The possible significance of variations in the mitotic systems of the aquatic fungi (phycomycetes)

351 BioSystems, 7 (1975) 351--359 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

THE POSSIBLE SIGNIFICANCE OF VARIATIONS IN THE MITOTIC SYSTEMS OF THE AQUATIC FUNGI (PHYCOMYCETES) I. BRENT HEATH Biology Department, Yorl" University, Toronto, Canada

1. Introduction With the aid of the electron microscope it has become possible to describe in considerable detail structural features of the mitotic apparatus of many diverse primitive organisms. From this increasing body of knowledge it should be possible to obtain information of relevance to the broad topics of cell biology and phylogeny in addition to the basic contribution to the knowledge of each organism. Pickett-Heaps (1972), Margulis (1974) and Kubai (1975) have recently reviewed the information available on mitosis in the primitive organisms studied so far and have attempted to reconstruct the possible line of evolution of the mitotic apparatus, whilst Pickett-Heaps and Marchant ( 1972) have used mitotic details to clarify the phylogeny of the green algae. The purpose of this paper is to focus in more detail on mitosis in the primitive aquatic fungi ( " P h y c o m y c e t e s " ) from two somewhat different viewpoints, namely: (a) what do the observations tell us about the mechanics of mitosis in general? and (b) what do they tell us about the phylogeny and taxono m y of this rather confused and obscure assemblage of organisms ?

2. Mitotic mechanism

Detailed descril:,tions of mitosis in the aquatic fungi {herein restricted to the Oomycetes, Chytridiomycetes, Hyphochytridiomycetes and Plasmodiophcromycetes) have been hampered at the light microscope level because of

the small size of the mitotic apparatus, but this has proven to be an advantage at the electron microscope level because it makes serial reconstructions of the entire apparatus a relatively simple proposition. However, before the detailed observations obtained by this technique can be discussed in the broader context of possible universal mitotic mechanisms, it must be established that there are indeed likely to be similar mechanisms operating in the aquatic fungi and higher plants and animals. As seen in Table 1 all appropriately examined aquatic fungal spindles contain both chromosomal and continuous microtubules. At least in Thraustotheca (Heath, 1974a), the continuous tubules comprise both tubules which truly run from pole to pole and others which extend from one pole to some point over half way along the spindle, a situation also found in animal spindles (Brinkley and Cartwright, 1971; McIntosh and Landis, 1971). Morphologically the fungal microtubules are comparable to those of other organisms and whilst Saprolegnia is highly resistant to colchicine and other anti-microtubule agents (Slifkin, 1967; Heath, 1975a) this resistance is not necessarily evidence for major differences in the microtubules themselves ( Heath, 1975a,b). During mitosis of all reported aquatic fungi the chromosomes are arranged at approximately the equator of the spindle prior to their poleward movements. The poleward movements involve both spindle elongation and a decrease in chromosome to pole distance. This behaviour is again c o m m o n to many higher organisms. Thus on these fundamental features there is considerable similarity between mitosis

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353 in the aquatic fungi and higher organisms. Furthermore analogy with other major cellular processes also suggests that there will be a basically similar mitotic mechanism for all eukaryotic organi..~ms. For example the force generating part of the flagellum, the axoneme, is highly conserved but with minor variations in basal body structure and flagellar appendages. Likewise the basic pathways of photosynthesis, respiration, transcription and translation are again highly conserved in the eukaryotes but with minor variations in many enzyme structures and co-factors. Thus it seems probable that there will be basic uniformity in mitotic mechanisms rathe:c than numerous fundamentally different force generating systems. What, then, can we dectuce about this mechanism from studies of the fungi listed in Table 1? Since there are numerous extant hypothetical models of mitotic force production, perhaps it is easiest t~ consider the contributions from the aquatic fungi to these models. Inoue and Sato (1967} and Dietz (1972} essentially propose that appropriate elongation and shortening of microtubules by variations of the polymerized versus depolymerized equilibrium of the tubulin subunits causes chromosome movements. Since such length changes do occur, the problem becomes one of differentiating between cause and effect and to date there is no information from the fungi, or elsewhere, which unequivocally resolves this question. Since force generation by tubule elongation presumably requires something for each end of the elongating tubule to act against the role of the in~rdigitating microtubules in Thraustotheca (Heath, 1974a) is hard to ascertain on the Inoue and Sato {1967} hypothesis, but once an interaction with a gel-like spindle matrix is allowed (Dietz, 1972) they could elongate the nucleus by pushing against the gel during simple elongation. However, any length change hypothesi,,', for force generation is hard to invoke in other microtubule associated movement systems (see below). Thus it is certainly worth seeking alternate hypotheses even though the above are not invalidated by observations on tl=e fungi.

McTntosh et al. (1969} suggested that mitotic movements could be accounted for by mechanochemical cross bridges causing sliding movements between appropriately oriented microtubules. Nicklas (1971} presented modifications of this basic hypothesis and certainly the closely packed overlapping microtubules of the mid-bodies in animal cells (e.g. McIntosh and Landis, 1971} and the spindle equator of Lithodesmium (Manton et al., 1970} support some aspects of this model. However in Thraustotheca (Heath, 1974a), Saprolegnia (Heath and Greenwood, 1968} and probably Entophylctis (Powell, 1975} and Thraustochytrium (Kazama, 1974) the intertubule distances, especially in the interzone during anaphase-telophase, appear to be too large to make intertubule cross bridges possible. Whilst it has been suggested (Heath, 1974a) that in the Oomycetes part of the force for nuclear elongation comes from an interaction between the nuclear envelope and cytoplasmic microtubules, it seems unlikely that the interzonal microtubules would develop for no function. Yet their involvement in force generation by intertubule cross bridges seems precluded by distance, thus suggesting that the model of McIntosh et al. (1969) cannot be universal. When it is possible to analyse the distribution of almost all microtubules in the spindle as in Thraustotheca (Heath, 1974a), it becomes apparent that the distribution of microtubules required by Bajer's (1973) "zipper" hypothesis does not exist. Furthermore it is hard to envisage the "zipper" hypothesis operating successfully in spindles containing only one microtubule per kinetochore (Table 1 ). Using the probable universality of actinmyosin interactions as agents of cellular movement and the recent observations of actin in some mitotic spindles, Forer {1974)has argued that actin is involved in mitotic force production and that the roles of the microtubules are cytoskeletal {continuous microtubules) and moderators of movement generated by actinmyosin type interactions (chromosomal microtubules}. Only one investigation seeking actin in the aquatic fungi is known (Heath, 1975c)

354 and although the results were negative they clearly carry little weight due to the problems of ensuring penetration of the heavy meromyosin through the cell wall and the possibly low quantity of actin needed. However the role of the interdigitating microtubules of Thraustotheca (Heath, 1974a) is hard to account for on Forer's (1974) model since they can hardly be moderators of chromosome movement and to be cytoskeletal they would need to be clearly anchored at both ends. However, apart from these tubules, the role of actin in the mitotic system and the moderating role of the chromosomal microtubules remain viable hypotheses based on evidence from aquatic fungi. Margulis (1974) has recently discussed a possible mechanism for mitosis in which she postulates anaphase chromosome movements being due in part to the elongation of interchromosomal microtubules pushing the chromosomes apart. Again, Thraustotheca (Heath, 1974a) contains a spindle which mitigates against this model since the necessary interchromosomal microtubules cannot be detected. A feature of higher mitotic systems which is included in many hypothetical schemes is the metaphase plate configuration. This is usually explained as resulting from a balance of equally opposed forces acting on the paired kinetochores of each chromosome. Clearly the existence of equatorially located, unpaired kinetochores in Thraustotheca (Heath, 1974a) and Saprolegnia (Heath, unpublished) indicates that any theory relying on equally opposed, balanced forces, to explain metaphase is inadequate as a universal hypothesis. In response to the above objections to various hypotheses it is possible to describe a concept which is compatible with all aquatic fungal observations and can be extended as a universal concept for microtubule associated movement systems. This concept is in fact a detailed compilation of components from each of the extant hypotheses mentioned above, plus parts of Subirana's (1968) model, and is an a t t e m p t to demonstrate the underlying similarity of all microtubule based systems on which

detailed, possibly phylogenetically significant, modifications have been imposed. The microtubule is basically conceived as having a shear force generating " m o t o r " located on its surface. This " m o t o r " could occupy the entire length of a tubule and would generate movement by interacting with other cellular components so that the type of movement observed would be a resultant of the anchoring of the microtubule and the cellular environment with which it was interacting. Thus in mitosis the matrix of the spindle is considered, at least in part, as a mechanically rigid gel (cf. Dietz, 1972) through which both chromosomal and interdigitating microtubules are able to propel themselves {cf. Subirana, 1968}. Clearly the rate, and timing (i.e. end of metaphase}, of chromosome movement would be controlled, at least in part, by the rate of chromosomal microtubule depolymerization (cf. Forer, 1974}. That there are at least two components involved in the mitotic force generation system is of course established (Forer, 1969, 1974; Oppenheim ct al., 1973) and the concept of a substantial mechanical integrity of the spindle exclusive of the microtubules is demonstrated by the large a m o u n t of non-tubule material (Forer, 1969, 1974) which can retain spindle shape in the absence of tubules (Bibring and Baxandall, 1971). The mode of force generation by the continuous microtubules (pole to pole) may be by simple elongation but likewise could involve an interaction with the matrix with concomitant synchronous polymerization maintaining the true pole to pole connections. In other cellular locations the same concept, with different tubule anchoring, can explain other types of motion. For example in the nuclear horns of the oomycetes (Heath, 1974a,b) and the other " a b n o r m a l " mitotic systems reviewed by Kubai (1975) and Heath, (1974c} the " m o t o r " on the microtubule could be interacting with the cytoplasm, the nuclear envelope and the chromatin (see Heath, 1974c for discussion of alternatives). In the case of organelle motility in the fungi (Heath, 1975c), synaptic vesicle transport as discussed by Ochs

355 (1972) and Schmitt (1968), and vesicle transport in Paramecium and other organisms (Allen, 1975 and refe::ences therein) the motor on the static microtubules would interact with the unattached organelles to bring about their movements. Situations where the evidence for inter-tubule sliding is good, e.g. Saccinobaculus axostyles (McIntosh, 1973) and flagellar axonemes (Summers and Gibbons, 1971} would be example,; where the two " m o t o r s " of adjacent microtubules interact. A similar explanation would hold for the closely spaced mammalian mid-body ( McIntosh and Landis, 1971) and Lithodesmium mitosis (Manton et al., 1970) microtubules if it is demonstrated that active sliding does occur in these regions. Clearly the important question is then, if such a universal " m o t o r " exists, what is it? Morphologically it can be represented as having two parts, both the "clear zone" c o m m o n l y seen surrounding microtubules and the crossbridges recently reviewed by McIntosh (1974). However, at the molecular level it is most likely to involve actin and myosin. Forer (1974) has recently reviewed the evidence for the association of actin with microtubules and for the universality of actin-myosin interactions as agents of cellular movements. There is little which needs to be added here. Thus the basic underlying similarl.ty in all mitotic systems is that the microtubules are able to move themselves, and any attached structures such as chromosomes, th:cough the spindle matrix probably by means of tubule-linked actin interacting with matrix-linked myosin. In other situations the tubule-linked actin could move membrane bound structures by interacting with membrane-linked myosin. The only difficulty with this ,;implest version of the hypothesis is the proolem of intertubule actions. Since actin is unlikely to move by working against actin one must postulate the ability of myosin to also link to microtubules, a concept for which there is as yet little evidence (however see Tilney, 1975). If this concept proves to be the underlying basic mechanism c o m m o n to all mitotic systems, what then can the detailed variations tell us of the phylogeny and taxono m y of the aquatic fungi?

3. Phylogenetic considerations Within the aquatic fungi it is clear from Table 1 that there is considerable homogeneity of the listed characteristics within the traditional taxonomic groupings, especially at the class level. The exception to this is Thraustochytrium. Consideration of the mitotic apparatus indicates removal of this genus from the Oomycetes, a conclusion which has been arrived at by Kazama {1974) and which had been formerly suggested by Darley et al. {1973) and Porter {1974) using different criteria. It should be noted that the listed 90 ° orientation of the centrioles may in fact not be strictly comparable to the rest of the listings because Kazama {1974) was working with material which was close to zoospore production. In the allied Labyrinthula Perkins (1970) has reported 180 ° orientation during part of the life cycle but, as in the Oomycetes (reviewed by Heath, 1975d), centriole reorientation to approximately 90 ° occurs prior to zoosporulation. Using time of centriole migration and absence of " h o r n s " the Leptomitales appear to be somewhat unrelated to the otherwise homogeneous Oomycetes, a separation supported by certain biochemical characteristics (reviewed by Le John, 1974) such as % GC content of their DNA, some aspects of their glutamic dehydrogenases and, at least for Apodachly, their ability to synthesize chitin. On nuclear criteria there is no basis for Sparrow's (1971, 1973) suggestion of splitting the two families of the Leptomitales (as represented by Apodachlya and Sapromyces respectively) to the Saprolegniales and Perenosporales. However, since it could be argued, on the basis of chitin containing cell walls and more specialized zoospores, that the Chytridiomycetes are more advanced than the Oomycetes, it could also be argued that the Leptomitales, with their centriole migration pattern and chitin containing cell walls, could be the most advanced Oomycetes, as previously suggested by Cantino (1955). However, such a criterion as time of centriole migration should be used with extreme caution since the Ascomycetes are simi-

356 lar to the Saprolegniales in having centriole equivalent (plaque) migration during spindle formation (Moens and Rapport, 1971 ), yet few would suggest a close relationship between these groups. Within the Chytridiomycetes, aspects of mitosis seem to correlate fairly well with other features such as zoospore structure. Thus the Blastocladiales can be separated from the rest of the chytrids by having no polar fenestrae or perinuclear endoplasmic reticulum and also having characteristic zoospores (Fuller and Calhoun, 1968; Chong and Barr, 1974). The Harpochytriales appear to be unique among the chytrids in not losing the central part of the nucleus at telophase. The mitotic system in the Monoblepharidales should prove interesting since their zoospores are distinctly different from the rest of the chytrids. The phylogenetically uncertain Hyphochytridiomycetes are clearly aligned closer to the chytrids than the Oomycetes based on the criteria used here, an affinity supported by 25 s ribosomal RNA molecular weights but not glutamic dehydrogenase types (Le John, 1974). However, as with other features, the Plasmodiophoromycetes, whilst retaining some affinities with the Oomycetes (see below), are clearly well isolated from them in most aspects of mitosis. At present it is clear that details of mitosis show sufficient correlation, en masse, with other taxonomic criteria to give enough confidence to embark on a reexamination of other criteria should an organism prove to have an apparently anomalous type of division. This would be the reverse of the actual sequence followed in removing Thraustochytrium from the Oomycetes (see above). If finer criteria than those selected in Table 1 are considered there is less homogeneity than noted above. Consideration of features such as the origin of the daughter nuclear envelopes and the position of the centrioles relative to the polar fenestrae has led Powell (1975) to suggest that Phlyctochytrium and Entophlyctis, both in the same family, have diverse ancestry from the algae and protozoa respectively. Since she

notes that other characteristics support this suggestion it may prove to be valid, in which case an increasingly detailed consideration of the variations in mitotic details may be warranted. However, as mentioned by Powell (1975) and others, the substantially different mitotic systems of the different life cycle stages of Physarum (Aldrich, 1969) make the most forceful argument for caution in the use of mitotic characters as phylogenetic markers. With the above caution in mind it is perhaps early to speculate on the possible ancestry of the aquatic fungi and clearly to consider all characters which may bear on their ancestry is beyond the scope of this work. However, there are a few pointers which emerge from a consideration of mitosis and centrioles which are seldom used by workers interested in phylogeny and which are perhaps worth mentioning here. The arrangement of the paired centrioles at 180 ° to each other, at least during some phase of the life cycle, is relatively rare. It is hard to believe that this arrangement confers some highly selected advantage on the possessing organism, thus suggesting that it is more likely to indicate common ancestry than convergent evolution. This arrangement of centrioles thus places the Oomycetes and Plasmodiophoromycetes on an evolutionary branch with such organisms as Nitella (Turner, 1970), Labyrinthula (Perkins, 1970}, Anthoceros and Marchantia (Moser and Kreitner, 1970), Nowellia (Simone, 1973) and Lycopodium (Robbins and Carothers, 1975). Clearly these organisms are not closely related to the fungi but it is interesting to note that Pickett-Heaps and Ott (1974) place the bryophytes and higher land plants at the end of the line containing the Charales (Nitella) and starting among the Prasinophyceae. By use of another somewhat rare character of apparently little functional significance, the concertina-like collar found at the base of the flagella in many Oomycetes (Heath and Greenwood, 1971, and references in Heath, 1975d), it is also possible to relate the Oomycetes to the Prasinophyceae, or at least to Pyramimonas

357 ( M o e s t r u p and T h o m s e n , 1 9 7 4 ) . U n f o r t u n a t e ly, as n o t e d b y Pickett-Heaps ( 1 9 7 5 ) Pyramim o n a s has an o p e n spindle whilst P e d i n o m o has, a n o t h e r p m s i r t o p h y t e , has a closed spindle which would be m o r e in line with the O o m y c e tes. H o w e v e r P e d i n o m o n a s has the typical green algal star shaped s t r u c t u r e at t h e base o f its flagellum ( P i c k e t t - H e a p s and Ott, 1974 ) ! C o n s i d e r a t i o n o f o t h e r organisms in which the concertina-like collar is r e p o r t e d relates the O o m y c e t e s to the X a n t h o p h y c e a e (Massalski, 1969), C h r y s o p h y t e s such as O c h r o m o n a s (Bouck, 1 9 7 1 ) a r d S p h a l e r o m a n t i s ( M a n t o n and Harris, 1 9 6 6 ) , and the E u s t i g m a t o p h y c e a e ( H i b b a r d and Leedale, 1 9 7 2 ) . A possible relationship b e t w e e n the O o m y c e t e s and the X a n t h o p h y c e a e ha~ long been d e b a t e d on o t h e r criteria (Klein and Cronquist, 1 9 6 7 ) and the closed spindle o f Vaucheria ( O t t and B r o w n , 1972) s u p p o r t s the relationship. U n f o r t u n a t e l y the c e n t r i o l e a r r a n g e m e n t , loss o f the i n t e r z o n e region o f the nucleus at telophase and configuration o f the m i c r o t u b u l e s possibly involved in nuclear m o t i l i t y in Vaucheria ( O t t and B r o w n , 1972) d o n o t s u p p o r t such a relationship! Likewise mitosis in O c h r o m o n a s (Slankis and Gibbs, 1 9 7 2 ; B o u c k and B r o w n , 1973) is totally d i f f e r e n t f r o m t h a t in the O o m y c e t e s (at least on the characteristics used here), thus indicating little relationship. U n d o u b t e d l y similar correlation,,; and c o n t r a d i c t i o n s to those given above can, or will be, f o u n d b e t w e e n o t h e r aquatic fungi and unicellular protists. Until far m o r e i n f o r m a t i o n on the details o f m i t o t i c systems and o t h e r aspects o f c e n t r i o l e and m i c r o t u b u l e based systems b e c o m e s available (and such i n f o r m a t i o n is hard to obtain) it will be impossible to m e a n i n g f u l l y evaluate their roles as p h y l o g e n e t i c indicators. T h e p h y l o g e n y o f the fungi indicated b y Fuller ( 1 9 7 5 ) , which s o m e w h a t sidesteps the q u e s t i o n b y deriving each o r d e r o f the aquatic fungi f r o m u n i d e n t i f i e d " a n c e s t r a l flagellates", m a y prove to be the best t h a t can be d o n e for m a n y years to c o m e , especially since the interrelationships and groupings o f the e x t a n t ancestral t y p e flagellates are still highly u n c e r t a i n .

Acknowledgements I should like to a c k n o w l e d g e n u m e r o u s helpful discussions with Dr. A. F o r e r c o n c e r n i n g the first part o f this w o r k , the e x c e l l e n t secretarial assistance o f D o r o t h y Gunning, and a grant f r o m the N.R.C. o f Canada which supports the u n p u b l i s h e d w o r k r e f e r r e d t o here.

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