1 - Endosymbiosis, Cell Evolution, And Speciation

8/9/2019 1 - Endosymbiosis, Cell Evolution, And Speciation

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Theory in Biosciences 124 (2005) 1–24

Endosymbiosis, cell evolution, and speciation

U. Kutscheraa,, K.J. Niklasb

aInstitut für Biologie, Universität Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, GermanybDepartment of Plant Biology, Cornell University, Ithaca, NY 14853, USA

Received 22 November 2004; accepted 21 April 2005

Abstract

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

Introduction

In his now classic textbook Lectures on the Physiology of Plants, Sachs (1882)

stated that the ‘‘chlorophyll bodies’’ (chloroplasts) behave like independent,

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

doi:10.1016/j.thbio.2005.04.001

Corresponding author.

E-mail addresses: [email protected] (U. Kutschera), [email protected] (K.J. Niklas).

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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 thatchloroplasts 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 (1855–1921) who hypothe-

sized 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 (Mereschkows-

ky, 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

modified 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, weoutlined 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 defined, both historically and currently.

Symbiosis and endocytobiology: basic definitions

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 (Hornschuhet 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 beneficial or harmful to one or more of the organisms

involved.

For example, in everyday parlance, the term ‘‘symbiosis’’ is often used to

denote beneficial 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 scientific 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

closely.

These two meanings trace their origins to a lecture presented by the German

mycologist A.H. de Bary (1831–1888). At a meeting of European naturalists and

physicians, De Bary defined 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 definition of symbiosis. Hence, the first formal definition 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

benefit or harm each other. The second more modern definition is found in textbooks

published around 1915 in which symbiosis is defined as the ‘‘union of two organisms

whereby they mutually benefit’’ (Wilkinson, 2001). Clearly, the ‘‘classical’’ definition

of de Bary includes parasitism, commensalism, and mutualism (de Bary, 1878),

whereas the more ‘‘modern’’ definition is restricted to the phenomenon of mutualism. Conflation of the two definitions of the word ‘‘symbiosis’’ has

engendered considerable confusion among professionals and students alike, because

de Bary’s definition spans the entire gamut of biological cost/benefit relationships,

i.e., cost effects (parasitic symbiosis), no cost or benefit effects (commensal

symbiosis), and beneficial 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 beneficial relationship that involves two or more biological

partners. In this context, it is important to bear in mind that formerly beneficial

relationships may evolve into pathological ones. Indeed, Hentschel et al. (2000) have

summarized data showing that the molecular mechanisms mediating the commu-nication 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 reflect 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 inthe 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 observa-

tions, Altmann concluded that mitochondria are modified 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, Altmann’s book was largely ignored (see, however, Wallin, 1927, who

adhered to some of Altmann’s ideas). One consequence of this ‘‘ejection of the babywith the bath water’’ was that Altmann’s contemporaries continued to believe that

organelles such as chloroplasts and mitochondria were intrinsic components of the

first 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 Altmann’s important work, the young

Russian biologist Mereschkowsky (1905) challenged this popular belief in a seminal

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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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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 hetero-

trophic 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): cyanobacter-

ia invade and live in heterotrophic cells; and (4) Zoochlorella–host associations

(Amoeba viridis or Hydra viridis) are analogous to the chloroplast/plant cell

relationships. On the basis of these data, Mereschkowsky concluded that plant cellsare ‘‘animal cells with invaded cyanobacteria’’. This basic idea serves as basis for the

endosymbiotic theory of the origin of plastids.

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Fig. 3. Title page of C. Mereschkowsky’s classic paper published in the journal Biologisches

Centralblatt Vol. 25, 593–604, 1905. (Adapted from the original publication.)

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.)



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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

Altmann’s (1890) erroneous concept that the nucleus is a union of ‘‘bioblasts’’,Mereschkowsky curiously did not accept this author’s 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 Mereschkowsky’s original paper (Fig. 3) were not

generally accepted as a serious contribution to cell biology (Wilson, 1925;

Ho ¨ xtermann, 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 cyanobac-

teria 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 non-

organelle-bearing cell (see Gray, 1992; Niklas, 1997). It was not until the revival of

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

plant lineages

Group Chlorophylls Mitochondrial cristae Plastid origin

Embryophytes (272,000) a and b Flattened Primary

Chlorophytes (17,000) a and b Flattened Primary

Charophytes (3400) a and b Flattened Primary

Glaucophytes (13) a and b Flattened Primary

Rhodophytes (6000) a and c Flattened Primary

Euglenoids (900) a and b Disk-shaped Secondary (green)

Cryptomonads (200) a and c Flattened Secondary (red)

Stramenopiles (14,000) a and c Tubular Secondary (red)

Haptophytes (300) a and c Tubular Secondary (red)

Dinoflagellates (2000) a, various Tubular Tertiary (various)

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



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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 symbioticresidency in their host cells simultaneously but rather did so in two discrete stages or

historical ‘‘events’’.

The evolutionary processes by which eukaryotic cells first 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 first endosymbiotic event involved those endosymbionts that were

the precursors of proto-mitochondria (Fig. 4). This key process, which prefigured or

attended the appearance of the first 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 first host cells were

prokaryotic Archaebacterial-like or eukaryotic in the sense of being membrane-

bound 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 flexible 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 first quarter of the

Mesoproterozoic (see Whitefield, 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 O2level had occurred by 2320 mya, i.e., before the presumed first 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 Mu ¨ ller, 1998; Lopez-Garcia and Moreira, 1999;

Martin et al., 2003; Martin and Russel, 2003). What is far more certain as aconsequence 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 Mesoproter-

ozoic era (Whitefield, 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 (Whitefield, 2004) with key events in prokaryotic and

eukaryotic cell evolution (Tice and Lowe, 2004). The two major endosymbiotic events givingrise 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 significantly lower than today. For example,

based on ion microprobe analyses of the carbon isotopes in individual Mesoproter-

ozoic acritarchs extracted from North China, Kaufmann and Xiao (2003) concludethat the atmospheric concentration of CO2 1400 mya was 10–100 times that of

today’s (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 Earth’s history (Fig. 4).

New evidence for a classical theory

The process of plastid and mitochondrial division (plastidokinesis and mitochon-driokinesis), 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; Fro ¨ hlich 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 endosymbiotichypothesis. In an important series of papers, Sitte (1989, 1991, 1994, 2001)

summarized eight documented facts that were not available to Sachs, Schimper,

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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 byarrows. cw¼ cell wall, cy¼ cytoplasm, mi ¼mitochondrion, p ¼ proplastid, s ¼ starch.

Bar ¼ 1 mm (M. Fro ¨ hlich and U. Kutschera, unpublished results).



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Altmann, and Mereschkowsky: (1) The presence of organelle-specific 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 aresimilar 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-proteobac-

teria 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 haveshown 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 (multisubunitmembrane-pigment–protein complexes named photosystems I and II) (Ben-

Shem et al., 2003).

4. Crystallographic analysis of cytochrome b6f (which is a major protein complex

that mediates the flow of electrons between PS II and I) indicates that the cyt b6f

complexes of cyanobacteria and chloroplasts have almost the same molecular

structure (Ku ¨ hlbrandt, 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 flagellate 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 eubacterialprogenitors.

7. 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.

8. 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 cyanobacter-

ium. Glaucophytes represent an intermediate form in the transition from

endosymbiont to plastid, because they have retained the prokaryotic peptido-

glycan layer between their two membranes (Keeling, 2004).

9. 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 withthose isolated from cyanobacteria (see Niklas, 2004). Likewise, the ultrastruc-

tural appearance of membrane-bound cellulose synthase proteins in cyanobac-

teria, 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

lineages.

10. The dynamin-related guanosine triphosphatase protein Fzo1 ( fuzzy onions or

mitofusin) is pivotal to the metabolic machinery responsible for mitochondria

fusion and fission (Meeuson et al., 2004). Molecular phylogenetic analysis

indicates that Fzo1 is likely derived from the eubacterial endosymbiotic genomethat 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 fission 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.

11. 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 nuclear-

encoded chloroplast protein, Light-dependent NADPH-protochlorophyllide

oxidoreductase (LPOR), catalyses the light-mediated reduction of protochlor-ophyllide 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 Altmann’s (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 (Ho ¨ xtermann, 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 first 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 modernmethanogenic 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 Earth’s 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-nucleuswas 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 foldedand 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 thevirus. 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 first 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-sizedribosomes 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 nucleo-

morph 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-sufficient, 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

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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 exemplified by the dinoflagellates (Table 1, Fig. 7). Although

many dinoflagellates 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

dinoflagellates 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 epifluorescence 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 dinoflagellates (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 knownas root nodules (Whitehead and Day, 1997). The best-studied symbiotic (nitrogen-

fixing) association is that between plants of the family Fabaceae and members of the

Gram-negative Rhizobiaceae. Three genera of soil bacteria, Rhizobium, Bradyrhi-

zobium and Azorhizobium, specifically 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-fixing

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.

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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 verticallytransmitted endosymbiotic bacteria and conclude that the bacteria have lost their

ability to live freely in the marine environment. This complete animal–bacteria

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 findings that

document the apparent transition of symbiotic bacteria into mitochondria-like

organelles in near-hydrothermal inhabitants (bivalves) of the underwater Mid-

Atlantic 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 specific 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 deficient 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, theinsect–bacteria association is essential for both partners. The Buchnera –aphid

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 first formal definition 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 fromchlorophyll (see Muscatine and Greene, 1973). Although this finding 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 specific layer of cells surrounding the

digestive tract (Figs. 6A and B). The chloroplasts are then distributed throughout the

animal’s 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 fixation. The chloroplasts remain active for a limited amount of time ( Rumpho

et al., 2000). Repeated feedings on algae therefore are required to maintain apopulation of ‘‘living’’ chloroplasts within the animal’s body. Nevertheless,

laboratory studies indicate that the ‘‘solar-powered sea slugs’’ were able to live

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over 9–10 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

unanswered.

Endosymbiosis, macroevolution, and speciation

The evolutionary integration of the proto-mitochondrial and nuclear genomes

that presaged the appearance of the first bona fide animal cells and the subsequent

integration of proto-plastids that was required to produce the first 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 generatedtwo deep (albeit not necessarily permanent) phyletic wedges in the tree of life, one

that continues to distinguish prokaryotes from eukaryotes and another that

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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 ¼ 1cm (A), 2 mm (B).)



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separated the most ancient eukaryotic heterotrophic lineages from their photo-

autotrophic counterparts. That these primary endosymbiotic events cast a long

shadow and continued to play an important role in life’s history is evident from the

subsequent (and in some case very recent) appearance of novel unicellular eukaryoticlineages 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

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Fig. 7. Diagrammatic rendering of primary, secondary and tertiary endosymbiotic eventsleading to novel unicellular body plans (macroevolution) in the phylogeny of various algae

(kingdom Protoctista). (Adapted from Knoll, 2003.)



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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 Permian–Triassic

boundary (Koositra et al., 2002; Armbrust et al., 2004). Likewise, based on current

morphological evidence, the dinoflagellates 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 Earth’s

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 defies most species

definitions (particularly the biological species concept; see Mayr, 2001), (3) mutation

is canonically insufficient to generate new species, and (4) endosymbiosis is primarily

responsible for speciation across most if not all of life’s 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 pro-positions (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 itself yleads

to speciation’’ (Margulis and Sagan, 2002, p. 29), flouts 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 overemphasizemutation 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

prefigured much of eukaryotic history. But the relative importance of symbiosis

compared to mutation (or sexual genomic recombination) once again rests onwhether we are willing to ignore the evolutionary history of eukaryotes simply

because it is comparatively brief compared to that of prokaryotes. Arguably, the

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

evolution.

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, algae-

eating 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 conflation 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 functionalequivalents 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 Sagan’s 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 witheukaryotes), 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’’ havebeen described in the literature and each is highly questionable (Thompson, 1987;

Saffo, 1992).

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Conclusions

Primary endosymbiotic events between archaeal-like host cells and a-proteobac-

teria are responsible for the appearance of the most ancient eukaryotic heterotrophiclineages (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 mito-

chondria vary widely across eukaryotic lineages and they possess features that make it

extremely difficult 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 rhodo-

phytes). 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 dinoflagellates) (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-sufficient 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-specific protein-synthesis elongation factor) resides in charophycean

nuclei and those of all embryophytes (i.e., members of the ‘‘green lineage’’, seeNiklas, 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 dinoflagellates) 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 hetero-

and 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 scientific theory (sensu

Mahner and Bunge, 1997) that is supported by a large body of empirical data (Sitte,

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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.

Acknowledgements

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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