The microbes' contribution to evolution

The microbes' contribution to evolution

266 BioSystems 7 (1975) 266--292 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands THE MICROBES' CONTRIBUTION TO EVOLUTION*...

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266 BioSystems 7 (1975) 266--292

© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands THE MICROBES' CONTRIBUTION TO EVOLUTION* LYNN MARGULIS Department of Biology, Boston University, 2 Cummington Street, Boston, Mass. 02215, USA

1. I n t r o d u c t i o n We light microscopists, w h e t h e r we call ourselves microbiologists, p r o t o z o o l o g i s t s , cell biologists, m i c r o p a l e o n t o l o g i s t s , c y t o g e n e t i c ists, histologists or m e m b e r s o f some o t h e r artificial discipline are direct d e s c e n d a n t s o f the D u t c h school o f m i c r o b i o l o g y and its' great ancestor f r o m Delft, A n t o n i van L e e u w e n h o e k . Insatiably curious, he c o n s i d e r e d all o f n a t u r e and especially its " a n i m a l c u l e s " w o r t h y o f a t t e n t i o n . T o van L e e u w e n h o e k the world was an organic whole e x t e n d i n g into smaller places; it was never divided into p e r m i t t e d and forbidden territories--botanical, zoological, protozoological or bacteriological. In this p a p e r I h o p e to revive van L e e u w e n h o e k ' s view t h a t e v e r y t h i n g small is w o r t h y o f study. It is m y belief t h a t if o n e follows the example of van L e e u w e n h o e k and excludes n o n e o f the living m i c r o b e s f r o m c o n s i d e r a t i o n b u t recognizes all as p r o d u c t s o f the neo-Darwin* On March 26 and 27th, 1975, The Royal Academy of the Netherlands (Koninklijke Akademie van Wetenschappen) celebrated the 300th anniversary of the work of their illustrious countryman, Antoni van Leeuwenhoek. This contribution to that occasion was the only one that, unfortunately, was not delivered in the Dutch language. Because of the nature of the meeting, which called together an active, diverse group of biologists and physicians, the paper is a review of the present status of the symbiosis theory of cell evolution, some aspects of which have appeared before (Margulis, 1975a and b). The entire symposium ("De microbiologie drie eeuwen na Antoni van Leeuwenhoek") will be published by Pudoc, Wageningen.

ian e v o l u t i o n a r y process, o n e is led d i r e c t l y t o the realization t h a t the organelles o f e u k a r y o t i c cells originated t h r o u g h a s e q u e n c e o f hereditary e n d o s y m b i o t i c association. One reason is simple: the processes p o s t u l a t e d to have occurred during the e v o l u t i o n o f e u k a r y o t e s recurred m a n y times and gave rise to m a n y strange microbeasts, some which were first witnessed b y t h e draper. C o n t i n u e d s t u d y , van L e e u w e n h o e k style, has led to r e o b s e r v a t i o n o f these organisms with far finer tools than he had available, and t o a r e a w a k e n i n g o f interest in the t h e o r y I am advocating. This p a p e r presents the "serial e n d o s y m b i o sis t h e o r y " ( T a y l o r , 1974) in its m o s t c u r r e n t f o r m and shows h o w r e e x a m i n a t i o n o f these microbial e c o s y s t e m s o f van L e e u w e n h o e k (e.g., microbes o f the m o u t h , sea water and ponds, d e c a y i n g m a t t e r ) leads to discoveries o f p r e c e d e n t s and analogs for all o f the processes h y p o t h e s i z e d as steps in the origin of n u c l e a t e d cells {Margulis, 1 9 7 0 ) . It has t a k e n until this second half o f the 2 0 t h C e n t u r y to bring van L e e u w e n h o e k ' s observations into a c o h e r e n t whole. He described h u m a n and animal sperm, placoid scales o f e l a s m o b r a n c h fish, insect appendages, oral and fecal spirochaetes, fossil f o r a m i n f e r o u s limestone, cell division and sexual c o n j u g a t i o n in ciliates. Indeed, m o s t m o d e m biologists have never gazed u p o n m a n y o f the objects he saw. If t h e y had, t h e y would perhaps have n o t f o u n d it so startling t h a t c o m p l e x i n t e r a c t i o n b e t w e e n species is a fact o f life, t h a t m u c h i n t e r a c t i o n leads to symbioses and t h a t symbioses include t h o s e o f the h e r e d i t a r y t y p e . T h e h e r e d i t a r y or

267 cyclical type of symbiosis is a relationship in which two partners of different species are regularly associated throughout the life cycle of both. Some 200 years after the death of van Leeuwenhoek came the publication of the little book by Kluyver and van Niel (1956) in continuation of the Dutch school of microbiology. This may have been the most important step toward the integration of the bacterial "animalcules" into the conceptual framework of biological thought. Followed by van Niel and Stanier's classic paper {1962), it led to the firm recognition of the prokaryotic nature of bacteria and blue green algae. Although the word " p r o k a r y o t e " was probably first coined by Chatton*, the concept of the prokaryote discontinuity was developed and brought to the rest of Europe and the USA by the descendants of the Dutch school (Stanier and van Niel, 1962; Stanier, 19,34). In the late 1950's, with the publication of The Microbial World (Stanier et al., 1957) and, more recently, Brock's {1970) elegant tome Biology of Microorganisms, it became clear what the "animalcules" are and how they differ from each other and from larger, more familiar organisms. However, the knowledge that microbes, like metazoans and green plants, are products of neo-Darwinian evolution is still in process of realization. That evolution in microbes has happened by changes of gene frequencies in natu::al populations with time at specific places and periods in the history of the Earth is a concept still under development. The field of evolutionary microbiology is right now emerging, and we are witnessing an enormously energetic intellectual synthesis in this direction. However, :.1o one yet calls himself a microbial evolutionist. It is possible that the first seven-eighths of earth's history (from about 3.5 to about 0.6 billion years ago) was the "Age of the Prokaryote Microbes" (Barghoorn, 1974). Entire fields * This is reputed to be in E. Chatton, 1937, Titres et travaux scientifique. E. Sottano sete Quatro. 407 p., but I have been unable to locate the book.

of study are being seen as relevant to the evolution of microorganisms on the early Earth. For some references to these developments see Barghoorn (1971); Ponnamperuma (1971); Ponnamperuma and Buvet (1971); Schopf (1974); Dose et al. (1974); Walters (1976). In spite of fantastic new methodological advances -- transmission and scanning electron microscopy; gas chromatograph-mass spectrometry; the ultracentrifuge; the use of radioisotopes; column chromatography and electrophoresis; and so forth -- it is the tool of van Leeuwenhoek himself that still occupies the most central role in the analysis of objects seen for the first time by him. For comparisons of live microorganisms, for the study of microbial fossils trapped in cherts (e.g., Tyler and Barghoorn, 1954; Schopf and Blacic, 1971; see Schopf, 1974 for review) and for the study of chromosomes, the light microscope has no equal.

2. Outline of the theory of the origin of eukaryotic cells by hereditary serial endosymbiosis What follows is admittedly the " e x t r e m e " version of the serial endosymbiotic theory (S.E.T.) in that all three classes of organelles (mitochondria, photosynthetic plastids and flagella/cilia, i.e. 9 + 2 homologues made of microtubules) are hypothesized to have originated by hereditary endosymbiosis. For critical discussion of these points see Taylor (1974). Although many are willing to accept the S.E.T. for plastids and mitochondria (e.g., Schnepf and Brown, 1971; Lipmann, 1974; Taylor, 1974; John and W~atley, 1975), others feel that the evidence for the symbiotic origin of mitochondria is inconclusive or unconvincing (Raff and Mahler, 1972; Avers, 1974; Uzell and Spolsky, 1974; Raff, 1975) and very few (if any) serious scientists share my belief that the eukaryote cilia-flagella system of microtubules originated by hereditary endosymbiosis. Even though it may not be correct, the extreme version of the S.E.T. is both explicit and testable and will therefore be included in the

268 subsequent discussion on the status of germane evidence. All organisms on earth have anaerobic, fermentative, heterotrophic, prokaryotic ancestors. The triplet nonoverlapping nucleotide base sequence that serves as the universal genetic code determining amino acid sequences in proteins was present in the bacterial population ancestral to all extant forms of life. In the early precambrian, selection pressures led to extensive adaptive radiation among prokaryotes, primarily on the metabolic level. Among others, the following cell types evolved: mycoplasm-like fermenters (catabolizing glucose to pyruvate via anaerobic glycolysis using the Embden Meyerhof metabolic pathway); spirochaetes; photosynthetic oxygen-eliminating prokaryotes (coccoid blue green algae); and aerobic gram negative eubacteria that oxidized small organic acids via the Krebs cycle (Sagan, 1967; Margulis, 1970; 1971b; 1972a,b; 1975a,b). According to the S.E.T., the first step in the origin of eukaryotes from prokaryotes was the acquisition of protomitochondria. This event could have occurred when a fermentative anaerobe (host) was invaded by Krebs cycle-containing gram negative eubacteria (protomitochondria); stabilization of this initially predatory association led to the formation of the mitochondria-containing amoeboids, from which all other eukaryotes derive (Hall, 1973). Because pinocytosis and phagocytosis are virtually unknown in prokaryotes (Stanier, 1970) and because recently characterized Bdellovibrio-like organisms provide such a fine model for the penetration of bacterial hosts by aerobic gram negative eubacteria, I now feel the first prokaryotic-prokaryotic symbiotic step probably occurred by invasion, modification and stabilization of Bdellovibrio-like behavior. (See Starr and Seidler, 1971, and Starr, 1975, for details concerning BdeUovibrio.) Alternatively because the electron transport chain of Paracoccus denitrificans is so similar to that of mitochondria, the first step could have been the acquisition of an intracellular endosymbiont comparable to the facultative aerobe

Paracoccus ( Micrococcus) denitrifricans. The general similarity of the cytochromes, quinones, inhibitor sensitivities and electron transport chain between Paracoccus and mitochondria is overwhelming (John and Whatley, 1975}. The differences between them can be interpreted as adaptations to an intracellular role (e.g., loss of the constitutive nitrate reductase pathway; transport of ATP to the surroundings; and so forth}. Perhaps a Paracoccus with predatory behavior might be sought as ancestor to mitochondria. The nucleus and other endoplasmic membranes probably evolved autogenously, after the presence of protomitochondria provided the way for the steroid biosynthetic pathway. Selection pressures for the formation of the nuclear membrane probably involved the segregation of newly synthesized DNA on endomembrane (Leibowitz and Schaechter, 1974} and/or the sequestering of nucleoplasm DNA to protect it from the more oxidizing conditions of the cytoplasm surrounding the mitochondria. The second symbiotic step is hypothesized to be the acquisition by the mitochondria-containing amoeboids of highly motile anaerobic surface bacteria (very likely spirochaetes}. Many mutations and intracellular transfer of genes from bacteria to host preceded the origin of amoeboflagellates, ciliates and many other protists. The surface spirochaete bacteria evolved into the "9 + 2" homologues: basal bodies, cilia, flagella, and other microtubulebased structures. Mitosis, as a process, evolved in many lines of organisms as the motile bacteria merged with the amoeboid host cell, and the morphogenetic processes of the bacteria eventually were utilized in the formation of the "achromatic apparatus" (mitotic spindle) of mitosis. (See Margulis, 1970, 1974b, and Pickett-Heaps, 1974, for detailed but different expositions of the possible steps involved in the origin of mitosis.) The final symbiotic step involved the acquisition of photosynthesis by different populations of eukaryotic heterotrophs. Ingestion, without digestion, of blue green algae by various protist hosts under nutrient-poor condi-

269

tions led to the establishment of stable, heritable, intracellular symbioses. The prokaryotic algae eventually became the obligatory symbiotic photosynt:netic plastids in the origin of various lines of nucleated algae and, eventually, chlorophytes anti archegoniate multicellular green plants. Thus, according to the S.E.T., all eukaryotes are at least digenomic (contain two independently derived genomes: host and mitochondria); according to the extreme S.E.T., most heterotrophic eukaryotes were originally trigenomic (host, mitochondria, 9 + 2 homologue and plastids) as illustrated in Fig. 1. I agree with Taylor's (1974) contention that

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"cell" is an inadequate term, and follow his suggestion that in referring to cells we use words that reflect the number of membrane bound protein synthesizing units; for example, the use of "monad" for prokaryote, "dyad" for heterotrophic eukaryotes and "triad" for photosynthetic eukaryotes. (If the spirochaete origin for the flagellar-mitotic system is correct, it is clear that the once independent protein synthesizing system of the motile bacteria must have become completely integrated into the complex eukaryotic host system. Thus Taylor's terminology can be applied to eukaryotes no matter which version of the serial endosymbiotic theory is finally accepted.) Multicellular

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270 units, then (according to Taylor, 1974) become: polymonads (e.g., filamentous and mycelial prokaryotes such as Gardnerula, Polyangium or Myxococcus); polydyads (e.g., slime molds, ascomycetes, metazoa); and polytriads (e.g., bryophytes, tracheophytes, brown and red eukaryotic seaweeds); and so forth. Application of Taylor's analyses to both cells and extant associations has already proven useful (Margulis, 1975b).

3. Historical theories: on the nature of the evidence It is never possible to rigorously prove after the fact that a unique series of events did occur in any historical context. Evolutionary biologists, like historians, deal with series of complex, irreversible phenomena. They can only present arguments based on the assumption that of all the plausible historical sequences, one is more likely to be a correct description of past events than another. The probability that one particular historical reconstruction is more accurate than another increases as the number of explanations for existing observations increase. An evolutionary theory can also be judged by its predictive power. In this context, some experimental studies already generated by the extreme S.E.T. are described below, and a number of potential areas of investigation are presented in Section 5. These studies and their predicted conclusions do not necessarily follow from alternative monophyletic and/or partial symbiotic concepts for the origin of eukaryotic cells. In his remarkably complete and well-argued paper Taylor (1974) explores several alternatives to the endosymbiotic view of the origin of mitochondria and plastids. He enumerates the logical problems with most endogenous or "pinching o f f " hypotheses and considers new data from molecular and cellular biology in the light of nonsymbiotic theories (Taylor, 1976). Since his presentation of non-S.E.T, alternatives is far less impassioned and biased than mine, the reader is referred to his reviews. In

my opinion, the strongest advocates of the nonsymbiotic view (Allsopp, 1969; Raff and Mahler, 1972 and Raff, 1975; Perlman and Mahler, 1970; Uzzell and Spolsky, 1974) ignore certain critical bodies of literature and facts (see Margulis, 1975a, for discussion). The burden of detailing the nonsymbiotic theory predictions must lie with its advocates. 4. Experimental studies and explanations generated by the serial endosymbiotic theory

4.1. Genetic behavior of organelles Three distinct patterns of inheritance can be detected in the ciliate protist Paramecium aurel/a: nuclear (Mendelian); caryonidal (a specialized modified nuclear mode); and cytoplasmic (Sonneborn 1947; Barnett, 1966). For many years the inheritance of the killer trait has been the standard example of the behavior of cytoplasmic genes (Sonnenborn, 1959). The killer p h e n o t y p e was found to depend on the presence of cytoplasmic kappa particles. Traits correlated with the presence of kappa particles are inherited independently of the nucleus, yet kappa requires several Mendelian genes for its maintenance (K genes) and is negatively affected by several other nuclear alleles (S genes). The complete solution of the problem is at hand. Kappa (Fig. 2) is a class (only one of many, Beale et al., 1969) of hereditary endosymbiotic bacteria that harbors virus (Preer et al., 1974). The similarity between the genetic behavior of kappa and similar endosymbiotic bacteria and mitochondria and photosynthetic plastids is striking. An obvious explanation is that both kappa and these organelles began as free-living prokaryotes and, with time, have become hereditary endosymbionts more and more dependent on products of nuclear genes. In fact, the hypothesis that mitochondria and plastids originated as prokaryotic symbionts has generated some fine experiments and an immense literature (see, for example, Nass, M.M.K., 1969, 1971; Nass, S., 1969; Cohen, 1970, 1973 for review; Pigott and Carr, 1972; Ebringer, 1972). Let us take just one limited

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272 but extremely exciting example of the usefulness of the S.E.T. in explaining the recently discovered "sexual" behavior of mitochondria and chloroplasts. The evidence that recombination of nonMendelian (cytoplasmic) markers occurs between differently marked mitochondria entering a cross in yeast is now overwhelming (Mounolou et al., 1967; Thomas and Wilkie, 1968; Wilkie and Thomas, 1973; see Kujon et al., 1974 and Gillham, 1974, 1976 for details.) Comparable organellar sexual recombination phenomena are probably occurring after zygote formation in Chlamydomonas chloroplasts as well (Sager, 1972; Gillham, 1974). The best model for the nature of the recombination process in organelles is a potentially multiparent, unidirectional (polarized) model {Gillham et al., 1974; Gillham, 1975). Thus, the detailed comparison of the genetic behavior of organelles (mitochondria and plastids) with prokaryotic recombination systems has proven fruitful. If these organelles had originated by differentiation of cytoplasm within a primitive eukaryote, the independent origin of prokaryote-type recombination in organelles and free-living bacteria and their viruses is difficult to account for. Genetic interaction between organisms that are clearly recognizable symbiotic partners is not well understood. There is no doubt that heritable modification of symbionts occurs with time. The gradual development of dependence of the host nuclei on symbiont products has been directly observed (by Jeon, 1972, for example) in new bacterial-amoebae associations (Fig. 3). Because of the usefulness of such information in understanding the processes that have led to the evolution of organelles from free-living symbionts, I have recently reviewed this literature (see Margulis, 1975b). 4.2. Protist phylogeny The contradictory classification systems devised by zoologists and botanists for the "lower eukaryotes" are notorious. The recognition of "anastomosing phylogenies," i.e., that certain groups such as lichens and chlorophyte

algae are products of symbioses, has begun to clarify the relationships among these organisms. Single consistent phylogenies and taxonomic schemes for the "lower eukaryotes" can now be drawn. Although symbiosis may not be a factor in the origin of most higher taxa, it has been decidedly significant in many cases {Fig. 4). (See Whittaker, 1969, 1975; Margulis, 1974a,c for details and Leedale, 1974, for discussion.) 4.3 The microfossiliferous Preca m brian It has long been recognized that there is no catastrophic environmental gap in the fossil record between the precambrian and the Phanerozoic. The "sudden appearance" of animal fossils at the base of the Cambrian has been puzzling, but the recognition that, fundamentally, the precambrian was the "Age of the Prokaryote Microbes" and the Phanerozoic the "age of Eukaryotes" clarifies this discrepancy (Barghoorn, 1974; Margulis, 1972c). Of course this is an oversimplification not addressed to the origin of shelled metazoans (Cloud, 1968) and the role of oxygen and other environmental variables shaping the selection pressures on the biota(Cloud, 1974). However, the symbiotic theory is consistent with the idea that prokaryotic microorganisms, including oxygeneliminating photosynthesizers, preceded by hundreds of millions of years the origin of the eukaryotic metazoan animals and green plants (senso stricto ). The recognition that precambrian times were dominated by prokaryotes has led to fruitful collaboration between blue green algal mat ecologists and precambrian sedimentary geologists (e.g., Waiters, 1976; Golubic, 1973), the identification of Entophysalis-like coccoid blue green algae in 2500 million year old rocks from Belcher Island, Canada for example (Golubic and Hofmann, 1975). 4.4. Additional examples The recognition that microbes are products of neo-Darwinian evolution, with selection act-

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Fig. 3. The effect o:[ nuclear transplant on the maintenance of symbiotic microorganisms in amoebae. These amoebae contain mitochondria, the "DNA containing bodies", and a second population of more recently acquired endosymbiotic bacteria. (a) Electron micrograph of normal Amoeba discoides showing the normal morphology of the DNA-containing bodies that happen to be clustered together, individually surrounded by membranes. (X 37,000) (b) Electron micrograph of Ameoba discoides infected with microorganisms of two kinds. Shown are the more recent symbionts which, unlike the "DNA-containing bodies" and the mitochondria, tend to look less adversely affected by intraspecific nuclear transplants. (c) Electron micrograph of nucleocytoplasmic hybrid (A. proteus nucleus and A. discoides cytoplasm} showing abnormal morphology of a mitochondrion and that of DNA-containing bodies. Since proteus nucleus is incompatible with cytoplasm of discoides, this hybrid is not viable. Courtesy of K. Jeon, University of Tennessee.

ing m a i n l y on m e t a b o l i c r a t h e r t h a n m o r p h o l ogic traits, has aided in placing in a t e m p o r a l sequence certain steps in t h e h i s t o r y o f t h e

a t m o s p h e r e (Cloud, 1 9 7 4 ; L o v e l o c k and Margulis, 1 9 7 4 a , b ; Margulis and L o v e l o c k , 1 9 7 4 ; Walker, 1976).

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The concept that complex (9 + 2) microtubule-based flagella regeneration is a process homologous to the movement of chromosomes in mitosis has led to the discovery of mitotic arrest by melatonin and other specific actions of certain microtubule inhibitors (Banerjee and Margulis, 1973; Margulis, 1973; Banerjee et al., 1975). The awareness of the homology between the flagella and cilia systems of eukaryotes has suggested a new theory of sensory transduction {primarily olfactory, gustatory and auditory, Atema, 1973).

Of course, the concept that mitochondria and plastids originated as bacteria has led to widespread use of specific antibiotics (e.g., streptomycin and other aminoglycosides, chloramphenicol, cycloheximide) in investigating the role of the organellar protein synthetic system relative to the " h o s t " eukaryote (Ebringer, 1972). The assumption that plant cells are the products of hereditary endosymbiosis explains the presence, for example, of redundant chloroplast and nonplastid metabolic pathways (e.g., polyunsaturated fatty

275 acids, Jacobson et al., 1973). It also clarifies the discovery of a very skewed distribution of qualitatively recoverable types of mutants in plants and Chlamydomonas relative to animal cells and fungi (L: et al., 1967).

5. Some predicted results of potential experiments generated by the S.E.T. The serial endosymbiosis theory has the advantage of generating a large number of experimentally verifiable predictions. Some are listed here.

5.1. Lack of mitosis in blue green algae No blue green algae that show trends towards the evolu;ion of mitosis will ever be found (e.g., with centriotes, microtubules or histone-containing chromosomes). All blue green algal developmental and sexual systems will prove to be similar to those of bacteria.

5.2. Free-living homologues: plastids Since coccoid blue green algae are hypothesized to be ancestral to photosynthetic plastids, the primary sequence of many proteins in the photosynthetic a:3paratus of plastids will turn out to be homologous to those from the appropriate coccoid algae.

5.3. Free-living homologues: mitochondria Since certain aerobic bacteria (e.g., Paracoccus denitrificans; John and Whatley, 1975) are hypothesized to be ancestral to mitochondria, the primary sequences of many of the proteins in the mitochondria will show homologies to those from the appropriate respiring bacteria (e.g., superoxide dismutase, Fredovich, 1975; c y t o c h r o m e 550 and c y t o c h r o m e oxidase).

5.4. Free-living homologues: spirochetes Since certain ,;pirochaetes (e.g., Pillotinas, Hollande and Gharagozlou, 1967) are hypothe-

sized to be ancestral to the 9 + 2 flagella/cilia homologues, the primary sequence of some spirochaete proteins will show homologies to those of the flagellar/ciliary axonemes and/or matrix. It is predicted that spirochaetes that contain authentic microtubes with colchicinebinding microtubule protein (perhaps even in a 9 + 2 array) will be found. Primary amino acid sequence of the eukaryotic flagella proteins (tubulins) will show greater similarities to whatever protein(s) is/are intrinsically motile in spirochaetes than to any other prokaryotic proteins.

5. 5. Basal body RNA and spirochetes RNA of the lumen of eukaryotic basal bodies will be found homologous to RNA of appropriate free-living spirochaetes. The replication (polymerization) of this RNA (Hartman et al., 1974} will be required for new basal body formation, whether in centrioles, kinetosomes or other de novo production of 9 + O's (Sonneborn, 1974). This RNA should also be homologous to "division centre" RNA in Physarum (Laane and Haugli, 1974).

5. 6. Phylogenetic significance of mitotic variations Striking variations on the theme of mitosis will be found in "lower eukaryotes." In opposition to statements in the classical literature (Wilson, 1925), these will turn out to be of phylogenetic significance and generally independent of the presence of photosynthetic plastid-related traits. (Pickett-Heaps, 1974, has already shown the phylogenetic significance of mitotic variations in certain green algae; Kubai, 1973, and Heath, 1974, have done the same for protists and flagellated fungi.) Meiosis will be shown to have originated independently in several preadapted lines of protists (e.g., ciliates, slime molds, hypermastigotes) just as h o m e o t h e r m y originated independently in several preadapted vertebrate lines.

276 5.7. Derivation of the red algae thallus from a mycelium with cyanelles

extant forms of red algae, fungi and other eukaryotes that have mitosis but lack flagella.

The photosynthetic plastids of red algae will be found to have many metabolic pathways and primary amino acid sequences in common with blue green algae; non-plastid cytoplasmic proteins in red algae will turn out to be more like certain heterotrophic protists and primitive zygomycetes or ascomycetes (amastigote conjugating organisms).

5.11. Eukaryote transformation and polygenomic control of metabolic pathways

5.8. Lack of autonomy as the advanced state Organisms containing well developed Mendelian genetic systems (e.g., yeasts, metazoans, metaphytes) will be found to have less autonomous protein synthetic systems in their mitochondria and photosynthetic plastids. Conversely, certain "lower eukaryotes" that have no eumitotic sexual systems will be shown to contain organelles with greater autonomy. Attempts to "culture" organelles will be more successful in organisms that show no or idiosyncratic sexuality (e.g., euglenids, dinoflagellates, etc; see Margulis, 1974b and PickettHeaps, 1974 for discussion of some of these forms). 5. 9. Bdellovibrio-like symbionts Bdellovibrio-like bacteria will be found that maintain stable relationships with their "prey." 5.10. Tubulins Because of their hypothesized common origin, ultimately from the ancestral spirochaetes, the amino acid sequence of mitotic spindle tubulin proteins from all eumitotic organisms will show striking homologies with those from the (9 + 2) flagellar and ciliary axonemes. It will be shown that flagellar proteins (a and fl tubulins) are used directly in the formation of the mitotic spindle microtubes in many if not all eumitotic eukaryotes. This protein will not be found in blue green algae. However, homologous tubulin proteins will be detected in

In certain intracellular associations, gene transfer from symbionts (bacteria) to hosts (eukaryotes) will be proved. With respect to organelles, the particular genes transferred from the mitochondria and/or plastids in any lineage will tend to be the same within that lineage and tend to be different outside that lineage (see Fig. 4). Many gene products (e.g., proteins) will be cytoplasmic recombinant products requiring two or more non-homologous genomes for their formation (e.g., of ribolosediphosphocarboxylase and leghemoglobin -- the former requires both nucleocytoplasmic and chloroplast protein synthesizing systems, the latter requires both symbiotic bacteroid and plant cell systems.) End products of high selective advantage produced by complex metabolic pathways {e.g., certain alkaloids, toxins, steroids, lichenic acids) will tend to be products of more than one genome; that is, the nucleic acids coding for the polypeptides of the proteins of these pathways will reside in part in the organelle(s) as well as in the nucleus. The "biological clocks," or circadian rhythms so characteristic of and limited to eukaryotic organisms may well turn out to be the result of interaction between gene products and metabolites of the multigenomic systems making up eukaryotic cells (e.g., dyads and triads). 5.12. In vivo growth of organelles Photosynthetic plastids and mitochondria will be cultured in vitro. The growth requirements for their culture will tend to be gene products (proteins, peptides or coenzymes) and/or metabolites supplied to them by the in vivo cycloheximide sensitive 80S ribosomal system. Other organeUes (those that presumably had an endogenous rather than exogenous origin, e.g., Golgi apparatus; pigment granules;

277 lysosomes; nucle:i) will not be culturable. Organelles that originated by hereditary endosymbiosis will piove to be intrinsically replicating units, unlike the endogenous structures, which presumably differentiated from the rest of the cell.

greatly enhanced by an understanding of eukaryotic organellar genetics.

6. Implications of the S.E.T. for the overall classification of living organisms into five kingdoms

5.13. Protein homologies of the 80S ribosome By amino acid sequence analysis, eukaryotic 80S ribosomes will show protein homologies, indicating that they have more than one prokaryotic ancestor (e.g., anaerobic fermenter and heterotrophic eubacteria, host and protomitochondrion, respectively; anaerobic fermenter and spirochaete, host and flagellum, respectively; and so forth). Perhaps proteins of the 80S ribosomes have direct ancestry in certain spirochaete ribosomal proteins.

5.14. Anastomosing phylogenies Only after acceptance of the symbiotic theory for the origin of organelles, will we be able to construct consis'~ent phylogenies at higher taxonomic levels {such as those available for tracheophytes and chordates) that are acceptable to botanists, zoologists and microbiologists.

5.15. Hybridization between organelles and free-living microorganism DNAs Direct nucleic acid hybridization studies will reveal homologies between organisms and organelles as outlined in Fig. 4. The a m o u n t of unique sequence transcribable DNA per organelle will turn out to be an approximate inverse function of the age of the association. This hypothesis can also be applied to the nucleic acids of l:,artners in clear-cut examples of symbioses.

5.16. Organellar genetics and physiology: models for infection Understanding of the genetic interactions in symbioses and pathogenic relations will be

Whittaker (1959, 1969), on ecological grounds, has devised a sound scheme for the classification of all living organisms into five groups, one prokaryote and four eukaryote. The five-kingdom system of Whittaker was originally published with only the inclusion of the highest taxa: kingdoms and phyla. By applying the concepts generated by the symbiotic theory, Whittaker's scheme could be modified to form an overall classification consistent with information generated by genetic and developmental studies. This detailed classification as published in Margulis ( 1 9 7 4 c ) a n d in more detail in Margulis (1974a) is schematically drawn in Fig. 1, which outlines the hypothesized role of symbiosis in the origin of higher taxa as well as other familiar groups that have traits strongly influenced by regular symbiotic associations. Although Wallin {1927) certainly overstated the case, it is obvious that "symbionticism" has been a mechanism in the origin of new taxa; examples may be found on every level from species to phylum. The basic modified Whittaker five-kingdom scheme can be summarized as follows: monerans (all prokaryotes, or monads); protists {dyads and triads, diverse groups of asexual and eumitotic eukaryotes, ploidy levels and mitotic systems vary); fungi (dyads: amastigote haploid or dikaryotic forms, zygo, asco- and basidiospore formers that grow by absorptive nutrition); animals (dyads: diploid metazoans that develop from blastulas and grow by ingestive nutrition); plants (archegoniate e m b r y o p h y t e triad photoautotrophs). (For discussion of the monad, dyad and triad terminology, see Taylor 1974 and Margulis, 1975b.) This is not the place to detail the modified Whittaker classification but only to urge you to look up your organism in it. Is the system

278 consistent with y o u r own experience with the living form? Any comments would be appreciated. An alternative logical two-kingdom, prokaryote and eu k ar yot e , system has been suggested by Taylor (1974). This and several other possibilities have been discussed by Leedale (1974). Following Whitehouse (1969), however, it seems to me both logical and respectful of systematic tradition to keep the p r o k a r y o t e (chromonemal) versus eukaryote (chromosomal) distinction at the superkingdom level. Thus the plant and animal kingdoms would remain in a restricted form supplemented by three other kingdoms: monerans, protists and fungi. For the first time the mycologists themselves have suggested the t r e a t m e n t of fungi as a kingdom (or at least a subkingdom, they have hedged a bit) in their most recent t a x o n o m i c treatise (Ainsworth et al., 1973), although they have not separated out flagellated " l o w e r " forms from the amastigote line.

7. Evolutionary convergence in the microbial world

7. 1. The lesson o f Mesodinium: acquisition of photosyn thesis by eukaryotes It is a well know observation in evolutionary biology that severe environmental restrictions produce, by convergence, similar structures in r e m o tely related organisms, for example: wings for aerial flight in bats, birds and insects; spores resistant to desiccation in clostridia, m y x o c o c ci, cellular slime molds and ascomycetes; spines and thorns for p r o t ect i on in roses and lobsters; protective bold black and white color patterns in clownfish and zebras; stromatolites by filamentous blue green and green algae; and so forth. Two o f the most significant sources of selection pressure in nature are (1) lack of carbohydrate and other organic food sources and (2) lack o f sufficient motility to escape potential predators and to migrate from inadequate to optimal habitats. Those organisms best

equipped to photosynthesize are often the least equipped to move. The formation of a motile p h o t o s y n t h e t i c unit by the joining of a fastmoving organism to a p h o t o a u t o t r o p h has repeatedly happened t h r o u g h o u t the course of evolution. In certain cases, such as the photosynthetic paramecia, hydroids or mollusks (Fig. 5), it is obvious that the host is fundamentally heterotrophic, and the motile-photosynthetic complex is a p r o d u c t of symbiosis. In other cases, such as Cyanidium, Cyanophora and all eukaryotic algae, the m et hod of origin of the motile photosynthesizer is not so clear. What has becom e increasingly evident in recent years, primarily due to ultrastructural analyses, is the prevalence of the p h e n o m e n o n of the acquisition of photosynthesis in motile organisms by symbiosis in unrelated forms. Table 1 summarizes some of this information. Mesodinium (Fig. 5b), a functionally photosynthetic ciliate, provides us with an example• Since it reddens the sea water and its distribution is ubiquitous, it has often been seen. This highly successful microorganism was noted by Charles Darwin off the coast of Chile in about 1839. It is possible that it was first seen by van Leeuwenhoek at Scheveningen, July 27, 1676 (Blackbourn et al., 1973). I went to the seaside at S c h e v e n i n g e n . . . and viewing some of the Sea-water very attentively . . I saw in it a little animal that was blackish, looking as if it had been made up of two globules• This creature had a peculiar motion, after the manner as when we see a very little flea leaping upon a white paper (yet at every leap it moved only about the length of a coarse sand grain); so that it might very well be called a Water-flea; but it was by far not so great as the eye of that little animal which Dr. Swammerdam calls the Water-flea [Daphnia]. Letter 18, 1676, Dobell (1958) Unlike Paramecium bursaria, Stentor polymorphous and Paraeuplotes tortugensis, (which are all photosynthesizing ciliates), Mesodinium does not contain chlorella-algae. Rather, every individual of the species rubrum contains what appear to be partial crypto•

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279 monad photosynthetic symbionts. Mesodinium rubrum fu:¢thermore seems to be swimming backwards and eating little (compared to other mesodinia), suggesting that the association with the provident photosynthesizer has led to hereditary alterations in the lifestyle of M. rubrum. The reduction of the ingestion apparatus and t:ne tendency towards greater autotrophy (relative to their nonphotosynthetic relatives} has happened convergently (where known) in most of the organism listed in Table 2. The acquisition of photosynthesis by heterotrophic flagellates that subsequently became

the various groups of eukaryotic algae may be seen as one example of a c o m m o n process. The evolutionary origin of Mesodinium (as well as the other entries in Table 1) are further examples. The merging of photosynthetic and motile organisms to form new complexes has happened at many times and places from the precambrian until recent times. 7.2. Natural history repeats itself: bacterial attachment sites

The basal b o d y and root system of cilia and flagella has been an enigma since its discovery

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Fig. 5. Functionally photosynthetic organism derived from nonphotosynthetic motile and photosynthetic immotile ancestors. (a) Pelochromatium, bacterial consortium in which large flagellated heterotrophic bacteria are covered with small photosynthetic bacteria. (structure at the limit of resolution of the light microscope) (Courtesy of James Staley) (b) Mesodinium rubrum, a ciliate, harboring "incomplete photosynthetic cryptomonads." (Courtesy of F.J.R. Taylor, see Taylor, 1974). (c) Elysia viridis, a gastropod mollusk, harboring functional chloroplasts (below) ultimately derived from siphoneous food algae. (Courtesy of Robert Trench).

in t h e late 1 9 t h c e n t u r y (see Wilson, 1925 f o r review o f early w o r k ) . All e u k a r y o t e cilia and flagella, w h e t h e r o f s e n s o r y cells, ciliates or s p e r m , originate f r o m these characteristic 9 + 0 basal bodies. L w o f f {1950) t h o u g h t t h a t these organelles divide a u t o n o m o u s l y . Careful elect r o n m i c r o s c o p y (Allen, 1 9 6 9 ; Dippell, 1 9 6 8 ,

1 9 7 5 ; a m o n g o t h e r s ) has s h o w n w i t h o u t question t h a t m o s t basal b o d i e s d o n o t divide. T h e y are d i f f e r e n t i a t e d f r o m a m o r p h o u s " g r a n u l a r fibrillar" m a t e r i a l in various f o r m s a n d n u m bers, d e p e n d i n g o n the o r g a n i s m . In s o m e cases (e.g., a m o e b o f l a g e l l a t e s ) basal b o d i e s a p p e a r " d e n o v o , " t h a t is, f r o m no p r e e x i s t i n g recog-

283 'FABLE 2 E v o l u t i o n a r y s t r u c t u r a l and f u n c t i o n a l c o n v e r g e n c e s in microbial systems. .

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Type of Environmerit

Convergence

E x a m p l e s of Organisms

spores

soil; tissues and cells myxoeocci of o t h e r organisms clostridia; bacilli; slime molds; s p o r o z o a ; a p i c o m p l e x a ; fungi

sporebearing struttures

m y x o c o c c i (e.u:., Polya ngiu m ) ; colonial ciliate; fungi

bacterial attachm e n t sites

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Selection Pressures .

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References

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desiccation; nutrient depletion

Brock, 1970

soil

desiccation and dispersal

Olive, 1 9 7 5 ; Brock, 1970

spirochaetes; unidentified b a c t e r i a with o t h e r bacteria or protists

hindl~uts o f roaches and t e r m i t e s ; sand grains; ( p s a m m o phile) ciliates; w o r m cuticles

hold fasts ( s e p a r a t i o n from h o s t )

Raikov, 197,t; B h ) o d g o o d , 1974

mycelia

slime m o l d s ; actinom y c e t e s fungi

soil; cells and tissues of o t h e r organisms

p e n e t r a t i o n ; increase of surface area

stromatolites

blue green algae; c h l o r o p h y t e and o t h e r e u k a r y o t i c algae

intertidal p h o t i c z o n e

s t a b i l i z a t i o n of local e n v i r o n m e n t against t u r b u l e n c e , etc.

G o l u b i c , 1973; A w r a m i e et al., 1975

nutrient depletion

Table 1

a c q u i s i t i o n of photosynthesis by motile organisms

bacterial c o n s o r t i a ; P a r a m e c i u m bursaria, Glaucocystis: Mesodiniu m

lake m u d s ; o p e n

sessile " a d u l t " b u t motile "larva" (hold fast.s)

Caulobacter; s u c t o r i a n ciliates; c h y t r i d s , (e.g. Blast oclad icl la ) mollusks ; t u n i c a t e s

surfaces

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o c e a n

nizable structure. In others they appear at regular positions in regular relations to preexisting basal bodies (e.g., the ciliate cortex). In some cases there is one old basal body for each newly emerging c ne. In other cases they appear as multiples; for example, in the formation of some plant sperm hundreds to thousands may be made at once. There is no evidence that the "reproduction" of basal bodies is an event that requires DNA synthesis (Younger et al., 1972); however, new RNA synthesis is required, and it is thought by some that a specific class of basal body RNA exists (For discussion of these complex issues see Hartman et al., 1974; Younger et al., 1972 and Frankel, 1974).

altering e n v i r o n m e n t a l cond i t i o n s (larval dispersion to o p t i m a l e n v i r o n m e n t for adult development)

The basal bodies and their axonemes (cilia and flagella shafts) have been hypothesized to have originated from free-living spirochaetelike microbes; it is this postulate that distinguishes the "extreme S.E.T." from all other versions of the cell symbiotic theory. The evidence for this concept has been stated previously (Sagan, 1967; Margulis, 1971) and will not be repeated here. This discussion centers on the principle that nature repeats itself, that given the appropriate environmental selection pressures, comparable convergent structures will evolve. Based on this principle, one would expect that organelles of motility have evolved from free-living spire-

284

chaete bacteria by convergence several times, although perhaps in just one ancient population of microbes ancestral to eukaryotes did

:4,

mitosis and the typical basal body (9 + 2) complex originate. Many protists form associations of greater or lesser intimacy with motile bac-

285

Fig. 6. Bacterial att~chments to protists. (a) Unidentified bacteria on the surface of Urinympha, a hypermastigote from the hindgut of a termite. (Courtesy of Robert A. Bloodgood, Yale University) (b) Bacteria on the surface of Pyrsonympha, a polymastigote from the termite Reticulitermes flavipes. (Courtesy of Robert A. BIoodgood, Yale University) (c) Unidentified spirochaete attached to the surface of Pyrsonympha. (Courtesy of Robert A. Bloodgood, Yale University) (d) Rootlet of the spirochaete, thought to anchor it to Pyrsonyrnpha. Part o f t h e microtubular axostyle of the host can be seen. (Courtesy of Harriet Smith, University of Chicago) (e) Spirochaete attached to surface of Pyrsonympha from Reticulitermes tibialis showing the specialized attachment region (A). (Courtesy of Robert A. BIoodgood, Yale University) (f) Bacterial attachment sites on the surface of host protist Barbulanympha. (g) Detail of Fig. 6f. (Courtesy of Dr. A. Hollande, Laboratoire de l'Evolution des etres organises, Paris) (h) Unidentified bacteria attached to the sand ciliate Kentrophoros (Courtesy of I.B. Raikov, Institute of Cytology, Leningrad)

teria. Examples of bacterial attachment structures (which clearly evolved by convergence) are shown in Fig. 6. These may be analogous to

ancestral basal bodies. In this connection, the similarity between the spirochaete attachment "rootlets" on Pyrsonympha (Fig. 6 c and d)

286 and eukaryotic cilia rootlets (e.g., gill cilia Fig. 7 a,b) is striking. Basal bodies (which are t h o u g h t to have subsequently evolved into mitotic centrioles for reasons outlined in Margulis, 1970) began perhaps as bacterial attachment sites. In some groups of organisms (e.g., Mixotricha) continued association led to the acquisition o f motility, conferred on the host by the symbionts. In the line of organism leading to eukaryotes, it is postulated that this association went much farther: the microtubules of the microtubule-containing spirochaete were utilized for axopod, axonem e and axostyle structure, sensory behavior, mitosis, intracellular pigment transport and many other microtubule-based eukaryotic cell prosesses. Again, the first description of symbiotic spirochaetes was given by van L eeuw e nhoe k: My e x c r e m e n t being so thin, I was at divers times persuaded to examine it; and at each time I kept in mind what f ood I had eaten, and what drink I had drunk, and what I found afterwards: but to tell all my observations here would make all too long a story... I have also seen a sort of animalcule that had the figure of the river eels: these were in very great plenty, and so small withal, that I deemed 500 or 600 of 'em laid out end to end would not reach to the length of a full grown eel such as there are in vinegar. These had a very nimble motion, and bent their bodies serpentwise, and shot through the stuff as quick as a pike does through the water. Letter 34, 1681, Dobell (1958) Accordingly, I t o o k (with the help of a magnifying mirror) the stuff of f and from between the teeth further back in my m o u t h where the heat o f the coffee c o u l d n ' t get at it. This stuff I mixt with a little spit out of my m o u t h (in which there were no air bubbles), and I did all this in the way I've always done: and then I saw with as great a w o n d e r m e n t as ever before, an inconceivably great n u mbe r of little animalcules.., the whole stuff seemed alive and a-moving... And I saw, too, sundry animalcules... These moved their bodies in great bends so swift a motion, in swimming first forwards and then

backwards, and particularly with rolling around on their long axis, that I coul dn't but behold t hem again with great w o n d e r and delight: the more so because I hadn't been able to find t hem for several years, as l've already said. Letter 75, 1692, Dobell (1958) The hypothesis that basal bodies originated as spirochaete a t t a c h m e n t sites and that the ciliary root l et systems arose from bacterial root l et systems analogous to those of the spirochetes of Pyrsonympha (Smith and Arnott, 1974) is certainly testable (Fig. 7) as noted above. For example, I predict that the RNA of basal bodies will show hom ol ogy in the primary sequence of nucleotides to one of the RNAs of the appropriately chosen population of spirochaetes.

7.3. Evolutionary convergences: cysts, spores and fruiting bodies The many mechanisms by which groups of organisms resist desiccation and insure dispersion are well known. That only very distantly related microorganisms arrive by convergence at the same sorts of "fruiting b o d y " structures can be seen directly by examination of Fig. 8. Again van Leeuwenhoek was the first to observe the adaptions o f microbial life to drying and rewetting. Now since we see that these animalcules can lie bedded so long in dry matter, as before described, and then on coming into water can sweel out their bodies, and swim off, we may therefore conclude that in all pools and marshes, which have water standing in them in winter but which dry up in summer, many kinds of animalcules ought to be found; and even though there were none at first in such waters, they would be brought thither by water fowl, by way of the mud or water sticking to their feet and feathers. Letter 147, April 1702, Dobell (1958) The discussion in this section illustrates that under the same sorts of selection pressures, convergent structures, behaviors and complexes of organisms originate. The microbial world contains processes precisely analogous to those occurring among larger organisms,

287

Fig. 7. Electron micrographs of cilia and ciliary rootlets from the gill of the fresh water mollusk Elliptio (a) X 51,000 (b) X 90,000. (Courtesy of Fred D. Warner, Dept. of Biology, Syracuse University)

288

Fig. 8. Convergent evolution of fruiting structures in microorganisms. Light microscope views of live material. (a) Rhizopus sporangium (zygomycetous fungus, eukaryote). (b) Myxococcus fulvus (fruiting bacterium, prokaryote). (Courtesy M. Dworkin and H. Reichenbach, University of Minnesota) (c) Chondromyces apiculatus (fruiting bacterium, prokaryote). (Courtesy M. Dworkin, University of Minnesota) (d) Unidentified colonial ciliate (eukaryote). (Courtesy Lindsay Olive, University of North Carolina).

including precedents for those events hypothesized to occur in the serial endosymbiotic origin of eukaryotes.

8. Summary The purpose of a scientific theory is to unite apparently disparate observations into accurate generalizations with predictive power. Historical theories, which necessarily treat complex, irreversible events, can never be tested directly. However, they certainly can lead to predictions. The " e x t r e m e " version of the serial endosymbiotic theory states that three classes of eukaryotic organelles: mitochondria; basal

bodies/flagella/cilia [(9 + 2) homologues] ; and photosynthetic plastids all were derived from free-living ancestors. This hypothesis is supported by microbial precedents. The symbiotic origin of photosynthesis in motile heterotrophic microorganisms is hypothesized to have happened convergently several times, as did the development of specialized attachment sites for motile spirochaetes. Even if the cell symbiosis theory turns out to be wrong, it has the advantage of generating a large number of unique and experimentally verifiable predictions. The collection of such new information can do no less than add to our knowledge of the evolution of microorganisms, many of which were first seen by van Leeuwenhoek.

289

Acknowledgements M a n y ideas here were d e v e l o p e d in discussions with J.K. Kelleher, S. G o l u b i c , C. Harw o o d , L. To, R o b e r t T r e n c h and m e m b e r s o f m y symbiosis class. I t h a n k M. Karakashian f o r excellent c o m m e n t s on the m a n u s c r i p t and J.R. Williams and B. Miranda f o r editorial assistance. I a c k n o w l e d g e with gratitude research s u p p o r t f::om N A S A ( N G R - 0 0 4 - 0 2 5 ) and travel s u p p o r t to a t t e n d the van Leeuwenh o e k S y m b o s i u m w h i c h c a m e f r o m the Koninklijke A k a d e m i e van W e t e n s c h a p p e n , Amsterdam.

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