremely difficult phylogenetic problem (see

Philippe, ch. 7 in this vol.), and this location for the universal

root still needs to be regarded with caution.

Domain Bacteria

Bacteria are highly variable, and there are few general rules

about them that are not violated somewhere. Sizes average

1–5 gm but range from 0.1 to 660 gm. Most have a peptidoglycan

cell wall sandwiched between an inner and outer

cell membrane composed of ester-linked lipids, but the cell

wall, the outer membrane, or both may be absent. A variety

of internal and external structures are found in bacteria, but

these are rarely membrane bound. Multicellular assemblages

are common, sometimes with terminally differentiated cell

types, and complex life cycles are found, sometimes including

several developmental stages. Motility is by means of

flagella, gliding, or adjustable buoyancy using gas-filled

vacuoles, and warfare is waged using a wide assortment of

“antibiotics.” Habitats seem to be any where there is water,

even small or sporadic amounts. These include everything

from deep crustal groundwater to natural gas deposits, volcanoes,

oil spills, clouds, and many, many more (Madigan

et al. 1997, Paustian 2003).

Bacterial genomes are most commonly organized into a

single circular chromosome with a single origin of replication,

very little repetitive DNA, many genes organized into

operons, and introns extremely rare. The chromosome is

located in a nuclear region (nucleoid) that is rarely membrane

bound, and proteins are synthesized nearby on 70S riboThe

Tree of Life 45

somes such that transcription and translation are simultaneous

(coupled). Extrachromosomal DNA minicircles (plasmids)

are common, carry a variety of genes often including

ones for antibiotic synthesis and resistance, and vary widely

in size. Gene expression is regulated by diffusible RNA polymerase

subunits, called sigma factors, that bind directly to

specialized promoter elements immediately preceding their

genes. Cells are generally haploid in lab culture, but most are

probably haplodiploid in nature, with large stretches of the

chromosome existing in multiple copies (Madigan et al. 1997).

Photosynthesis is common and usually anoxygenic, using

photosystem I or II (PSI, PSII). Only cyanobacteria use

both PSI and PSII, which, when coupled, can split water and

release oxygen (i.e., perform oxygenic photosynthesis). A

wide diversity of bacteria are thermophilic (prefer or require

high temperatures). It therefore appears that thermophily

must have evolved multiple times, probably aided by lateral

transfer of critical genes such as DNA gyrase (Forterre 2002).

Adaptations to thermophily include positive supercoiling of

DNA and its packaging with histone-like proteins, increased

guanine + cytosine (G+C) content in catalytic RNAs (but not

in protein-encoding DNA), and on-demand production of

heat-labile small molecules. Parasitism and symbioses are

widespread, mostly with eukaryotes. However, bacteria can

parasitize other bacteria or members of Archaea and also form

sometimes extremely complex symbioses or highly coordinated

commensal relationships with them (Madigan et al.

1997).

Because Bacteria are too biochemically and morphologically

plastic to be classified by such characters, their higher

order systematics and the entire field of bacterial evolution

did not really exist before molecular phylogeny. The first true

phylogenetic treatment of bacteria was Carl Woese’s now

classic 1987 paper in which he placed all the major groups

of cultured taxa into 12 “classical” groups, some predicted

and others still without phenotypic justification. More are

being added from existing culture collections—SSU rRNA

sequences exist for fewer than half of these taxa—and the

Figure 4.1. The Tree of Life. The tree shown is our current best guess on the major groups of life and their relationships to each

other. Solid bars indicate groupings for which there is considerable molecular phylogenetic support. Shaded bars indicate tentative

groupings with moderate, weak, or purely ultrastructural support.

46 The Origin and Radiation of Life on Earth

exploration of new habitats. However, the biggest revolution

in our appreciation of bacterial diversity has come from ciPCR

studies (Hugenholtz et al. 1998). These suggest the possible

existence of 10–20 or more new groups, some of them widespread

and diverse and probably important components of

a variety of ecosystems (see Pace, ch. 5 in this vol.). The study

of bacterial evolution, and bacteriology in general, has been

further revolutionized by the advent of rapid whole-genome

sequencing, revealing the entire genetic inventory of diverse

bacterial species. There are ~70 completed bacterial genomes

listed at the National Center for Biotechnology Information

genomics server (Benson et al. 2004) and severalfold more

in progress (Bernal et al. 2001). There are also probably as

many again in the private domain, particularly from medically

and commercially important taxa.

Molecular phylogenies of SSU rRNA and other universal

genes seem, for the most part, to define the major groups of

bacteria but not the relationships among them. This is because

of an assortment of problems, including the antiquity

of these relationships, lack of sequence data from important

taxa, and LGT (see Doolittle, ch. 6 in this vol.). The extent

of LGT in bacterial evolution has only recently been recognized,

and although informational (transcription and translation

component encoding) genes seem less susceptible (Jain

et al. 1999), no gene appears to be immune (see Doolittle,

ch. 6 in this vol.; see also Asai et al. 1999). Analyses of multigene

CDSs now show some consistent strong resolution of

major deep branches. However, these studies are still few in

number, only include taxa with completely sequenced genomes,

are somewhat overlapping in gene content, and may

not always be free of LGT-induced artifacts. Therefore, figure

4.2 shows a somewhat optimistic view of higher order

bacterial systematics, and many newly described lineages are

missing because of a general lack of information on them.

The following adheres to the standardized bacterial nomenclature

as proposed in the most recent edition of Bergey’s

Manual of Systematic Bacteriology (2001).

Hyperthermophiles: Thermotogae and Aquificae

Thermotogae

Thermotogae (fig. 4.2, node 2) are nonphotosynthetic rodshaped

hyperthermophilic (65–95°C) anaerobes that consume

organic compounds and generate hydrogen gas and

hydrogen sulfide. Besides being phenotypically narrow, the

group as a whole is not particularly large or widespread, as

ciPCR studies indicate, and so far they are almost exclusively

restricted to geothermal habitats (Hugenholtz 1998). The

only well-characterized taxon is Thermotoga maritima, originally

isolated from geothermal marine sediments and named

for its loose “toga-like” outer membrane. This taxon is usually

among the deepest, if not the deepest, branch in phylogenetic

trees within Bacteria. This seemed to be supported

by initial analyses of its completed genome sequence, which

showed that 24% of its genes are more similar to homologs

in Archaea than to those in Bacteria (Nelson et al. 1999).

However, this number appears to be considerably overestimated

(Ochman 2001) because it is based on a simple database

search strategy (“blastology”; see Doolittle, ch. 6 in this

vol.), and in-depth analyses of the remaining archaea-like

genes show that at least some are probably the result of relatively

recent LGT (Nesbo et al. 2001).

Aquificae (Aquifex/Hydrogenobacter Group)

Isolated from hot springs, volcano calderas, and marine hydrothermal

vents, Aquificae (fig. 4.2, node 2) thrive at 86–

95°C, making them some of the most thermophilic bacteria

known. Like the Thermotogae, members of this group appear

to be restricted to geothermal habitats (Hugenholtz et al.

1998), where they live by splitting hydrogen gas or hydrogen

sulfide and fixing carbon dioxide for carbon, all abundant

in geothermal volcanic gases (Hjorleifsdottir et al. 2002).

The Aquificae are more diverse than are Thermotogae and

include halophiles, isolated from saline hot springs, and an

acidophile, isolated from an acidic solfatar (sulfur deposits,

e.g., volcanoes; Takacs et al. 2001). The best characterized

are species of Aquifex, a blue filament and currently the most

thermophilic bacteria known. The completely sequenced, relatively

small (1.55 megabases) genome of A. aeolicus lacks many

metabolic pathways, consistent with the organism’s obligate

chemolithotrophic lifestyle (Deckert et al. 1998). New genera

belonging to this group have recently been described (Huber

et al. 2002a). These new taxa significantly extend the phylogenetic

diversity of the group (according to SSU rRNA divergence)

but not particularly its physiological diversity, because

all are hyperthermophilic chemolithoautotrophs.

Phylogeny

Thermotogae and Aquificae are the most consistently basal

branches in bacterial trees, both in CDS and single gene analyses

(fig. 4.2, node 2). However, they are found in a variety of

arrangements either together as a group (Olsen et al. 1994,

Bocchetta et al. 2000, Wolf et al. 2001, Daubin et al. 2002)

or as adjacent branches and in alternating order (Olsen et al.

1994, Brown et al. 2001). On the other hand, Brochier and

Philippe (2002) suggest that the basal branching of these two

taxa in SSU rRNA trees at least is due to a long-branch attraction

artifact. This is the tendency in phylogenetic trees for

highly divergent sequences, that is, those with long terminal

branches, to group together and/or be drawn toward the base

of the tree when a distant outgroup is used to root it.

Green Nonsulfur Bacteria (Chloroflexi)

The green nonsulfur (GNS) bacterial group is currently defined

solely on the basis of SSU rRNA phylogeny. Members

of the group are found diverse habitats, sometimes in abundance

(Hugenholtz et al. 1998, Bjornsson et al. 2002), and

The Tree of Life 47

the group appears as an early branch of Bacteria in some

phylogenetic trees (Oyaizu et al. 1987). Four major subdivisions

are defined, with most described taxa falling within

a single subdivision, Chloroflexi. Two entire subdivisions,

including Subdivision 1, the most divergent (by SSU rRNA

analysis), are known only from ciPCR. Recently an isolate

belonging to Subdivision 1 has been obtained from activated

sludge (Sekiguchi et al. 2001), although it has not been characterized

in detail.

Contrary to the group name, not all members are green

and sulfide intolerant. The group is metabolically diverse and

includes thermophilic sulfur-intolerant green phototrophs

(Chloroflexi), thermophilic red phototrophs (Heliothrix),

mesophilic sulfur-tolerant green phototrophs (Oscillochloris),

thermophilic heterotrophs (Herpetosiphon, Dehalococcoides,

Thermoleophilum), Thermomicrobium, Sphaerobacter, and

probably many more. Morphologically, the group appears to

be rich in filamentous representatives (Bjornsson et al. 2002),

including all of the described species with the exceptions of

the rod-shaped Thermomicrobium and Sphaerobacter, and

coccus-shaped Dehalococcoides.

Chloroflexi

Chloroflexi contain the best characterized of the GNS bacteria.

They are superficially similar but apparently unrelated

to the green sulfur bacteria (GSB; described below). Members

of Chloroflexi are moderately thermophilic (35–72°C)

gliding green filaments that leave a characteristic slime trail.

They are metabolically versatile but generally act as facultative

anoxygenic phototrophs and are common in microbial

mats, sometimes forming massive accumulations in hot

springs. In fact, it may have been GNS bacteria rather than

cyanobacteria that formed the large >3 Byr old continuous

microbial mats known as stromatolites (Oyaizu et al. 1987).

Figure 4.2. Support for deep branches in the bacterial tree. (A) shows data supporting the consensus phylogeny of major bacterial

groups shown in (B). Bootstrap values (% BP) from individual data sets supporting the numbered nodes are indicated by circles in black

(75–100% BP), gray (60–75% BP), or white (<60% BP). Rejection of nodes by individual data sets is measured by the strongest bootstrap

support for any conflicting grouping and is indicated by a circled cross (65–100% BP) or bar (<65% BP). For SSU rRNA phylogeny,

white or black circles indicate only presence or absence in trees, respectively. Data sets used are SSU rRNA, combined SSU and LSU

(Brochier et al. 2002), 14 assorted conserved genes (Brown et al. 2001), 57 translational proteins (Brochier et al. 2002), 32 ribosomal

proteins (Wolf et al. 2001), and supertree analysis of 121 genes (Daubin et al. 2002). Most of the new ciPCR-only groups are not

included because of space limitations and their current omission from combined data sets, as indicated by the dotted line below Op11.

48 The Origin and Radiation of Life on Earth

Their photosynthesis is a hybrid of features of both GSB (light

is harvested in cylindrical organelles called chlorosomes using

bacteriochlorophyll) and proteobacteria (electron transport

occurs across the cell membrane using PSI).

Other GNS Bacteria

The other GNS bacteria exhibit a wide variety of phenotypes.

Herpetosiphon is a mesophilic gliding bacterium that is poorly

characterized but maybe very common in soil. Other nonphotosynthetic

members of the group have very unusual

growth substrates. Thermoleophilum lives at 60°C and can

grow on wax using a novel respiratory naphthoquinone.

Species of Dehalococcoides can thrive on chlorohydrocarbons

such as tetrachloroethane, which are toxic, highly persistent,

and ubiquitous environmental contaminants (Adrian et al.

2002). This makes them potential bioremediation agents.

However, little else is known about these taxa.

Phylogeny

The chimeric nature of the chloroflexan photosynthetic apparatus

led to early speculation that the GNS bacteria might

be very ancient and represent the ancestral bacterial photosynthetic

apparatus. This is consistent with SSU rRNA phylogeny,

which places them among the deepest branches in

the bacterial tree (fig. 4.2; Woese 1987, Pace 1997). However,

with the addition of environmental clades, the relative

branching order among major bacterial groups now appears

to be unresolved, and no deep branches can be identified with

certainty (Hugenholtz et al. 1998). There are very few other

molecular data on the group, and no completed genome

sequences, so they have not yet been included in CDS or

supertree analyses. However, sequencing of at least two GNS

bacterial genomes is in progress (Bernal et al. 2001).

Planctomycetes

Planctomycetes (fig. 4.2, node 1) are perhaps the most phenotypically

unique group of bacteria known, with a whole

series of unusual although possibly interrelated features. They

are aquatic, appendage forming (prosthecate) aerobes. Cells

anchor themselves to various substrates using stalks, and

when anchored to each other, they form rosettes. Probably

because of this morphology, they divide by asymmetrical

budding rather than binary fission, as is also the case in other

stalk-forming bacteria such as b-proteobacteria (described

below; Hallbeck et al. 1993). This means that cells are dimorphic

with distinct mother and daughter cells, resulting in a

colonial genealogy. Daughter cells or “swarmers” are motile

by means of flagella that are often lost when the cells develop

stalks and settle down to become mother cells.

Unique among bacteria, Planctomycetes have peptidoglycan-

free proteinaceous cell walls that are covered with distinctive

pits of unknown function. Most striking of all is

the presence in many of the Planctomycetes of a single- or

double-membrane-bound nucleoid, reminiscent of the membrane-

bound nuclei of eukaryotes. The recently described

taxon Candidatus “Brocadia anammoxidans” also has a

membrane-bound anammoxosome region that performs

anaerobic ammonia oxidation (anammox; Lindsay et al. 2001).

The membrane of this “organelle” consists of biologically unprecedented

ladderane lipids that make the anammoxosome

highly impermeable and thus protect the cell from the anammox

intermediates (Sinninghe Damste et al. 2002).

Because of their distinctive morphology, many

Planctomycetes-like species have been described, but few

have been successfully cultured. They are well represented

in the majority of ciPCR studies, including those of geothermal

vents, fresh and marine waters, and deep subsurface

habitats (Hugenholtz et al. 1998). Their SSU rRNA sequences

are highly usual; although clearly a monophyletic group, they

exhibit very low sequence similarity to all other bacteria. This

is consistent with their being either very odd (i.e., rapidly

evolving) or very old (Woese 1987). Speculation has arisen,

at various times, that they might represent an extremely early

divergence from the common bacterial root. This idea is not

supported by most SSU rRNA phylogenies, where they do

not branch particularly deeply and tend to have an affinity

for Chlamydia and Verrucomicrobia, although without strong

statistical support. However, a recent reanalysis of SSU rRNA

data using only the most slowly evolving positions places

them back at the base of the Bacteria with moderate support

(Brochier and Philippe 2002), although the merits of this

approach are not proven. Additional molecular data are

needed to test this possibility.

CFB Group (Bacteroidetes) and GSB (Chlorobi)

CFB Group (Cytophagas, Flexibacteria, Flavobacteria,

and Bacteroides)

The CFB group (fig. 4.2, node 5) is a group without phenotypic

justification, that is, lacking common defining features,

perhaps in part because the different taxa have been studied

in very different ways (Woese 1987). They are generally rod

shaped but pleomorphic (variable), may form sheets, and

may move, either by gliding or with flagella. They tend to

have peptidoglycan-free cell walls and unusual membrane

lipids. All are organochemotrophs, that is, are nonphotosynthetic

and non-carbon-fixing. Most can degrade large

complex macromolecules such as cellulose and chitin, and

they are common animal commensals. Bacteroides thetamicrobium

is the most abundant organism in human gut (~1010

cells/g body weight), and in its membranes it has sphingolipids,

lipids that are otherwise largely restricted to mammalian

nerve cells.

Flavobacteria and Cytophagas

Flavobacteria, named for their characteristic carotene-induced

yellow color, are obligately aerobic nonmotile rods

with a mitochondria-like electron transport chain. They are

found in soils and aquatic environments and receive conThe

Tree of Life 49

siderable attention as opportunistic pathogens of fish in

crowded conditions such as farms or aquaria. Obligate intracellular

parasites/symbionts of the amoebozoan Acanthamoeba

are also known. The cytophagas, including flexibacteria, are

essentially gliding flavobacteria. They occur in similar habitats

and are especially noted for their ability to degrade complex

macromolecules such as DNA, cellulose, chitin, and agar,

suggesting that they may have important roles in natural

nutrient recycling.

Bacteroidetes

The Bacteroides constitute the third major group within the

CFB phylum. All are obligate anaerobes and capable of living

freely, but they are most commonly encountered in the

mammalian gut. Here they are extremely abundant and

highly diverse (Ramsaka et al. 2000). They possess thick

polysaccharide coats that permit them to survive in these

environments, where they break down host-indigestible

materials such as cellulose and pectin. Some of these breakdown

products may be absorbed by the host, but their primary

benefit to humans may be in rendering the gut

inhospitable to potential pathogens and also, by sheer force

of numbers, physically blocking the latter from attaching.

Porphyromonas species are associated with dental disease in

humans, although whether as cause or effect is not known.

ciPCR has identified a complex assemblage of Prevotolla species

in the guts of ruminants (Ramsaka et al. 2000).

CFB Group Phylogeny

The CFB group of bacteria as a whole appear to be ubiquitous.

ciPCR studies find them in every habitat examined so

far and often in abundance (Hugenholtz et al. 1998). Phylogenetically

the group is diverse and poorly understood. No

completed genome sequences exist at this time, so they are

not included in current CDS studies, but the genomes of

Bacteroides fragilis, Porphyromonas gingivalis, and Cytophaga

hutchinsonii will be completed soon. Although SSU rRNA

phylogeny strongly supports the group as a whole, it does

not support the integrity of any of the three subgroups, and

major reclassification is underway (Olsen et al. 1994, Maidak

et al. 2001). In addition, new, apparently basal lineages within

the group have been described recently such as Rhodothermus

(Andresson and Fridjonsson 1994).

Green Sulfur Bacteria (GSB) Group (Chlorobi)

The cultivated members of the GSB (fig. 4.2, node 5) are

obligately anaerobic, sometimes gliding but mostly nonmotile,

green or brown phototrophs. They thrive in high-sulfur,

low-light habitats such as sulfur-rich muds, where they oxidize

sulfur and excrete sulfate. This gives rise to their characteristic

large, iridescent extracellular sulfate globules. The

cultivated GSB tend to be rod-shaped, often twisted into

a variety of shapes, including crescents, rings, ovals, or

spheres, or aggregated into long, sometimes spiral chains.

Chlorobi are extremely efficient photosynthesizers, requiring

approximately one-quarter the light intensity required

by other phototrophs. They include Chlorochromatium aggregatum,

which is a consortium consisting of a Chlorobium

and an unidentified host that is heterotrophic (i.e., obtains

nutrients by nonphotosynthesis means; Overmann and van

Gemerden 2000). The large, motile, light-seeking host provides

transport for the many small chlorobi attached to its

surface. These may in turn provide their host with photosynthate.

The whole consortium divides synchronously, although

the relationship appears to be obligate only for the

host (Overmann and van Gemerden 2000).

GSB photosynthesis superficially resembles that of GNS

bacteria, with whom they were once thought to be closely

related. Both use similar pigments (bacteriochlorophyll b, c,

or d) and have at least superficially similar cylindrical light

harvesting organelles (chlorosomes). However, the structures

of these organelles may be substantially different between the

two groups. Also, GSB use PSII and fix carbon dioxide with

a reverse Krebs (tricarboxylic acid) cycle, whereas GNS bacteria

use PSI and the Calvin cycle, at least primarily (Hanson

and Tabita 2001). The two groups are also clearly separated

in SSU rRNA phylogenies (e.g., Woese 1987, Hugenholtz

et al. 1998; see also Pace, ch. 5 in this vol.).

The cultivated representatives of the GSB appear as a

closely related group in SSU rRNA trees. The majority of

sequence diversity in the group appears to be represented

by as yet uncultivated lineages detected in environmental SSU

rRNA surveys. These ciPCR studies also indicate GSB-type

bacteria in diverse habitats, including subsurface layers completely

devoid of light (Hugenholtz et al. 1998). The latter

suggests that the phenotypic diversity of the group may also

be much broader than the currently recognized anaerobic

phototrophy and may include nonphotosynthetic members.

Phylogeny

The robust grouping of the GSB and CFB groups (fig. 4.2,

node 5), always excluding the GNS group, was first noted

based on SSU rRNA sequence signatures, structural features,

and phylogeny (Woese 1987). This relationship has continued

to hold strongly throughout the massive expansion of

the SSU rRNA database (Pace 1997). Because the genome

sequence of Chlorobium tepidum was only released this year,

representatives of the CFB and GSB groups have been included

in only two CDS studies to date (fig. 4.2A, line 5).

These both strongly support a GSB + CFB clade and suggest

that ultimately the two should be combined as a single major

bacterial group.

Chlamydiae and Spirochaetes

Chlamydiae

The few described species of Chlamydiae (fig. 4.2, node 6)

are a closely related, highly specialized, medically important

group of obligate intracellular parasites. They cause a num50

The Origin and Radiation of Life on Earth

ber of significant human diseases such as pelvic inflammatory

disease and trachoma, the leading cause of preventable

blindness. Their cell walls lack peptidoglycan, but they retain

the necessary enzymes to make it and are therefore sensitive

to b-lactam antibiotics (which target cell-wall biosynthetic

enzymes; Stephens et al. 1998). The life cycle consists of a

desiccation-resistant infective form (elementary bodies) that

“germinates” upon entering the host to form reticulate bodies

sequestered in intracellular vacuoles or “inclusions.” Upon

maturing, they revert to elementary bodies that escape by

lysing the host cell.

Known clinical isolates probably account for only a small

subset of the Chlamydiae, and even many clinical isolates

remain uncharacterized. They have also recently been identified

as intracellular parasites of amoebae. ciPCR has indicated

as many as four additional subgroups of Chlamydiae

(Horn and Wagner 2001), although they have not been found

in many habitats other than soil (Hugenholtz et al. 1998).

Four Chlamydia genomes have been completely sequenced.

They are extremely reduced with around 1000 protein-coding

genes and lack many biosynthetic pathways, including

those for basic small molecules. Rough sequence matching

(blastology) suggests that they have acquired an unprecedented

~35 genes from their hosts (Stephens et al. 1998).

Spirochaetes

Most Spirochaetes (fig. 4.2, node 6) are free-living or harmless

commensals, part of the normal host bioflora, but a

number are important obligate intracellular parasites. Known

free-living species are chemorganotrophs, obtaining both

carbon and energy from organic compounds. Parasitic species

include the causative agents of syphilis, Lyme disease,

leptospirosis, and relapsing fever. Spirochaetes have a very

distinctive spiral morphology and corkscrew-like movement.

This is the result of paired polar flagella that extend toward

each other and intertwine along the midline of the cell. Unusually,

the flagella lie within the periplasmic space rather

than outside the cell. Therefore, when they beat they turn

like a rotor, spinning the cell within its outer membrane

sheath and propelling it forward.

ciPCR studies show that members of the Spirochaetes

occur in a wide variety of habitats, including thermophilic,

but apparently not marine environments (Hugenholtz et al.

1998). A large diversity of Spirochaetes, mostly of the genus

Treponema, have been found by ciPCR to the hindgut of termites

(Lilburn et al. 1999). Their role here appears to be in

fixing nitrogen for their hosts and the parabasalid protists,

also found exclusively in this habitat (Lilburn et al. 2001;

described below).

Phylogeny

There is no phenotypic resemblance between Chlamydiae

and Spirochaetes (fig. 4.2, node 6). Although both include a

number of obligate intracellular parasites with highly reduced

genomes, these are undoubtedly correlated characters that

have evolved independently many times. Nonetheless, the

two taxa group together in nearly all CDS analyses with

moderate to strong statistical support (fig. 4.2A, line 6). Their

grouping is also suggested weakly (Pace 1997), although not

consistently (Brochier et al. 2002), in SSU rRNA trees. Although

Chlamydiae may have heightened levels of LGT, acquiring

genes particularly from their hosts (Subramanian

et al. 2000, Stephens et al. 1998), Spirochaetes may have

much lower levels of LGT (Dykhuizen and Baranton 2001).

The postulated origin of eukaryotic flagella from endosymbiotic

Spirochaetes (Sagan and Margulis 1987) has found no

molecular support.

Deinococcus-Thermus Group and Cyanobacteria

Deinococcus-Thermus Group

Deinococci (fig. 4.2, node 9) are aerobic, nonmotile, redpigmented,

tetrad-forming chemorganotrophic rods or cocci.

They are extremely “tough” and occur in some of the most

inhospitable environments known: Antarctic dry valleys,

dust, cloud droplets, irradiated food, and medical instruments.

They can tolerate, among other things, high levels of

ultraviolet and gamma-irradiation (up to 1500 kilorads),

extreme desiccation and starvation, and mutagens such as

hydrogen peroxide. All of these conditions can cause doublestranded

breaks in DNA. In Escherichia coli, two or three such

lesions are lethal, but Deinococcus radiodurans can rapidly and

accurately repair 1000 or more. It does this by encoding every

pathway for DNA protection and accurate repair known and

maintaining its genome in multiple copies (White et al. 1999,

Makarova et al. 2001). Although unrelated to “true” grampositive

bacteria, Deinococci have thickened gram-positivestaining

cell walls. All of these characters make them attractive

targets to engineer for bioremediation, and variants of these

bacteria can clean up mercury and toluene. The National

Aeronautics and Space Administration also plans to use D.

radiodurans as a model in simulations used to guide the search

for life on Mars.

The Thermus group includes three described genera, all

hyperthermophiles isolated from hot springs. Thermus

aquaticus is particularly noteworthy as the source of Taq

polymerase (which is named after it), the most widely used

enzyme for DNA amplification by thermocycling (PCR). The

Deinococcus–Thermus grouping was unsuspected before

SSU rRNA phylogeny (Woese 1987), and there is still no

phenotypic justification for the group. However, it is unambiguously

supported by a large body of molecular sequence

data (White et al. 1999). Although still not publicly available,

the genome sequence of T. aquaticus was completed probably

years ago by private industry hoping to mine it for more

heat-stable enzymes.

Cyanobacteria

Formerly known as the blue-green algae, cyanobacteria (fig.

4.2, node 9) comprise a large, distinct, well-characterized

The Tree of Life 51

group. They are ubiquitous, occurring anywhere there is light

and even tiny amounts of transient moisture and can survive

long periods of desiccation and dormancy. Habitats include

between ice crystals in frozen water, in hot springs up

to 70°C (the photosynthetic limit), and on or within desert

rocks and soil. They are the only oxygenic photosynthetic

bacteria and use a variety of pigments to trap (harvest) light,

resulting in a range of colors from blue-green to red-brown.

Many also fix nitrogen, often in separate terminally differentiated

thick-walled cells (heterocysts). Morphologies range

from single cells or small colonies to macroscopic filaments

and mats.

Cyanobacteria can be extremely abundant and form

large macroscopic filaments and mats. On the other hand,

tiny Prochloron (0.2–0.7 gm) may be the most abundant

creature on the planet and our single greatest source of

oxygen (Chisholm et al. 2002). Cyanobacteria are also the

most frequent photosynthetic component of lichens and a

frequent source of color in reef animals. The oldest recognizable

fossils appear to be cyanobacteria, and they are the

original source of atmospheric oxygen. They also probably

at least helped build the oldest known living structures, the

stromatolites, although recent evidence suggests these may

consist largely of GNS bacteria (Oyaizu et al. 1987; described

above).

Cyanobacteria are the only bacteria with chlorophyll a

and both photosystems (PSI and PSII), which allows them

to generate enough energy to split water and thereby release

oxygen in the form of O2. The accessory pigments and proteins

for capturing the light to do this vary among species.

This led to theories that eukaryotic photosynthesis originated

multiple times, with the differently pigmented eukaryotic

algae acquiring their plastids from different cyanobacteria

(Urbach et al. 1992). However, there is now considerable

molecular data on eukaryotic plastids, and these strongly

support a single common endosymbiotic origin for all of them

(Douglas 1998).

Phylogeny

A large supergroup consisting of Deinococcus-Thermus,

Cyanobacteria, and Actinobacteria is found in all CDS analyses

except those using SSU + LSU (large ribosomal subunit),

often with strong statistical support (65–100% bootstrap; fig.

4.2, node 8). Within this supergroup, Deinococcus-Thermus

and Cyanobacteria are most often found together, a grouping

that is also found by SSU + LSU (fig. 4.2A, line 8). As

further support of this relationship, these two taxa also appear

to exclusively share a large insertion in protein synthesis

elongation factor Tu genes (Gupta 2001). CDS analyses

are currently limited by the fact that there is only a single

published genomic sequence each for Deinococcus-Thermus

and Cyanobacteria. Better resolution should be possible with

the five or more cyanobacterial genomes currently in progress

(CyanoBase 2003) and release of any completed Thermus

sequences.

Actinobacteria and Firmicutes (High- and

Low-G+C Gram-Positive Bacteria)

Actinobacteria (High-G+C Gram-Positive Bacteria)

The Actinobacteria consist of five major subdivisions:

Actinobacterae, Acidimicrobidae, Rubrobacteridae, and

Coriobacteridae. The Actinobacterae include most of the

well-characterized taxa. These are chemorganotrophic, often

filamentous, mostly aerobic bacteria with ~70% G+C

in their genomes. They are speciose and often highly abundant.

ciPCR studies find them in every habitat sampled and

particularly plentiful in soil and freshwater (Hugenholtz

et al. 1998). Shapes vary from rods to straight or branching

filaments and mycelia, and many form highly resistant,

potentially long-lived spores. Streptomycetes and Actinomycetes

were once mistaken for fungi because of their

branching aerial hyphae.

Most Actinobacteria are free-living or harmless animal

commensals or, at the most, opportunistic pathogens. However,

the group also includes Corynebacterium diptheriae

(diphtheria), Mycobacterium leprae (leprosy), and M. tuberculosis,

the single most lethal infectious agent of humans (Cole

2002). Mycobacteria are particularly problematic because

they have complex, lipid-rich cell walls resistant to various

environmental insults, including many antibiotics. Important

beneficial species include Proprionibacteria, used in cheese

production, and Streptomycetes and Actinomycetes, producers

of more than two-thirds of all naturally occurring antibiotics.

Arthrobacter is possibly the single most common

cultivated soil organism and an important natural herbicide

degrader, as are some Actinomycetes.

Firmicutes (low-G+C Gram-Positive Bacteria)

Sometimes referred to as the Bacillus-Clostridium group, members

of the Firmicutes (fig. 4.2, node 11) are chemorganotrophic,

often anaerobic, non-filament-forming taxa with

~30% G+C in their genomic DNA. Like the Actinobacteria,

they are widespread, found so far in all but geothermal habitats,

and predominate in both soil and wastewater (Hugenholtz

et al. 1998). There are three subgroups; endospore formers,

lactic acid bacteria (anaerobic fermenters), and cocci.

Endospore formers are primarily soil inhabitants. Notable

members include Bacillus anthrasis (anthrax), Clostridia (tetanus,

botulism, gas gangrene), and Bacillus thuringiensis (commercial

source of the powerful insecticide Bt toxin). Other

Bacilli are important sources of industrial enzymes such as

amylases and proteases. Endospores are formed from the

entire cell contents and have a dense outer coating. This

makes them highly resistant and potentially very long-lived,

possibly surviving many millions of years (Cano and Borucki

1995, Vreeland et al. 2000) and perhaps even space travel.

Lactic acid fermenters are anaerobic but oxygen tolerant.

They produce vast quantities of lactic acid, probably the earliest

preservative, and various Lactobacilli are still used in the

production of buttermilk, yogurt, and pickles. The cocci

52 The Origin and Radiation of Life on Earth

include Heliobacterium and Mycoplasma. Heliobacteria, the

only photosynthetic members of Firmicutes, occur in rice

paddy soils, where they are important fixers of nitrogen. The

cell-wall-free mycoplasmas are among the smallest independent-

living organisms known in terms of both physical size

and the size of their genomes (Fraser et al. 1995). They are

metabolically simple, often parasitic, and the only noneukaryote

with cholesterol, which they use to strengthen their

membranes. Mycoplasma pneumoniae is the causative agent

of “walking pneumonia,” which has a slower onset than other

bacterial forms (Chlamydia pneumoniae, Streptococcus pneumoniae,

Klebsiella pneumoniae).

Sporomusa

This is an intriguing, relatively new taxon containing what

were previously considered to be a diversity of species

(Willems and Collins 1995, Janssen and O’Farrell 2002). All

possess classical gram-negative cell walls and an outer cell

membrane (Kuhner et al. 1997). They are sometimes listed

as a third division of “gram-positive” bacteria, but SSU rRNA

trees generally place them at the base of the Firmicutes group

(Willems and Collins 1995, Janssen and O’Farrell 2002). This

suggests the possibility that the gram-positive cell wall could

have evolved independently in Firmicutes and Actinobacteria.

This possibility would, in turn, lend further support

to the growing idea that the “gram-positive bacteria” may

not be a true phylogenetic group, as indicated by all current

molecular trees (fig. 4.2, node 7).

Phylogeny

The two groups of traditional gram-positive bacteria are

united by the shared absence of an outer cell membrane and,

except for mycoplasmas, the presence of a thick gram-stainretaining

cell wall. However, a thickened cell wall is not a

complex structure, leaving open the possibility that it could

have evolved twice independently. Pressure for this could be

common because it probably helps cells resist high salt and

desiccation. This is consistent with its independent presence

in the highly desiccation-resistant Deinococci and Methylbacterium,

a proteobacterium. Therefore, the only unique

character uniting the Firmicutes and Actinobacteria groups

is a single shared loss, that of the outer cell membrane. However,

although phylogenetic trees show no tendency to unite

these two groups, these analyses are hampered by the extreme

differences in the G+C content of these taxa, which

affects even amino acid substitution patterns in proteins

(Cole 2002).

Proteobacteria (Purple Bacteria)

Proteobacteria (fig. 4.2, node 12) are, more or less, the traditional

“gram-negative” bacteria and the single largest group

of described bacteria. The a, b, and g subgroups each have

more described taxa than all other bacteria groups combined

except cyanobacteria and are found in every habitat type

sampled, predominating in many (Hugenholtz et al. 1998).

The group is highly diverse and difficult to define but unambiguously

monophyletic in molecular trees (fig. 4.2, node

12). Nearly every described bacterial morphology is found

and in nearly every subgroup, apparently switching rapidly

over evolutionary time and with changing growth conditions.

Purple photosynthesis is dispersed throughout and was probably

the ancestral state, but with multiple losses (Woese

1987). Many also fix carbon dioxide. The five subgroups were

originally identified on the basis of SSU rRNA and given

provisional Greek letter names, which seem to have stuck

(Woese 1987, Olsen et al. 1994).

g-Proteobacteria

This is a large, diverse, metabolically rich group and, together

with the g subdivision, the most widespread (Hugenholtz

et al. 1998). The group abounds with symbionts, commensals,

and parasites, including many complex symbioses, most

notably the eukaryotic mitochondrion. Well-known species

include Agrobacterium (used in plant genetic engineering),

Rhizobium and Bradyrhizobium (nitrogen-fixing symbionts of

legumes), and Rickettsia (typhus, Rocky Mountain spotted

fever). Rickettsia-like proteobacteria are probably the closest

living relatives of the mitochondrion. Morphologies vary from

rods to spirals to budding stalks, the latter being complex

extensions of the cytoplasm. Similar to the Planctomycetes,

a-proteobacteria may form stalks anchored to the substrate

or each other (rosettes) and reproduce by asymmetrical budding.

Most are chemorganotrophic, but there are also purple

nonsulfur (high-sulfur-intolerant) phototrophs and extracellular

and obligate intracellular parasites (Rickettsia).

b-Proteobacteria

Also morphologically and biochemically diverse and widely

distributed (Hugenholtz et al. 1998), the group includes

Neisseria gonorrhoea, Neisseria meningitidis, Bordetella pertussins

(whooping cough), and Thiobacillus. Nitrosomonas

plays an important ecological role by completing the final step

in nitrogen recycling. Recent molecular phylogenetic data

show these taxa to be a subgroup within the g-proteobacteria

(e.g., Brochier et al. 2002).

g-Proteobacteria

This group consists of another bewildering array of phenotypes,

and representatives have been identified in most ciPCR-sampled

habitats. The group includes purple sulfur phototrophs (e.g.,

Chromatium); enterics such as Escherichia coli and Salmonella

(mild to severe food poisoning, typhoid); human pathogens

such as Legionnella (Legionnaire’s disease), Vibrio cholerae (cholera),

Haemophilus influenza, Yersinia pestis (plague), and Proteus

vulgaris (cystitis); and a whole host of bizarre phenotypes, including

bioluminescent (Vibrio), fluorescent (Pseudomonas),

metal reducing (Shewanella), methane consuming (Methylomonas),

nitrogen fixing (e.g., Azotobacter), plant pathogenic

(Xanthomonas), magnetotrophic, and many more.

The Tree of Life 53

d-Proteobacteria

These include two major subdivisions, Myxococcus and

Desulfovibrio. Members of Desulfovibrio are morphologically

diverse, aquatic or moist soil inhabiting chemolithotrophs

that are capable of oxidizing metals such as underground

pipes. Bdellovibrios include bacterial parasites that invade

and reproduce within the periplasm of their bacterial host.

Members of Myxococcus also prey on other bacteria. Under

conditions of nutrient starvation they aggregate to form

motile multicellular “slugs.” These mature into stalked fruiting

bodies carrying a head of spores, a sort of miniature cellular

slime mold (described below).

e-Proteobacteria

This is the most restricted subgroup, mostly inhabiting extreme

environments such as hydrothermal vents and acid

lakes. They include Helicobacter pylori (causative agent of

peptic ulcers), Campylobacter jejuni (gastroenteritis), and

Thiovolum (symbiont of hydrothermal vent invertebrates).

Phylogeny

This huge, highly diverse, and well-studied taxon is probably

more appropriately treated as a supergroup. Nonetheless,

its monophyly is strongly supported in all CDS trees (fig.

4.2A, line 12). Only the d division, which SSU rRNA trees

place as the deepest branch in the group, are omitted so far

from the CDS trees because of the lack of a complete genome

sequence.

Bacteria: Recent Additions

The preceding are the “classic” bacterial groups, originally

defined by SSU rRNA analyses in 1987 (Woese 1987) and

including all well-studied taxa. What follows are descriptions

of the largest and most robust of the newly identified major

groups. Pace (ch. 5 in this vol.) now estimates a total of about

40 “phylum-level” groups of bacteria. This is based on continued

SSU rRNA characterization of already-collected strains,

isolation of new strains using traditional and new advanced

culturing techniques, and, especially, ciPCR. The latter indicate

that some of these new groups may be diverse, widespread,

and abundant (Hugenholtz et al. 1998). As many as

20 of them are identified solely by their ciPCR sequences,

although some have been subsequently confirmed by fluorescence

in situ hybridization or even isolated and cultured.

A word of warning: any classification based on a single gene

must be treated with caution. Even the best phylogenetic

methods cannot always distinguish genuinely distinctive

sequences from rapidly evolving ones that may nonetheless

belong to well-established groups.

Acidobacteria are currently the largest and most diverse

newly recognized group with at least eight major subdivisions

(Hugenholtz et al. 1998). They were first identified in acid

environments such as acid bogs and mine drainage. However,

ciPCR studies show all subdivisions represented in 43

separate soil samples (Barns et al. 1999), and fluorescent

ciPCR-based probes indicate a diversity of morphological

types (Ludwig et al. 1997). Because the group is genetically

and metabolically diverse and environmentally widespread,

it is probably of significant ecological importance (Barns et al.

1999). Only three cultured members are described, Acidobacterium

capsulatum, Holophage foetida, and Geothrix fermentans,

representing two subdivisions of the Acidobacteria.

Recently, representatives of a third Acidobacteria subdivision

have been isolated from soil (Sait et al. 2002).

Verrucomicrobia are a large, diverse, and widespread

group with five or six subdivisions currently indicated

(Hugenholtz et al. 1998). Division 1 includes most of the

cultured taxa, such as Verrucomicrobia and the appendaged

Prosthecobacter species (Hedlund et al. 1996). Division 2

includes a ciliate ectosymbiont, which defends its host by

ejecting proteinaceous “spines.” These spines appear to contain

the closest bacterial homologs yet found of the eukaryotic

protein tubulin (Petroni et al. 2002). Division 3 includes

the ciPCR taxon EA25, thought to constitute up to 10% of

the bacteria in some soils. Division 4 includes Ultramicrobium,

the smallest bacterium known (0.1 mm3 in volume) and able

to pass through the normal bacteria-excluding 0.2 mm filters.

This taxon may account, at least in part, for earlier reports

of a surprising abundance of viruses in the open ocean.

OP11 is a purely “environmental lineage,” a major group

of the bacteria with no known cultivated members. There are

five subdivisions indicated, all known entirely from ciPCR

sampling. OP11 “phylotypes” seem to be present and abundant

in most habitats and a major constituent of subsurface

environments. All the OP11 phylotype sequences have long

branches in SSU rRNA trees, which suggests that they have

undergone accelerated evolution. If so, this would make it

difficult to identify cultured relatives if they exist (Hugenholtz

et al. 1998).

Other Newly Proposed Groups

These tend to have fewer representatives. However, most

contain of at least two subdivisions and have been identified

in a number of ciPCR studies. Others consist of recently

characterized species that have been successfully cultured but

seem to lack close relatives in SSU rRNA trees. All of these

small possible new groups should be considered provisional

until further data are available because they could simply be

taxa with highly divergent SSU rRNA sequences obscuring

their true affinities. This list does not include the roughly onethird

of all ciPCR groups that still lack any cultivated representatives

and have not yet been formally named.

Coprothermobacter is a moderate thermophile (55°C). Its

SSU rRNA sequence shows some affinity for the Hyperthermophiles

(described above), which would make it the first

mesophilic member of the group (Etchebehere and Muxi

2000). The latter may also include Dictyoglomus. This hyperthermophile

is popular as a source of proteins for crystallography

and new thermostable enzymes (Ding et al. 1999).

54 The Origin and Radiation of Life on Earth

Fibrobacter is one of the two most abundant genera of cellulose

degraders within the complex biota in the rumen of grazing

animals. It may be related to the GSB group of bacteria

(described above; Gordon and Giovannoni 1996; but see also

Pace 1997). The flexistipes include mesophiles and mild

thermophiles that nonetheless seem to be largely restricted

to geothermal habitats.

The Fusobacteria include major constituents of tooth

plaque, with highly distinct phylotypes seeming to specialize

on different mammalian taxa (Foster et al. 2002). The

Nitrospira, including Leptospirillum, Nitrospira, Magnetobacterium,

and Thermodesulfovibrio, were previously assigned to

the d-proteobacteria but have highly distinct SSU rRNA sequences

(Pace 1997). As the names imply, this a metabolically

very diverse collection of taxa. Nitrospira species are

nitrite oxidizers and include the principle natural detoxifiers

of freshwater aquaria (Hovanec and DeLong 1996). The

synergistes include six genera, all strictly anaerobic.

Synergistes species are relatively scarce rumen bacteria that

can break down dihydroxypyridine, the compound that renders

legumes poisonous to grazing mammals.

The Thermodesulfobacteria are low-G+C thermophiles

isolated from hot springs, hydrothermal vents, and oil platforms.

They are the only bacteria that appear to have archaealike

membrane lipids. Gemmatimonadetes was formerly

classified as candidate division “BD” or “KS-B” and consisted

solely of ciPCR sequences. A single strain has recently been

isolated from sludge processed under enhanced biological

phosphorus removal conditions (Zhang et al. 2003) and

appears to accumulate polyphosphate. Other members of the

group have been identified in soil and marine samples (Zhang

et al. 2003). TM7 is known only from ciPCR but has been

partially characterized nonetheless as streptomycin-resistant,

sheathed filaments. It also appears to have a typical grampositive

cell envelope and may have some bearing on discussions

of the mono- versus polyphyletic origin of this trait

(Hugenholtz et al. 2001). Full taxon lists for each of these

groups can be found at the National Center for Biotechnology

Information taxonomy server (available at http://www.

ncbi.nlm.nih.gov/Taxonomy/).

Domain Archaea

The Archaea are easily the least understood of the three domains,

with orders of magnitude fewer described species

compared with members of Bacteria or Eucarya. Most of the

characterized taxa are extremophiles inhabiting some of the

harshest environments imaginable, but ciPCR suggests there

are as many or more mesophiles. Before 1970 bacteriologists

dispersed them across various classical bacterial taxa, and it

was not suspected that such diverse organisms could be related

to each other. However, even early SSU rRNA analyses

showed clearly and strikingly both that Archaea form a coherent

group and that they constitute a third domain of life

(Woese and Fox 1977).

Circumscription of Archaea is qualified by the fact that

very few taxa have been studied in culture, and there is almost

no information on the many, sometimes major ciPCRindicated

subdivisions. Even most cultivated taxa are poorly

characterized, partly because of technical problems such as

working with organisms that cannot survive below the melting

point of agar or prefer corrosive media such as 0.1 M

sulfuric acid. Therefore, there are no archaeal genetic systems,

and much of what we know comes from recent genomic

sequencing (Bernal et al. 2001). There is also a lack

of incentive: Archaea includes no known pathogens, and

their ecological roles and economic potential are largely

unknown.

Members of Archaea are perhaps best described as a mix

of bacterial and eukaryotic features: essentially eukaryotic

“brains” in bacterial cells, living bacterial lives (Coulson et al.

1991). Morphologically and metabolically they are bacterial,

with small (0.5–5 mm) cells, lacking internal membranebound

organelles, and usually surrounded by rigid cell walls,

albeit ones made of protein rather than of peptidoglycan.

Their genomes are small (~1.5–3 Ч 106 base pairs), mostly

closed circles, probably with a single origin of replication.

Genes tend to be in operons often structurally identical to

those of bacteria. Known taxa are autotrophs or chemorganotrophs,

sometimes photosynthetic, often sulfur-dependent,

frequently fixing carbon dioxide. A variety of symbioses

and commensalisms are also known (Madigan et al. 1997).

On the other hand, information processing (i.e., RNA

transcription and translation) is eukaryotic both in overall organization

and in individual component sequences (Kyrpides

and Woese 1998, Edgell and Doolittle 1997). Even before the

genes were sequenced, it was noted that archaeal RNA polymerases

had a subunit composition similar to those of eukaryotes

(Huet et al. 1983). This was later confirmed by striking

similarities in their protein sequences and structures. Archaeal

RNA polymerases also require the eukaryotic-type transcription

factors TBP, TFIIB, and TFIID to bind their promoters

(Bell and Jackson 2001). This is unlike the much smaller bacterial

RNA polymerase that can bind DNA on its own and uses

small exchangeable subunits called sigma factors to identify

its promoters.

Likewise, the components of archaeal protein synthesis

are mostly either uniquely shared with eukaryotes or are

eukaryotic versions of universal ones (Kyrpides and Woese

1998). Many are encoded in canonical bacterial operons but

have strikingly eukaryotic sequences and structures (Garrett

et al. 1991) and are functionally interchangeable with eukaryotic

factors in vitro. Members of Archaea also have fundamentally

eukaryotic DNA replication (Myllykallio et al. 2000, She

et al. 2001, Bohlke et al. 2002), and euryarchaeotes (but no

known crenarchaeotes) use histones to package their DNA

in nucleosome-like structures (Sandman and Reeve 2001).

The Tree of Life 55

The possibility of members of Archaea having given rise

to either Bacteria or Eucarya or both has long been a contentious

issue (Rivera and Lake 1992, Baldauf et al. 1996).

However, with more data now available, most analyses, including

CDS trees, unambiguously support their being a

monophyletic group (Brown et al. 2001, and S. L. Baldauf

and J. Cockrill, unpubl. obs.). Members of Archaea are also

distinct in possessing highly unique membrane lipids that

appear to be restricted to them, with the possible exception

of the thermodesulfobacteria (described above). These membranes

consist of isoprenyl lipids ether-linked to D-glycerol,

distinctly different from the ester-linked fatty acid lipids and

L-glycerol of members of Eucarya and Bacteria. Archaeans are

also the only known organisms with lipid monolayer membranes.

These are common in hyperthermophiles, where they

probably provide membrane stability at high temperatures

and account for these cells’ inability to live at lower temperatures

(Coulson et al. 2001).

All characterized members of Archaea and most ciPCR

phylotypes fall cleanly into two distinct groups, the Crenarchaeota

and the Euryarchaeota (fig. 4.3). The cultured Crenarchaeota

or “thermoacidophiles” are phenotypically narrow,

whereas the Euryarchaeota are extremely diverse. However,

ciPCR suggests that both groups are much broader, particularly

the Crenarchaeota. ciPCR also indicates the possible

existence of two additional major divisions of Archaea (fig. 4.3),

the Korarchaeota, known only from ciPCR studies (Barns et al.

1996), and the extremely small (0.4 mm diameter) Nanoarchaeota

(Huber et al. 2002b). However, the classification of these

taxa as major new archaeal lineages is based entirely on SSU

rRNA trees and is not unambiguously supported even by them.

Crenarchaeota

All cultured Crenarchaeota (fig. 4.3, nodes 13 and 14) are

hyperthermophiles, including some of the most thermophilic

Figure 4.3. Support for deep branches in the archaeal tree. (A) shows support for the labeled nodes in (B) (same symbols as in fig.

4.2A). Data sets used are SSU, SSU + LSU and 53 ribosomal proteins (Matte-Tailliez et al. 2002), 14 housekeeping genes (Brown et al.

2001), universal ribosomal proteins (Wolf et al. 2001), 14 random clusters of orthologous groups of proteins (COGs) (J. Cockrill and

S. L. Baldauf, unpubl. obs.). Lowercase letters within open circles indicate where a single taxon interrupts an otherwise strongly

supported grouping, as indicated in (B). In (B), boldface is used to indicate the taxa used in combined analyses (i.e., taxa for which

complete genome sequence data are available). Major differences between SSU and all other analyses are indicated by lightly dotted

lines. Two potential major new archaeal lineages are attached to the base of the tree by dashed lines.

56 The Origin and Radiation of Life on Earth

and acidophilic organisms known. Optimum growth temperatures

range from 75°C to 100°C, with some cells surviving temperatures

as high as 115°C and most unable to survive below

70°C. They have a wide range of pH tolerances but may flourish

at pH 1–2 and still live and grow at pH 0. Most cells are

flagellate, but shapes vary widely from simple disks (Thermodiscus),

nearly rectangular rods (Thermoproteus, Pyrobaculum),

irregularly lobed cocci (Sulfolobus), or extremely long thin filaments

(Thermofilum), to grapelike aggregates (Staphylothermus)

and large fibrous networks (Pyrodictium; Barns and Burggraf

1997). Many species are acidophilic, using sulfur or sulfur

compounds as electron donors, acceptors, or both. Carbon may

be acquired from organic compounds (chemorganotrophy) or

by fixing carbon dioxide using ribulose biphosphate carboxylase

or a reverse tricarboxylic acid cycle.

Although all the cultured members of Crenarchaeota are

hyperthermophilic and often anaerobic, ciPCR indicates the

existence of mesophilic aerobic taxa throughout the group.

Unlike the crenarchaeal thermophiles, many of the mesophiles

appear to be widespread. Their habitats range from

shallow sediments (Hershberger et al. 1996) to the open

ocean (DeLong 1992, Fuhrman et al. 1992). Some may also

be extremely abundant, including dominating the ocean’s

interior, “the world’s largest biome” (Karner et al. 2001). They

are found throughout the water column, increasing in abundance

with depth until they constitute ~39% of all the microbial

cells (DeLong 1992, Fuhrman et al. 1992).

The Crenarchaeota are the least-characterized major division

of living organisms, and no taxonomic group has been

more fundamentally revised by ciPCR data. These now indicate

that there are two major divisions of Crenarchaeota,

referred to in the following as Divisions I and II. Division II

was discovered only in the last 5 years and consists entirely

of ciPCR phylotypes plus Cenarchaeum, an obligate symbiont

of a marine sponge. General references for the following are

Madigan et al. (1997), Barns and Burggraf (1997), and Brown

(2002).

Crenarchaeota Division I

Thermoproteales. Thermoproteales (fig. 4.3, node 13)

currently include six described genera: Caldivirga, Pyrobaculum,

Thermocladium, Thermofilum, Thermoproteus, and

Vulcanisaeta. These are generally rod shaped thermophiles

living at near neutral pH. Thermoproteus (60–96°C, optimum

85°C) cells are long rods that reproduce by budding and are

very common in solfatars (sulfur deposits). Thermofilum species

(70–95°C) form extremely long, thin (1–100 (0.15–0.3

mm) filaments and are common in deep-sea hydrothermal

vents, as are Pyrobaculum species (74–115°C, optimum

100°C), which are rods or flattened cocci. The latter can

grow well at temperatures up to 115°C, making them, together

with Methanopyrus (described below), the most thermophilic

organisms known (note: at these depths 115°C is

well below the boiling point of water). Pyrobaculum species

are high-sulfur-intolerant denitrifiers, using oxygen instead

of sulfur as a terminal electron acceptor (aerobic respiration).

This is unusual for crenarchaeotes, which are generally obligate

or at least facultative anaerobes.

Sulfolobales. This group currently includes five genera:

Acidianus, Methanosphaera, Sulfolobus, Sulfurisphaera, and

Stygiolobus. This is an entire group of organisms whose natural

habitat is essentially boiling sulfuric acid. All are coccoidshaped

thermophilic acidophiles with growth optima of

75–95°C and pH 1.0–3.5. The most extreme is Acidianus,

which can grow at pH 0. Sulfolobus species (55–87°C, optima

75–85°C, pH 2–3) thrive in thermoacidic environments

such as solfatars, boiling mud pots, and hot acid mine drainage,

where they often grow in great abundance and in near

monoculture. They reproduce by budding, which produces

characteristic irregularly shaped lobes that may also function

in adhesion and for which they get their name. Both Sulfolobus

and Acidianus (65–95°C, optima 85–90°C pH 2) have extremely

low genomic G+C content (37% and 31%, respectively),

demonstrating once again that thermophilic adaptation

does not require elevated genomic percent guanine + cytosine

(G+C) (She et al. 2001).

Desulfurococcales. Desulfurococcales (fig 4.3, node 11) is

currently the largest group of crenarchaeotes, with 11 described

genera divided into two groups. All are coccoid or rod

shaped, neutrophilic (living at neutral pH) hyperthermophiles.

Group 1 includes Aeropyrum, Desulfurococcus, Ignicoccus,

Staphylothermus, Stetteria, Sulfophobococcus, Thermodiscus, and

Thermosphaera. Staphylothermus species (65–98°C, optimum

92°C) are cocci that grow in grapelike clusters. Thermodiscus

(75–98°C, optimum 90°C) cells are, not surprisingly, diskshaped.

Ignicoccus includes the host of a newly discovered

symbiont thought to represent a new subdivision of Archaea,

the Nanoarchaeota (described below). Aeropyrum are unusual

in being strictly aerobic hyperthermophiles (optimum 90–

95°C).

Members of Group 2 (Hyperthermus, Pyrodictium, and

Pyrolobus) are all found in shallow submarine volcanic habitats.

Pyrodictium species (62–110°C, optimum 100°C) are

disk-shaped cells held in networks by hollow, proteinaceous

fibers growing in “moldlike” layers on suspended sulfur crystals.

The fibers maybe arranged in regular patterns similar to

the protein in bacterial flagella (Madigan et al. 1997). Their

mature rRNA molecules have an unusually high percentage

of modified bases, presumably to stabilize their structure and

hence their activity at high temperatures. The other cultured

members of the group are Pyrolobus (90–113°C, optimum

105°C), with lobed cocci, and Hyperthermus (95–106°C,

optimum 108°C).

Crenarchaeota Division II (Cenarchaeum Group)

This second major division of the Crenarchaeota (fig. 4.3,

node 14) includes a wide variety of ciPCR phylotypes indicating

mesophiles, thermophiles, and hyperthermophiles.

The Tree of Life 57

Some of the mesophiles are also widespread and/or extremely

abundant. The only characterized member of the division is

Cenarchaeum symbiosum, an obligate symbiont of the marine

sponge Axinella species (Preston et al. 1996). The association

between the sponge and symbiont appears to be stable, widespread,

and highly specific, and the symbiont is highly abundant

within its host (Preston et al. 1996). This is the only

known eukaryote–archaean symbiosis involving a crenarchaeote.

Although the symbiont cannot be separated from

its host, large fragments if its genome have been characterized

using whole-organism genomic libraries (Schleper et al.

1998).

Crenarchaeote Phylogeny

ciPCR data suggest the need for major revision of the

Crenarchaeota, but further information is needed on at least

some of these “taxa.” Nonetheless, it now appears clear that

there are at least two deeply separated subdivisions within

the group, referred to here as Divisions I and II. Division I

includes all the cultured hyperthermophiles, which form a

tight cluster nested within several layers of deeply diverging

ciPCR “groups” (fig. 4.3, node 13; see Pace, ch. 5 in this vol.).

Division II is almost entirely composed of ciPCR phylotypes,

including many mesophiles. There are also several groups of

ciPCR phylotypes that do not clearly belong to either division,

and these may indicate additional distinct lineages.

Because the latter are mostly hyperthermophiles, this is still

thought to be the ancestral condition of the group, with

mesophily derived multiple times within it.

Within Division I, the major branching patterns are fairly

well resolved for the taxa with completed genome sequences:

Sulfolobus solfataricus, Pyrobaculum aerophilum (Thermoproteales),

and Aeropyrum pernix (Desulfurococcales). CDS

trees for these taxa agree with SSU rRNA trees, grouping

Sulfolobus and Aeropyrum together (fig. 4.3, node 12) to the

exclusion of Pyrobaculum. Because Sulfolobus is the only acidophile

of the three, this suggests that acidophily was derived

from neutrophily, rather than ancestral as originally

postulated. However, these studies are starkly taxonomically

narrow, which makes any broad conclusions premature.

Because Division II, with the exception of Cenarchaeum, is

known only from ciPCR phylotypes (fig. 4.3, node 14), all

information on the group comes from SSU rRNA trees, which

support it strongly.

Euryarchaeota

The Euryarchaeota (fig. 4.3, node 10) are an extremely diverse

group including mesophilic, thermophilic, and hyperthermophilic

methanogens; thermoacidophiles; sulfur-reducing

thermophiles; and extreme halophiles. These tend to form

roughly seven robust subgroups, three of which are methanogenic

(fig. 4.3). All of these have a broad sampling of cultured

representatives. Euryarchaeotes include a number of

environmentally important organisms, both beneficial and

harmful.

Archaeoglobi

Archaeoglobus species are sulfate-reducing, obligately anaerobic

hyperthermophiles (60–90°C, optimum 83°C) with

irregularly spherical, flagellated cells. They are found in

hydrothermal environments and subsurface oil fields, where

the iron sulfide they produce may corrode oil and gas mining

equipment (Klenk et al. 1997). None of the current

molecular phylogenetic data place them even close to

the Thermococci, the other thermophilic sulfate-reducing

Euryarchaeotes (fig. 4.3). Thus, the thermoacidophilic

habit appears to have arisen in Euryarchaeota at least twice

independently.

Halobacteria

These extreme halophiles require a minimum of 1.5–2.0 M

NaCl (or equivalent) and many can survive in saturated or

near-saturated brine (up to 5 M NaCl). They are probably

present in all high-salt environments, including salted fish,

and are common in hypersaline seas, salterns (shallow salt

evaporation pools), and subterranean salt deposits. They

photosynthesize using the purple retinal pigment conjugated

to bacteriorhodopsin. This results in the pink hue of salt

evaporation pools, the purple of the Dead Sea, and the red

in salted herring (the famous “red herring”).

Unlike other halophilic organisms, members of Halobacteria

are isosmotic with their environment, so there is no

osmotic pressure on their cell walls. This allows for unusual

morphologies such as Haloarcula, which has ultrathin, 0.1–

mm-thick cells that form perfect squares, rectangles, and even

triangles, mostly with tufts of flagella at their apexes. These are

the only phototropic archaeans, and they use a mechanism

fundamentally different from any bacterial photosynthesis. The

light-harvesting machinery is embedded in the cell membrane

itself, and absorbed light is used directly to pump protons

across the membrane. This creates a proton-motive force that

is then coupled to ATP synthesis (Lanyi and Luecke 2001).

Methanogens

The methanogenic members of Archaea include at least six

major groups (fig. 4.3, nodes 1, 6, 7, and 8), and they are

almost certainly not monophyletic. Nonetheless, all are obligately

anaerobic and methanogenic (methanogenic enzymes

are oxygen intolerant) and possess a unique fluorescent cytochrome,

F420, that is found nowhere else. They are widely

dispersed in nature and are found in sediment, soil, wastewater

treatment ponds, landfills, subterranean oil deposits,

and animal intestinal flora. They are the source of swamp gas

and intestinal methane and are important components of the

rhizosphere, the plant root environment.

Three of the methanogen groups, represented by Methanosarcina,

Methanospirilla, and Methanococcus, are currently

58 The Origin and Radiation of Life on Earth

placed together in the Methanococcales. However, CDS trees

tend to split them up, placing the first two taxa with the

Halobacteria (fig. 4.3, node 2) and the latter some distance

away with Methanobacteria and Methanothermus (fig. 4.3,

node 7). Because Methanopyrus also appears to be closely

associated with the latter group (Slesarev et al. 2002), this

means that all currently known methanogenic archaeans can

probably be assigned to two well-defined groups.

Methanogen Group 1 (MG1): Methanosarcina + Methanospirillum.

Methanosarcina + Methanospirillum (fig. 4.3,

node 1) produce methane from a variety of substrates, including

acetate, methylamines, and methanol, an unusual

metabolic diversity for methanogens. They occur in diverse

habitats from anaerobic lake bottoms and muds to cattle

rumen, where they are responsible for methane production,

a significant source of global greenhouse gas (Deppenmeier

et al. 2002). Methanosarcina species are mildly thermophilic

(40–55°C) and common in soils, sediment, swamps and

wastewater treatment sludge, where they play an essential

role in the early stages of sewage processing. The group also

includes cold-adapted species that survive temperatures as

low as –10°C and are found in Antarctic lakes and cold deepmarine

sediments (Thomas and Cavicchioli 1998).

Methanogen Group 2 (MG2). MG2 includes Methanococci

+ Methanobacteria + Methanopyri (fig. 4.3, nodes 7 and

8). This tentative group includes mesophiles, thermophiles,

and hyperthermophiles. Most are highly self-sufficient, metabolically

speaking, and some are pure prototrophs, that is,

capable of living on only hydrogen gas, carbon dioxide, and

either nitrogen gas or ammonium ions. Methanococci are

irregular flagellated cocci. They include borderline mesophiles

to hyperthermophiles (48–98°C) isolated from marine and

freshwater sediments and the deep-sea hydrothermal vents

known as “black smokers” (described below). Methanobacteria

are nonmotile rods or filaments. They include mesophilic

to moderately thermophilic (40–70°C, optimum

65°C) pure prototrophs common in animal colon and rumen

and also isolated from sewage sludge and sea sediments and

as symbionts of animals, plants, and protists. In the termite

hindgut, they form symbioses or endosymbioses with cellulose-

digesting protists, from whom they get hydrogen gas

(Tokura et al. 2000). Methanopyrus kandlerii, the sole cultivated

member of the Methanopyri, is a “gram-positive” hyperthermophile

(80–110°C) first isolated from a 2000-m-deep

black smoker. It has an internal salt concentration of 1.1 M,

probably part of the means by which they maintain enzymatic

activity in extreme heat.

Methanogen Phylogeny. Most of the phylogeny of archaeal

methanogens is based on SSU rRNA trees, which split them

into numerous separate groups and tend to place Methanopyrus

as the deepest euryarchaeote branch. However current

CDS analyses tend to restrict the methanogen to only two

distinct groups (MG1 and MG2; fig. 4.3, nodes 1 and 8),

including Methanopyrus as sister to a Methanococcus + Methanobacterium

clade (MG2; fig. 4.3, node 8; Slesarev et al. 2002).

Furthermore, neither methanogen group appears to be

among the deepest branches in the euryarchaeote tree. On

the other hand, CDS data strongly reject recent claims that

the archaeal methanogens are monophyletic; MG1 is nested

within a substantial group of nonmethanogens, at some distance

from MG2 (fig. 4.3, nodes 2–5).

Thermococci

These include Thermococcus and Pyrococcus, which are thermophilic/

hyperthermophilic (75–100°C, pH ~7) flagellated

cells commonly found, along with other thermophilic members

of Archaea, in and around the “black smokers” formed

by deep-sea hydrothermal vents. Black smokers are mineral

chimneys formed by the buildup of sulfides deposited by the

mineral-rich waters spewing from the vents and giving the

appearance of belching black smoke. The warm hydrogen

sulfide–rich habitat around these vents supports a rich fauna,

essentially oases of life along the otherwise largely barren

seafloor. These communities are dependent on energy from

the oxidation of sulfide rather than light; that is, they are

lithotrophic rather than phototrophic.

Thermococci and Pyrococci together form a distinct, tight

phylogenetic group in all phylogenetic trees. The group appears

to be quite shallow, but this may reflect a slow rate of

molecular sequence evolution, which is seen in many hyperthermophiles.

This may be due to the restrictive amino acid

requirements of thermostable proteins; thermococcalean

proteins tend to have highly biased amino acid use and favor

nonpolar amino acids over polar ones by a ratio of around

3:1 (Howland 2000). Unlike most other major subdivisions

of Archaea, uncultured Thermococci do not display a large

diversity in any of the habitats sampled by ciPCR analyses

(Maidak et al. 2001).

Thermoplasmata (Thermoplasma, Ferroplasma,

and Picrophilus)

These are all thermophilic extreme acidophiles (growth

optima 40–60°C, pH 0.5–2.0), the only organisms able to

survive, never mind thrive, at pH < 0 (Ruepp et al. 2000,

Edwards et al. 2000, Schleper et al. 1995). Cells are small

(0.2–5 mm), spherical, and sometimes flagellated and, unlike

all other archaeans, lack a cell wall. Despite this, they

survive external pHs of 0–4 while maintaining an internal

pH ~7 (Ruepp et al. 2000). Natural habitats include hot solfatars

and coal refuse, which are rich in highly toxic metals such

as copper, arsenic, cadmium, and zinc. It appears that they

make their “living” by scavenging complex organics released

by cells that are killed by these extreme conditions. Ferroplasma

is responsible for the acidification of coal mine drainage,

the primary environmental problem associated with

mining (Edwards et al. 2000).

Thermoplasma acidophilum has an extremely small genome

(~1.6 megabases), and, in an apparent case of massive LGT,

it shares 17% of it exclusively with the crenarchaeote Sulfolobus

(Ruepp et al. 2000). The shared sequence resides in

The Tree of Life 59

approximately five large blocks and codes for many of the

transport and metabolic pathways needed for this unique

lifestyle, which requires importing a variety of complex organic

compounds. Consistent with this, Thermoplasmata and

Sulfolobales often co-occur in habitats that they share almost

exclusively except for a few species of Bacillus. ciPCR indicates

that Thermoplasmata is much larger and broader than

the currently known taxa, although it still appears to be restricted

to hot acid environments (Maidak et al. 2001). The

lack of a cell wall in thermoplasmas has led to speculation

that the group might include the direct ancestor of Eucarya.

However, a large body of molecular phylogenetic data now

soundly reject this (fig. 4.3, node 5).

Euryarchaeote Phylogeny

Euryarchaeota is an ancient, large, and extremely diverse

group, and resolving relationships within it will be difficult.

This is also complicated not only by LGT but also, perhaps

more important, by the strong biases in the amino acid composition

of their proteins. These are required for adaptation

to extremes of salt, pH, and temperature. Therefore, inclusion

of sequence data from the mesophilic taxa indicated by ciPCR

phylotypes could potentially improve resolution considerably.

Nonetheless, certain trends can be identified at this point

with some confidence. The Euryarchaeota are almost certainly

a monophyletic group (fig. 4.3, node 10); theories that

bacteria might have originated from euryarchaeote ancestors

have been largely abandoned (Lake 1988). Within the euryarchaeotes

it appears unlikely that the methanogens form a

monophyletic group. Recent claims to the contrary are not

supported by analyses with fuller taxonomic representation

(Slesarev et al. 2002).

The earliest branch of the euryarchaeotes appears to be

the Thermococcoides (fig. 4.3, node 9). This is followed by

a number of ciPCR lineages that may or may not form a single

group. Data beyond SSU rRNA sequences are needed before

any more conclusions on these taxa can be drawn. The remaining

euryarchaeotes appear to split into two groups: MG1

and their allies (MG1+; fig. 4.3, node 7) and MG2 (fig. 4.3,

node 8). The inclusion of Methanopyrus in MG2 is still tentative;

the grouping is only weakly supported by the only CDS

study with Methanopyrus in it, and it is strongly rejected by

SSU rRNA trees, in which Methanopyrus SSU shows no clear

affinity for any other euryarchaeote sequence and tends to

fall toward the base of the tree.

MG1+ (fig. 4.3, node 5) is a surprisingly robust clade. The

group is further supported by the shared presence of cytochrome

b and/or c, which are found among members of

Archaea only in Methanomicrobiales, Halobacteria, and

Thermoplasmas. The most problematic taxon within MG1+

is the Halobacteria, probably because of their extreme,

uniquely biased amino acid use (Ng et al. 2000). Nonetheless,

they group together strongly with MG1 in a number of trees

(fig. 4.3, node 5), and when they do not it is often because

they are found in highly unlikely positions, such as at the base

of the entire Archaea domain (Slesarev et al. 2002). A grouping

of Halobacteria + MG1 is also consistent with the fact that

the latter includes Methanohalophilus species, the only known

halophilic methanogens. The exact position of Archaeoglobus

within MG1+ is also very unstable, suggesting that there may

have been considerable LGT during its evolution (fig. 4.3A).

Korarchaeota and Nanoarchaeota

Two additional major subdivisions of Archaea have been

suggested recently, the Korarchaeota and Nanoarchaeota.

The Korarchaeota were originally identified in a ciPCR study

of a Yellowstone Park hot spring (74–93°C). Two phylotypes

were found that formed a distinct group that was clearly

archaeal but not specifically related to either Crenarchaeota

or Euryarchaeota. The group, provisionally named “Korarchaeota”

(Barns et al. 1996), has since been detected in

Icelandic sulfide hot springs (Hjorleifsdottir et al. 2002) and

geothermal effluent (Marteinsson et al. 2001), sometimes in

abundance (Hjorleifsdottir et al. 2002). This indicates that

the group at least is real, but additional data are still needed

to test their classification as a unique archaeal subdivision.

Caution is warranted also by the fact that their position as a

unique branch among archaeal SSU rRNA sequences varies

depending on the taxon composition of the data set and

the analytical method used. Nonetheless, korarchaeote SSU

rRNA sequences lack features generally associated with phylogenetic

artifact; that is, they do not form long branches and

lack strong percentage G+C bias.

The “Nanoarchaeota” were described even more recently

and have been encountered so far only once (Huber et al.

2002b). They are hyperthermophiles (70–98°C) from an Icelandic

coastal hot submarine vent and were found attached to

cells of Igniococcus, a desulfurococcalean crenarchaeote (described

above). Everything about them is small, including their

cells (0.4 mm diameter) and their genomes (500 kilobases),

which is near the theoretical limit for a “free-living organism”

(Huber et al. 2002b). So far, they can be cultured only when

attached to a live host. However, they are probably not parasites

because the host grows equally well with or without them.

Nanoarchaeum SSU rRNA is clearly archaeal but otherwise

highly divergent and shows no specific affinity for any currently

known archaeal group (Huber et al. 2002b). However, unlike

the korarchaeote SSU rRNAs, these sequences do possess features

associated with phylogenetic artifact; that is, they are

extremely divergent and lack a number of otherwise universally

conserved nucleotides. This suggests they may belong to

a rapidly evolving lineage rather than an ancient one.

Summary of Archaebacteria

The Archaea include the most extremophilic organisms

known, and more than for any other group of taxa, our

understanding of them is being fundamentally rewritten by

ciPCR and whole-genome data. Genomic sequencing is the

60 The Origin and Radiation of Life on Earth

only way to study many of them in any detail, and ciPCR

studies indicate major mesophilic components and additional

new groups at all taxonomic levels. Some consistent resolution

seems to be emerging from the still very limited CDS

trees, but little can be said with confidence. Resolving the

archaeal tree will require more genes and more taxa representing

the true diversity of the group, as well as careful attention

to confounding factors such as LGT. Protein gene

sequences from mesophilic taxa may be the key to circumventing

the systematic phylogenetic artifact caused by the

highly skewed amino acid composition of extremophile sequences.

Although these taxa have largely escaped cultivation,

recent progress in genomic analyses of uncultured taxa

could circumvent this limitation (Schleper et al. 1998).

Domain Eucarya

Eukaryotes are defined first and foremost by the presence of

a nucleus surrounded by a double membrane punctuated

with large highly complex pores. The nuclear membrane is

part of a larger endo-membrane system that also includes the

endoplasmic reticulum and Golgi apparatus, which synthesizes

membranes and processes, sorts, and packages proteins

for distribution or export. Other organelles are also usually

present, most notably mitochondria and chloroplasts (more

correctly “plastids”), both of which are descended from bacterial

endosymbionts (Alberts et al. 2002). Mitochondria in

particular, but possibly also plastids (Andersson and Roger

2002), originated early in eukaryotic evolution, and no premitochondrial

eukaryotes appear still to exist. However,

mitochondria have been lost, reduced, or converted to

fermentative, hydrogen-gas-producing organelles (hydrogenosomes;

Dyall and Johnson 2000) several times independently

over the course of eukaryote evolution.

Eukaryotic cells vary widely in size (from <1.0 to 100

mm in diameter) and often form colonies or multicellular

structures. They have numerous other unique features,

many probably correlated with the advent of membranebound

nuclei and the invention of endocytosis, such as the

actin cytoskeleton probably derived from bacterial celldivision

protein ftsA (van den Ent and Lowe 2000). Eukaryotic

flagella are large, complex multiprotein structures

unrelated to bacterial flagella, which are composed almost

exclusively of flagellin. The eukaryotic flagellum was probably

derived early in eukaryotic evolution, possibly from

the cytoskeleton, and is clearly not of endosymbiotic origin

(Cavalier-Smith 2002). Sequestering DNA into a membrane-

bound nucleus spatially separates transcription

(copying of DNA into RNA) from translation (decoding of

RNA into protein), unlike in bacteria, which have the two

processes coupled and possibly coregulated. However, it

now appears that a significant amount of eukaryotic translation

may occur in the nucleus, coupled with transcription

as in bacteria (Iborra et al. 2002).

Eukaryotic information processing is essentially an expanded

version of the archaeal system. Transcription uses

archaea-like RNA polymerases, and gene expression is controlled

with the same basic machinery, although with many

eukaryote-unique factors layered on top (Bell and Jackson

2001). Eukaryotes are still unique in having large operonfree

genomes on multiple linear chromosomes, packaged

around histones, usually containing large amounts of repetitive

DNA. Introns tend to be much more common, to the

point of being highly abundant in some plants, animals,

fungi, and amoebozoans. These introns are mostly of the

spliceosomal type; that is, they require a large multiprotein

complex (the “spliceosome”) to remove them from the premessenger

RNA transcript, unlike the self-splicing introns of

the members of Bacteria and Archaea (Logsdon 1998). Eukaryotes

are also the only organisms known to have true

diploidy (and polyploidy) and sex (meiosis). However, these

rules are nearly all broken somewhere among eukaryotes, and

more exceptions will undoubtedly be found.

Eukaryotes are a highly derived, unquestionably monophyletic

group. More so than for members of either Archaea

or Bacteria, most of what we know about them is based on

SSU rRNA trees. These define most of the major groups, some

of which were not previously suspected. The overall structure

of this SSU rRNA tree also led to the influential

“Archezoa” (Cavalier-Smith 1987) and “Crown Radiation”

(Sogin and Gunderson 1987, Sogin 1991) hypotheses, which

have since been disproved. The former was based on the

observation that the deepest eukaryote SSU branches, admittedly

largely parasitic, lacked mitochondria and most other

internal structures, and therefore might represent primitive

pre-mitochondrial lineages. The latter hypothesis suggested

that the clustering of most of the other “more advanced” lineages

meant that they arose comparatively late in eukaryote

evolution, perhaps in a single explosive radiation (see

Philippe, ch. 7 in this vol.). A fairly large body of data now

agrees that both phenomena are different aspects of the same

artifact: fast-evolving (long-branched) taxa (members of the

Archezoa) being drawn toward the base of the tree and causing

the remaining taxa (the crown) to appear as a dense cluster

(Morin 2000, Philippe and Germot 2000).

Fourteen major eukaryotic groups are currently defined

based on molecular phylogenetic data (fig. 4.4; Cavalier-

Smith 1998, Baldauf et al. 2000). These include most of

Patterson’s (1999) 60 “ultrastructural types.” The most thorough

reference on eukaryote morphology and fine-level taxonomic

diversity is The Illustrated Guide to the Protozoa (Lee

et al. 2000). All unreferenced material in the following sections

is derived from that book, which relies heavily on the

work of Patterson, Brugerolle, and colleagues or of Hausmann

and Hьlsmann (1996).

However, it is now apparent that this description is far

from representing the true diversity of eukaryotes. In culture

collections alone, more than 200 taxa are without known

relatives, and several major groups of amoebae lack even SSU

The Tree of Life 61

rRNA sequences (Patterson 1999). More important, recent

ciPCR studies suggest the existence of major undiscovered

eukaryotic lineages (Amaral-Zettler et al. 2002, Dawson and

Pace 2002, Moriera and Lopйz-Garcia 2003). These

“nanoeukaryotes,” cells less than 2–3 mm in diameter, have

previously escaped detection because they are all but indistinguishable

from bacteria under the light microscope. Some

of the new taxa appear to represent major new subdivisions

of established groups (e.g., Alveolates; fig. 4.4) or perhaps

even the first known representatives of entire new lineages.

Major revisions in the eukaryotic tree are to be expected in

the very near future (Moriera and Lopйz-Garcia 2002).

Excavates

Excavates 1: Amitochondriate Excavates (fig. 4.4, node 1)

Among the best candidates for the earliest diverging Eukaryotes

are the group of taxa recently united as the Excavata.

This is a diverse assemblage of single-celled organisms most

of which possess a conspicuous “excavated” ventral feeding

groove (Cavalier-Smith 2002, Simpson and Patterson 1999,

2001). However, the group as a whole lacks material molecular

phylogenetic support. For convenience they are treated

as two somewhat arbitrary subgroups, (1) “amitochondriate”

excavates, which lack classical mitochondria, and (2) “mitochondriate”

excavates.

The best known amitochondriate excavates are diplomonads.

These typically exhibit a “doubled” morphology, with

duplicate nuclei, sets of flagella, and cytoskeletons arranged

back to back in each cell. The intestinal parasite Giardia

intestinalis is a major human diarrheal agent, whereas Spironucleus

includes some serious fish parasites. Some other

diplomonads are free-living and are common in low-oxygen

habitats (Bernard et al. 2000). Retortamonads are broadly similar

to diplomonads but have a single nucleus, flagellar cluster,

and feeding groove per cell. Most are intestinal commensals.

Figure 4.4. Support for deep branches in the eukaryote tree. (A) shows support for the labeled nodes in (B) (same symbols as in

fig. 4.2A). Data sets used are combined SSU and LSU rRNA (Van der Auwera et al. 1998), combined mitochondrial proteins (Burger

et al. 1999), four combined proteins (Baldauf et al. 2000), 123 combined proteins (Bapteste et al. 2002), and individual gene

phylogenies for SSU rRNA (Van de Peer and De Wachter 1997, Sogin 1991), actin (Bhattacharya and Weber 1997, Keeling 2001),

and b-tubulin (Keeling and Doolittle 1996, Keeling et al. 2000). Alternative rootings of the tree are indicated by dashed lines and

arrows: “a,” for the molecular phylogenetic root using archaeal outgroup sequences, and “b,” as indicated by the fusion of the genes

for DHFR and TS (described in text; Stechmann and Cavalier-Smith 2002). The asterisk (*) indicates that nodes 1 and 4 in the SSU +

LSU CDS are interrupted by aberrant deep branching of microsporidian and lobosan sequences.

62 The Origin and Radiation of Life on Earth

Oxymonads are flagellated symbionts from the intestinal tracts

of animals, mostly termites. Some attach to the gut wall by stalk

or “holdfast,” others squirm using an internal motile cytoskeleton,

and still others are free-swimming cells. Diplomonads,

retortamonads, and oxymonads all seem to lack any cellular

structure that may be homologous to mitochondria.

Parabasalids are a diverse group almost entirely comprised

of parasites and symbionts united by the presence of a

parabasal apparatus, which is a complex of Golgi stacks and

striated cytoskeletal elements. In place of mitochondria, parabasalids

have organelles called hydrogenosomes that anaerobically

generate ATP from pyruvate, liberating hydrogen gas

in the process (Rotte et al. 2000). Some parabasalids from termites,

for example, hypermastigids, are huge multiflagellated

cells, hundreds of micrometers long and covered in

ectosymbiotic bacteria, whereas most “trichomonad” parabasalids

are small teardrop-shaped cells with four to six flagella.

Trichomoniasis, caused by the trichomonad parabasalid

Trichomonas vaginalis, is the most common human sexually

transmitted infection affecting ~170 million people worldwide

(Mьller 1988). Trimastix and Carpediemonas are free-living,

groove-bearing, bacterivorous flagellates that inhabit low-oxygen

environments (Bernard et al. 2000). Although neither has

classical mitochondria, both have small organelles that superficially

resemble the hydrogenosomes of parabasalids.

Phylogeny. The amitochondriate excavates have been

central to exploring the origin and early diversification of

eukaryotic cells. On the strength of early SSU rRNA phylogenies,

diplomonads, retortamonads, oxymonads, and parabasalids

were widely thought to be among the earliest

branching eukaryotes, diverging before the acquisition of the

bacterial symbiont that became the mitochondrion (Cavalier-

Smith 1987, Sogin 1991). This deep-branching placement

is also seen with protein-coding genes (e.g., Baldauf et al.

1996, Roger 1999, Bapteste et al. 2002). However, several

genes of mitochondrial origin have since been found in parabasalid

and diplomonad nuclear genomes (Roger 1999,

Tachezy et al. 2001), suggesting that both groups originally

had a mitochondrial symbiont (in parabasalids, this symbiont

is preserved as the hydrogenosome).

Recent phylogenetic evidence also demonstrates that

diplomonads and retortamonads are very closely related to

Carpediemonas and parabasalids, whereas oxymonads are

close to Trimastix (Dacks et al. 2001, Simpson and Patterson

2001, Silberman et al. 2002, Embley and Hirt 1998,

Simpson et al. 2002). Although mitochondrial origins of the

hydrogenosome-like organelles of Trimastix and Carpediemonas

have not been proven, it seems very likely that all

amitochondriate excavates have ancestors that bore mitochondrial

symbionts. It is also argued that the basal placements

of diplomonads and parabasalids in many molecular

phylogenetic trees could be analysis artifacts caused by aberrant

(especially accelerated) gene sequence evolution in

these groups (Embley and Hirt 1998, Philippe and Adoutte

1998). Therefore, the relevance of amitochondriate excavates

to understanding early eukaryotic history is now uncertain,

although they remain fascinating organisms for exploring the

biochemical diversity and potential of eukaryotic cells.

Excavates 2: Mitochondriate Excavates (Discicristates,

Jakobids, and Malawimonas)

The most important and best known mitochondrion-bearing

excavates are the Discicristates (fig. 4.4, node 3). They

are among the most recent of major eukaryotic groups to

be confirmed by strong molecular phylogenetic support

(Baldauf et al. 2000). Discicristates include the Euglenozoa

and the Heterolobosea, which share the unusual characteristic

of having mitochondria whose cristae are discoid in

shape. These infoldings of the inner mitochondrial membrane

are the site of electron transport and ATP production.

Other mitochondriate excavates are the more obscure jakobids

and Malawimonas.

Euglenozoa contain two major supergroups: kinetoplastids

and euglenids. Kinetoplastids are small uni- or

biflagellated cells with a distinctive and baroque mitochondrial

genome organization. The mitochondrial DNA is condensed

into a large mass or masses called the kinetoplast, and

many of the messenger RNAs for mitochondrial genes require

extensive RNA editing (mediated by other smaller RNA

molecules called guide RNAs) before they encode functional

proteins (Sollner-Webb 1996). The kinetoplastids include

the trypanosomatid parasites, among which are the agents

of several deadly human diseases: sleeping sickness, Chagas

disease, and leishmaniases. Many other kinetoplastids are also

commensals or parasites, but free-living forms are abundant

consumers of bacteria and small eukaryotes.

Euglenids are usually free-living uni- or biflagellate cells

enclosed by a thickened pellicle made longitudinal proteinaceous

strips. Most of the diversity of euglenids are free-living

osmotrophs, or phagotrophs that are often able to consume

large eukaryotic cells, although the most famous euglenids

are the photosynthetic forms, such as Euglena. The photosynthetic

euglenids have chloroplasts that are of secondary

origin—they are derived from an eukaryotic green algal cell

that was ingested by a nonphotosynthetic euglenid ancestor.

Heterolobosea (fig. 4.4, node 3). These are mostly amoebae,

although many have flagellate phases in their life cycles

(Patterson and Sogin 2000). Heteroloboseids differ in appearance

from lobose amoebae in their “eruptive” formation of

pseudopodia. Most are soil or freshwater bacterivores, although

one, Naegleria fowleri, is a rare but often fatal facultative

human pathogen. A subgroup, the acrasids, are slime

molds that form fruiting bodies, but they are unrelated to

the “true” mycetozoan slime molds (Roger et al. 1996).

Jakobids (i.e., core jakobids) are small free-living bacterivores.

They have the most bacteria-like mitochondrial

genomes known, having retained genes apparently lost, relocated,

or replaced in other studied eukaryotes (Lang et al.

1997). A final small group, Malawimonas, is superficially similar

to jakobids but might be more closely related to some or

The Tree of Life 63

all amitochondriate excavates (O’Kelly and Nerad 1999,

Simpson et al. 2002).

Jakobids are also interesting because their bacteria-like

mitochondrial genomes may represent a primitive state for

living eukaryotes (Lang et al. 1997). However, the recent

resolution of the broad-scale eukaryotic tree using the

dihydrofolate reductase–thymidylate synthase gene fusion

suggests that Excavata might be closer to Plantae than to

Opisthokonta (Stechmann and Cavalier-Smith 2002) and

thus not especially deeply branching after all (assuming that

Excavata is, in fact, a natural group).

Phylogeny. The monophyly of excavates is currently

contentious. The cytoskeleton supporting the cell is distinctively

similar in all of them. The exceptions, most notably

parabasalids, are convincingly related to at least one “good”

excavate in molecular trees (e.g., Baldauf et al. 2000). Thus,

morphology suggests that excavates descend from a similar

common ancestor (Simpson and Patterson 1999, Simpson

et al. 2002). By contrast, almost all molecular analyses place

excavates as multiple separated clusters distributed across the

diversity of eukaryotes (Simpson et al. 2002). However, different

groups of excavates exhibit drastic differences in evolutionary

rate for commonly used molecular markers, a

property known to complicate and confound phylogenetic

analysis (Philippe and Adoutte 1998). Resolving whether

excavates are a natural group using molecular markers promises

to be a difficult problem in eukaryotic phylogeny. One

theoretical possibility is that excavates currently represent an

ancestral grade for most or all living eukaryotes, rather than

a natural group (O’Kelly 1993).

Chromalveolates

Chromalveolates (fig. 4.4, node 5) are a broadly diverse group

of protists that includes the Chromista (fig. 4.4, node 7), comprising

the cryptophytes, haptophytes, and stramenopiles

(heterokonts) and the Alveolata (fig. 4.4, node 6), which

include the parasitic apicomplexans, ciliates, and dinoflagellates.

The chromalveolates were postulated primarily on the

basis of molecular phylogenetic analyses that unite particular

members of these disparate lineages (described below),

and the hypothesis that all taxa containing a chromophytic

plastid (i.e., containing chlorophyll c) share a common origin

(Cavalier-Smith 2000).

Chromalveolates 1: Chromists

The Chromista (Cavalier-Smith 1986) are a provisional group

including the cryptophyte, haptophyte, and stramenopiles.

These are largely marine, unicellular algae that are some of

the most important photosynthetic forms on the planet. The

stramenopiles have unambiguous molecular and ultrastructural

justification. Almost all groups within Heterokonta include

organisms with a “tinsillated” flagellum, and most have

a second, shorter, smooth flagellum. The shorter flagellum

is posteriorly directed and often associated with an eyespot.

The tinsillated flagellum is anteriorly directed and bears two

rows of stiff, tripartite hairs along its length. These hairs

reverse the flow around the flagellum so that the cell is

dragged forward although the medium, rather than pushed

along. The group is named for the structure of the flagellar

hairs (stramenopiles), but they are also often referred to as

heterokonts, which means “different flagella.” These characters

are not found in the cryptophytes or haptophytes, and

their phylogenetic affinity has so far been hard to resolve.

Stramenopiles (Diatoms, Kelps, Oomyetes, Labyrinthulids).

Stramenopiles are possibly the largest and most diverse group

of eukaryotes. They include opalinids (endocommensals,

mostly in cold-blooded vertebrates), oomycetes (including

water molds and downy mildews, previously classified as fungi),

bicosoecids (small heterotrophic biflagellates), labyrinthulids

(slime nets), and all the diverse types of chlorophyl a and c algae.

The latter include the diatoms, dominant marine photoautotrophs

that reside in lidded boxes made of silica (glass) called frustules.

There are ~11,000 recognized species, and millions of

undescribed ones by some estimates (Norton et al. 1996). Other

stramenopile algae include the multicellular kelps, which are

particularly widespread in temperate intertidal and subtidal

zones. These have true parenchyma and build “forests” in nearshore

environments that support complex ecosystems including

fish and marine mammals.

Oomycetes are important group of parasites or saprobes

(e.g., Phytophthora infestans, the cause of potato blight and the

great famine of Ireland). Although lacking a plastid, recent

sequence data suggest that these taxa may have once been

photosynthetic (Andersson and Roger 2002). There is also

a huge diversity of very small free-swimming phototrophic,

mixotrophic, and heterotrophic stramenopiles in most planktonic

systems (Moriera and Lopйz-Garcia 2002), for example,

the bicosoecid Cafeteria, perhaps the world’s most abundant

predator. Others, such as Blastocystis and opalinids, are commensals

in the guts of animals. Although lacking a plastid,

recent sequence data suggest that they may have once been

photosynthetic because they retain nuclear-encoded genes of

apparent cyanobacterial origin (Andersson and Roger 2002).

Haptophytes. Haptophytes get their name from the presence

of a unique anterior appendage, the haptonema, used

for adhesion and capturing prey. The group includes the

coccolithophorids, which build external coverings of calcium

carbonate scales (coccoliths) and tend to dominate open

oceanic waters worldwide. Emiliana huxleyi, in particular, has

received considerable attention because of its important role

in cloud production through dimethyl sulfoxide release, the

effects of its “blooms” on temperature and optical quality of

oceanic waters, and its role as a major carbon sink (Buitenhuis

et al. 1996). Coccoliths from dead cells accumulate as limestone

deposits on the ocean bottom, forming the largest inorganic

reservoir of carbon on Earth. The haptophyte

Chrysochromulina is an important source of toxic blooms.

Cryptophytes. The cryptophytes are perhaps the least

known of the chromists, being relatively small (mostly 2–10

64 The Origin and Radiation of Life on Earth

mm diameter) unicells and primarily found in cold or deep

aquatic environments. The group has been critical to our

understanding of plastid secondary endosymbiosis because

they have retained an intermediate stage in the process.

Current theory holds that all chromalveolates acquired their

plastid by a single event (Cavalier-Smith 2000), in which a

common ancestor of the group ingested a single-celled photosynthetic

eukaryote, in this case a red alga (vs. a green

alga in the case of euglenids). The host would have then transferred

the red algal nuclear genes required for plastid maintenance

into their own nuclear genome, and the original red

algal nucleus would have been lost. However, in the case of

cryptophytes a remnant of the red algal nucleus persists as a

“nucleomorph” that resides together with the plastid surrounded

by a double membrane—a kind of cell within a cell.

Analysis of the nucleomorph genome (e.g., Douglas et al.

1999, 2001) provided the first phylogenetic evidence for the

chimeric nature of algal cells by confirming the red algal origin

of the cryptophyte plastid.

Chromalveolates 2: Alveolates

The alveolates (fig. 4.4, node 6) represent another large assemblage

of protists with strong molecular and ultrastructural

justification. The group includes the dinoflagellates,

many of which are algae, the parasitic apicomplexans, and

the ciliates (Gajadhar et al. 1991). All members of the group

possess sacs or alveoli under the plasma membrane. The alveoli

form the pellicle in ciliates and surround the peripheral

armor plates in dinoflagellates.

Ciliates. Ciliates are mostly free-living aquatic unicells.

These well-known protists (e.g., Paramecium tetraurelia) are

characterized by an abundance of cilia on their body surface,

nuclear dualism, and the presence of a conjugation stage

during the sexual phase of the life cycle (Hausmann and

Hьlsmann 1996). Nuclear dualism refers to the maintenance

of two different types of nuclei in each cell. The smaller micronucleus

contains the diploid germ nucleus, whereas the

second much larger macronucleus contains thousands of

copies of only the physiologically active genes. Ciliate nuclear

genome organization is truly remarkable; genes are not only

fragmented by introns and short intervening sequences, but

the order of the gene fragments themselves may be scrambled

(Prescott 2000). Therefore, extensive editing can be required

during generation of the macronucleus in order to produce

the active working copy of the gene.

Dinoflagellates. This is a diverse, predominantly unicellular

group, characterized by having one transverse and

one longitudinal flagellum, resulting in a unique rotatory

swimming motion. Most are covered by often elaborate plates

or armor. Although the group was probably primitively photosynthetic

(described below), only about half of the extant

dinoflagellates still are, and many of these species are mixotrophs.

These ingest bacteria and other eukaryotes and are

notorious for acquiring temporary endosymbionts from

them, particularly plastids from a variety of algae. In fact, the

group appears to include the first known example of tertiary

endosymbiosis involving the secondary endosymbiosis of a

haptophyte, itself already secondarily endosymbiotic (described

above; Yoon et al. 2002a). Others, such as Symbiodinium

species, are themselves endosymbionts of corals, and

these and other dinoflagellates are a common source of phosphorescence

in marine waters. Under all trophic condition,

the dinoflagellates are an important component of marine

ecosystems as symbionts and primary producers. They also

produce some of the most potent neurotoxins known and

are the main source of toxic red tides and other forms of fish

and shellfish poisoning.

Apicomplexa. Closely related to the dinoflagellates are

the apicomplexans, which formerly constituted the bulk of

the “sporozoa.” They include some of the most important

protozoan disease agents of both invertebrates and vertebrates

and are the causative agents of malaria and toxoplasmosis.

All are obligate, mostly intracellular parasites

characterized by the presence of an intricate apical complex.

This is a system of organelles and microtubules situated at

the posterior of the cell that functions in the attachment and

initial penetration of the host. Their complex life cycles are

completed entirely within the host, and they exist outside it

only as spores or oocysts. The group appears to have been

derived from photosynthetic ancestors, and recent data show

that they retain a vestigial plastid (apicoplast) most likely of

red algal origin (Fast et al. 2001). Much research in malaria

is now being directed at finding drugs that target potential

functions of this organelle.

Phylogeny

Chromalveolates (fig. 4.4, node 5) are a broadly diverse group

of protists postulated primarily on the hypothesis that all taxa

containing a chromophytic (chlorophyll c) plastid share a

common origin (Cavalier-Smith 2000). Molecular phylogenetic

support for the heterokont and alveolate groupings is

generally strong, although these data sets are mostly very

taxon limited. However, support for the entire chromalveolates

grouping is still slight, although some analyses of

nuclear-encoded genes have shown moderate to moderately

strong support for a stramenopile + alveolate clade (e.g., Van

de Peer and De Wachter 1997, Baldauf et al. 2000; see also

Philippe, ch. 7 in this vol.).

More recently, a much more inclusive five-gene plastid

data set shows the first robust support for a single common

origin of chromist plastids, implying a monophyletic origin

for the chromalveolates (Yoon et al. 2002b). These trees show

the cryptophytes as the deepest branch in the group, implying

that the nucleomorph and phycobilin pigments are ancestral

characters lost before the divergence of haptophytes

and stramenopiles. Plastid loss after secondary endosymbiosis

must also have been common (e.g., the oomycetes). Molecular

clock analyses place the earliest date for the origin of

chromists at ~1.26 Byr ago. Thus, a single, ancient event, the

secondary endosymbiosis of a red algal plastid, appears to

The Tree of Life 65

have been a fundamental one in eukaryote evolution, giving

rise to an entire protist superassemblage, the chromalveolates.

Plantae

The Plantae (fig. 4.4, node 8) consists of the rhodophytes

(red algae), glaucophytes (glaucophyte algae), and Viridiplantae

or green plants (green algae + land plants). Rhodophytes

vary from large seaweeds to crustose mats that look

more like rocks than living plants. Their plastids have two

membranes and unstacked thylakoids. Light is harvested

primarily with chlorophyll a and phycoerythrins (red chromophores)

conjugated to phycobiliproteins. There are two

major subgroups, bangiophytes and florideophytes; the

former appears to be older and may have given rise to the

latter. Glaucophytes are a small but distinct group of unicellular

flagellates. They harvest light energy in plastids called

“cyanelles” using rhodophyte-like proteins and pigments.

Cyanelles have two membranes, unstacked thylakoids and,

most remarkably, bacteria-like peptidoglycan walls. Viridiplants

vary from single-celled flagellates to large marine

filaments to redwoods. Their plastids have two membranes

and stacked thylakoids, and they harvest light with chlorophylls

a and b attached to chlorophyll–a-b–binding proteins.

Virdiplantae includes the chlorophyte, ulvophyte, trebouxiophytes,

charophyte, and “prasinophyte” algae (see Delwiche

et al., ch. 9 in this vol.). Land plants were clearly derived from

charophyte algae, and the single-celled “prasinophytes” are

almost certainly para- or even polyphyletic (Turmel et al.

2002).

Phylogeny

Plantae are probably the only eukaryotic group to acquire

photosynthesis directly from cyanobacteria (primary endosymbiosis).

That this only happened once is most strongly

supported by the fact that their plastid genomes have a similar,

derived gene order and composition (Douglas 1998). It

is also consistent with the fact that these are all the eukaryotes

whose plastids have only two membranes, thought to

correspond to the inner and outer membrane of the original

cyanobacterial endosymbiont (Archibald and Keeling 2002).

All other algae have three or four outer plastid membranes,

believed to be the result of additional endosymbioses (see

Delwiche et al., ch. 9 in this vol.).

If their primary endosymbiosis only happened once in

eukaryotic evolution, then red green and glaucophyte plants

would be expected to form a clade. However, there are still

very few molecular data to test this; there are few nuclear gene

sequences for very few rhodophytes, and even fewer nuclear

and no mitochondrial data for glaucophytes. Actin and mitochondrial

sequence trees strongly support a monophyletic

red–green clade (Burger et al. 1999), as do some nuclear

markers (Hilario and Gogarten 1998). However, others still

appear to reject it strongly (Stiller et al. 2001). Actin trees

also tentatively place all three plant lineages together

(Bhattacharya and Weber 1997), as do combined sequence

data (Moreira et al. 2000, Baldauf et al. 2000). Clearly, more

data are needed from a more representative sampling of algal

lineages.

Cercozoa, Foraminifera, and Radiolaria

This is a heterogeneous assemblage of morphologically and

ecologically diverse forms (fig. 4.4, node 10), including cercomonads,

thaumatomonads, cryothecomonads, Spongomonas,

chlorarachniophytes, euglyphids, Gromia, plasmodiophorids,

and haplosporids, probably also the foraminiferans, and

possibly also the radiolarians. With the exception of the latter

two taxa, each group is currently represented by just a

few genera. The foraminiferans and radiolarians, on the other

hand, are large and well-characterized groups. Most members

of the Cercozoa produce filose pseudopodia (or axopodia) that

are used to capture food particles.

Chlorarachniophytes (genera Chlorarachnion, Lotharella,

Gymnochlora) are photosynthetic marine amoebae with reticulate

(anastomosing, networklike) pseudopodia and a

uniflagellate dispersal stage. Theirs is another example of

secondary endosymbiosis, and, similar to Cryptophytes (described

above), they retain a remnant of the primary endosymbiont

nucleus (nucleomorph). Euglyphids are testate

amoebae with filose pseudopodia, found commonly in freshwater

and in mosses. Their silica hard outer shells (test) are

composed of regularly arranged, secreted plates, which are

also used as characters for species identification. The gromids

are widespread marine protists characterized by filose pseudopodia

and a large (up to 5 mm) spherical to ovoid organic test

with a characteristic layer of honeycomb membranes. It has

a complex life cycle with a well-documented gamontic phase.

Cercomonads (genera Cercomonas, Heteromita, Massisteria)

are common, heterotrophic flagellates, with two naked flagella,

usually able to produce pseudopodia in their trophic stage.

Thaumatomonads (e.g., Protaspis, Thaumatomonas) are biflagellate

heterotrophic, mostly benthic flagellates. They maintain a

rigid cell profile but feed with ventral pseudopodia. Cryothecomonads

are flagellated planktonic predators known mostly

from polar oceans. Spongomonas are sessile flagellates that embed

into a spongy-walled matrix. Plasmodiophorids and haplosporids

are typically plasmodial endoparasites of other

eukaryotes. The Plasmodiophora members (10 genera) are

plant parasites, sometimes treated as fungi. They are characterized

by multinucleated plasmodia, unusual cruciform

nuclear division and zoospores with two anterior flagella. The

Haplosporidia members (three genera) cause diseases in freshwater

and marine invertebrates. They form large multinucleate

plasmodia with unusual organelles, called haplosporosomes,

of unknown function.

Foraminifera and Radiolaria

Compared with Cercozoa, Foraminifera and Radiolaria are

morphologically well-defined, large groups, composed of

66 The Origin and Radiation of Life on Earth

about 940 and 140 modern genera, respectively. Foraminiferans

are widely distributed in all types of marine environment,

but some also occur in freshwater and terrestrial

habitats. They are characterized by finely granular reticulated

pseudopodia (granuloreticulopodia) with bidirectional cytoplasmic

flow. Most members of Foraminifera possess a test,

which may be organic, agglutinated or calcareous, and composed

of single or multiple chambers. Many foraminiferans

have complex life cycles consisting of alternation of sexual

and asexual generations. Nuclear dimorphism has been observed

in a few species. Some calcareous foraminiferans live

in endosymbiosis with dinoflagellates, diatoms, green algae,

or red algae.

Radiolaria are characterized by the combination of internal

mineralized “skeletons” and axopodia—long, radiating,

unbranched processes stiffened by arrays of microtubules.

All are marine and pelagic, solitary or colonial. Some live in

symbiosis with different types of algae. Radiolarians consist

of three distinct classes: Acantharea, Phaeodarea, and Polycystinea.

Acantharia are characterized by delicate skeletons

that consists of radial spicules, composed of strontium sulfate,

joined at the center of the cell and emerging from the

cell surface in a regular pattern. Phaeodaria are characterized

by siliceous skeletons formed of hollow radial spines (not

always present) and a very thick capsular membrane. Polycystinea

is divided into Spumellaria, whose members possess

a spherical cell body plan, and Nassellaria, with members

having a nonspherical body plan and skeletons varying from

simple spicules to complex helmet-shaped structures. Both

foraminiferan and radiolarian (polycystine and phaeodarian)

skeletons contribute substantially to the microfossil record

in marine sediments extending back to the Cambrian. Their

fossilized tests are used in micropaleontology as biostratigraphic

markers and as paleoceanographic indicators to determine

ancient water temperature, ocean depths, circulation

patterns, and the age of water masses.

Phylogeny

The grouping of Cercozoa, Foraminifera, and Radiolaria is

based almost exclusively on molecular phylogenetic data.

Although the majority of the protists belonging to these

groups possess pseudopodia, this character is also present

in Amoebozoa (described below) and other, now clearly

unrelated groups. The cercozoan clade was originally demonstrated

by a series of SSU rRNA analyses progressively

adding more of the unusual members of the group (Bhattacharya

et al. 1995, Bulman et al. 2001, Wylezich et al.

2002), the most recent addition being the enigmatic soil

flagellate Proleptomonas faecicola (Vickerman et al. 2003)

and a marine filosean Gromia oviformis (Burki et al. 2002).

Cercozoan affinity was also suggested for Haplosporidia and

Marteilia (Paramyxea; Cavalier-Smith 2000), despite earlier

molecular study, which considered them as independent

eukaryotic phyla (Berthe et al. 2000). SSU rRNA trees always

place Plasmodiophora as the deepest “reliably placed” branch

in the clade and cercomonads sensu stricto as para- or polyphyletic

within the group.

The grouping of Chlorarachnion and Cercomonas has been

confirmed by a-tubulin (Keeling et al. 1998) and actin (Keeling

2001) gene phylogeny. The latter also first revealed the

close relation between Cercozoa and Foraminifera, contradicting

the previous rRNA-based analyses (Pawlowski et al.

1996), and is now confirmed by polyubiquitine structure

(Archibald et al. 2003) and analysis of RNA polymerase II

subunit 1 sequences (Longet et al. 2003). The latter also

shows that, among the Cercozoa, Gromia appears to be the

closest relative to the foraminiferans.

Neither the composition nor the overall phylogenetic

position of Radiolaria is well established. Early SSU rRNA

analyses including Acantharia and Polycystina suggested the

group was polyphyletic (Amaral-Zettler et al. 1997). However,

later analyses of the same data (Pawlowski et al. 1999)

or with the addition of new ciPCR sequences (Lopйz-Garcia

et al. 2003) showed that these two groups are actually closely

related, and the group as a whole to be related to the Cercozoa

(Cavalier-Smith 2002). There are currently no molecular data

for Phaedaria and their morphological distinctiveness has led

to suggestions that they might have an origin independent

from the other two groups.

There are also currently no published SSU rRNA data on

Heliozoa, and their inclusion in a Cercozoa + Foraminifera

+ Radiolaria clade is contradicted by morphological data indicating

that at least some of them (actinophryids) are related

to stramenopiles (Mikryukov and Patterson 2001).

However, Radiolaria and Cercozoa appear adjacent to each

other in some SSU rRNA trees (Lopйz-Garcia et al. 2003,

Cavalier-Smith 2002) and may form a very weak clade when

only the shortest branches are analyzed (A. G. B. Simpson,

unpubl. obs.).

Amoebozoa

Lobosa (Lobose Amoebae)

Approximately 14 amoeboid types (fig. 4.4, node 12) are

recognized. They appear to be scattered across the eukaryote

tree and may have arisen independently from flagellate

ancestors a number of times (Patterson et al. 2000, Cavalier-

Smith 1998, 2002). Traditional taxonomy of amoebae relies

mainly on pseudopodial morphology and, where present, the

morphology and composition of extracellular scales or shells

(tests). There are very few, if any, molecular data on most of

them and generally little indication of their place within the

larger eukaryote tree. Nonetheless, the phylogenetic positions

of the heterolobosean, foraminiferan, and euglyphid (all

described above) amoebae seem to be resolved, as is that of

the lobose amoebae.

The nontestate (naked) lobose amoebae (also known as

ramicristate or gymnamoebae) are now clearly placed with

the Mycetozoa based on mitochondrial genome synapomorphies

and phylogenetic trees (Bhattacharya and Weber

The Tree of Life 67

1997, Baldauf et al. 2000). They generally have one to many

lobose or tubelike pseudopods (lobopodia), usually a single

nucleus, and mitochondrial cristae that are tubular and

branched. Sizes range from a few micrometers to several

millimeters, and many smaller forms probably remain to be

discovered. They are cosmopolitan in distribution and important

as major bacterial predators. Some form cysts to

survive desiccation or other harsh conditions or to invade

hosts. The group consists largely of widespread free-living

species, but they also include animal commensals and opportunistic

pathogens, such as Acanthamoeba, which causes

eye infections in contact lens wearers. The naked lobose

amoebae may be related to the testate lobose amoebae

(Arcinellinids), but there are no molecular data on them.

Pelobionts and Entamoebae

Pelobionts are amoeboflagellates (possessing both amoeboid

and flagellate morphologies) that mostly live in low-oxygen

environments. They have one or many apical flagella and vary

widely in size; the most famous, Pelomyxa, is a massive

amoeba as long as 3 mm with numerous nonmotile flagella

on its surface. Entamoebae are small aflagellate amoebae, and

almost all are small commensals or parasites of animals. Several

species live in the mouth and intestinal tract of humans,

causing amoebic dysentery, and they sometimes invade the

liver (Entamoeba histolytica), resulting in serious illness. Both

pelobionts and entamoebas lack mitochondria and, partly on

this basis, were widely thought to represent very early diverging

eukaryotes (Cavalier-Smith 1987). However, they have

recently been shown to have mitosomes, small organelles of

mitochondrial origin (Tovar et al. 1999).

Mycetozoa (True Slime Molds)

The Mycetozoa contain the myxogastrid, dictyostelid, and

protostelid slime molds, although the latter may be paraphyletic

with respect to either or both of the former (Olive

and Stoianovitch 1975). Members of the three groups have

very different trophic (feeding stage) morphologies, as described

below. This has led to a long-running debate as to

whether they are related or not, and the myxogastrids and

dictyostelids have been variously classified as plants, animals,

and fungi in the ~150 years since they were first described.

However, they all have distinctly similar fruiting bodies consisting

of a cellulosic stalk supporting spore-bearing sori,

albeit of widely varying size, form, and complexity (Olive and

Stoianovitch 1975).

The myxogastrids (Myxogastridae) are also known as the

plasmodial, true, or acellular slime molds. The best known

is Physarum polycephalum, easily grown on agar plates in the

lab. These are amoeboflagellates, switching between amoeboid

and flagellate morphologies early in their life cycle before

maturing into large plasmodia with 10,000 or more

nuclei. Plasmodia are capable of a slow, creeping movement

propelled by cytoplasmic pulsations, even though they can

be 100 cm or more in diameter. The trophic stage of dictyostelids

(Dictyostelidae), on the other hand, is strictly amoeboid.

Under appropriate conditions the amoebae can aggregate,

although cells never fuse to form true plasmodia. As

many as 10,000 or more cells stream together to form a “slug,”

which surrounds itself with a single outer covering and acts

much like a very simple multicellular organism in that it has

a defined head and tail region and is mobile. In Dictyostelium

discoideum, cell fate is determined in the slug, and only

the cells in the tail region can form spores. Protostelids

(Protostelidae) were first described in 1960 and are almost

entirely microscopic. They can be either amoeboflagellate or

strictly amoeboid, sometimes among apparently closely related

taxa (Olive and Stoianovitch 1975). Thus, they seem

to bridge the “gap” between the dictyostelid and myxogastrid

morphologies (Olive and Stoianovitch 1975), and may be

paraphyletic with respect to them.

The possible monophyly of Mycetozoa has been debated

since their discovery in the late 1800s, based on the striking

differences in their trophic stages and striking similarities in

their fruiting bodies. This was not helped by early rRNA trees,

which separated D. discoideum (Dictyostelidae) and Physarum

polycephalum (Myxogastridae) widely. However, all other

molecular data tend to place them together, mostly with very

strong support (Baldauf et al. 2000). Only a single molecular

study includes a protostelid sequence (Planoprotostelium

aurantium), placing it as the sister group to a strong Physarum

polycephalum + Dictyostelium discoideum clade. This suggests

that not only are the myxgastrids and dictyostelids related,

but they are in fact only a subgroup of Mycetozoa (Baldauf

and Doolittle 1997).

Phylogeny

Although there are few molecular data from lobose amoebae,

and all from Acanthamoeba castellanii, based on these

sequences the monophyly of the Lobosa + Mycetozoa (fig.

4.4, node 12) is strongly supported by actin (e.g., Bhattacharya

et al. 1995) and combined (Baldauf et al. 2000) data.

Lack of support for this grouping from SSU rRNA is not

surprising because these data rarely even bring the Mycetozoa

together, much less support them strongly as a group.

Combined sequence data also support a strong grouping of

pelobionts and entamaebids and place together with the

Mycetozoa (Bapteste et al. 2002). Although Entamoebae

histolytica is represented in many single gene trees, it is almost

never united with the Mycetozoa. However, sequences

from this taxon also tend to be very divergent and to form

highly unstable long branches in phylogenetic trees (e.g.,

Keeling and Doolittle 1996).

Perhaps the most convincing data for Lobosa + Mycetozoa

are shared unique similarities in their mitochondrial

genomes (Ogawa et al. 2000), but because pelobionts and

entamaebids lack mitochondria, these data cannot be extended

to them. Morphologically, the Amoebozoa are united

by the presence of lobose pseudopodia moving in a smooth,

noneruptive manner and tubular mitochondrial cristae.

68 The Origin and Radiation of Life on Earth

Acrasids were until recently grouped with the dictyostelids

because they form similar-looking fruiting bodies. However,

they have discoidal mitochondrial cristae, form eruptive filose

pseudopodia, do not aggregate, and have now been unambiguously

reclassified as Heterolobosea (described above),

and molecular phylogeny seems to firmly link both groups

to each other and to the Mycetozoa (Bapteste et al. 2002).

Opisthokonta (Animalia and Fungi)

Animalia

Animals (fig. 4.4, node 15) are defined as multicellular heterotrophs

capable of complex and relatively rapid movement,

acquiring food by ingestion and digesting it in an internal

cavity. Their cells lack rigid cell walls, and all except sponges

are made up of cells organized into specialized tissues, which

are mostly further organized into specialized organs. Most

are diploid and reproduce sexually by means of differentiated

eggs and sperm. Animal development is characterized

by distinctive stages including a zygote, blastula, and gastrula

(see Eernisse and Peterson, ch. 13 in this vol.).

Fungi

Fungi (fig. 4.4, node 14) are single or multicellular heterotrophs,

acquiring their food by absorption after first digesting

it extracellularly with secreted hydrolytic enzymes. Cell walls

are generally present and composed of chitin; multicellular

forms consist of multinucleate filamentous tubes, termed

hyphae. There are five major subtypes: chytrids, zygomycetes,

ascomyetes, basidiomycetes, and microsporidians.

Thraustochytrids, oomycetes, mycetozoa, plasmodiophorids,

and labyrinthulids have all been removed from the group,

mostly to the heterokonts. The earliest branches of true fungi

are clearly chytrids, although neither they nor the members

of Zygomycetes are monophyletic (see Taylor et al., ch. 12

in this vol.). The microsporidia are often depicted as extremely

early-diverging lineages in molecular trees, but this

is now known to an artifact of their fast evolutionary rates

for most genes (fig. 4.4, Baldauf et al. 2000).

Animal–Fungus Allies (“Choanozoa”)

A diverse group of taxa have been recently assigned to the

opisthokont clade, although their various branching positions

within it are not generally well resolved. These include

choanoflagellates (aquatic uniflagellates), ichthyosporeans

(obligate intracellular parasites of aquatic animals), corallochytreans

(free-living saprophytes), and nucleariids

(cristidiscoidean amoebae). This is a diverse collection of

single-celled taxa with seemingly little in common. Ichthyosporeans

and corallochytreans are highly reduced morphologies.

Nucleariids lack both “diagnostic” features of

opisthokonts, that is, have no flagella, much less a single

basal one, and their mitochondrial cristae appear to be discoidal

rather than flattened (Zettler et al. 2001). Only

choanoflagellates are long-standing candidates for the sister

group to animals, because of their strong resemblance to

the collar cells of sponges. However, the reassignment of

these taxa to Opisthokonta, originally based on SSU rRNA

trees, has been confirmed for choanoflagellates (Monosiga)

and ichthyosporeans (Amoebidium) based on combined

mitochondrial gene trees (Burger et al. 2003). These trees

strongly place the choanoflagellate as the closest sister group

to animals and Amoebidium as a sister group to the

choanoflagellate–animal clade.

Phylogeny

The sisterhood of animals and fungi is now well accepted

among evolutionary protistologists (Cavalier-Smith 1998,

Patterson 1999) and is supported by all large, broadly taxonomically

sampled molecular data sets, including SSU rRNA,

LSU rRNA, HSP70 (70 KD heat shock protein), EF-1a (protein

synthesis elongation factor 1-a), a-tubulin, b-tubulin,

and actin, by combined analysis of 23 proteins using the sum

of likelihood scores method, and by all CDS trees (Baldauf

et al. 2000, Moreira et al. 2000, Bapteste et al. 2002). A small

number of morphologically synapormophies have been defined—

the unique combination of flattened mitochondrial

cristae and, when flagellate, the presence of a single basal

flagellum on reproductive cells (Cavalier-Smith 1998, 2002)

and similarities in the flagellar anchorage system (Patterson

1999). However, these characters are only sporadically found

among the various opisthokont allies (described above).

Nonetheless, the grouping is often not found in small, poorly

taxonomically sampled single-gene trees, probably because

of long-branch problems and hidden paralogy (Baldauf and

Palmer 1993).

Possible New Additions

There are more than 200 poorly known but distinct groups

of eukaryotes whose affinities are unclear (Patterson 1999).

Most of these are small free-living heterotrophic flagellates

or amoebae or are parasites of various kinds. Many will

doubtless turn out to fall within one or more of the groups

described above, but there are reasons for guessing that some

form distinct major groups. Apusomonads and Ancyromonas

are probably closely related, small gliding flagellates supported

by submembranous thecae. Some SSU rRNA trees

weakly suggest that they are closely related to opisthokonts.

Collodictyonids are free-swimming predators of other eukaryotic

cells that form no close relationships in SSU rRNA

trees (Brugerolle et al. 2002).

Heliozoa (“sun animals”) are a large, diverse collection

of cells that capture food particles using radiating stiffened

pseudopodia. They form at least four distinct groups and are

widely assumed to be polyphyletic, although actinophryid

heliozoa alone are thought to be descended from heterokonts.

Tenuous morphological considerations suggest pivotal

roles for Phalansterium and Multicilia (small flagellates)

for understanding the evolution of Amoebozoa, and possiThe

Tree of Life 69

bly all eukaryotes, but this is not confirmed with detailed

examinations or molecular data. Little is known of the positions

of kathablepharids, spironemids, or Telonema, to name

just a few.

The Eukaryote Root

Probably the single most outstanding question in eukaryote

evolution is the location of the root of tree. The predominant

theory until recently has been the Archezoa hypothesis

based on the observation that the deepest branches in the

eukaryotic SSU rRNA tree were mitochondrion-lacking

organisms, that is, microsporidia, diplomonads, and parabasalids

(described above; Cavalier-Smith 1987, Sogin 1991).

This led to the suggestion that these taxa diverged before

unique acquisition of the mitochondrial symbiont in eukaryotes.

However, nuclear-encoded mitochondria-like genes

have been found in representatives of each of these groups,

suggesting they once had at least the precursor of this organelle

(Roger 1999, Tachezy et al. 2001). Analyses of protein-encoding

genes also showed microsporidia to be members of Fungi

(Keeling et al. 2000, Hirt et al. 1999, Baldauf et al. 2000). The

deep placement of their sequences in SSU rRNA trees is an

extreme case of long-branch attraction (Embley and Hurt

1998; see also Philippe, ch. 7 in this vol.).

Most other molecular phylogenies, including CDS trees,

still place diplomonads and/or parabasalids as the most basal

eukaryote branches (Hashimoto et al. 1994, Philippe and

Adoutte 1998, Bapteste et al. 2002). However, these sequences

still tend to form very long branches in these trees,

and it can still be argued that their deep placement is simply

a long-branch artifact (see Philippe, ch. 7 in this vol.). Methods

designed to compensate for long-branch attraction, such

as transversion parsimony or covarion analyses, tend to show

the eukaryote tree without any deep resolution, which may

indicate that the major eukaryote groups arose by explosive

radiation (see Philippe, ch. 7 in this vol.), or simply that these

methods remove most of the information from a data set so

that nothing can be resolved.

A radically different placement of the eukaryote root is

suggested by a recently investigated a gene fusion involving

dihydrofolate reductase (DHFR) and thymidylate synthase

(TS). The genes for these proteins are separate in bacteria and

opisthokonts but fused in plants, cercozoans, chromalveolates,

apusomonads, centrohelids, and discicristates (Stechmann

and Cavalier-Smith 2002). If this root is correct, it

places the opisthokonts as one of, if not the, first extant

branch off the main line of eukaryote descent. If the excavates

are then taken as monophyletic, for which there is currently

no strong molecular phylogenetic support, this shift

in the root makes the amitochondriate excavates a relatively

recently derived group (sister group to discicristates). The

strength of this character rests on the assumption that gene

fusions are highly irreversible, which is not true and difficult

to evaluate here. The scenario is further complicated by

the fact that Dictyostelium, a pivotal taxon in this scheme,

lacks these genes entirely (Myllykallio et al. 2002), as do

members of the alternative deepest eukaryote branch, amitochondriate

excavates. Further data are clearly needed.

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