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Theory in Biosciences 124 (2005) 124

Endosymbiosis, cell evolution, and speciation

U. Kutscheraa,, K.J. Niklasb

Institut fur Biologie, Universitat Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, Germany
Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA

Received 22 November 2004; accepted 21 April 2005

In 1905, the Russian biologist C. Mereschkowsky postulated that plastids (e.g., chloroplasts)
are the evolutionary descendants of endosymbiotic cyanobacteria-like organisms. In 1927,
I. Wallin explicitly postulated that mitochondria likewise evolved from once free-living bacteria.
Here, we summarize the history of these endosymbiotic concepts to their modern-day derivative,
the serial endosymbiosis theory, which collectively expound on the origin of eukaryotic cell
organelles (plastids, mitochondria) and subsequent endosymbiotic events. Additionally, we
review recent hypotheses about the origin of the nucleus. Model systems for the study of
endosymbiosis in action are also described, and the hypothesis that symbiogenesis may
contribute to the generation of new species is critically assessed with special reference to the
secondary and tertiary endosymbiosis (macroevolution) of unicellular eukaryotic algae.
r 2005 Elsevier GmbH. All rights reserved.
Keywords: Algae; Chloroplasts; Cyanobacteria; Endosymbiosis; Mitochondria; Plastid
evolution; Speciation

In his now classic textbook Lectures on the Physiology of Plants, Sachs (1882)
stated that the chlorophyll bodies (chloroplasts) behave like independent,
Corresponding author.

E-mail addresses: (U. Kutschera), (K.J. Niklas).

1431-7613/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.


U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124

autonomous organisms that grow by division and adapt in number to the size of
expanding leaves. Eight years later, the German cytologist Altmann (1890)
demonstrated that cell granules (mitochondria) display the same staining
properties as bacteria. Thus, Sachs and Altmann explicitly concluded that
chloroplasts and mitochondria are semi-autonomous organelles displaying the
behaviour of independent forms of life. However, the actual evolutionary origins of
plastids and mitochondria remained unknown and highly contentious until a seminal
publication of the Russian botanist C. Mereschkowsky (18551921) who hypothesized that plastids are evolutionarily derived from once free-living cyanobacteria
(blue-green algae). This landmark paper, which was published one century ago in
Biologisches Centralblatt (the precursor of this journal), was followed by two
additional publications on symbiogenesis and the evolution of cells (Mereschkowsky, 1905, 1910, 1920). These papers provided profoundly important insights into the
evolution of eukaryotic organisms-insights that have been substantiated in manifold
ways by many researchers working in diverse disciplines. Additionally, the
discoveries and deductions of Sachs (1882), Altmann (1890), Mereschkowsky
(1905, 1910, 1920), and other more recent workers have been elaborated and
modied to give rise to the serial endosymbiosis hypothesis of the origin of
eukaryotes. This concept, which has been evaluated extensively by Sitte (1989,
1991, 1994, 2001), see also Taylor (1979), attempts to unify many of the insights
gained from evolutionary and cell biology in the context of repeated endosymbiotic
events involving eukaryotic as well as prokaryotic organisms.
In a previous article reviewing the modern theory of biological evolution, we
outlined the process of endosymbiosis and noted that it is pivotal to understanding
the history of life (Kutschera and Niklas, 2004). Here, we summarize in greater detail
the history of this subtheory of the expanded synthesis and we review the evidence
that has been used to verify the basic precepts of the endosymbiotic theory, with
particular reference to a series of papers authored by Sitte (1989, 1991, 1994, 2001).
We then discuss critically the more recent proposal that eukaryotic speciation has
been driven by symbiogenesis a hypothesis introduced by Wallin (1927) and
described at greater length by Margulis and Sagan (2002). However, to avoid any
ambiguity, we begin our treatment of endosymbiosis by exploring how some basic
terms and concepts are dened, both historically and currently.

Symbiosis and endocytobiology: basic denitions

Most if not all eukaryotes live in close association with microbes (bacteria) that
either inhabit certain tissues of their hosts, or live externally but nevertheless in close
physiological relationship. Examples include bacteria that live on the skin or within
the digestive tracts of animals, bacterial associations in the rhizosphere with the
roots of many seed plants, and the recently discovered growth-promoting
methylobacteria on the epidermal cells of bryophytes and angiosperms (Hornschuh
et al., 2002; Kutschera, 2002). These and many other relationships have been
historically categorized by biologists in a variety of ways that indicate whether a

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particular association is benecial or harmful to one or more of the organisms

For example, in everyday parlance, the term symbiosis is often used to
denote benecial associations between the smaller organisms (the symbionts)
and their hosts (animals and plants). However, Wilkinson (2001) points out
that the term symbiosis has two different scientic meanings, a classical and a
modern one. The distinctions between these two meanings have particular relevancy
to any discussion of the theory of endosymbiosis. Therefore, they must be evaluated
These two meanings trace their origins to a lecture presented by the German
mycologist A.H. de Bary (18311888). At a meeting of European naturalists and
physicians, De Bary dened symbiosis as the phenomenon in which unlike
organisms live together (Symbiose ist die Erscheinung des Zusammenlebens
ungleichnamiger Organismen) (de Bary, 1878). In this lecture, which provided
the gist for a subsequently published book, de Bary explicitly included parasitism in
his general denition of symbiosis. Hence, the rst formal denition stipulates a close
physical (and/or metabolic) association between two unlike organisms (usually
different species) and does not include a judgement as to whether the two symbionts
benet or harm each other. The second more modern denition is found in textbooks
published around 1915 in which symbiosis is dened as the union of two organisms
whereby they mutually benet (Wilkinson, 2001). Clearly, the classical denition
of de Bary includes parasitism, commensalism, and mutualism (de Bary, 1878),
whereas the more modern denition is restricted to the phenomenon of
mutualism. Conation of the two denitions of the word symbiosis has
engendered considerable confusion among professionals and students alike, because
de Barys denition spans the entire gamut of biological cost/benet relationships,
i.e., cost effects (parasitic symbiosis), no cost or benet effects (commensal
symbiosis), and benecial effects to both partners (mutualistic symbiosis).
To avoid any confusion in this article, we will use the word symbiosis in its modern
sense a mutually benecial relationship that involves two or more biological
partners. In this context, it is important to bear in mind that formerly benecial
relationships may evolve into pathological ones. Indeed, Hentschel et al. (2000) have
summarized data showing that the molecular mechanisms mediating the communication between bacteria and host cells in symbiotic and pathogenic interactions are
quite similar. This similarity draws attention to the continuum that exists across
symbiotic, commensal and parasitic interactions. Equally important, it provides the
caveat that the interactions we observe between two or more organisms today may
not reect the interactions among these organisms in the distant or even recent past.
Finally, we will use the word endosymbiosis in reference to cases where one
symbiont lives within the cytoplasm of its unicellular or multicellular partner. In
passing, we note that the term endocytobiology has been used in the context of
studies of intracellular symbionts (Margulis, 1990). Indeed, it is the title of a classical
monograph (Schwemmler and Schenk, 1980). However, this term is rarely used in
the current literature treating cell biology or evolution, and it conveys little that is
not communicated by the more frequently employed word endosymbiosis.


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Classical publications: Schimper, Altmann, Mereschkowsky, and Wallin

The concept of endosymbiosis and the origin of cell organelles (plastids,
mitochondria) has deep historical roots going back to the late 19th century. In a
series of publications, which were summarized in a major review article, Schimper
(1885) amply demonstrated that non-pigmented granules (plastids) develop into
chloroplasts in the embryos of higher plants. The observation that the relatively large
chlorophyll bodies always arose from pre-existing (colourless) plastids led
Schimper (1885) to conclude that the relationship between plant cells and
chloroplasts (or plastids, more generally) is symbiotic. This theory, which was
implicitly held by Sachs (1882) (Fig. 1), led Schimper (1885) to speculate that
symbiotic events may have been of great importance during the evolutionary history
of green plants.
Five years later, Altmann (1890) discovered that the granular bodies
(mitochondria) in the cytoplasm of plant and animal cells display the staining
properties of free-living microbes. Based on his many careful cytological observations, Altmann concluded that mitochondria are modied bacteria (Fig. 2).
Unfortunately, this important insight was diminished by his claim that mitochondria
represent the ultimate living units of the cell, which he called bioblasts.
Additionally, Altmann (1890) erroneously believed that the nucleus is an aggregation
of bioblasts, which was capable of a free-living existence. For these and other
reasons, Altmanns book was largely ignored (see, however, Wallin, 1927, who
adhered to some of Altmanns ideas). One consequence of this ejection of the baby
with the bath water was that Altmanns contemporaries continued to believe that
organelles such as chloroplasts and mitochondria were intrinsic components of the
rst cellular forms of life, i.e., the popular textbook opinion at the time favoured the
autogenous (self-generating) theory for the origin of organelles (Wilson, 1925;
Niklas, 1997).
Roughly 15 years after the publication of Altmanns important work, the young
Russian biologist Mereschkowsky (1905) challenged this popular belief in a seminal

Fig. 1. Chloroplasts in the cells of the moss Funaria hygrometrica (A) and stages in chloroplast
division (B). (Adapted from Sachs, 1882.)

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Fig. 2. Stained cell granules (mitochondria) in pancreas tissue of a mouse (Mus musculus) (A)
and symbiontic bacteria in cells of a root nodule of a leguminous plant (Coronilla glauca) (B).
(Adapted from Altmann, 1890.)

Fig. 3. Title page of C. Mereschkowskys classic paper published in the journal Biologisches
Centralblatt Vol. 25, 593604, 1905. (Adapted from the original publication.)

theoretical paper that argued for the xenogenous origin of organelles (Fig. 3).
Mereschkowsky postulated that plastids are reduced foreign microorganisms
(cyanobacteria or blue-green algae) that evolved as symbionts within heterotrophic host cells during the early phase of cell evolution (Mereschkowsky, 1905). In
this paper and those that followed, Mereschkowsky presented four arguments to
support his theory (Mereschkowsky, 1905, 1910, 1920): (1) According to Schimper
(1885) plastids never appear de novo, but are inherited; (2) These chlorophyll
bodies show structural, metabolic and reproductive resemblances to cyanobacteria;
(3) There are documented cases of intracellular symbioses (cytobioses): cyanobacteria invade and live in heterotrophic cells; and (4) Zoochlorellahost associations
(Amoeba viridis or Hydra viridis) are analogous to the chloroplast/plant cell
relationships. On the basis of these data, Mereschkowsky concluded that plant cells
are animal cells with invaded cyanobacteria. This basic idea serves as basis for the
endosymbiotic theory of the origin of plastids.


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Table 1. Chlorophyll and mitochondrial features, and postulated origin of plastids in major
plant lineages


Mitochondrial cristae

Plastid origin

Embryophytes (272,000)
Chlorophytes (17,000)
Charophytes (3400)
Glaucophytes (13)
Rhodophytes (6000)
Euglenoids (900)
Cryptomonads (200)
Stramenopiles (14,000)
Haptophytes (300)
Dinoagellates (2000)

a and b
a and b
a and b
a and b
a and c
a and b
a and c
a and c
a and c
a, various


Secondary (green)
Secondary (red)
Secondary (red)
Secondary (red)
Tertiary (various)

Approximate species-numbers in parentheses (adapted from Graham and Wilcox, 2000).

In his last two papers dealing with endosymbiosis, Mereschkowsky introduced the
hypothesis that different groups of cyanobacteria became endosymbionts such that
chloroplasts are polyphyletic (Mereschkowsky, 1910, 1920) an idea that resonates
with the two major chlorophyll compositions observed across extant algal lineages
(Chlorophyll a and b versus Chlorophyll a and c) (Table 1). Although he adopted
Altmanns (1890) erroneous concept that the nucleus is a union of bioblasts,
Mereschkowsky curiously did not accept this authors notion that mitochondria are
domesticated bacteria. This idea only gathered momentum with the publication of
a book by Wallin (1927), who recognized mitochondria as descendants of ancient
once free-living bacteria. As was the case with the ideas of Sachs, Schimper, and
Altmann, those expressed in Mereschkowskys original paper (Fig. 3) were not
generally accepted as a serious contribution to cell biology (Wilson, 1925;
Hoxtermann, 1998). For example, Famintzin (1907) argued that there is no
evidence for the occurrence of evolution in nature and vigorously attacked
Mereschkowsky by saying the claim that chloroplasts are incorporated cyanobacteria is without any empirical basis.

Serial primary endosymbiosis: the timing of historical events

Even though the bacterial-like nature of plastids and mitochondria was well
documented by Mereschkowsky (1905), Altmann (1890) and Wallin (1927), the
majority of scientists considered the endosymbiotic hypothesis as either too
speculative or downright wrong well into the 1970s (e.g., see Lloyd, 1974;
Cavalier-Smith, 1975) and continued to adhere to the alternative autogenous
hypothesis, which states that plastids and mitochondria arose de novo within a nonorganelle-bearing cell (see Gray, 1992; Niklas, 1997). It was not until the revival of

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the endosymbiosis hypothesis by Margulis (1970) that this important concept

received the attention that it deserved. Margulis also used the phrase serial
endosymbiosis theory (Margulis, 1993), a term originally coined by Taylor (1979),
to convey the idea that mitochondria and plastids did not acquire symbiotic
residency in their host cells simultaneously but rather did so in two discrete stages or
historical events.
The evolutionary processes by which eukaryotic cells rst appeared have been the
subject of extensive recent discussion and speculation (see de Duve, 1996; Niklas,
1997, 2004; Cavalier-Smith, 2000; Schopf, 1999; Kutschera, 2001; Martin et al.,
2001; Woese, 2002; Knoll, 2003; Keeling, 2004 and others). Several lines of evidence
indicate that the rst endosymbiotic event involved those endosymbionts that were
the precursors of proto-mitochondria (Fig. 4). This key process, which pregured or
attended the appearance of the rst heterotrophic unicellular eukaryotes, probably
occurred between 2200 and ca. 1500 million years ago (mya) (Dyall et al., 2004). It is
not known with certainty whether the genomes of the rst host cells were
prokaryotic Archaebacterial-like or eukaryotic in the sense of being membranebound and consisting of linear DNA molecules with histones (Martin et al., 2001).
The latter seems more likely because the capacity to engulf potential endosymbionts
requires a exible cell membrane (by virtue of sterols) and a specialized cytoskeleton,
both of which are absent in bacteria but present in many ancient unicellular
eukaryotic lineages.
It is also not clear whether this pivotal evolutionary event occurred under aerobic
or anaerobic conditions (Martin et al., 2003). The period between 2200 and ca.
1500 mya covers ca. 2/3 of the Palaeoproterozoic and rst quarter of the
Mesoproterozoic (see Whiteeld, 2004). Bekker et al. (2004) summarize evidence
indicating that the level of atmospheric oxygen (O2) was very low before 2450 mya
(during the Archaean) but reached considerable levels by 2200 mya. The rise in O2
level had occurred by 2320 mya, i.e., before the presumed rst endosymbiotic event.
These data support the aerobically driven origin of mitochondria (which in turn is
consistent with the fact that sterol biosynthesis requires molecular oxygen), although
the anaerobic-driven hypothesis cannot be ruled out due to the lack of an exact
timing of this process (Martin and Muller, 1998; Lopez-Garcia and Moreira, 1999;
Martin et al., 2003; Martin and Russel, 2003). What is far more certain as a
consequence of recent molecular comparisons among pro- and eukaryotic genomes
is that the ancestral prokaryotic lineage of modern-day mitochondria is related to
extant a-proteobacteria.
According to Dyall et al. (2004) biochemical, phylogenetic and structural studies
have documented that a single symbiotic association between an ancient
cyanobacterium and a mitochondria-carrying eukaryote led to the primary origin
of the plastids in green algae, land plants (embryophytes), rhodophytes, and
glaucophytes (Table 1). This event likely occurred between 1500 and 1200 mya, a
time interval that corresponds to the Ectasian and Calymmian of the Mesoproterozoic era (Whiteeld, 2004) (Fig. 4). Single-celled eukaryotic remains in the form of
acritarchs (i.e., resting cysts of eukaryotic algae) are known from ca. 1900 million
years old marine sediments (Schopf, 1999; Cowen, 2000; Knoll, 2003). These fossils


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Fig. 4. Updated geological time scale (Whiteeld, 2004) with key events in prokaryotic and
eukaryotic cell evolution (Tice and Lowe, 2004). The two major endosymbiotic events giving
rise to mitochondria and plastids are denoted as endosymbiosis 1 (which involved the
transition of a-proteobacteria-like organisms into proto-mitochondria) and endosymbiosis 2
(which involved the transition from cyanobacteria-like organisms into proto-plastids).
(Adapted from Kutschera and Niklas, 2004.)

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have been used to shed light on the composition of the Mesoproterozoic atmosphere
at a time when solar luminosity was signicantly lower than today. For example,
based on ion microprobe analyses of the carbon isotopes in individual Mesoproterozoic acritarchs extracted from North China, Kaufmann and Xiao (2003) conclude
that the atmospheric concentration of CO2 1400 mya was 10100 times that of
todays (ca. 400 parts per million, p.p.m.). It seems therefore that the second primary
endosymbiontic event responsible for the origin of modern-day chloroplasts may
have occurred during a CO2 peak in Earths history (Fig. 4).

New evidence for a classical theory

The process of plastid and mitochondrial division (plastidokinesis and mitochondriokinesis), which provoked Sachs, Schimper, and others to advance what was
subsequently called the endosymbiotic hypothesis, has been analysed recently by
means of transmission electron microscopy (e.g., Kutschera et al., 1990; Kutschera
and Hoss, 1995; Frohlich and Kutschera, 1994) (Fig. 5). Yet, in spite of the many
technological advances, the precise mechanism for plastid or mitochondrial division
has not been fully elucidated. We do know that plastids use proteins derived from the
ancestral cyanobacterial division machinery, whereas mitochondria have evolved a
separate (non-bacterial) mode of division. Likewise, both types of organelles require
dynamin-related guanosine triphosphatase to divide (Osteryoung and Nunnari, 2003).
Nevertheless, many additional lines of evidence support the endosymbiotic
hypothesis. In an important series of papers, Sitte (1989, 1991, 1994, 2001)
summarized eight documented facts that were not available to Sachs, Schimper,

Fig. 5. Transmission electron micrographs of transverse sections through a 1-day-old rye

coleoptile (Secale cereale). A dividing proplastid (A) and a mitochondrion (B) are indicated by
arrows. cw cell wall, cy cytoplasm, mi mitochondrion, p proplastid, s starch.
Bar 1 mm (M. Frohlich and U. Kutschera, unpublished results).


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Altmann, and Mereschkowsky: (1) The presence of organelle-specic DNA that is

naked (non-histonal) as in the cytoplasm of prokaryotes; (2) High degrees of
sequence homology between the DNA of chloroplasts and cyanobacteria and
between the DNA of mitochondria and proteobacteria; (3) Organelle ribosomes are
similar to those of prokaryotes but differ from those found in the cytoplasm of
eukaryotic cells (70 S- versus 80 S-type, respectively); (4) The 70 S-type ribosomes of
prokaryotes and organelles are sensitive to the antibiotic chloramphenicole, whereas
80 S-type ribosomes are not; (5) The initiation of messenger RNA translation in
prokaryotes/organelles occurs by means of a similar mechanism; (6) Organelles and
prokaryotes lack a typical (cytoplasmic) actin/tubulin system; (7) Fatty acid
biosynthesis in plastids occurs via Acylcarrier proteins (as in certain bacteria); (8)
Plastids and mitochondria are surrounded by a double membrane. In the
inner mitochondrial membrane the bacterial membrane lipid cardiolipin is abundant.
The cardiolipin content of eukaryotic biomembranes is close to zero (Gray, 1992;
Gray et al., 1999).
Subsequent work has provided other lines of supporting evidence:
1. DNA sequences indicate that extant free-living cyanobacteria and a-proteobacteria are the closest relatives of plastids and mitochondria, respectively (Douglas
and Raven, 2003; Martin et al., 2001, 2003; Logan, 2003).
2. Genome sequences reveal that plastids and mitochondria, which have retained
large fractions of their prokaryotic biochemistry, contain only remnants of the
protein-coding genes that their ancestors possessed. Experimental studies have
shown that DNA has been transferred from organelles to the nucleus of the host
cell (Martin, 2003; Timmis et al., 2004). Endosymbiotic gene transfer was
proposed years ago (Sitte, 1991) and is now a process that can be analysed by
molecular cell biologists. For instance, in the model plant Arabidopsis thaliana,
the chromosome 2 contains an entire copy of the 367-kb mitochondrial genome
close to the centromere (Timmis et al., 2004). This documents a massive transfer
of genes from the mitochondria into the nucleus.
3. Although plastids diverged from their cyanobacterial ancestors at least 1000 mya
(Fig. 4), the chlorophyll a=b arrangements in embryophyte chloroplasts and
the cyanobacterium Synechococcus are essentially the same (multisubunit
membrane-pigmentprotein complexes named photosystems I and II) (BenShem et al., 2003).
4. Crystallographic analysis of cytochrome b6f (which is a major protein complex
that mediates the ow of electrons between PS II and I) indicates that the cyt b6f
complexes of cyanobacteria and chloroplasts have almost the same molecular
structure (Kuhlbrandt, 2003).
5. A common origin for the enzymes of the oxidative branch of the Krebs cycle in a
free-living bacterium (Bacteoides sp.) and mitochondria is documented (Walden,
2002), indicating perhaps that a consortium of bacterial endosymbionts
contributed to the evolution of mitochondria.
6. Lang et al. (1999) discovered that the heterotrophic agellate Reclinomonas
americana contains an ancestral (minimally derived) mitochondrial genome with

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eubacterial-like operonic clustering. The Reclinomonas mt-DNA contains 62

protein-coding genes of known function, much more than the mitochondrial
genomes of humans (13) or yeast (8). It may form a connecting link between
the derived mitochondria of the metazoa and their ancestral eubacterial
Zhang et al. (2002) report that redox complexes in yeast mitochondria and
bacteria are preferentially assembled in regions rich in cardiolipin, which is a
minor phospholipid with a distribution limited to bacterial cytoplasmic and
organelle biomembranes.
The outer membranes of mitochondria and plastids are characterized by the
presence of beta-barrel-membrane proteins (bbps). In gram-negative bacteria,
the outer biomembrane also contains bbps. Paschen et al. (2003) have shown
that essential elements of the topogenesis (integration of the proteins into the
lipid bilayers and assemblage into oligomeric structures) of beta-barrel proteins
have been conserved during the evolution of mitochondria from free-living
prokaryotic ancestors. Plastids are surrounded by two membranes, which are
derived from the inner and outer membranes of a Gram-negative cyanobacterium. Glaucophytes represent an intermediate form in the transition from
endosymbiont to plastid, because they have retained the prokaryotic peptidoglycan layer between their two membranes (Keeling, 2004).
Nobles et al. (2001) documented the occurrence of cellulose biosynthesis in nine
species (ecotypes) of cyanobacteria. Cellulose synthase genes isolated from
various embryophyte and algal species have strong sequence homologies with
those isolated from cyanobacteria (see Niklas, 2004). Likewise, the ultrastructural appearance of membrane-bound cellulose synthase proteins in cyanobacteria, cellulose synthesizing proteobacteria, various stramenopile algal lineages,
and the embryophytes are very similar, suggesting that cellulose synthase genes
have been laterally transferred from cyanobacteria to a variety of eukaryotic
The dynamin-related guanosine triphosphatase protein Fzo1 (fuzzy onions or
mitofusin) is pivotal to the metabolic machinery responsible for mitochondria
fusion and ssion (Meeuson et al., 2004). Molecular phylogenetic analysis
indicates that Fzo1 is likely derived from the eubacterial endosymbiotic genome
that was the precursor of mitochondria. Fzo1 is also molecularly closely related
to a number of other dynamin-related guanosine triphosophatases that
commonly function in membrane ssion events, such as mitochondrial and
chloroplast division (Osteryoung and Nunnari, 2003). These molecular and
functional relationships provide another line of evidence relating the origins of
plastids and mitochondria to eubacterial endosymbiotes.
Vargas et al. (2003) isolated and characterized genes for the enzymes alkaline/
neutral invertases (A/N-Inv.) from a cyanobacterium (Anabaena sp.). A/N-Inv.
homologues were discovered in all cyanobacterial strains examined and in the
genomes of plants. A phylogenetic analysis led to the conclusion that A/N-Inv.
in plant cells originated from an ancestral A-Inv.-like cyanobacterial gene that
was transferred from the protochloroplast into the nucleus.


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12. About 18% of the nuclear genes in the plant A. thaliana seem to come from
ancient cyanobacteria (Martin, 2003). In green algae and plants, the nuclearencoded chloroplast protein, Light-dependent NADPH-protochlorophyllide
oxidoreductase (LPOR), catalyses the light-mediated reduction of protochlorophyllide to chlorophyllide. Yang and Cheng (2004) conducted a genome-wide
sequence comparison, combined with a phylogenetic analysis. The authors
conclude that photosynthetic eukaryotes obtained their LPOR homologues from
ancient cyanobacteria through endosymbiotic gene transfer.

The origin of the nucleus

Although Altmanns (1890) notion that the nucleus is a union of bioblasts (also
see Mereschkowsky, 1905, 1910, 1920) was never supported by unequivocal
cytological evidence and was later abandoned (Hoxtermann, 1998), the evolutionary
origin of the nucleus (see Fig. 4) remained highly problematic and contentious for
over half a century. Today, however, there are three contending hypotheses for the
origin of the nucleus (Hartman and Fedorov, 2002; Pennisi, 2004; Baluska et al.,
2004). The rst hypothesis notes that recent comparisons of fully sequenced
microbial genomes indicate that archaeal-like genes tend to run the eukaryotic
processes involving DNA and RNA functions, whereas bacterial-like genes are
responsible for the metabolic housekeeping cores. Additionally, some modern
methanogenic Archaea have genes encoding for histones, whereas Eubacterial
genomes do not. Assuming that the most ancient prokaryotic symbiotic relationship
involved methane-making Archaea living in Eubacteria cells (that relied on
fermentation), the hypothesis argues that Earths changing environmental conditions
may have prompted a shift in the relationship such that the Archaea gradually lost
their requirement for hydrogen, ceased making methane, and increasingly relied on
their Eubacteria hosts for other nutrients. In this scenario, the archaeal membrane,
which had been critical for methanogenesis, gradually became redundant but
subsequently invaginated to form a cellular compartment containing its DNA (but
excluded its mature ribosomes). The selective advantage for forming a proto-nucleus
was the uncoupling of DNA transcription from mRNA translation.
The second hypothesis for the origin of the nucleus argues that organisms with
proto-nuclei actually predate those lacking this organelle (i.e., nuclei-like bearing
prokaryotes predate eukaryotes). This scenario is based on a group of soil- and
freshwater prokaryotes known as planctomycetes, which have a cell wall far less rigid
than those of other Eubacteria. Detailed electron microscopic studies of two
planctomycetes (Gemmata obscuriglobus and Pirellula marina) reveal internal
membrane-bound structures, some of which hold a dense mixture of RNA and
DNA as well as DNA- and RNA-processing proteins (but no ribosomes).
Importantly, in one of these organisms (G. obscuriglobus), the membrane is folded
and discontinuous in ways that are reminiscent of the nuclear pores of eukaryotic
cells. This organism, depicted in a recent news report (Pennisi, 2004), may represent

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an intermediate form (connecting link) between a prokaryotic cell and a primitive

eukaryotic microbe.
The third hypothesis, which is perhaps the most radical, argues that the genomes
of viruses living in Archaea hosts merged with the DNA of their hosts inside the
virus. This scenario draws attention to the fact that most viral and eukaryotic DNA
is arranged linearly (whereas most bacterial DNA is circular), viruses and eukaryotic
nuclei transcribe DNA but do not translate mRNA, and that some poxviruses make
membranes around their DNA using the endoplasmic reticula of their host cells (de
Duve, 1996; Hartman and Fedorov, 2002; Pennisi, 2004; Baluska et al., 2004).
Although these three hypotheses are not mutually exclusive (in the sense that the
nucleus may have originated more than once in the history of life), no single
hypothesis has received even a conditional wide acceptance. It is nevertheless clear
that modern experimental techniques hold out the promise that we may one day
know with some certainty how the nucleus made its rst evolutionary appearance in
some lineages.

Secondary and tertiary endosymbiosis

Evidence for secondary endosymbiosis comes primarily from two sources: the
presence of two additional membranes surrounding the plastids of some host cells,
and the discovery of small structures containing DNA and eukaryotic-sized
ribosomes between these two membranes (see Fig. 7). The DNA-containing
structure, which has been called a nucleomorph, has been interpreted to be the
highly reduced nucleus of the photoautotrophic endosymbiont (Maier et al., 2000;
Keeling, 2004). Recent research supports this thesis in so far as that the genome of
cryptomonad nucleomorphs typically consists of three small chromosomes that
primarily contain only those genes encoding for the products necessary for the
maintenance of the nucleomorph itself.
For instance, the cryptomonad Guillardia theta contains a tiny 551 kb genome
with only 17 diminutive spliceosomal introns and 44 overlapping genes (Douglas et
al., 2001). These genes and their messenger RNAs show typical eukaryotic features,
which lend additional support to the thesis that the plastid is a highly reduced
eukaryotic photoautotrophic endosymbiont. Because the highly reduced nucleomorph genome (an enslaved algal nucleus) does not encode for any of the
products necessary for the maintenance of its original plastid and because the
genome of the original plastid is not self-sufcient, extensive lateral gene transfer
must have occurred from the original host nucleus (the nucleomorph) and the
nucleus of the secondary host cell (McFadden and Gilson, 1995; Douglas, 1998;
Douglas et al., 2001; Moreira and Philippe, 2001; Stoebe and Maier, 2002;
Bhattacharya et al., 2003; Armbrust et al., 2004).
As noted, many secondary endosymbiotic events involved rhodophyte plastids.
This bias is explicable by the fact that the genome of the red plastid retains a
complementary set of core genes that confer photosynthetic functionality. As


U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124

pointed out by Falkowski et al. (2004), this genome encodes for both the small and
large subunits of the important enzyme ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco). In contrast, the genes encoding for the small subunit of this
enzyme were transferred to the nucleus of the host cells with green plastids.
That the barriers to lateral gene transfers from plastids to nuclei have been
breached repeatedly is attested to by tertiary as well as secondary endosymbiotic
events, which are best exemplied by the dinoagellates (Table 1, Fig. 7). Although
many dinoagellates are plastid-free, a large number of species has acquired plastids
from phyletically diverse endosymbiotic eukaryotic donors whose plastids were
themselves of secondary endosymbiotic origin, e.g., cryptomonad and chlorophyte
endosymbionts that were reduced functionally to mere plastids (Douglas, 1998;
Moreira and Philippe, 2001). Repeated endosymbiosis is further illustrated by those
dinoagellates living within the gastrointestinal cells of scleractinian corals. These
endosymbionts or zooxanthellae can provide as much as 100% of the
carbohydrates and low-molecular weight lipids required to sustain their cnidarian
hosts whose growth is limited primarily by nitrogen availability. In this regard, a
recent electron and epiuorescence microscopy study of the coral Montastraea
cavernosa indicates that this limitation to growth can be reduced or wholly
eliminated by the presence of endosymbiotic cyanobacteria living within their coral
host cells side by side with endosymbiotic dinoagellates (Lesser et al., 2004). In a
very real sense, M. cavernosa is a community of extraordinarily diverse pro- and
eukaryotic partners.

Model systems for the study of endosymbiosis

The endosymbiotic theory for the origin of plastids and mitochondria receives
additional support from a variety of examples of symbiotic relationships between
pro- and eukaryotic organisms that can be observed directly and subjected to
experimental manipulation. These model systems provide some insight into the
ancient primary endosymbiotic events that led to the evolution of two cell organelles,
chloroplasts and mitochondria.
Legumes respond to bacterial inoculation by developing unique structures known
as root nodules (Whitehead and Day, 1997). The best-studied symbiotic (nitrogenxing) association is that between plants of the family Fabaceae and members of the
Gram-negative Rhizobiaceae. Three genera of soil bacteria, Rhizobium, Bradyrhizobium and Azorhizobium, specically associate with legumes. Rhizobia enter the
root via an ingrowth of the cell wall (infection thread) and are taken up by
endocytosis of the membrane, forming an endocytotic vesicle. The membrane and
the enclosed bacteria form a symbiosome; domesticated rhizobia are called
bacteroids. Whitehead and Day (1997) conclude that symbiosomes (i.e., bacteroids,
enclosed by the peribacteroid membrane) can be interpreted as special N2-xing
organelles within the root cells (see Fig. 2B).
The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae)
harbours a sulfur-oxidizing proteobacterium in the specialized cells of its gill tissues.

U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124


A number of studies have shown that this clam species depends on these symbiotic
bacteria for its nutrition. Importantly, these bacteria are transmitted like
mitochondria via the eggs of the animal (Yaffe, 1999; Logan, 2003). Hurtado et
al. (2003) analysed the association between vesicomyid clams and their vertically
transmitted endosymbiotic bacteria and conclude that the bacteria have lost their
ability to live freely in the marine environment. This complete animalbacteria
interdependence may parallel ancient evolutionary processes by which eukaryotic
cells acquired mitochondria and plastids. In a series of studies, Kuznetsov and
Lebkova (2002) report electron microscopic and histochemical ndings that
document the apparent transition of symbiotic bacteria into mitochondria-like
organelles in near-hydrothermal inhabitants (bivalves) of the underwater MidAtlantic ridge. These investigators analysed gill tissues of bivalves of the genera
Nucula, Conchocele and Calyptogena and obtained similar results: the molluscs
depend strictly on endosymbiotic bacteria that show an ultrastructure very similar to
that of the mitochondria in ordinary eukaryotic cells.
Many endosymbiotic relationships exist between specic bacteria and invertebrate
hosts (Insecta) that appear to be the result of ancient infections followed by vertical
transmission within host lineages. The best-characterized insect endosymbiont is the
bacterium Buchnera amphidicola, a mutualist of aphids (Insecta: Homoptera)
(Moran and Baumann, 2000). Aphids suck phloem sap that is rich in many nutrients
but decient in amino acids that are provided by Buchnera, which are intracellular
and restricted to the cytoplasm of one insect cell type. As in other previous examples,
these endosymbionts are maternally inherited via the aphid ovary. Thus, the
insectbacteria association is essential for both partners. The Buchneraaphid
mutualism is obligatory. Douglas and Raven (2003) point out that the intracellular
Buchnera resemble endosymbiotic bacteria at the proto-organelle grade of
evolution and may aid in understanding how ancient proteobacteria became
mitochondria as residents of eukaryotic cells (see Fig. 4).
Perhaps the most impressive model system for the study of the origin and
evolution of eukaryotic organelles was described in 1876, just 2 years before the
publication of the rst formal denition for symbiosis (de Bary, 1878): the
discovery that the green pigment in many marine hermaphroditic sea slugs in the
ophistobranch order of Gastropods (Ascoglossa) was indistinguishable from
chlorophyll (see Muscatine and Greene, 1973). Although this nding led to the
erroneous conclusion that the sea slugs contained entire algal symbionts, we now
know that these animals feed by evacuating the cellular contents of siphonaceous
algae (Vaucheria litorea), transfer metabolically active chloroplasts into their bodies,
and engulf them phagocytotically into a specic layer of cells surrounding the
digestive tract (Figs. 6A and B). The chloroplasts are then distributed throughout the
animals body and become lodged only one cell layer beneath the epidermis. By these
means, the animals become green and capable of light-dependent photoautotrophic
CO2 xation. The chloroplasts remain active for a limited amount of time (Rumpho
et al., 2000). Repeated feedings on algae therefore are required to maintain a
population of living chloroplasts within the animals body. Nevertheless,
laboratory studies indicate that the solar-powered sea slugs were able to live


U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124

Fig. 6. Dorsal view of the green slug Elysia chlorotica, feeding on the green siphonous alga
Vaucheria litorea (A). Electron micrograph (B) of an endosymbiontic chloroplast within a
host cell of the digestive tract of the animal. (Adapted from Rumpho et al., 2000).
Bars 1 cm (A), 2 mm (B).)

over 910 months like plants without uptake of organic substances. It has
been suggested that the unique chloroplast symbiosis may represent tertiary
endosymbiosis (i.e., macroevolution) in action (Rumpho et al., 2000), but many
questions as to the interaction between the chloroplast and the host tissue are

Endosymbiosis, macroevolution, and speciation

The evolutionary integration of the proto-mitochondrial and nuclear genomes
that presaged the appearance of the rst bona de animal cells and the subsequent
integration of proto-plastids that was required to produce the rst plant cells were
macroevolutionary events in every sense of the word (Kutschera and Niklas, 2004).
They not only heralded the appearance of entirely new species. They also generated
two deep (albeit not necessarily permanent) phyletic wedges in the tree of life, one
that continues to distinguish prokaryotes from eukaryotes and another that

U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124


separated the most ancient eukaryotic heterotrophic lineages from their photoautotrophic counterparts. That these primary endosymbiotic events cast a long
shadow and continued to play an important role in lifes history is evident from the
subsequent (and in some case very recent) appearance of novel unicellular eukaryotic
lineages resulting from secondary and tertiary endosymbiotic events (Table 1,
Fig. 7). For example, molecular clock studies indicate that diatoms (a
stramenopile lineage that is given divisional status by some workers and belongs

Fig. 7. Diagrammatic rendering of primary, secondary and tertiary endosymbiotic events

leading to novel unicellular body plans (macroevolution) in the phylogeny of various algae
(kingdom Protoctista). (Adapted from Knoll, 2003.)


U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124

to the Heterokontophyta) may have evolved as a result of secondary endosymbiotic

events as early as the Upper Jurassic but certainly no later than the PermianTriassic
boundary (Koositra et al., 2002; Armbrust et al., 2004). Likewise, based on current
morphological evidence, the dinoagellates likely evolved during Mesozoic times
(Moreira and Philippe, 2001; Morden and Sherwood, 2002).
Although the importance of endosymbiosis in evolutionary history is clearly
evident, particularly among unicellular eukaryotic lineages, it can be overstated. For
example, in their book Acquiring Genomes: A Theory of the Origin of Species,
Margulis and Sagan (2002) correctly point out that the vast majority of Earths
biological history occurred during the Precambrian during which prokaryotes were
the dominant life forms (Tice and Lowe, 2004). However, these authors then argue
that (1) the more recent and comparatively brief history of eukaryotic life is
overemphasized in most textbooks, (2) the biology of prokaryotes dees most species
denitions (particularly the biological species concept; see Mayr, 2001), (3) mutation
is canonically insufcient to generate new species, and (4) endosymbiosis is primarily
responsible for speciation across most if not all of lifes history. For example,
Margulis and Sagan argue that yrandom mutation, a small part of the
evolutionary saga, has been dogmatically overemphasized. The much larger part
of the story of evolutionary innovation, the symbiotic joining of organismsyfrom
different lineages, has systematically been ignored by self-proclaimed evolutionary
biologists (Margulis and Sagan, 2002, p. 15).
To a certain extent, the third proposition (i.e., that mutation is unimportant
to speciation) is logical legerdemain, because it emerges directly from propositions (1) and (2). If prokaryotic evolution dominated life history and if
prokaryotes are not species sensu stricto, then it follows that mutation is
not responsible for the majority of speciation events. However, this logic,
which is clearly expressed by statements like No evidence in the vast literature
of heredity change shows unambiguous evidence that random mutation itselfyleads
to speciation (Margulis and Sagan, 2002, p. 29), outs the many well-documented
cases of new bacterial forms of life resulting from mutation, the fact that
different prokaryotic taxa do not exchange genomic materials helter-skelter,
and that many species concepts are as appropriate for bacteria as for vertebrates.
In passing, we also think it unfair to say that most textbooks overemphasize
mutation when dealing with evolutionary theory. Indeed, most emphasize genomic
recombination attending sexual reproduction, which provides genomic rates
of variation that may be required to cope with the comparatively low mutation
rates observed for multicellular eukaryotic organisms (Niklas, 1997; Kutschera,
2001, 2003).
Likewise, the fourth premise of their argument (i.e., that symbiosis is far more
important than mutation) emerges logically from propositions (1) and (2). Certainly,
all of the evidence reviewed here indicates that primary endosymbiotic events
pregured much of eukaryotic history. But the relative importance of symbiosis
compared to mutation (or sexual genomic recombination) once again rests on
whether we are willing to ignore the evolutionary history of eukaryotes simply
because it is comparatively brief compared to that of prokaryotes. Arguably, the

U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124


history of eukaryotes is brief, but it nevertheless remains an important episode in cell

Additionally, we believe that a sharp distinction must be drawn between
symbiosis and endosymbiosis. This distinction is important, because none of
the biological examples used by Margulis and Sagan (2002) to explore how new
species evolve as a result of symbiosis are convincing. For example, when discussing
lichens as the classic example of symbiogenesis, Margulis and Sagan state that
the alga and the fungus are both easily seen with low-power microscopy, so neither
can be studied without simultaneous study of the other (Margulis and Sagan, 2002,
p. 14). Clearly, the implication is that lichens are species that have evolved as a result
of symbiosis. However, this line of reasoning ignores the fact that the phyco- and
mycobiontic components of many lichen associations are capable of an independent
existence (and have been frequently studied as isolates under laboratory conditions),
i.e., most if not all lichens are not true species (Friedl and Bhattacharya, 2001).
Similarly, when discussing green sea slugs (Fig. 6), Margulis and Sagan state that all
such species are permanently and discontinuously different from the grey, algaeeating ancestors (Margulis and Sagan, 2002, p. 13). Yet, no evidence is provided
that the ability of these animals to retain living chloroplasts in their cells is the trait
that precludes sexual reproduction among grey and green related species.
These two examples illustrate what we believe is an injudicious conation of the
meaning of symbiosis with endosymbiosis, particularly in the context of speciation
and macroevolution (Meyer, 2002). In our view, symbiotic associations of organisms
are not species. At best, they are more appropriately seen as the functional
equivalents of communities. For this reason, the examples of symbio-speciation
discussed by Margulis and Sagan (2002) are unconvincing (see Thompson, 1987;
Saffo, 1992, who present a more balanced view of this topic). In contrast, examples
of lateral gene transfers attending endosymbiosis clearly show that new species and
even new clades can evolve after genomic integration. The failure to draw this
distinction does not diminish Margulis and Sagans basic message that symbiosis and
endosymbiosis are important phenomena, nor does it distract from the claim that the
biological species concept is ill equipped to describe the origins and early history of
bacterial life. However, by diminishing the importance of mutation (when dealing
with bacteria), ignoring sexual genomic recombination (when dealing with
eukaryotes), and by arguing that ymost self-described evolutionary biologists
disregard cell biology, microbiology, and even the geological rock record, Margulis
and Sagan (2002) have misrepresented the status of current evolutionary thinking in
what appears to be an overzealous effort to educate those few individuals who are
still unaware of the importance of prokaryotes in modern-day ecosystems or
evolutionary history.
In two recent books, current theories on the modes of speciation are described
in detail (Schilthuizen, 2001; Coyne and Orr, 2004). It should be noted that the
views and concepts of Margulis and Sagan (2002) are not discussed by these
authors. To our knowledge, only about a dozen symbio-speciation events have
been described in the literature and each is highly questionable (Thompson, 1987;
Saffo, 1992).


U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124

Primary endosymbiotic events between archaeal-like host cells and a-proteobacteria are responsible for the appearance of the most ancient eukaryotic heterotrophic
lineages (e.g., diplomonads and microsporidians, kingdom protoctista) (Fig. 4).
Three lines of evidence provide the strongest support for this hypothesis. First,
mitochondria possess eubacterial-like DNA and transcription/translation systems
(e.g., ribosomes similar in size to those of prokaryotes); second, proteobacteria
possess infolded membranes similar to the cristae of mitochondria; and, third, strong
molecular sequence similarities, particularly those of 16S rRNA genes, between
mitochondria and a-proteobacteria genomes. Nevertheless, the genomes of mitochondria vary widely across eukaryotic lineages and they possess features that make it
extremely difcult to trace the evolutionary history of this organelle (Lang et al.,
1999). Subsequent endosymbiotic events involving the incorporation of coccoid
cyanobacteria-like endosymbionts within ancient eukaryotic host cells (Fig. 4) gave
rise to the most ancient photoautotropic lineages (e.g., chlorophytes and rhodophytes). Some of the lines of evidence for this hypothesis include the similarities in
cyanobacterial and plastid gene sequences, similarities in 16S rDNA and various
protein-coding sequences, and the presence of a self-splicing Group I intron in a
leucine transfer RNA gene of the cyanobacterium Anabaena, which has a similar
sequence and position to an intron found in the plastid genome (Xu et al., 1990).
Secondary and tertiary endosymbiotic events gave rise to evolutionarily more
recent algal lineages (e.g., euglenoids, cryptomonads, and dinoagellates) (Fig. 7).
Evidence for this hypothesis comes from the pigment compositions of the various
algal groups, the presence of additional membranes surrounding their plastids, and
the presence of nucleomorphs (nucleus-like structures) between the two outer
membranes. Among these recent algal lineages, those with red predominate,
perhaps because the red plastid genome is more self-sufcient in terms of
photosynthetic functionality. Lateral gene transfer from the mitochondrial and
plastid genomes to the nuclear genome occurred during primary, secondary, and
tertiary endosymbiotic events. For example, the gene tufA, which encodes for Tu (a
chloroplast-specic protein-synthesis elongation factor) resides in charophycean
nuclei and those of all embryophytes (i.e., members of the green lineage, see
Niklas, 2000; Scherp et al., 2001), but it remains encoded in the plastid genomes of
other groups of algae (Baldauf and Palmer, 1990). Lateral gene transfer is likely
responsible for the widespread phyletic distribution of the capacity to synthesize
cellulose (e.g., in chlorophytes, tunicates, oomycetes, and dinoagellates) as well as
chitin (e.g., in oomycetes, diatoms, and some chlorophytes). The integration of
endosymbiotic and host cell genomes into one functional unit is therefore responsible
for many macroevolutionary events and phenomena, not the least of which was the
division between pro- and eukaryotic organisms and the division between heteroand photoautotrophic eukaryotic lineages.
Clearly, the hypothesis of Mereschkowsky published one century ago in this
journal (Fig. 3) has evolved over the past decades into a solid scientic theory (sensu
Mahner and Bunge, 1997) that is supported by a large body of empirical data (Sitte,

U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 124


1989, 1991, 1994, 2001). In spite of the importance of endosymbiosis in the history of
life (Kutschera and Niklas, 2004), the relevance of symbiogenesis in the generation
of new species in the eukaryotic world of macroorganisms has been grossly
overestimated by some scientists. The currently popular book of Margulis and Sagan
(2002), which is quoted by many anti-evolutionists around the world, delivers the
basic message that genomic variation and natural selection are of subordinate
importance in the process of speciation. This erroneous conclusion is not based on
solid empirical evidence and it has provided cannon fodder to an anti-Darwinian
ideology that has no place in modern science.

This review article is dedicated to Prof. Dr. Dr. h.c. P. Sitte on the occasion of his
75th birthday. The cooperation of the authors was initiated by the Alexander von
Humboldt-Stiftung (AvH, Bonn, Germany).

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