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Theory in Biosciences 124 (2005) 1–24

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Endosymbiosis, cell evolution, and speciation

U. Kutscheraa,�, K.J. Niklasb

aInstitut fur Biologie, Universitat 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,

see front matter r 2005 Elsevier GmbH. All rights reserved.

.thbio.2005.04.001

nding author.

dresses: [email protected] (U. Kutschera), [email protected] (K.J. Niklas).

www.elsevier.de/thbio

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U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 1–242

autonomous organisms that grow by division and adapt in number to the size ofexpanding leaves. Eight years later, the German cytologist Altmann (1890)demonstrated that ‘‘cell granules’’ (mitochondria) display the same stainingproperties as bacteria. Thus, Sachs and Altmann explicitly concluded thatchloroplasts and mitochondria are ‘‘semi-autonomous’’ organelles displaying thebehaviour of independent forms of life. However, the actual evolutionary origins ofplastids and mitochondria remained unknown and highly contentious until a seminalpublication 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 inBiologisches Centralblatt (the precursor of this journal), was followed by twoadditional publications on symbiogenesis and the evolution of cells (Mereschkows-ky, 1905, 1910, 1920). These papers provided profoundly important insights into theevolution of eukaryotic organisms-insights that have been substantiated in manifoldways by many researchers working in diverse disciplines. Additionally, thediscoveries and deductions of Sachs (1882), Altmann (1890), Mereschkowsky(1905, 1910, 1920), and other more recent workers have been elaborated andmodified to give rise to the ‘‘serial endosymbiosis hypothesis of the origin ofeukaryotes.’’ This concept, which has been evaluated extensively by Sitte (1989,1991, 1994, 2001), see also Taylor (1979), attempts to unify many of the insightsgained from evolutionary and cell biology in the context of repeated endosymbioticevents 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 understandingthe history of life (Kutschera and Niklas, 2004). Here, we summarize in greater detailthe history of this subtheory of the ‘‘expanded synthesis’’ and we review the evidencethat has been used to verify the basic precepts of the endosymbiotic theory, withparticular reference to a series of papers authored by Sitte (1989, 1991, 1994, 2001).We then discuss critically the more recent proposal that eukaryotic speciation hasbeen driven by symbiogenesis – a hypothesis introduced by Wallin (1927) anddescribed at greater length by Margulis and Sagan (2002). However, to avoid anyambiguity, we begin our treatment of endosymbiosis by exploring how some basicterms 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) thateither inhabit certain tissues of their hosts, or live externally but nevertheless in closephysiological relationship. Examples include bacteria that live on the skin or withinthe digestive tracts of animals, bacterial associations in the rhizosphere with theroots of many seed plants, and the recently discovered growth-promotingmethylobacteria on the epidermal cells of bryophytes and angiosperms (Hornschuhet al., 2002; Kutschera, 2002). These and many other relationships have beenhistorically categorized by biologists in a variety of ways that indicate whether a

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U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 1–24 3

particular association is beneficial or harmful to one or more of the organismsinvolved.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 outthat the term ‘‘symbiosis’’ has two different scientific meanings, a classical and amodern one. The distinctions between these two meanings have particular relevancyto any discussion of the theory of endosymbiosis. Therefore, they must be evaluatedclosely.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 andphysicians, De Bary defined symbiosis as the phenomenon in which ‘‘unlikeorganisms live together (Symbiose ist die Erscheinung des Zusammenlebensungleichnamiger Organismen)’’ (de Bary, 1878). In this lecture, which providedthe gist for a subsequently published book, de Bary explicitly included parasitism inhis general definition of symbiosis. Hence, the first formal definition stipulates a closephysical (and/or metabolic) association between two unlike organisms (usuallydifferent species) and does not include a judgement as to whether the two symbiontsbenefit or harm each other. The second more modern definition is found in textbookspublished around 1915 in which symbiosis is defined as the ‘‘union of two organismswhereby they mutually benefit’’ (Wilkinson, 2001). Clearly, the ‘‘classical’’ definitionof de Bary includes parasitism, commensalism, and mutualism (de Bary, 1878),whereas the more ‘‘modern’’ definition is restricted to the phenomenon ofmutualism. Conflation of the two definitions of the word ‘‘symbiosis’’ hasengendered considerable confusion among professionals and students alike, becausede Bary’s definition spans the entire gamut of biological cost/benefit relationships,i.e., cost effects (parasitic symbiosis), no cost or benefit effects (commensalsymbiosis), 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 biologicalpartners. In this context, it is important to bear in mind that formerly beneficialrelationships may evolve into pathological ones. Indeed, Hentschel et al. (2000) havesummarized data showing that the molecular mechanisms mediating the commu-nication between bacteria and host cells in symbiotic and pathogenic interactions arequite similar. This similarity draws attention to the continuum that exists acrosssymbiotic, commensal and parasitic interactions. Equally important, it provides thecaveat that the interactions we observe between two or more organisms today maynot 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. Inpassing, we note that the term ‘‘endocytobiology’’ has been used in the context ofstudies of intracellular symbionts (Margulis, 1990). Indeed, it is the title of a classicalmonograph (Schwemmler and Schenk, 1980). However, this term is rarely used inthe current literature treating cell biology or evolution, and it conveys little that isnot 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 aseries of publications, which were summarized in a major review article, Schimper(1885) amply demonstrated that ‘‘non-pigmented granules’’ (plastids) develop intochloroplasts in the embryos of higher plants. The observation that the relatively large‘‘chlorophyll bodies’’ always arose from pre-existing (colourless) plastids ledSchimper (1885) to conclude that the relationship between plant cells andchloroplasts (or plastids, more generally) is symbiotic. This theory, which wasimplicitly held by Sachs (1882) (Fig. 1), led Schimper (1885) to speculate thatsymbiotic events may have been of great importance during the evolutionary historyof green plants.Five years later, Altmann (1890) discovered that the ‘‘granular bodies’’

(mitochondria) in the cytoplasm of plant and animal cells display the stainingproperties 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 mitochondriarepresent the ultimate ‘‘living units’’ of the cell, which he called ‘‘bioblasts’’.Additionally, Altmann (1890) erroneously believed that the nucleus is an aggregationof ‘‘bioblasts’’, which was capable of a free-living existence. For these and otherreasons, Altmann’s book was largely ignored (see, however, Wallin, 1927, whoadhered 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 thatorganelles such as chloroplasts and mitochondria were intrinsic components of thefirst cellular forms of life, i.e., the popular textbook opinion at the time favoured theautogenous (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

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. 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.)


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). Inthis paper and those that followed, Mereschkowsky presented four arguments tosupport his theory (Mereschkowsky, 1905, 1910, 1920): (1) According to Schimper(1885) plastids never appear de novo, but are inherited; (2) These ‘‘chlorophyllbodies’’ 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 cellrelationships. On the basis of these data, Mereschkowsky concluded that plant cellsare ‘‘animal cells with invaded cyanobacteria’’. This basic idea serves as basis for theendosymbiotic 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

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).


In his last two papers dealing with endosymbiosis, Mereschkowsky introduced thehypothesis that different groups of cyanobacteria became endosymbionts such thatchloroplasts are polyphyletic (Mereschkowsky, 1910, 1920) – an idea that resonateswith the two major chlorophyll compositions observed across extant algal lineages(Chlorophyll a and b versus Chlorophyll a and c) (Table 1). Although he adoptedAltmann’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 ofa book by Wallin (1927), who recognized mitochondria as descendants of ancientonce free-living bacteria. As was the case with the ideas of Sachs, Schimper, andAltmann, those expressed in Mereschkowsky’s original paper (Fig. 3) were notgenerally accepted as a serious contribution to cell biology (Wilson, 1925;Hoxtermann, 1998). For example, Famintzin (1907) argued that ‘‘there is noevidence for the occurrence of evolution in nature’’ and vigorously attackedMereschkowsky 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 welldocumented by Mereschkowsky (1905), Altmann (1890) and Wallin (1927), themajority of scientists considered the endosymbiotic hypothesis as either toospeculative 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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the endosymbiosis hypothesis by Margulis (1970) that this important conceptreceived the attention that it deserved. Margulis also used the phrase ‘‘serialendosymbiosis 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 orhistorical ‘‘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 evidenceindicate that the first endosymbiotic event involved those endosymbionts that werethe precursors of proto-mitochondria (Fig. 4). This key process, which prefigured orattended the appearance of the first heterotrophic unicellular eukaryotes, probablyoccurred between 2200 and ca. 1500 million years ago (mya) (Dyall et al., 2004). It isnot known with certainty whether the genomes of the first host cells wereprokaryotic 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 endosymbiontsrequires a flexible cell membrane (by virtue of sterols) and a specialized cytoskeleton,both of which are absent in bacteria but present in many ancient unicellulareukaryotic 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.1500mya covers ca. 2/3 of the Palaeoproterozoic and first quarter of theMesoproterozoic (see Whitefield, 2004). Bekker et al. (2004) summarize evidenceindicating that the level of atmospheric oxygen (O2) was very low before 2450mya(during the Archaean) but reached considerable levels by 2200mya. The rise in O2level had occurred by 2320mya, i.e., before the presumed first endosymbiotic event.These data support the aerobically driven origin of mitochondria (which in turn isconsistent with the fact that sterol biosynthesis requires molecular oxygen), althoughthe anaerobic-driven hypothesis cannot be ruled out due to the lack of an exacttiming 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 aconsequence of recent molecular comparisons among pro- and eukaryotic genomesis that the ancestral prokaryotic lineage of modern-day mitochondria is related toextant a-proteobacteria.According to Dyall et al. (2004) biochemical, phylogenetic and structural studies

have documented that a single symbiotic association between an ancientcyanobacterium and a mitochondria-carrying eukaryote led to the primary originof the plastids in green algae, land plants (embryophytes), rhodophytes, andglaucophytes (Table 1). This event likely occurred between 1500 and 1200mya, atime interval that corresponds to the Ectasian and Calymmian of the Mesoproter-ozoic era (Whitefield, 2004) (Fig. 4). Single-celled eukaryotic remains in the form ofacritarchs (i.e., resting cysts of eukaryotic algae) are known from ca. 1900 millionyears 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 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 atmosphereat 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 1400mya was 10–100 times that oftoday’s (ca. 400 parts per million, p.p.m.). It seems therefore that the second primaryendosymbiontic event responsible for the origin of modern-day chloroplasts mayhave 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 wassubsequently called the endosymbiotic hypothesis, has been analysed recently bymeans of transmission electron microscopy (e.g., Kutschera et al., 1990; Kutscheraand Hoss, 1995; Frohlich and Kutschera, 1994) (Fig. 5). Yet, in spite of the manytechnological advances, the precise mechanism for plastid or mitochondrial divisionhas not been fully elucidated. We do know that plastids use proteins derived from theancestral cyanobacterial division machinery, whereas mitochondria have evolved aseparate (non-bacterial) mode of division. Likewise, both types of organelles requiredynamin-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 ¼ cell wall, cy ¼ cytoplasm, mi ¼ mitochondrion, p ¼ proplastid, s ¼ starch.

Bar ¼ 1mm (M. Frohlich 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 ofsequence homology between the DNA of chloroplasts and cyanobacteria andbetween the DNA of mitochondria and proteobacteria; (3) Organelle ribosomes aresimilar to those of prokaryotes but differ from those found in the cytoplasm ofeukaryotic cells (70 S- versus 80 S-type, respectively); (4) The 70 S-type ribosomes ofprokaryotes and organelles are sensitive to the antibiotic chloramphenicole, whereas80 S-type ribosomes are not; (5) The initiation of messenger RNA translation inprokaryotes/organelles occurs by means of a similar mechanism; (6) Organelles andprokaryotes lack a typical (cytoplasmic) actin/tubulin system; (7) Fatty acidbiosynthesis in plastids occurs via Acylcarrier proteins (as in certain bacteria); (8)Plastids and mitochondria are surrounded by a double membrane. In theinner 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 (Douglasand Raven, 2003; Martin et al., 2001, 2003; Logan, 2003).
2.
Genome sequences reveal that plastids and mitochondria, which have retainedlarge fractions of their prokaryotic biochemistry, contain only remnants of theprotein-coding genes that their ancestors possessed. Experimental studies haveshown that DNA has been transferred from organelles to the nucleus of the hostcell (Martin, 2003; Timmis et al., 2004). Endosymbiotic gene transfer wasproposed years ago (Sitte, 1991) and is now a process that can be analysed bymolecular cell biologists. For instance, in the model plant Arabidopsis thaliana,the chromosome 2 contains an entire copy of the 367-kb mitochondrial genomeclose to the centromere (Timmis et al., 2004). This documents a massive transferof genes from the mitochondria into the nucleus.
3.
Although plastids diverged from their cyanobacterial ancestors at least 1000mya(Fig. 4), the chlorophyll a=b – arrangements in embryophyte chloroplasts andthe 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 complexthat mediates the flow of electrons between PS II and I) indicates that the cyt b6fcomplexes of cyanobacteria and chloroplasts have almost the same molecularstructure (Kuhlbrandt, 2003).
5.
A common origin for the enzymes of the oxidative branch of the Krebs cycle in afree-living bacterium (Bacteoides sp.) and mitochondria is documented (Walden,2002), indicating perhaps that a consortium of bacterial endosymbiontscontributed 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 62protein-coding genes of known function, much more than the mitochondrialgenomes of humans (13) or yeast (8). It may form a ‘‘connecting link’’ betweenthe derived mitochondria of the metazoa and their ancestral eubacterialprogenitors.

7.
Zhang et al. (2002) report that redox complexes in yeast mitochondria andbacteria are preferentially assembled in regions rich in cardiolipin, which is aminor phospholipid with a distribution limited to bacterial cytoplasmic andorganelle biomembranes.
8.
The outer membranes of mitochondria and plastids are characterized by thepresence of beta-barrel-membrane proteins (bbps). In gram-negative bacteria,the outer biomembrane also contains bbps. Paschen et al. (2003) have shownthat essential elements of the topogenesis (integration of the proteins into thelipid bilayers and assemblage into oligomeric structures) of beta-barrel proteinshave been conserved during the evolution of mitochondria from free-livingprokaryotic ancestors. Plastids are surrounded by two membranes, which arederived from the inner and outer membranes of a Gram-negative cyanobacter-ium. Glaucophytes represent an intermediate form in the transition fromendosymbiont 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 ninespecies (ecotypes) of cyanobacteria. Cellulose synthase genes isolated fromvarious 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 geneshave been laterally transferred from cyanobacteria to a variety of eukaryoticlineages.
10.
The dynamin-related guanosine triphosphatase protein Fzo1 (fuzzy onions ormitofusin) is pivotal to the metabolic machinery responsible for mitochondriafusion and fission (Meeuson et al., 2004). Molecular phylogenetic analysisindicates that Fzo1 is likely derived from the eubacterial endosymbiotic genomethat was the precursor of mitochondria. Fzo1 is also molecularly closely relatedto a number of other dynamin-related guanosine triphosophatases thatcommonly function in membrane fission events, such as mitochondrial andchloroplast division (Osteryoung and Nunnari, 2003). These molecular andfunctional relationships provide another line of evidence relating the origins ofplastids 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 thegenomes 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 thatwas 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 fromancient cyanobacteria (Martin, 2003). In green algae and plants, the nuclear-encoded chloroplast protein, Light-dependent NADPH-protochlorophyllideoxidoreductase (LPOR), catalyses the light-mediated reduction of protochlor-ophyllide to chlorophyllide. Yang and Cheng (2004) conducted a genome-widesequence comparison, combined with a phylogenetic analysis. The authorsconclude that photosynthetic eukaryotes obtained their LPOR homologues fromancient cyanobacteria through endosymbiotic gene transfer.
The origin of the nucleus

Although Altmann’s (1890) notion that the nucleus is a union of ‘‘bioblasts’’ (alsosee Mereschkowsky, 1905, 1910, 1920) was never supported by unequivocalcytological evidence and was later abandoned (Hoxtermann, 1998), the evolutionaryorigin of the nucleus (see Fig. 4) remained highly problematic and contentious forover half a century. Today, however, there are three contending hypotheses for theorigin of the nucleus (Hartman and Fedorov, 2002; Pennisi, 2004; Baluska et al.,2004). The first hypothesis notes that recent comparisons of fully sequencedmicrobial genomes indicate that archaeal-like genes tend to run the eukaryoticprocesses involving DNA and RNA functions, whereas bacterial-like genes areresponsible for the metabolic ‘‘housekeeping cores’’. Additionally, some modernmethanogenic Archaea have genes encoding for histones, whereas Eubacterialgenomes do not. Assuming that the most ancient prokaryotic symbiotic relationshipinvolved methane-making Archaea living in Eubacteria cells (that relied onfermentation), the hypothesis argues that Earth’s changing environmental conditionsmay have prompted a shift in the relationship such that the Archaea gradually losttheir requirement for hydrogen, ceased making methane, and increasingly relied ontheir Eubacteria hosts for other nutrients. In this scenario, the archaeal membrane,which had been critical for methanogenesis, gradually became redundant butsubsequently invaginated to form a cellular compartment containing its DNA (butexcluded 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 bearingprokaryotes predate eukaryotes). This scenario is based on a group of soil- andfreshwater prokaryotes known as planctomycetes, which have a cell wall far less rigidthan those of other Eubacteria. Detailed electron microscopic studies of twoplanctomycetes (Gemmata obscuriglobus and Pirellula marina) reveal internalmembrane-bound structures, some of which hold a dense mixture of RNA andDNA 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 eukaryoticcells. 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 primitiveeukaryotic 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 DNAis arranged linearly (whereas most bacterial DNA is circular), viruses and eukaryoticnuclei transcribe DNA but do not translate mRNA, and that some poxviruses makemembranes around their DNA using the endoplasmic reticula of their host cells (deDuve, 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 singlehypothesis has received even a conditional wide acceptance. It is nevertheless clearthat modern experimental techniques hold out the promise that we may one dayknow with some certainty how the nucleus made its first evolutionary appearance insome lineages.

Secondary and tertiary endosymbiosis

Evidence for secondary endosymbiosis comes primarily from two sources: thepresence 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-containingstructure, which has been called a nucleomorph, has been interpreted to be thehighly reduced nucleus of the photoautotrophic endosymbiont (Maier et al., 2000;Keeling, 2004). Recent research supports this thesis in so far as that the genome ofcryptomonad nucleomorphs typically consists of three small chromosomes thatprimarily contain only those genes encoding for the products necessary for themaintenance 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 etal., 2001). These genes and their messenger RNAs show typical eukaryotic features,which lend additional support to the thesis that the ‘‘plastid’’ is a highly reducedeukaryotic photoautotrophic endosymbiont. Because the highly reduced nucleo-morph genome (an ‘‘enslaved’’ algal nucleus) does not encode for any of theproducts necessary for the maintenance of its original plastid and because thegenome of the original plastid is not self-sufficient, extensive lateral gene transfermust have occurred from the original host nucleus (the nucleomorph) and thenucleus 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 acomplementary 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 andlarge subunits of the important enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In contrast, the genes encoding for the small subunit of thisenzyme 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 endosymbioticevents, which are best exemplified by the dinoflagellates (Table 1, Fig. 7). Althoughmany dinoflagellates are plastid-free, a large number of species has acquired plastidsfrom phyletically diverse endosymbiotic eukaryotic donors whose plastids werethemselves of secondary endosymbiotic origin, e.g., cryptomonad and chlorophyteendosymbionts that were reduced functionally to mere plastids (Douglas, 1998;Moreira and Philippe, 2001). Repeated endosymbiosis is further illustrated by thosedinoflagellates living within the gastrointestinal cells of scleractinian corals. Theseendosymbionts or ‘‘zooxanthellae’’ can provide as much as 100% of thecarbohydrates and low-molecular weight lipids required to sustain their cnidarianhosts whose growth is limited primarily by nitrogen availability. In this regard, arecent electron and epifluorescence microscopy study of the coral Montastraea

cavernosa indicates that this limitation to growth can be reduced or whollyeliminated by the presence of endosymbiotic cyanobacteria living within their coralhost cells side by side with endosymbiotic dinoflagellates (Lesser et al., 2004). In avery real sense, M. cavernosa is a community of extraordinarily diverse pro- andeukaryotic partners.

Model systems for the study of endosymbiosis

The endosymbiotic theory for the origin of plastids and mitochondria receivesadditional support from a variety of examples of symbiotic relationships betweenpro- and eukaryotic organisms that can be observed directly and subjected toexperimental manipulation. These ‘‘model systems’’ provide some insight into theancient 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 (nitrogen-fixing) association is that between plants of the family Fabaceae and members of theGram-negative Rhizobiaceae. Three genera of soil bacteria, Rhizobium, Bradyrhi-

zobium and Azorhizobium, specifically associate with legumes. Rhizobia enter theroot via an ingrowth of the cell wall (infection thread) and are taken up byendocytosis of the membrane, forming an endocytotic vesicle. The membrane andthe enclosed bacteria form a symbiosome; domesticated rhizobia are calledbacteroids. Whitehead and Day (1997) conclude that symbiosomes (i.e., bacteroids,enclosed by the peribacteroid membrane) can be interpreted as special N2-fixingorganelles 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 symbioticbacteria for its nutrition. Importantly, these bacteria are transmitted – likemitochondria – via the eggs of the animal (Yaffe, 1999; Logan, 2003). Hurtado etal. (2003) analysed the association between vesicomyid clams and their verticallytransmitted endosymbiotic bacteria and conclude that the bacteria have lost theirability to live freely in the marine environment. This complete animal–bacteriainterdependence may parallel ancient evolutionary processes by which eukaryoticcells acquired mitochondria and plastids. In a series of studies, Kuznetsov andLebkova (2002) report electron microscopic and histochemical findings thatdocument the apparent transition of symbiotic bacteria into mitochondria-likeorganelles in near-hydrothermal inhabitants (bivalves) of the underwater Mid-Atlantic ridge. These investigators analysed gill tissues of bivalves of the generaNucula, Conchocele and Calyptogena and obtained similar results: the molluscsdepend strictly on endosymbiotic bacteria that show an ultrastructure very similar tothat 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 verticaltransmission within host lineages. The best-characterized insect endosymbiont is thebacterium Buchnera amphidicola, a mutualist of aphids (Insecta: Homoptera)(Moran and Baumann, 2000). Aphids suck phloem sap that is rich in many nutrientsbut deficient in amino acids that are provided by Buchnera, which are intracellularand 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–aphidmutualism is obligatory. Douglas and Raven (2003) point out that the intracellularBuchnera resemble ‘‘endosymbiotic bacteria at the proto-organelle grade ofevolution’’ and may aid in understanding how ancient proteobacteria becamemitochondria 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 thepublication of the first formal definition for ‘‘symbiosis’’ (de Bary, 1878): thediscovery that the green pigment in many marine hermaphroditic sea slugs in theophistobranch order of Gastropods (Ascoglossa) was indistinguishable fromchlorophyll (see Muscatine and Greene, 1973). Although this finding led to theerroneous conclusion that the sea slugs contained entire algal symbionts, we nowknow that these animals feed by evacuating the cellular contents of siphonaceousalgae (Vaucheria litorea), transfer metabolically active chloroplasts into their bodies,and engulf them phagocytotically into a specific layer of cells surrounding thedigestive tract (Figs. 6A and B). The chloroplasts are then distributed throughout theanimal’s body and become lodged only one cell layer beneath the epidermis. By thesemeans, the animals become green and capable of light-dependent photoautotrophicCO2 fixation. The chloroplasts remain active for a limited amount of time (Rumphoet 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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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 1 cm (A), 2mm (B).)


over 9–10 months like plants without uptake of organic substances. It hasbeen suggested that the unique chloroplast symbiosis may represent tertiaryendosymbiosis (i.e., macroevolution) in action (Rumpho et al., 2000), but manyquestions as to the interaction between the chloroplast and the host tissue areunanswered.

Endosymbiosis, macroevolution, and speciation

The evolutionary integration of the proto-mitochondrial and nuclear genomesthat presaged the appearance of the first bona fide animal cells and the subsequentintegration of proto-plastids that was required to produce the first plant cells weremacroevolutionary 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, onethat continues to distinguish prokaryotes from eukaryotes and another that

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separated the most ancient eukaryotic heterotrophic lineages from their photo-autotrophic counterparts. That these primary endosymbiotic events cast a longshadow and continued to play an important role in life’s history is evident from thesubsequent (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 (astramenopile 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.)

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to the Heterokontophyta) may have evolved as a result of secondary endosymbioticevents as early as the Upper Jurassic but certainly no later than the Permian–Triassicboundary (Koositra et al., 2002; Armbrust et al., 2004). Likewise, based on currentmorphological 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. Forexample, 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’sbiological history occurred during the Precambrian during which prokaryotes werethe dominant life forms (Tice and Lowe, 2004). However, these authors then arguethat (1) the more recent and comparatively brief history of eukaryotic life isoveremphasized in most textbooks, (2) the biology of prokaryotes defies most speciesdefinitions (particularly the biological species concept; see Mayr, 2001), (3) mutationis canonically insufficient to generate new species, and (4) endosymbiosis is primarilyresponsible for speciation across most if not all of life’s history. For example,Margulis and Sagan argue that ‘‘yrandom mutation, a small part of theevolutionary saga, has been dogmatically overemphasized. The much larger partof the story of evolutionary innovation, the symbiotic joining of organismsyfromdifferent lineages, has systematically been ignored by self-proclaimed evolutionarybiologists’’ (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 ifprokaryotes are not ‘‘species’’ sensu stricto, then it follows that mutation isnot responsible for the majority of speciation events. However, this logic,which is clearly expressed by statements like ‘‘No evidence in the vast literatureof heredity change shows unambiguous evidence that random mutation itselfyleadsto speciation’’ (Margulis and Sagan, 2002, p. 29), flouts the many well-documentedcases of new bacterial forms of life resulting from mutation, the fact thatdifferent 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 genomicrecombination attending sexual reproduction, which provides genomic ratesof variation that may be required to cope with the comparatively low mutationrates 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 eventsprefigured much of eukaryotic history. But the relative importance of symbiosiscompared to mutation (or sexual genomic recombination) once again rests onwhether we are willing to ignore the evolutionary history of eukaryotes simplybecause 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 cellevolution.Additionally, we believe that a sharp distinction must be drawn between

‘‘symbiosis’’ and ‘‘endosymbiosis’’. This distinction is important, because none ofthe biological examples used by Margulis and Sagan (2002) to explore how newspecies evolve as a result of symbiosis are convincing. For example, when discussinglichens 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 neithercan 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 resultof symbiosis. However, this line of reasoning ignores the fact that the phyco- andmycobiontic components of many lichen associations are capable of an independentexistence (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 allsuch species are ‘‘permanently and discontinuously different from the grey, algae-eating ancestors’’ (Margulis and Sagan, 2002, p. 13). Yet, no evidence is providedthat the ability of these animals to retain living chloroplasts in their cells is the traitthat 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 speciationand macroevolution (Meyer, 2002). In our view, symbiotic associations of organismsare 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, examplesof lateral gene transfers attending endosymbiosis clearly show that new species andeven new clades can evolve after genomic integration. The failure to draw thisdistinction does not diminish Margulis and Sagan’s basic message that symbiosis andendosymbiosis are important phenomena, nor does it distract from the claim that thebiological species concept is ill equipped to describe the origins and early history ofbacterial life. However, by diminishing the importance of mutation (when dealingwith bacteria), ignoring sexual genomic recombination (when dealing witheukaryotes), and by arguing that ‘‘ymost self-described evolutionary biologistsdisregard cell biology, microbiology, and even the geological rock record’’, Margulisand Sagan (2002) have misrepresented the status of current evolutionary thinking inwhat appears to be an overzealous effort to educate those few individuals who arestill unaware of the importance of prokaryotes in modern-day ecosystems orevolutionary 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 theviews and concepts of Margulis and Sagan (2002) are not discussed by theseauthors. 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, proteobacteriapossess infolded membranes similar to the cristae of mitochondria; and, third, strongmolecular sequence similarities, particularly those of 16S rRNA genes, betweenmitochondria and a-proteobacteria genomes. Nevertheless, the genomes of mito-chondria vary widely across eukaryotic lineages and they possess features that make itextremely difficult to trace the evolutionary history of this organelle (Lang et al.,1999). Subsequent endosymbiotic events involving the incorporation of coccoidcyanobacteria-like endosymbionts within ancient eukaryotic host cells (Fig. 4) gaverise to the most ancient photoautotropic lineages (e.g., chlorophytes and rhodo-phytes). Some of the lines of evidence for this hypothesis include the similarities incyanobacterial and plastid gene sequences, similarities in 16S rDNA and variousprotein-coding sequences, and the presence of a self-splicing Group I intron in aleucine transfer RNA gene of the cyanobacterium Anabaena, which has a similarsequence 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 variousalgal groups, the presence of additional membranes surrounding their plastids, andthe presence of nucleomorphs (nucleus-like structures) between the two outermembranes. Among these recent algal lineages, those with ‘‘red’’ predominate,perhaps because the red plastid genome is more self-sufficient in terms ofphotosynthetic functionality. Lateral gene transfer from the mitochondrial andplastid genomes to the nuclear genome occurred during primary, secondary, andtertiary endosymbiotic events. For example, the gene tufA, which encodes for Tu (achloroplast-specific protein-synthesis elongation factor) resides in charophyceannuclei 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 ofother groups of algae (Baldauf and Palmer, 1990). Lateral gene transfer is likelyresponsible for the widespread phyletic distribution of the capacity to synthesizecellulose (e.g., in chlorophytes, tunicates, oomycetes, and dinoflagellates) as well aschitin (e.g., in oomycetes, diatoms, and some chlorophytes). The integration ofendosymbiotic and host cell genomes into one functional unit is therefore responsiblefor many macroevolutionary events and phenomena, not the least of which was thedivision 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 (sensuMahner 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 oflife (Kutschera and Niklas, 2004), the relevance of ‘‘symbiogenesis’’ in the generationof new species in the ‘‘eukaryotic world of macroorganisms’’ has been grosslyoverestimated by some scientists. The currently popular book of Margulis and Sagan(2002), which is quoted by many anti-evolutionists around the world, delivers thebasic message that genomic variation and natural selection are of subordinateimportance in the process of speciation. This erroneous conclusion is not based onsolid empirical evidence and it has provided cannon fodder to an anti-Darwinianideology 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 his75th birthday. The cooperation of the authors was initiated by the Alexander vonHumboldt-Stiftung (AvH, Bonn, Germany).

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