The evolutionary origin and phylogeny of microtubules, mitotic spindles and eukaryote flagella

BioSystems, 10 (1978) 93--114 © Elsevier/North-Holland Scientific Publishers Ltd.

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THE E V O L U T I O N A R Y ORIGIN AND P H Y L O G E N Y OF MICROTUBULES, MITOTIC SPINDLES AND E U K A R Y O T E F L A G E L L A T. C A V A L I E R - S M I T H

Department of Biophysics, University of London King's College, 26--29 Drury Lane, London WC2B 5RL, England

1. Introduction Microtubules and organelles composed of them are one of the fundamental distinguishing featues of eukaryotes and are thought to be absent in prokalTotes (Stanier, 1970). I have suggested (Cavalier-Smith, 1975) that the evolution of microtubules and o f the actinmyosin system was the key event in the origin of eukaryotes. Microtubules consist mainly of a single protein, tubulin, which can undergo selfassembly in the absence of other macromolecules to form a microtubule (Stephens and Edds, 1976; Goldman et al., 1976; Jacobs and Cavalier-Smith, 1977). (Structures superficially similar to microtubules have been reported in a few prokaryotes (e.g., Jensen and Ayala, 1976) b u t there is no reason to think that they are homologous with eukaryote tubulin,containing microtubules). As predicted by Porter (1966) microtubule assembly in cells is regulated by specific nucleation sites, on which microtubules are assembled in a polar fashion. In this paper I discuss the evolution o f microtubules and microtubule nucleation sites with special reference to the evolution of the mitotic spindle and eukaryote cilia and flagella. My basic argument is that microtubule nucleation centres evolved from the RNA cores of prokaryote chromosomes and that spindle microtubules evolved before eukaryote flagella and not vice versa. Nucleation centres and microtubules both underwent evolutionary diversification as a result of a series of gene duplications and formed numerous different organelles including eukaryote 9 + 2 flagella. Though at this stage one cannot

expect to p u t forward a detailed phylogeny of eukaryote microtubular organelles that has much chance of standing up in the face of the discoveries likely in the next decade or two, the hypothesis proposed here is sufficiently specific to be refuted by future observations, and may help to focus attention on some key problems. Many previous authors have discussed the evolution of mitosis (e.g., Pickett-Heaps, 1969, 1974, 1975; Margulis, 1970, 1974a and as Sagan, 1967; Kubai, 1975) but there are no previous detailed discussions of the evolution o f e u k a r y o t e flagella. Wallin (1927) postulated that they arose from symbiotic bacteria and Sagan (1967) suggested not only that ectosymbiotic spirochaetes, like those living on the flagellate Mixotricha, could have become transformed into eukaryote flagella, b u t that spindle and other microtubules (as well as centrioles and other mitotic centres) all evolved secondarily from eukaryote flagella. Though no detailed explanation was given of h o w spirochaetes could be transformed into flagella, or o f how flagella could give rise to non-flagellar microtubules, the symbiotic theory of the evolution of flagella and the spindle has, despite some criticism (Taylor 1974, Pickett-Heaps, 1974, Hartman, 1975}, become widely a d o p t e d (e.g., Maynard-Smith, 1975). It is therefore necessary to begin by explaining w h y I consider it untenable.

2. Difficulties of the symbiotic theory o f flagellar origins First it must be stressed that even if one

94 could show homologies between spirochaet axial filaments and/or bacterial flagella on the one hand, and eukaryote flagella on the other, this would not constitute proof of the symbiotic theory. This is because non-symbiotic and symbiotic theories b o t h postulate a prokaryote ancestor for eukaryotes; thus the persistence of any prokaryote feature in eukaryotes would not be suprising and can offer no evidence for or against either theory. A non-symbiotic theory deriving eukaryote flagella by direct descent from a flagellated bacterium or spirochaete is in principle possible; such a theory would differ from the symbiotic one by supposing that the nucleus

100nm I I 9"1-2 structure

was derived from the s a m e organism as the flagella; a possible ancestor for a non-symbiotic eukaryote origin, i f there were tubulinflagellin homologies, would be an ancestor of blue-green algae still retaining bacterial flagella (like a photosynthetic bacterium) but having evolved a blue-green algal photosynthesis. Anyone attempting to derive eukaryote flagella from bacterial flagella or spirochaete axial filaments, whether through a symbiotic o r a non-symbiotic mechanism, must explain how this could have occurred. But these prokaryote and eukaryote flagella differ so profoundly in structure, chemistry and

~ central f ~ , i ~ p . ~ k flagellar pair outer ~f l~l~h/~t •.~1 surface I /doublet it ~ ' . ~ . ' ~ l! coat spokesI armsA-~ "~'~!~:rJ/ ~ l/ l~'~"

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Fig. 1. Diagram of the flagellum, transitional region and basal body of the green alga Chlamydomonas reinhardii. The 9 + 2 axoneme and basal body structures are typical of eukaryotes. The transitional region 9-pointed star is found only in the Haptophyta, Chlorophyta, Bryophyta and Tracheophyta. (From Cavalier-Smith, 1974b).

95 9 + 2 region alone, excluding the transitional region and basal bodies) there are 130 different polypeptides (100 in the axoneme itself) (Piperno et al., 1977), presumably each specified by a different gene. Though methods are now available for isolating basal bodies (Wolfe, 1972) and transitional regions, very little is known of their chemistry; one would expect them to contain many additional proteins. By contrast bacterial flagella are very simple, the filament usually consisting of a single polypeptide, flagellin. The hook consists of a second polypeptide and the bacterial basal body of only 9 polypeptides (Hilmen and Simon, 1976). Some spirochaete axial filaments also consist of only one polypeptide While others have three or six, though it is not known whether more than one peptide is found in individual filaments (Canale-Parola, 1977). The physiological differences are equally profound. Eukaryote flagella work by microtubule sliding, mediated by crossbridges projecting from the outer doublets (the arms and spokes); this is powered by ATP hydrolysed by the ATPase dynein, which forms the arms; the membrane is not directly necessary for motility (Warner, 1976). Bacterial flagella work by rotation, have no crossbridges and lack ATPase on the filament {Adler, 1976}. Escherichia coli flagella cannot use ATP at all, being powered directly by an intermediate of oxidative phosphorylation generated in the plasma membrane in which the basal body is embedded; but purely fermentative bacteria,

physiology that no relationship between them can be discerned. The eukaryote flagellumbasal body complex consists of an axoneme that is fundamentally intracellular, being on the cytoplasmic side of the flagellar membrane, an extension of the plasma membrane which encloses both the :[lagellar axoneme proper and the transitional[ region (see Fig. 1). By contrast neither bacterial flagella nor the spirochaete axial filament are intracellular; the two organelles axe homologous; each consists of a long helical flagellar filament, a short hook (both outside the plasma membrane), and a short basal body which is partly outside the plasma membrane and partly embedded iin it (see Fig. 2). The major difference between ordinary bacterial flagella and the filaments forming the spirochaete axial filament is that in spirochaetes the hook and filament are found within the periplasmic space (i.e., between the plasma membrane and the outer cell wall layers), whereas in ordinary bacterial flagella they emerge to the exterior through a hole in the cell wall. Though flagella have very little, structurally and topologically, in common with eukaryote axonemal microtubules, they do resemble slightly the extracellular tubular mastigonemes that clothe the external surface of many eukaryote flagella (Bouck,1972); but in view of the many detailed differences this resemblance al,;o is probably quite superficial. The chemistry of eukaryote flagella is complex. In Chlamydomonas flagella (the

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Fig. 2. (a) S t r u c t u r e o f t h e basal e n d o f a b a c t e r i a l flagellum a n d its i n s e r t i o n in t h e cell m e m b r a n e a n d cell wall. (b) S t r u c t u r e o f t h e basal e n d o f a c o m p o n e n t f i l a m e n t o f a s p i r o c h a e t e axial f i l a m e n t .

96 including anaerobic spirochaetes, must presumably use ATP. Spirochaete motility is thought to result from rotation of the filaments composing the axial filament (Berg, 1976). Tubulin is a heterodimer of two different polypeptide chains, a and/3, each of mol wt. 55 000. The similarities o f the properties and amino acid sequences (Luduena and Woodward, 1975) on the a- and ~-chains show that they must have resulted from a gene duplication long ago. Since all tubulins so far studied have a- and ~-chains this gene duplication must have been one of the earliest events in evolution o f microtubules. The tubulin sequence data are still t o o meagre for proper comparison with possible ancestral molecules such as flagellin or actin, both of which consist of single polypeptide chains. The short sequence so far available has no obvious homologies with either of these. Tubulin shows no close similarities with flagellin (e.g., tubulin contains bound guanine nucleotides and its assembly is favoured by GTP and Mg 2+ and inhibited by Ca 2+ above 2 mM or by colchicine; none of these is true of flagellin), though there are certain similarities with actin, notably the ability to stimulate myosin ATPase activity (Alicea and Renaud, 1975). In view of the apparent lack of homology between flagellin and tubulin, and the fundamental difference in the structure and physiology of eukaryote and prokaryote flagella, the suggestion that a periplasmic spirochaete axial filament coded by 1--6 genes directly gave rise to an intracellular eukaryote flagellar axoneme coded by 150 different genes raises a great many problems, without solving any. If instead one tries to derive the flagellum from a whole spirochaete the problems are equally great. The only resemblance between a flagellum and a spirochaete is that they are similar in diameter, have a membrane and b o t h wiggle. To postulate that a spirochaete lost its wall, fused its membrane with the host plasma membrane, lost its ribosomes and DNA (possibly transferring some of the latter

to the host cell's chromosome) and either internalised its axial filaments and used them as a basis for a flagellar axoneme or lost them and developed 9 + 2 axonemes from some hypothetical intracellular proteins, greatly complicates rather than simplifies the problem of eukaryote flagella origin. All the 'predictions' of the symbiotic theory mentioned by Margulis (1975), e.g., homology of tubulin from flagella and spindles, are equally compatible with non-symbiotic theories.

3. What were the evolutionary precursors o f flagella? I assume that cytoplasmic microtubules evolved before 9 + 2 flagella (henceforth flagella will mean eukaryote flagella unless otherwise specified) and that flagella subsequently evolved from them b y a gradual process involving many stages. The strongest argument that the direction of evolution was from microtubules to flagella and n o t vice versa is that a flagellum is a b o u t 100 times as complicated as a microtubule. If allowance is made for proteins specific to the transitional region and basal b o d y , it is likely that the flagellum/basal b o d y complex contains over 200 different polypeptides (and therefore is coded by over 200 different genes), whereas the basic structure of a microtubule consists of only t w o polypeptides (a- and ~-tubulin) which are clearly descended from a single ancestral polypeptide. (Though microtubules do contain additional proteins, these are n o t essential to their basic structure though they may be important for function or controlling their assembly.) The outer doublet microtubules of flagella a r e structurally more complex than singlet microtubules; furthermore the ~- and ~-tubulins in the A-tubule (the aA~A tubulin) both have a different primary structure from those in the B-tubule (~B~B tubulin) and therefore must be coded by different genes; the A- and B-tubulins are, however~ so closely related that they must be derived by gene duplication from a c o m m o n

97 ancestor (Stephens and Edds, 1976). If the existence of doublet tubules depends on a prior gene duplication, then singlet microtubules must be ancestral to doublet microtubules. At some stage therefore there must have been evolution from singlet to doublet and triplet microtubules. When, and in what organisms, did this occur? No tubulin-containing cytoplasmic microtubules, whe~Lher singlet, doublet or triplet have been observed in any prokaryotes, so it is unlikely (though n o t impossible) that it occurred before the origin of eukaryotes. Singlet microtubuh;s have been observed, at least in the spindle, in all eukaryotes that have been studied carefully. Therefore singlet microtubules must either have originated in the very first eukaryotes, or all early eukaryotes that failed to evolve microtubules became extinct. In any c ~ e , it must have occurred very early in eukaryote evolution (CavalierSmith, 1975). By contrast, flagella with their doublet microtubules and basal bodies and centrioles with their triplet microtubules, are not found in all eukaryotes and are totally lacking in several major groups. This could be either because they have been lost during evolution or because the ancestors of the present organisms never had them. In some cases ,(e.g., gymnosperms other than the cycads, and the angiosperms) it is certain that t h e y have been lost as an adaptation to terrestrial life. But is it sensible to explain their absence in all groups in this way? Much eukaryote diversification could have occurred before the evolution of flagella and centrioles; the absence of flagella and centrioles in m a n y groups shows that neither is essential for basic eukaryote cellular processes. Since these organelles are so m u c h more complex than microtubules, and there is no evidence for their occurrence in prokaryotes, it is unlikely that they could have evolved very quickly. Nor would their evolution necessarily lead to the extinction o f all eukaryotes lacking flagella, since we know that m a n y non-flageUate organisms can compete effectiw.,ly with flagellates. We

might therefore expect that primitively nonflagellate eukaryotes should still survive. However, unless we can build up an accurate phylogeny based on characters other than flagella we cannot show that this is really the case. There are several eukaryote groups where there is, so far, no solidly based evidence for a flagellate ancestry: (1) Red algae (Rhodophyta), (2) ascomycete fungi, (3). basidiomycete fungi; (4) zygomycete fungi; (5) cellular slime moulds (Acrasida); (6) Cnidospora (myxosporean and microsporean protozoa). All other groups of non-flagellate eukaryotes (except perhaps some non-flagellate amoebae) clearly have flagellated relatives so could easily have become non-flagellate through loss of flagella. In these six groups however the uniform absence o f flagella suggests that t h e y each evolved from a nonflagellated ancestor. Though it is possible that one or more of these ancestors were themselves derived at some stage from flagellates by loss of flagella, it is simpler to assume that they were primitively non-flagellate, at least until there is clear evidence to the contrary. Christensen's (1962) division of algae into the primarily non-flagellate Aconta and the flagellate Contophora should be extended to non-photosynthetic organisms to become the primary subdivision of all eukaryotes.

4. The origin of microtubules and microtubule nucleation sites Symbiotic (Stanier, 1970) and non-symbiotic (Cavalier-Smith, 1975) theories of eukaryote origins both assume that a key step in the evolution of eukaryotes was the loss of the prokaryote cell wall and the origin of cytoplasmic motility based on actin and myosin. The loss of a cell wall would have led to strong selection pressures in favour of an improved internal cytoskeleton to stabilise the cell against environmental stresses. In modern eukaryote cells 6 nm actin micro-

98 filaments together with 24 nm microtubules are major components of the cytoskeleton (Goldman et al., 1976), though other proteins like the 10 nm skeletin filaments (Small and Sobieszek, 1977) are probably also important. I suggest that the initial function o f both actin and tubulin was cytoskeletal (Pollard, 1976) and that their involvement in active motility came later. The basic molecular requirements for a cytoskeleton are much simpler than for motility: the molecules need only to be able to polymerise to form a structure able to withstand mechanical stress and to be able to attach to specific sites. Simple F-actin filaments or microtubules consisting only of tubulin could act as a cytoskeleton, b u t additional components are required for motility. An ATPase (myosin or dynein respectively) is needed to provide energy and mechanochemical coupling, and proteins (e.g., troponin) for control o f motility. The ability to polymerise into a microfilament or microtubule is not a demanding requirement for a protein; a suitable geometrical arrangement of hydrophobic residues on the surface of the m o n o m e r is all that is needed. A variety of proteins other than actin can form filaments (e.g., flagellin, spectrin, spasmoneme filaments, skeletin) and molecules other than tubulin can polymerise as tubules (e.g., TMV coat protein, bacterial F-pili, mastigoneme proteins (Bouck, 1972), or even haemoglobin (Murayama, 1974). Many prokaryote proteins probably have the potential to mutate quite independently to make such structures; superficially similar 'microfilaments' and 'microtubules' need have no direct relationship. Prokaryotes do have a simple cytoskeletal matrix: though a detailed chemical analysis of this might reveal the ancestors of actin and/or tubulin (Neimark, 1976, has isolated an actin-like protein from Mycoplasma) their ancestry might lie in some non-skeletal prokaryote protein (e.g., elongation factor Tau, Rosenbusch et al., 1976). Whatever their ancestry, and whether or not tubulin and actin are related (Alicea and

Renaud, 1975), their origin presents no insuperable difficulties. The mechanical properties of microfilaments and microtubules are different and partly complementary. Microfilaments are good at bearing tension (and so would be an economic w a y of holding a cell together in the face of forces that might cause swelling or bursting) and can exert a pull; b u t individual ones cannot resist sizeable compressive or bending forces so cannot exert a push, though bundles of microfilaments can (Tilney, 1976). Because of their tubular design individual microtubules are more rigid and can resist compressive or bending forces with a maximum e c o n o m y of material, as well as being able to exert pulling forces, though with less e c o n o m y of material than microfilaments. In the postulated wall-less ancestor of eukaryotes there would have been strong selection pressures in favour o f a cytoskeleton with elements that could resist tension, compression and bending. Actin and tubulin may be universal in eukaryotes not because they are fundamentally better suited for their role than other possible proteins but simply because they were the first intraceUular proreins to evolve with these capacities. Selection for an efficient cytoskeleton would lead to mechanisms for controlling the assembly and disassembly of micro filaments and microtubules and the evolution of other molecules to form specific attachment sites (e.g., a-actinin, the usual attachment site for actin filaments). Both structures show polarity, so attachment sites for their two ends must differ; c o m m o n l y they are attached only at one end. There is evidence that microtubules grow b y the assembly o f tubulin monomers at their free ends (Stephens and Edds, 1976). After the establishment o f these basic properties, the way would have been open for the evolution o f proteins specifically involved in motility like myosin or dynein, the new selective pressures thus opened up leading to the evolution of the eukaryote cell (Cavalier-Smith, 1975). The early involvement

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Fig. 3. Suggested divL,;ion mechanism in the ancestor of eukaryotes prior to the evolution of the nucleus and separate chromosomes. The two daughter chromosomes are pushed apart by the elongation of microtubules growing from their RNA cores (primitive precursors of centrosomes and kinetochores, still attached to the protonuclear envelope deriw-~d by endocytosis from the plasma membrane) (Cavalier-Smith,1975). The arrowheads show the direction of microtubule growth.

of microtubules in chromosome segregation (Pickett-Heaps, 1975; Cavalier-Smith, 1975) can have been possible only if they were attached to the chromosomes in some way. I suggest that their attachment was n o t to the DNA itself b u t to the R N A core upon which a bacterial chromosome is folded (Worcel and Burgi, 1972). The R N A chromosome core would serve both as an attachment site and as a nucleation site for microtubule assembly, which could reguhte the number, location and orientation of microtubules in the cell. Even if the growing end was n o t attached to any specific structure, and no dynein or other crossbridges had y e t evolved, the chromosomes could be segregated b y the force generated by the g r o w t h of the microtubules (Inou~ and Sato, 1967) as they push against the cell surface, so long as the cytoplasm contained an actin meshwork or other gel structure (Porter, 1976) that could preserve the orientation of the R N A cores and microtubules as shown in Fig. 3. A prerequi,+ite for such a mechanism is the initial antiparallel arrangement of RNA cores. There is evidence for the presence of R N A associated with kinetochores, basal bodies, and centrosomes (Hartman, 1975; Dippell, 1976) artd that the R N A of basal bodies is essential for their ability to nucleate aster formation in Xenopus eggs (Heidemann et al., 1977).

5. The evolution o f mitosis Because the bacterial plasma membrane is thought to be involved in chromosome segregation*, there has been a tendency to assume (Pickett-Heaps, 1975; Kubai, 1975) that in modern eukaryotes attachment of kinetochores, or of mitotic spindle pole bodies, to the nuclear envelope, and the persistence of the nuclear envelope during mitosis, are primitive features. There has also been a tendency to try to place m o d e m mitotic variants in a linear progression (e.g., Kubai, 1975; Margulis, 1970) from primitive to advanced; I suspect that this approach is mistaken. In other areas there is evidence that evolution is not a linear progression b u t involves divergent adaptive radiations and specialisations, which may include loss and simplification, and partial reversal of evolutionary trends, as well as increased complexity. The complex and patchy taxonomic distribution of the various mitotic characters summarised by Kubai is just what one would expect from such a view. This does n o t mean that mitotic characters have no phylogenetic significance (the evidence * Evidence for the release of chromosomes from the membrane after the end of replication (Jones and Donachie, 1974) is not easy to reconcile with this idea.

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is quite the reverse), but that we cannot build a phylogeny and identify ‘primitive’ characters by considering mitosis alone or by imposing a linear pattern of evolution on the data. A phylogeny should be consistent with the taxonomic distribution of all characters. In trying to construct such a phylogeny (section 8) I have been driven, against my initial preconceptions, to the conclusion that the simplest evolutionary hypothesis is that all present day eukaryotes are descended from an organism having both chromosomal and polar microtubules. As Kubai points out, the apparent absence of chromosomal microtubules is often because of technical difficulties in revealing them. The only entire phyla apparently lacking them are the (related) Euglenophyta and Kinetoplastida; if careful serial section studies confirm their absence here, it could be more simply explained by loss than by supposing these groups to be closest to the ancestral eukaryotes. Thorough examination of mitosis in simple eukaryotes (e.g., Peterson and Ris, 1976; Heath and Heath, 1976) often reveals chromosomal microtubules. All mitotic mechanisms can be derived by modification of that shown in Fig. 4. In some closed spindles the centrosome is attached to the nuclear envelope (e.g., yeast (Byers and Goetsch, 1974), probably the most primitive condition), in others the kinetochore is (e.g., dinoflagellates (Kubai, 1975)): in the many organisms with open spindles, or with polar holes in the nuclear envelope, neither is. There are many indications that nuclear envelope behaviour is an evolutionarily labile character (Kubai, 1975; Fuller, 1976). Primitively, no centrioles would have been associated with the centrosomes, but after the evolution of flagella (see below) basal bodies commonly became secondarily attached to the centrosomes during mitosis as centrioles (Pickett-Heaps, 1975). The mutually perpendicular arrangement of parent and daughter centrioles is simply a relic of the arrangement first evolved in biflagellate algae.

Fig. 4. Model for the basic mitotic mechanism found in eukaryotes since the evolutionary duplication of microtubule and nucleation centre genes to produce separate kinetochores and centrosomes, and kinetochore and centrosomal microtubules. The microtubules (shown by double lines) grow away from their respective nucleating centres in the direction shown by the arrowheads and shorten in the reverse direction. Anaphase spindle elongation is produced by the relative sliding of centrosomal microtubules mediated by crossbridges and/or tubule growth (in contrast to flagellar sliding, the interacting tubules here have opposite polarity); Metakinesis and the poleward anaphase movements of the chromosomes (where these occur, they are not universal in eukaryotes) are produced by sliding interactions between kinetochore microtubules and actin filaments attached to each centrosome with the same polarity; the rate of poleward movement is limited by the disassembly of the kinetochore tubules. (Considerations of microtuble polarity, and of symmetry of the two centrosomes, require different mechanisms for spindle elongation and poleward anaphase chromosome movement; there is also experimental evidence for this, McIntosh et al, 1976). Kinetochores and centrosomes would be duplicated in interphase. Such a spindle could have evolved before or after the nuclear envelope, shown here only as small membrane fragments attached to the centrosomes.

Since the precise manner of association of centrioles with the spindle poles varies in protists, it is likely that this attachment evolved several times. I suggest that the modem form of mitosis (as postulated in Fig. 4) evolved from the primitive eukaryote one (Fig. 3) concomitantly with the splitting of the single ancestral chromosome into several chromosomes (Cavalier-Smith, 1974a, 1975). This splitting

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could n o t have been successful in the absence of a corresponding multiplication of the R N A cores (or part of them) to form kinetochores in addition to the primitive centrosomes, because the chromosomes could not otherwise be segregated. The original association with the nuclear envelope would be retained b y the centrosomes but lost b y the kinetochores (I regard the association of kinetochores and nuclear envelope in dinoflageUates (including Syndinium ) and Trichonympha as a consequence of the re,evolution of a closed mitosis b y an ancestor already having basal bodies and an open spindle).

6. Diversification of microtubules and nucleation centres

Even in a single species there can be many kinds of microtubule that differ n o t only in location b u t in subtle structural, chemical or physiological ways. Though some of these differences could be attributed to posttranslational modification (Stephens and Edds, 1976), there is growing evidence for the idea that there are several species of tubulin. Apart from the differences between the Aand B-tubulins, Fulton and Simpson (1976) have evidence for structural differences between Naegleria flagellar and cytoplasmic tubulins, and Bibring et al. (1976) found differences between ciliar and flagellar tubulins. The differences in the properties and behaviour of kinetochore and interpole spindle microtubules can be easily explained if they consist of different tubulins. The same is true of the differences between the stable cortical microtubules found in many protists and the more labile spindle microtubules and between the central pair and the outer doublet microtubules of flagella. I predict that these and the, C-tubules of basal bodies will all turn o u t to have different primary structures and that even a simple protist like Chlamydomonas will turn o u t to have at least 7 different kinds of tubulin corresponding to the 7 classes o f microtubule: flagellar A-,

B- and central pair (cp) tubulins, basal b o d y C-tubulins, kinetochore and interpolar spindle tubulins, and stable cortical tubulins: the phycoplast and cytokinetic tubulins might be an eighth kind. Organisms like Paramecium with highly complex cortices could have many more. If, as preliminary evidence for the A- and B-tubulins suggests, the various tubulins of a single eukaryote species differ in both their a- and H-chains, then the original a- and ~genes must have undergone simultaneous duplications at various points in early eukaryote evolution. This could come about by duplication of the whole genome (CavalierSmith, 1975; Sparrow and Nauman, 1976). A more attractive explanation is that the aand H-genes have remained adjacent to each other on the chromosome ever since the original duplication that formed them. I suggest that this is so and that they form a single operon; sharing a single messenger would simply ensure that a- and ~-tubulins are produced in equimolar amounts. Duplication of the entire tubulin operon would allow the divergent evolution of the different tubulins. Divergence and the efficient functioning of different microtubule systems would depend not only on mutations in the tubulin structural genes, b u t also on divergence in the regulatory parts of the operons (e.g., flagellar differentiation in Naegleria specifically switches on transcription of flagellar tubulin genes) and in mutations in non-tubulin genes affecting the assembly and functioning of the microtubules. These other genes would include those coding for minor constituents of microtubules (e.g., microtubule associated proteins, tan factor (Jacobs and CavalierSmith, 1977)), for the numerous accessory structures that reach their highest development in flagella (e.g., dynein and nexin (Warner, 1976; Gibbons et al., 1976; Piperno et al., 1977)), and for the nucleating centres; many of these genes probably underwent duplication also. Microtubule nucleation sites are of key importance in understanding the inheritance

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of eukaryote cell structure (Beisson and Sonneborn, 1965) and division (Jacobs and Cavalier-Smith, 1977). They are necessary to control the location, orientation and numbers of microtubules and microtubule-based organelles like basal bodies (Cavalier-Smith, 1967, 1974b). I postulate that each kind o f microtubular structure has a distinct nucleation structure, b u t that all such structures are homologous with the RNA core of prokaryote chromosomes and are descended from it by gene duplication and divergence. Nucleation sites probably contain proteins as well as RNA, both being coded by nuclear DNA. RNA is ideally suited to form the core o f a nucleation structure. If a single molecule forms the core, the number of nucleation sites can be controlled simply by controlling the number of R N A molecules in the cell. It is in principle easier to control the number of RNA molecules than of protein molecules (or any other molecules apart from DNA; double stranded DNA would be unsuitable because, unlike single-stranded R N A it cannot fold up on its own to form specific and complex 3D structures). The number o f centrioles or other centrosomal structures and basal ~bodies in a cell normally doubles exactly every cell cycle. Many protists have a constant association between a pair (or tetrad) of basal bodies and a nucleus which can be very stable during mutation (Warr, 1968) and evolution (e.g., the evolution of multinucleate polymastigotes like Calonympha, or of multinucleate dinoflagellates like Polykrikos (Chadefaud, 1960), where the nuclei, basal bodies and other structures increase in direct proportion). This could be explained if there is a very tight coupling between replication and the transcription of nucleation centre RNA. I suggest that this coupling evolved in bacteria where RNA synthesis (possibly of the RNA chromosomal core) precedes and is an essential prerequisite for the initiation of DNA replication and that it simply persisted in eukaryotes after the chromosomal R N A core became transformed into the centrosome, the kinetochores and basal b o d y nucleation

sites. In animals centriole duplication occurs near the beginning of S phase; in yeast spindle plaque duplication precedes and is an essential prequisite for the initiation of chromosome replication (Byers and Goetsch, 1974). Furthermore, the yeast spindle plaque is twice as big in diploid as in haploid yeasts. My model would predict that permanent diploid strains of Chlamydomonas would have twice as much nucleation centre R N A as haploids. Once the basic mechanisms of microtubule assembly and nucleation had evolved there could be considerable adaptive radiation of microtubule-based structures. Apart from spindles, modern eukaryotes may have 9 + 2 flagella, non-9 + 2 flagella, haptonemata, axopodia, axostyles, pharyngeal baskets, suctorian tentacles and cortical microtubules (Tucker, 1977). The evolution of each of these would depend on coordinated changes in each of the following: (a) a specific tubulin; (b) a specific nucleating site; (c) specific accessory proteins forming part of the structure; (d) factors involved in controlling assembly and disassembly; (e) regulatory genes. Duplications and subsequent divergence of nucleation site genes would have been of k e y importance in allowing a great diversity of microtubule arrangements to evolve.

7. The Evolution o f 9 + 2 flagella Nine plus t w o flagella are so complex that their evolution must have involved many stages, new components being successively added to and modifying the preexisting structure. A rational explanation o f the origin of flagella must show h o w each stage could lead to the next and also h o w each stage functions and what its selective advantage to the organism is. In our present state of ignorance about the nature and functions of each flagellar component, one can only attempt this in outline.

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I suggest that flagella initially need not have been motile, but were slender cell extensions strengthened by a skeleton of ninesingle microtubules, ancestors of the A-tubules of m o d e m basal bodies and flagella. The evolution of such a protoflagellum would have been quite simple for an early eukaryote that has already evolved an extensive system of stable cytoskeletal microtubules in the cell cortex. Microtubules, nucleation centres and methods of attachment to the membrane by other structures would already have been present. The most basic change would be in the structures attaching the microtubules to the membrane; they must have mutated to allow attachment perpendicular to the membrane instead of being parallel as before. This would automatically ensure that microtubule growth would produce a finger-like projection from the membrane, a primitive filopodium. (a)

I assume that the first eukaryotes were benthic, phagocytic and photosynthetic 'protoalgae' (Cavalier-Smith, 1975), lacking both prokaryote flagella and gas-vacuoles and being able to move only by a lobopodial type of amoeboid motion. Though some would be washed into the open ocean, they could not colonise it effectively unless they could avoid too rapid sinking below the euphotic zone. Filopodial projections could greatly slow their sinking rate enabling greater photosynthetic production, and also serve to catch prey as in many m o d e m foraminifera, radiolaria, heliozoa and Chrysophyta, and would be strongly selected for; they might also help uptake of dissolved nutrients. The filopodia and the axopodia of these organisms, the haptophyte haptonema, and the suctorian tentacle are living examples of the use of strengthening arrays of singlet microtubules (b)

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Fig. 5. Possible intermediate stages of flagella evolution shown in longitudinal and transverse sections. (a) the protoflagellum, (b) a later stage after the evolution of arms, sliding motility and the centre pair, but before the evolution of doublet zad triplet microtubules.

104

to form slender cell extensions. Once the precambrian oceans were colonised b y such planktonic eukaryotes an extensive adaptive zone would have been opened up leading inevitably to an extensive adaptive radiation (Simpson, 1953). Organisms would evolve with a great variety of axonemal structures based on the single microtubule and on a variety o f mechanisms of attachment to the cell membrane, as a result of the selection of mutations occurring in the microtubules, attachment mechanisms, and in the nucleation sites. Nucleation site mutations could alter the number and arrangement of microtubules. The protoflagellum was simply one of these with a nucleation centre having sites for the assembly of a ring of 9 microtubules. It is interesting that some h a p t o n e m a have nine single microtubules at their base (Sleigh, 1973) though these are not in a circle. Though there is nothing very unlikely about the number of nine, it is conceivable that haptonemata are phylogenetically related to the protoflagella postulated here and shown in Fig. 5a.; in the relaxed state they are tightly coiled, b u t extend when activated. Flagella axonemes have a strong tendency to coil up within the flagella membrane when slightly damaged; many o f the complex features o f flagella and basal bodies are clearly adaptations to give t h e m greater rigidity w i t h o u t impairing their motility. Flagella motility depends on microtubule sliding mediated b y crossbridges having ATPase activity. It is likely, though evidence is so far lacking, that axostyle and haptonemal motility depend on similar crossbridges; it is also possible that this is true for the sliding that occurs during anaphase spindle elongation (McIntosh et al., 1976), b u t anaphase sliding, unlike the other examples, would involve microtubules of opposite polarity. Present evidence is consistent with the idea that dynein crossbridges and ATP
(Gibbons et al., 1976), evolved b y gene duplication and diverging selection from a single dynein species that m a y have been associated with the microtubules even before the evolution of the nine~singlet protoflagellum. Nexin, which cross-links the outer doublets, could also have been present then. The evidence from axostyles and haptonemata suggests that even such a simple structure consisting of single microtubules, crosslinks and ATPase could propagate bending waves and so be a functional flagellum. Subsequent selection for greater mechanical stability and rigidity, and ease of control of motion, could have led to the addition of B-tubules to form 9 outer doublets and o f a central pair o f tubules. The fact that the various outer d o u b l e t accessory structures (2 arms, radial spoke and nexin) are attached to the A-tubules only (Warner, 1976), and that the B-tubule (unlike the A-tubule) is n o t a complete 13 subunit microtubule, fits in with the idea that the B-tubule was a fairly late evolutionary addition. In the basal b o d y t o o it is the A-tubules that bear the A-tubulefeet to which the cartwheel spokes are attached; these structures could have evolved as a strengthening of the basal b o d y before the B- and C-tubules were formed (Fig. 5b). During basal b o d y development A-tubules are c o m m o n l y assembled first (Cavalier-Smith 1967, 1974b; Dippell 1968) and the young Atubules may be held together b y a temporary link until the A- and C-tubules are formed; a similar link could have been a permanent feature o f the nine-singlet protoflagellum. The central pair and its various projections could also have evolved before the B- and Ctubules; a new nucleation site would have been needed in the flagellar transitional region, which could have evolved from one of the cytoplasmic nucleation sites. It is notew o r t h y that there is cytochemical evidence for R N A in the ciliate axosome (Dippell, 1976) whose location is exactly what one would expect for a central pair nucleation site. The bald-2 flagella m u t a n t of Chlamydomonas (Goodenough and St. Clair, 1975) that lacks B- and C-tubules, b u t still makes an

105 otherwise complete basal b o d y and a defective transition region, is similar to that postulated in Fig. 5b except for its instability; presumably this is the secondms~ result of adaptation of the A-tubules to the evolution of B- and Ctubules. Mutations in the basal b o d y nucleation site, and one or t w o more tubulin gene duplications, would have allowed the evolution of B- and C-tubules; until then the flagellum would have been rather fragile and less rigid than today. The addition of B-tubules must have caused selection to shorten and modify dynein I arms, n o w interacting with the Btubule, whereas the; radial spokes could have remained unchanged. As evolving flagella became more vigorously motile stronger attachments for basal bodies would have been selected; transitional fibres, striated fibers and microtubular rootlets probably arose very early. Too little is known about the functions o f the various minor components of flagella for more detailed speculation to be worthwhile. The sequence of events I have suggested is only one of several possible; I have simply tried to show that iit is n o t difficult to suggest h o w flagella could have evolved gradually from ordinary cytoplasmic microtubules b y a non-symbiotic mechanism. Once microtubules, microtubule nucleation sites, dynein and crossbridges had evolved eukaryotes were preadapted for the evolution o f flagella, which could have been rapid, given suitable selection pressures. If flagella evolved, as suggested here, from microtubule-containing filopodial cell extensions in primitive planktonic eukaryotes, there would from the beginning have been strong selection pressures for the ability to swin actively, and to control the direction of swimming in relation to environmental stimuli. It is likely that mechanisms of phototaxis (and perhaps also geotaxis and chemotaxis) evolved simultaneously with flagella; the prime need would have been to keep at the correct level in the euphotic zone. Adaptive taxes require a sensor and a means for the sensor to control the flagella. Such

mechanisms are found in prokaryotes, so it is likely that even the first eukaryote had the physical machinery preadapting it to the control of flagellar motility in response to environmental stimuli. In cilia of both Paramecium and the mussel Mytilus calcium controls the beat (Kung, 1976). An active calcium p u m p extrudes Ca 2+ from the cell maintaining a low internal level of calcium. Noxious stimuli open a voltage-sensitive Ca 2+ gate or channel (presumably in the flagellar and/or plasma membrane) allowing a passive influx of Ca 2÷, which is propagated as an action potential. In Paramecium this reverses, and in My tilus it stops, the ciliary beat. Margulis et al. (1976) suggested that calcium extrusion evolved "primarily for the intracellular stabilisation of the mitotic apparatus and other somatic microtubules". This is unlikely because active Ca 2÷ extrusion is found in E. coli (Tsuchiya and Rosen, 1975) and is probably a general property of all cells Whether prokaryote or eukaryote. Thus all cells are preadapted for the control of intracellular events b y Ca 2+ release, which is used to control the actin-myosin system and exocytosis, and perhaps also cell division (Berridge, 1976}, as well as ciliary motion. The inhibition of microtubule assembly by mM Ca 2+ probably simply results from the fact that they evolved in an intracellular milieu with very low Ca 2+ concentrations maintained b y the plasma membrane Ca 2÷ pump: Taxes require coupling between the sensors and the fiagellar/plasma membrane Ca 2+ channel. Most algal eyespots (Dodge, 1974) are part of the chloroplast; in many algae they are close to the flagella, b u t in green algae they are not--I suggest that communication is through a gap junction between the chloroplast envelope and plasma membrane. Eyespot structure in Chlamydomonas (Cavalier-Smith, 1976), in which Ca ~+ has been shown to couple flagellar reversal to photostimulation (Schmidt and Eckert, 1976) is consistent with this suggestion. Euglenoid and e,~stigmatophyte eyespots are n o t in the chloroplast and so may have originated independently of

106 those found in most other algae. Chemotaxis receptors are n o t obvious microscopically but could be located in the flagellum membrane itself, or even in the axoneme. Inhibition of animal flagella and Paramecium cilia by CO2 (Brokaw and Simonick, 1976} could be a vestige of a chemotactic mechanism evolved in primitive phytoflagellates to enable them to accumulate in areas rich in COs. Significantly CO2 inhibits only flagellar bending and the associated dynein activity; it does n o t inhibit microtubule sliding, or dynein in vitro, or d y n e i n activity that it is n o t associated with bending (Brokaw and Simonick, 1976); if it did it might inhibit division also.

8. Diversification of flagella and eukaryote

phylogeny In the absence of fossils showing intracellular detail, the phylogeny of eukaryote protists must be reconstructed from extant organisms and invented "missing links". There is little prospect of identifying most such missing links in the fossil record, but a phylogenetic scheme that postulates particular missing links can be tested indirectly by making further observations on living organisms. Whether fossils are known or not, there are only three criteria of any importance in judging the scientific merit of a phylogeny: 1. consistency with the known facts; 2. simplicity; 3. definiteness. Consistency with the known facts is of obvious overriding importance, but on its own is not enough because numerous contradictory phylogenies can be proposed that are consistent with known facts. If consistency is the only criterion, we can never choose between competing phylogenies because they can always be modified to fit in with new facts by making extra assumptions. To avoid the confusing proliferation o f unnecessarily complex phylogenies we must insist that assumptions be kept to a minimum and that phylogenies be made as simple as possible. If

two theories are both compatible with the facts we should prefer the simpler one as our working hypothesis. But we cannot judge the relative complexity of two competing theories unless the main evolutionary steps in each are explicitly formulated. If we wish to indulge in phylogenetic speculation at all, we must not be afraid to speculate in enough detail for our theories to be criticised by others and tested by new observations. We must not try to protect our pet theories by vagueness or by refraining from a detailed exposition of the successive evolutionary steps that our theories imply. Other possible phylogenetic criteria are really combinations or special cases of the three I have mentioned e.g., explanatory power is a combination of one and two. Ability to make predictions and to be tested depend on three. An important aspect of the simplicity criterion is that one should not postulate the independent origin of complex characters dependent on m a n y genes, though one can do this for characters dependent on only a few genes, provided there is no evidence for detailed sequence homologies o f the macromolecules in question. Phylogenies should therefore be based on (a) complex characters dependent on m a n y genes, and (b) primary sequence homologies of RNA and protein molecules. There are four distinct phylogenetic processes: 1. direct descent w i t h o u t modification; 2. direct descent with modification; 3. divergence from a c o m m o n ancestor; 4. partial or complete merging o f two genomes through hybridization, symbiosis or virus infection. The first three are of general importance and have occurred repeatedly; the fourth can in principle occur and must be considered as a real possibility in eukaryote protistan phylogeny. Gain o f an organelle (e.g., a chloroplast) by symbiosis is often an alternative phylogenetic explanation to the evolutioncry loss of the organelle. L w o f f (1951) emphasised the importance of evolution by loss. Studies of cave dwelling organisms

107

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108

(Barr, 1968) show h o w rapidly even a complex organ like an eye can be lost in the absence of continued selection for its presence. The loss of photosynthetic ability by phagotrophic or mixotrophic algae and of the ability to synthesize vitamins and amino acids by heterotrophs is very frequent. Evolution by loss can be very rapid, can be initiated b y a single mutation and can occur independently many times in unrelated organisms (Lwoff, 1951). By contrast, the gain of an organelle by symbiosis would require many complex changes and mutual adjustments. We should therefore only postulate it where it can explain the facts more simply than alternative hypotheses, even allowing for these complex changes. Neither symbiosis nor loss should be postulated without considering the selection pressure involved. The simplest phylogeny is one where complex organelles depending on many genes evolved only once, and were passed by direct descent, with or without modification and/or loss, to the phyletic lines descended from the original ancestor. Since the loss of plastids b y algae that are n o t obligate p h o t o t r o p h s is so easy in comparison with their gain by non-photosynthetic heterotrophs, it is better to assume loss, unless there is clear evidence to the contrary. Fig. 6 shows a phylogeny for eukaryotes based on the above principles, which is more detailed than (and differs in several respects from) m y earlier a t t e m p t (Cavalier-Smith, 1975). Some of the suggested relationships are more tentative than others, and I shall be grateful for criticisms and suggested modifications. In looking for hypothetical c o m m o n ancestors for diverse groups sharing certain basic features, I have often looked for or invented organisms representing the lowest c o m m o n multiple of characters present in the apparently related groups, rather than the highest c o m m o n factor. This is justifiable if divergent evolution in protists commonly involves loss o f characters and makes possible a fairly simple phylogeny that neither involves multiple evolution of complex structures nor (in contrast to Marguhs, 1970) acquisition by multiple symbiosis.

The characteristics of flagellar and cytosketetal structures are of key importance for eukaryote phylogeny for four reasons: 1. Most of the adaptive radiations that led to the evolution of the main groups of eukaryotes shown in Fig. 6 occurred in unicellular flagellates (Blackman, 1900; Smith, 1933; Sleigh, 1973; Hanson, 1976). Therefore if one wishes to understand the selective forces that caused eukaryote diversification one needs to understand the ecological and intracellular forces that shaped the cell structure of flagellates. 2. Flagella and the various cytoskeletal structures are sufficiently complex and stable during evolution to serve as reliable indicators of evolutionary relatedness (Manton, 1965; Taylor, 1976). 3. They are one of the few characters that axe found in most eukaryotes regardless of whether their m e t h o d of nutrition is photosynthetic, saprotrophic, phagocytic, parasitic or a mixture of t w o or more of these. It is clear that shifts from one nutritional adaptive zone to another have been a very c o m m o n feature o f eukaryote evolution and one which considerably modifies many cytological characters. For this reason the nature of nutrition-related characters, e.g., chloroplasts, is often very helpful in showing relations within phyla or smaller taxa and differences between phyla, but is much less helpful in indicating the relationships between photosynthetic and non-photosynthetic phyla. 4. They are little altered when unicellular organisms give rise to multicellular organisms. Evidence from comparative cytology indicates that multiceUular organisms have evolved on many separate occasions so that the division between Protista and macroorganisms is often arbitrary and does not group together closely related organisms. The three evolutionary forces leading to large size and multicellularity are: (a) competition for light b y photosynthesizers (e.g., macroalgae, land plants); (b) competition for prey by predators (e.g., sponges, worms, jellyfish);

109

(c) dispersal of spores by air currents (higher fungi, slime moulds). These forces have led to the convergent evolution of multicellularity and other adaptations by a variety of distantly related eukaryotes (Smith, 1933; Bonnet, 1958; Hanson, 1958, 1976; Whittaker, 1969; Cain, 1972), which obscures evolutionary relationships. If one wishes to construct a realistic phylogeny, relatiwfly stable characteristics that straddle severs] phyla are especially important. Basal body and 9 + 2 axoneme characters are the most stable, but even these can undergo secondary modification as is shown by insect sperm (Phillips, 1970), the 9 + 0 flagellum of the diatom Lithodesmium (Manton and yon Stosch, 1966), and the 6 + 0 flagella of a sporozoan (Schrevel and Besse, 1975) (though the two latter might conceivably be vestiges of early stages in flagellar evolution, this is rendered unlikely by the sporadic occurrence of such aberrant flagella in species belonging to groups which otherwise have quite normal 9 + 2 axonemes). The characteristics of the transitional region, flagellar roots and mastigonemes are less stable, but still ofl~n straddle several phyla (Manton, 1965; Taylor 1976), thus showing how they are related; for example the ninefold star found in the transitional region of flagella in Tracheophyta, Bryophyta, Prasinophyta, Chlorophyta and Haptophyta is of such complexity (Fig. 1B) that it must indicate a close relationship between these phyla. The phylogeny shown in Fig. 6 was constructed by giving special emphasis to such "straddling" characters. I chose the Haptophyta as the most primitive flagellates mainly because their two equal smooth flagella and nine-fold star in the transitional region (Fig. 1) show them to be related to the green algae (Prasinophyta, Chlorophyta) and archegoniate plants, whereas their chloroplast characters and the presence of chloroplast endoplasmic reticulum show close relationships with the chromophytes. The simplest explanation of this is that they are the relatively unmodified descendants of the ancestors of both groups. A second, and weaker, reason is the presence of the hapton-

ema; its similarity with the protoflagellum postulated above suggests a common ancestry. It is simpler to assume that it has been retained by haptophytes but lost by other algae, than that it arose independently of flagella; duplication of at least some of the genes coding for the protoflagellum would have been necessary to allow the divergent evolution o f the haptonema and flagella. One would also expect the first flagellate to have identical flagella, since repetition of identical structures must precede their divergent evolution. If the Haptophyta are the most primitive flagellates, it is possible that the nine-fold star structure of the transitional region, which rigidly joins the A-tubules together, evolved even before the B-tubules as a means of strengthening the flagellar base, (perhaps also incorporating a nucleation centre for the central pair), and could be dispensed with following the evolution of B-tubules. The biggest omission for this scheme is the Glaucophyta (Skuja, 1954). If their 'cyanelles' are indeed descendants of symbiotic blue-green algae, as may well be true for Cyanophora, then their "hosts" could be located in the appropriate place in the scheme i.e., among the histone-containing dinoflagellates (Hollande, 1975). But if the cyanelles of any of those that have flagella are regarded as true plastids, they would belong close to the red algae as offshoots from the first flagellates before the loss of phycobilins, or else be regarded as red algae that have flagella! Another possibility is that they are the result of a symbiosis between a histone~ontaining dinoflagellate and the plastid of a unicellular "blue-green" red alga such as Porphyridium aerugineum (Gantt et al., 1968). Most of the protist taxa indicated in Fig. 6 are sufficiently distinct to merit treatment as phyla. But how should they be grouped into kingdoms? It has long been clear that the simple dichotomy into animal and plant kingdoms is inadequate (Copeland, 1956). The importance of the primary division of living organisms into prokaryotes and eukaryotes is now generally accepted, despite dis-

110

agreement as to how ~he evolutionary transition from prokaryotes to eukaryotes actually occurred (Uzzell and Spolsky, 1974). It seems sensible to regard the Prokaryota and Eukaryota as two super-kingdoms and to subdivide the Eukaryota into a number of kingdoms. Whittaker's (1969) division of eukaryotes into four kingdoms is unsatisfactory as a natural classification because they are based not on the structure of organisms but on broad (largely nutritional) adaptive zones; as a result his Plantae, Animalia and Fungi are probably polyphyletic, and his Protista a highly diverse mixture. Later modifications (Margulis, 1970, 1974b) considerably reduce this polyphyly, but at the expense of making the Protista even more of a rag bag. To avoid these problems Leedale (1974) suggested 18 kingdoms. This seems the right approach, though the number of kingdoms can probably be reduced if real relationships can be found between some of them. On the basis of the phylogeny of Fig. 6, I suggest seven, possibly monophyletic, eukaryote kingdoms: 1. Aconta (6 phyla: the Rhodophyta and their descendants), 2. Haptophyta (1 phylum), 3. Cryptophyta (1 phylum); 4. Heterokonta (15 phyla, as shown in Fig. 6); 5. Corticoflagellata (Dinoflagellates, Sporozoans, Metamonads, Opalinids, Ciliates, Mesozoa, plus the Eumetazoan animal phyla), 6. Euglenoida (2 asexual phyla: Euglenophyta and Kinetoplastida); 7. Chlorophyta (Chlorophyte algae, Prasinophyta, Bryophyta, Tracheophyta). This classification does not correspond with the traditional academic adaptive zones of taxonomists (i.e., botanists, zoologists, my cologists, protozoologists and phycologists) as well as does Whittaker's, but I hope this will not prevent careful consideration of its merits. I suspect that many or even most biologists would accept the monophyletic nature of five of these groups, but that some would be unhappy with my groups Aconta and Cortico-

flagellata. Demoulin (1974) and Kohlmeyer (1975) have reviewed evidence for a relationship between Ascomycetes and red algae. Though it is arguable that the zygomycetes, Acrasida and other amoebae lacking flagella (not shown in Fig. 6 through lack of space) evolved by the loss of flagella there is no evidence for this and it is simpler to assume they never had any. Clearly more comparative research is needed into characters other than plastids and flagella to establish their trde relationships. The same is true of the Cnidospora which could be grouped with the Aconta because of their lack of flagella, or (as I have here) with the corticoflagellata because of the resemblance of polar capsules and nematocysts. I have grouped the Corticoflagellata together on the basis of a highly developed cortical microtubule system, a phagocytic mode of nutrition, a strong tendency to evolve repeated cortical structures and also multiple nuclei, genomes or cells and the absence of the traditional region star and of tubular mastigonemes. A relationship between Dinoflagellates and Ciliates has been argued by Chadefaud (1960), between ciliates and Eumetazoa by Had~i (1963) and Hanson (1958, 1976). Greenberg (1959) has argued for a separate origin for Coelenterates, Ctenophores and Platyhelminths. It would not be difficult to derive both Coelenterates and Ctenophores by subdivision of large multinucleate non-photosynthetic predatory dinoflagellates like Polykrikos, the former could have come from a radially symmetrical dinoflagellate with nematocysts and the latter from one having biradial symmetry and colloblast precursors. The chief objection to a dinoflagellate ancestry for the various corticoflagellate groups is the usual absence of histones and their bizarre mitotic mechanisms, which have led many people to regard them as primitive eukaryotes. However since dinoflagellates have normal eukaryote sexual mechanisms (Loeblich, 1976), a thoroughly eukaryote sequence organisation in their nuclear DNA (Allen et al., 1975), and an

111

exceedingly well ,developed and complex cytoplasm, this seems very dubious. Since a few do have histones in one phase of their cycle but lack them at others (Kubai, 1975), it seems highly probable that their ancestors once had histones:, which were later lost. Recent work (Oaldey and Dodge, 1974) shows that their mitosis resembles that of other eukaryotes in having both kinetochore and interpole microtubules. It is therefore not unreasonable to postulate the existence about 700 million years ago of cortico-flagellates having most of the features of modern dinoflagellates, but having normal eukaryote chromatin and mitosis and two unequal flagella arranged as in Prorocentrum (Loeblich, 1976) (and not in grooves as in most dinoflagellates).Such an organism would have been a suitable ancestor for all groups I have included in the Corticoflagellata. Though m y Kingdom Heterokonta groups together groups traditionally thought of as algae, fungi and protozoa, the sharing of c o m m o n flagellar characteristics suggests that they axe a natural group. The Chrysophyta like some o f their :probable derivatives often use silica as an extracellular skeleton, frequently feed phagocytically, and have filopodial as well as flageUar motility. A rational eukaryote t a x o n o m y and nomenclature necessitates the merging of the Botanical and Zoological Codes of Nomenclature. Flagellar and cytosketetal characters are especially valuable for drawing up preliminary phylogenies, because electron microscopy can show much detail (dependent on many genes) and be used fairly rapidly for broad surveys. But det~dled tests of alternative phylogenies require more laborious primary sequence data for several key proteins in each major organelle in organisms from two or more of the phyla of each of the seven Kingdoms; molecular evolutionists need to devote more attention to the algae and less to animals. A clear prediction of m y phylogeny is that meiotic synaptinemal complex proteins should be homologous in all organisms.

Margulis' (1970) phylogeny, which postulates numerous independent origins for meiosis, would be refuted ff this were true. The timing of the various radiations in Fig. 6 could only be known if there were a better fossil record. Since rates of evolution can vary over many orders of magnitude depending on the selective pressures, assigning a time to the various events in almost pure guesswork. The short generation time of protists would allow very rapid evolution. I see no reason in principle w h y the evolution of a new organelle like a flagellum could not occur in well under a million years given (a) the right starting materials and (b) the right selection pressures, or w h y all the suggested changes could n o t in principle have occurred in 50 million years or less. I argued previously (1975) that mitosis and meiosis evolved simultaneously during the early evolution of eukaryotes, and see no biological reason w h y a long delay of hundreds of millions of years between the origin o f mitosis and sexuality, or between sexuality and the evolution of multicellular organisms (Schopf, 1974), m u s t be postulated. Intracellular preservation is so poor in fossils that many or even all of the so-called eukaryotes found in the period 1500--700 million years ago could well be prokaryotes. We should look critically at the fossil record and consider seriously the possibility that eukaryotes evolved only about 700 million years ago and that eukaryote diversification into the various modern phyla occurred in the following 100 million years. References Adler, J., 1976, Some aspects of the structure and function of bacterial flagella, in: Cell Motility, R. Goldman, T. Pollard and J. Rosenbaum (eds.) (Cold Spring Harbor Laboratory) pp. 29--33. Allen, J.R., T.M. Roberts, A.R. Loeblich and L.C. Klotz, 1975, Characterisation of the DNA for the Dinoflagellate Crypthecodinium cohnii and implications for nuclear organisation. Cell 6, 161--169. Alicea, H.A. and F.L. Renaud, 1975, Actin-tubulin homology revisited. Nature 257,601--602.

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