Endosymbiosis-Cell Evolution and Speciation

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ELSEVIER Theory in Biosciences 124 (2005) 1 24 www.elsevier .de/thbio

Endosymbiosis, cell evolution, and speciation

U. Kutschera a'*, K.J. N i k l a s b

alnstitut fffr Biologic, Universitdt Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, Germany bDepartment of Plant Biology, Cornell University, Ithaca, N Y 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. @ 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 "chlorophyl l bodies" (chloroplasts) behave like independent,

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

1431-7613/$-see front matter @ 2005 Elsevier GmbH. All rights reserved. doi: 10.1016/j.thbio.2005.04.001

2 u. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 1-24

autonomous organisms that grow by division and adapt in number to the size of expanding leaves. Eight years later, the German cytologist Altmann (1890) demonstrated that "cell granules" (mitochondria) display the same staining properties as bacteria. Thus, Sachs and Altmann explicitly concluded that chloroplasts and mitochondria are "semi-autonomous" organelles displaying the behaviour of independent forms of life. However, the actual evolutionary origins of plastids and mitochondria remained unknown and highly contentious until a seminal publication of the Russian botanist C. Mereschkowsky (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, we outlined the process of endosymbiosis and noted that it is pivotal to understanding the history of life (Kutschera and Niklas, 2004). Here, we summarize in greater detail the history of this subtheory of the "expanded synthesis" and we review the evidence that has been used to verify the basic precepts of the endosymbiotic theory, with particular reference to a series of papers authored by Sitte (1989, 1991, 1994, 2001). We then discuss critically the more recent proposal that eukaryotic speciation has been driven by symbiogenesis - a hypothesis introduced by Wallin (1927) and described at greater length by Margulis and Sagan (2002). However, to avoid any ambiguity, we begin our treatment of endosymbiosis by exploring how some basic terms and concepts are defined, both historically and currently.

Symbiosis and endocytobiology: basic definitions

Most if not all eukaryotes live in cl6se association with microbes (bacteria) that either inhabit certain tissues of their hosts, or live externally but nevertheless in close physiological relationship. Examples include bacteria that live on the skin or within the digestive tracts of animals, bacterial associations in the rhizosphere with the roots of many seed plants, and the recently discovered growth-promoting methylobacteria on the epidermal cells of bryophytes and angiosperms (Hornschuh et al., 2002; Kutschera, 2002). These and many other relationships have been historically categorized by biologists in a variety of ways that indicate whether a

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particular association is 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 Ba r f 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 in the current literature treating cell biology or evolution, and it conveys little that is not communicated by the more frequently employed word "endosymbiosis".

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Classical publications: Schimper, Altmann, Mereschkowsky, and Wallin

The concept of endosymbiosis and the origin of cell organelles (plastids, mitochondria) has deep historical roots going back to the late 19th century. In a series of publications, which were summarized in a major review article, Schimper (1885) amply demonstrated that "non-pigmented granules" (plastids) develop into chloroplasts in the embryos of higher plants. The observation that the relatively large "chlorophyll bodies" always arose from pre-existing (colourless) plastids led Schimper (1885) to conclude that the relationship between plant cells and chloroplasts (or plastids, more generally) is symbiotic. This theory, which was implicitly held by Sachs (1882) (Fig. 1), led Schimper (1885) to speculate that symbiotic events may have been of great importance during the evolutionary history of green plants.

Five years later, Altmann (1890) discovered that the "granular bodies" (mitochondria) in the cytoplasm of plant and animal cells display the staining properties of free-living microbes. Based on his many careful cytological 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 baby with 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

A ~ B

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

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A. Animal tissue B. Root nodule

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.)

Biologisches Centralblatt, XXlT. B d . 15. September 1905 . ~ 18.

Ober Natur und Ursprung der Chromatophoren im Pflanzenreiehe.

Von C. Mereschkowsky, Privatdozent an der Kais. Universit~it in Kasan.

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.)

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


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 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; H6xtermann, 1998). For example, Famintzin (1907) argued that "there is no evidence for the occurrence of evolution in nature" and vigorously attacked Mereschkowsky by saying "the claim that chloroplasts are incorporated cyanobacteria is without any empirical basis".

Serial primary endosymbiosis: the timing of historical events

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


the endosymbiosis hypothesis by Margulis (1970) that this important concept received the attention that it deserved. Margulis also used the phrase "serial endosymbiosis theory" (Margulis, 1993), a term originally coined by Taylor (1979), to convey the idea that mitochondria and plastids did not acquire symbiotic residency in their host cells simultaneously but rather did so in two discrete stages or historical "events".

The evolutionary processes by which eukaryotic cells 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. 1500mya 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 (02) was very low before 2450 mya (during the Archaean) but reached considerable levels by 2200 mya. The rise in O2 level had occurred by 2320 mya, i.e., before the presumed 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 Mfiller, 1998; Lopez-Garcia and Moreira, 1999; Martin et al., 2003; Martin and Russel, 2003). What is far more certain as a consequence of recent molecular comparisons among pro- and eukaryotic genomes is that the ancestral prokaryotic lineage of modern-day mitochondria is related to extant ~-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 1200mya, 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

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

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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 e-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.)


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) conclude that the atmospheric concentration of CO2 1400mya 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; Fr6hlich and Kutschera, 1994) (Fig. 5). Yet, in spite of the many technological advances, the precise mechanism for plastid or mitochondrial division has not been fully elucidated. We do know that plastids use proteins derived from the ancestral cyanobacterial division machinery, whereas mitochondria have evolved a separate (non-bacterial) mode of division. Likewise, both types of organelles require dynamin-related guanosine triphosphatase to divide (Osteryoung and Nunnari, 2003).

Nevertheless, many additional lines of evidence support the endosymbiotic hypothesis. In an important series of papers, Sitte (1989, 1991, 1994, 2001) summarized eight documented facts that were not available to Sachs, Schimper,

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* , , ~ v v ,-,.e~.,- " ' ~ ' ; ~ : ~ : "

Fig. 5. Transmission electron micrographs of transverse sections through a l-day-old rye coleoptile (Secale cereale). A dividing proplastid (A) and a mitochondrion (B) are indicated by arrows, cw = cell wall, cy = cytoplasm, mi = mitochondrion, p ----- proplastid, s = starch. Bar = 1 pm (M. Fr6hlich and U. Kutschera, unpublished results).

l0 u. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) 1-24

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 are similar to those of prokaryotes but differ from those found in the cytoplasm of eukaryotic cells (70 S- versus 80 S-type, respectively); (4) The 70 S-type ribosomes of prokaryotes and organelles are sensitive to the antibiotic chloramphenicole, whereas 80 S-type ribosomes are not; (5) The initiation of messenger RNA translation in prokaryotes/organelles occurs by means of a similar mechanism; (6) Organelles and prokaryotes lack a typical (cytoplasmic) actin/tubulin system; (7) Fatty acid biosynthesis in plastids occurs via Acylcarrier proteins (as in certain bacteria); (8) Plastids and mitochondria are surrounded by a double membrane. In the inner mitochondrial membrane the bacterial membrane lipid cardiolipin is abundant. The cardiolipin content of eukaryotic biomembranes is close to zero (Gray, 1992; Gray et al., 1999).

Subsequent work has provided other lines of supporting evidence:

1. DNA sequences indicate that extant free-living cyanobacteria and ~-proteobacteria are the closest relatives of plastids and mitochondria, respectively (Douglas and Raven, 2003; Martin et al., 2001, 2003; Logan, 2003).

2. Genome sequences reveal that plastids and mitochondria, which have retained large fractions of their prokaryotic biochemistry, contain only remnants of the protein-coding genes that their ancestors possessed. Experimental studies have shown that DNA has been transferred from organelles to the nucleus of the host cell (Martin, 2003; Timmis et al., 2004). Endosymbiotic gene transfer was proposed years ago (Sitte, 1991) and is now a process that can be analysed by molecular cell biologists. For instance, in the model plant Arabidopsis thaliana, the chromosome 2 contains an entire copy of the 367-kb mitochondrial genome close to the centromere (Timmis et al., 2004). This documents a massive transfer of genes from the mitochondria into the nucleus.

3. Although plastids diverged from their cyanobacterial ancestors at least 1000 mya (Fig. 4), the chlorophyll a/b - arrangements in embryophyte chloroplasts and the cyanobacterium Synechococcus are essentially the same (multisubunit membrane-pigment~rotein 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 1) indicates that the cyt b6f complexes of cyanobacteria and chloroplasts have almost the same molecular structure (Kfihlbrandt, 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 eubacterial progenitors.

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 cyanobacterium. 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 with those isolated from cyanobacteria (see Niklas, 2004). Likewise, the ultrastruc- tural appearance of membrane-bound cellulose synthase proteins in cyanobacteria, cellulose synthesizing proteobacteria, various stramenopile algal lineages, and the embryophytes are very similar, suggesting that cellulose synthase genes have been laterally transferred from cyanobacteria to a variety of eukaryotic lineages.

10. The dynamin-related guanosine triphosphatase protein Fzol (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 Fzol is likely derived from the eubacterial endosymbiotic genome that was the precursor of mitochondria. Fzol 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 protochlorophyllide to chlorophyllide. Yang and Cheng (2004) conducted a genome-wide sequence comparison, combined with a phylogenetic analysis. The authors conclude that photosynthetic eukaryotes obtained their LPOR homologues from ancient cyanobacteria through endosymbiotic gene transfer.

The origin of the nucleus

Although 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 (H6xtermann, 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 modern methanogenic Archaea have genes encoding for histones, whereas Eubacterial genomes do not. Assuming that the most ancient prokaryotic symbiotic relationship involved methane-making Archaea living in Eubacteria cells (that relied on fermentation), the hypothesis argues that 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-nucleus was the uncoupling of DNA transcription from mRNA translation.

The second hypothesis for the origin of the nucleus argues that organisms with proto-nuclei actually predate those lacking this organelle (i.e., nuclei-like bearing prokaryotes predate eukaryotes). This scenario is based on a group of soil- and freshwater prokaryotes known as planc'tomycetes, which have a cell wall far less rigid than those of other Eubacteria. Detailed electron microscopic studies of two planctomycetes (Gemmata obscuriglobus and Pirellula marina) reveal internal membrane-bound structures, some of which hold a dense mixture of RNA and DNA as well as DNA- and RNA-processing proteins (but no ribosomes). Importantly, in one of these organisms (G. obscuriglobus), the membrane is folded and discontinuous in ways that are reminiscent of the nuclear pores of eukaryotic cells. This organism, depicted in a recent news report (Pennisi, 2004), may represent

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

The third hypothesis, which is perhaps the most radical, argues that the genomes of viruses living in Archaea hosts merged with the DNA of their hosts inside the virus. This scenario draws attention to the fact that most viral and eukaryotic DNA is arranged linearly (whereas most bacterial DNA is circular), viruses and eukaryotic nuclei transcribe DNA but do not translate mRNA, and that some poxviruses make membranes around their DNA using the endoplasmic reticula of their host cells (de Duve, 1996; Hartman and Fedorov, 2002; Pennisi, 2004; Baluska et al., 2004).

Although these three hypotheses are not mutually exclusive (in the sense that the nucleus may have originated more than once in the history of life), no single hypothesis has received even a conditional wide acceptance. It is nevertheless clear that modern experimental techniques hold out the promise that we may one day know with some certainty how the nucleus made its 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-sized ribosomes between these two membranes (see Fig. 7). The DNA-containing structure, which has been called a nucleomorph, has been interpreted to be the highly reduced nucleus of the photoautotrophic endosymbiont (Maier et al., 2000; Keeling, 2004). Recent research supports this thesis in so far as that the genome of cryptomonad nucleomorphs typically consists of three small chromosomes that primarily contain only those genes encoding for the products necessary for the maintenance of the nucleomorph itself.

For instance, the cryptomonad Guillardia theta contains a tiny 551 kb genome with only 17 diminutive spliceosomal introns and 44 overlapping genes (Douglas et al., 2001). These genes and their messenger RNAs show typical eukaryotic features, which lend additional support to the thesis that the "plastid" is a highly reduced eukaryotic photoautotrophic endosymbiont. Because the highly reduced nucleomorph genome (an "enslaved" algal nucleus) does not encode for any of the products necessary for the maintenance of its original plastid and because the genome of the original plastid is not self-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

14 u. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) I 24

pointed out by Falkowski et al. (2004), this genome encodes for both the small and large subunits of the important enzyme ribulose-l,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 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 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 wa41 (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 ceils (see Fig. 2B).

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae) harbours a sulfur-oxidizing proteobacterium in the specialized cells of its gill tissues.


A number of studies have shown that this clam species depends on these symbiotic bacteria for its nutrition. Importantly, these bacteria are transmitted - like mitochondria - via the eggs of the animal (Yaffe, 1999; Logan, 2003). Hurtado et al. (2003) analysed the association between vesicomyid clams and their vertically transmitted endosymbiotic bacteria and conclude that the bacteria have lost their ability to live freely in the marine environment. This complete 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, the insect-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 from chlorophyll (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 a population of ~qiving" chloroplasts within the animal's body. Nevertheless, laboratory studies indicate that the "'solar-powered sea slugs" were able to live


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 = I cm (A), 2 pm (B).)

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 generated two deep (albeit not necessarily permanent) phyletic wedges in the tree of life, one that continues to distinguish prokaryotes from eukaryotes and another that


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

@

Heteroko~

Dinophyta 1/2/3

I

Haptophyta 2

ryptophyta " ~

.,rtiary ldosymb os s

R,o op,yta 0

/

I Euglenophyta

._ O ~ , j /

l | Chlor~r~c.hil ~ [ Ch/0~'arachin~

O~? I 1 ~ Chlorophytt

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C ~ ~ m Phagocytotic eukaryote

n - - - J

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.)


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 Acquirin9 Genomes." A Theory of the Oriqin 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 ofprokaryotes 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 ".. .random 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 organisms.., from different lineages, has systematically been ignored by self-proclaimed evolutionary biologists" (Margulis and Sagan, 2002, p. 15).

To a certain extent, the third proposition (i.e,, that mutation is unimportant to speciation) is logical legerdemain, because it emerges directly from propositions (1) and (2). If prokaryotic evolution dominated life history and if prokaryotes are not "'species" sensu stricto, then it follows that mutation is not responsible for the majority of speciation events. However, this logic, which is clearly expressed by statements like "No evidence in the vast literature of heredity change shows unambiguous evidence that random mutation itself.., leads 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 overemphasize mutation when dealing with evolutionary theory. Indeed, most emphasize genomic recombination attending sexual reproduction, which provides genomic rates of variation that may be required to cope with the comparatively low mutation rates observed for multicellular eukaryotic organisms (Niklas, 1997; Kutschera, 2001, 2003).

Likewise, the fourth premise of their argument (i,e., that symbiosis is far more important than mutation) emerges logically from propositions (1) and (2). Certainly, all of the evidence reviewed here indicates that primary endosymbiotic events prefigured much of eukaryotic history. But the relative importance of symbiosis compared to mutation (or sexual genomic recombination) once again rests on whether we are willing to ignore the evolutionary history of eukaryotes simply because it is comparatively brief compared to that of prokaryotes. Arguably, the


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 functional equivalents of communities. For this reason, the examples of "symbio-speciation" discussed by Margulis and Sagan (2002) are unconvincing (see Thompson, 1987; Saffo, 1992, who present a more balanced view of this topic). In contrast, examples of lateral gene transfers attending endosymbiosis clearly show that new species and even new clades can evolve after genomic integration. The failure to draw this distinction does not diminish Margulis and 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 with eukaryotes), and by arguing that ". . .most 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 ofprokaryotes in modern-day ecosystems or evolutionary history.

In two recent books, current theories on the modes of speciation are described in detail (Schilthuizen, 2001; Coyne and Orr, 2004). It should be noted that the views and concepts of Margulis and Sagan (2002) are not discussed by these authors. To our knowledge, only about a dozen "symbio-speciation events" have been described in the literature and each is highly questionable (Thompson, 1987; Saffo, 1992).

20 U. Kutschera, K.J. Niklas / Theory in Biosciences 124 (2005) l 24

Conclusions

Primary endosymbiotic events between archaeal-like host cells and c~-proteobacteria are responsible for the appearance of the most ancient eukaryotic heterotrophic lineages (e.g., diplomonads and microsporidians, kingdom protoctista) (Fig. 4). Three lines of evidence provide the strongest support for this hypothesis. First, mitochondria possess eubacterial-like DNA and transcription/translation systems (e.g., ribosomes similar in size to those of prokaryotes); second, proteobacteria possess infolded membranes similar to the cristae of mitochondria; and, third, strong molecular sequence similarities, particularly those of 16S rRNA genes, between mitochondria and c~-proteobacteria genomes. Nevertheless, the genomes of mitochondria 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 rhodophytes). Some of the lines of evidence for this hypothesis include the similarities in cyanobacterial and plastid gene sequences, similarities in 16S rDNA and various protein-coding sequences, and the presence of a self-splicing Group I intron in a leucine transfer RNA gene of the cyanobacterium Anabaena, which has a similar sequence and position to an intron found in the plastid genome (Xu et al., 1990).

Secondary and tertiary endosymbiotic events gave rise to evolutionarily more recent algal lineages (e.g., euglenoids, cryptomonads, and 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", see Niklas, 2000; Scherp et al., 2001), but it remains encoded in the plastid genomes of other groups of algae (Baldauf and Palmer, 1990). Lateral gene transfer is likely responsible for the widespread phyletic distribution of the capacity to synthesize cellulose (e.g., in chlorophytes, tunicates, oomycetes, and 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,

U. Kutschera. K.J. Niklas / Theory in Biosciences 124 (2005) 1 24 21

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.

A c k n o w l e d g e m e n t s

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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