ultrastructural aspects of basal body associated fibrous structures in green algae: a critical...

B/aSystems, 12 (1980) 85-104 85 © Elsevier/North-Holland Scientific Publishers Ltd.

U L T R A S T R U C T U R A L A S P E C T S OF BASAL BODY ASSOCIATED FIBROUS S T R U ~ S IN G R E E N A I ~ A E ~ A CRITICAL REVIEW

MICHAEL MELKONIAN

Botanisches Institut, Westfdlische Wiihelms-Universitdt, SchloBgarten 3, D-4400 Miinster, Federal Republic of Germany

(Received July 14th, 1979) (Revised version received September 9th, 1979)

Ultrastructural aspects of fibrous structures associated with basal bodies of green algae are critically discussed. It is apparent that variation among these structures is much greater than in microtubular flagellar root systems and it is therefore suggested that fibrous structures may be more useful than microtubular roots in elucidating phylogenetic relationships within the Chlorophyceae sensu Stewart and Mattex and the Prasinophyceae sensu Christensen.

Two main types of fibrous structures are distinguished: (1) Connecting fibres (these connect different basal bodies); (2) Fibrous roots (these originate at basal bodies and terminate somewhere else in the cell). Fibrous roots are of two types: (a) microtubular-root associated striated fibres (striation pattern 25-35 nm; system I-fibres); (b) striated fibres composed of a bundle of filaments (filament diameter: 5-10 nm; striation pattern greater than 80 nm; system II fibres). Numbers, disposition and substructure of connecting fibres and fibrous roots are variable in different genera of green algae. In the experimental section new observations on fibrous roots in the ulvalean genus Enteromorpha as well as preliminary information on fibrous structures in Carteria obtusa and Bryopsis lyngbyei are included. Functional and evolutionary aspects of fibrous structures associated with the flagellar apparatus of green algae are discussed.

1. Introduct ion

Ul t r a s t ruc tu r a l aspects of the f lagellar ap- pa r a tu s in the green a lgae have been increas- ingly used as impor t an t phylogenet ic indicators especially wi th respect to h igher p lan t evolut ion (recent s u m m a r y by Moestrup, 1978). Emphas i s was laid on the mic ro tubu la r pa r t of f lagellar roots in es tabl i sh ing two m a i n lines of evolut ion wi th in the green algae: one group of a lgae possessing a un i la te ra l root, which in most cases is proximal ly associated wi th an MLS-s t ruc tu re (the Charo- phyceae sensu S tewar t and Mattox), and the major i ty of green algae exhibi t ing a crucia te f lagellar root sys tem of the X-2-X-2-type (the Chlorophyceae sensu S tewar t and Mat tox and most genera of the Prasinophyceae sensu Chris tensen) .

Dur ing the pas t few years knowledge on

cruciate f lagellar root sys tems has g rea t ly in- creased (e.g. Moestrup, 1978; Goodenough and Weiss, 1978; Melkonian, 1978, 1979) and it has been shown t h a t micro tubule number s and dispositions are s l ight ly var iable in small sys temat ic categories (e.g. Birkbeck, 1976; Moestrup, 1978; Melkonian, 1978), while the genera l occurrence of two root types and specific root tubule configurat ions is surpris- ingly un i fo rm a m o n g diverse Chlorophyceae and Prasinophyceae. It was speculated t h a t this un i fo rmi ty of the crucia te root sys tem m a y indicate an impor t an t common funct ion of these systems, which is however at present u n k n o w n (Melkonian, 1979). F u r t h e r m o r e the lack of var ia t ion in the cruciate micro- t ubu la r root sys tem probably means t h a t it cannot be used for taxonomic or phylogenet ic considerat ions in the h igher t axa of the Chlorophyceae and Prasinophyceae.

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Fibrous structures associated with basal bodies of green algae have not been studied in the same detail as the microtubular root systems. In his recent review Moestrup (1978) listed available information on fibrous "roots" in a table, while fibrous structures interconnecting basal bodies were excluded from the review. Brown et al. (1976) in a study on the flagellar apparatus of PolytomeUa agilis poin- ted out the phylogenetic potential of connecting fibres within the green algae and stated that comparable data were mostly lacking. Several reasons may account for this apparent lack of information concerning fibrous structures associated with basal bodies of green algae: The conditions for optimal fixation of fibrous structures when compared to the well- known fixation schedules for microtubules are unknown. There is some evidence that the ionic environment during fixation effects morphology of some fibrous structures (e.g. Robenek and Melkonian, 1979). In addition some structures change their morphology during flagellar function (e.g. Watson, 1975; Melkonian, 1978; Salisbury and Floyd, 1978). Extensive serial sectioning is especially important for evaluation of positional relations of various interconnecting fibres. Also con- fusion exists with respect to terminology. Connecting fibres were named according to Ringo (1967), although his description applies only to chlamydomonad-like biflagellate cells. With respect to fibrous "roots" the term "rhizoplast" is not unequivocally defined and has sometimes been used for morphologically different fibrous structures.

In this review relevant information on fibrous structures of green algae is critically discussed and a terminology proposed, which hopefully permits easier identification of fibrous structures. In the light of the enormous variabili ty of fibrous structures associated with basal bodies of Chlorophyceae and Prasinophyceae, it is suggested that these structures might be more important as phylogenetic indicators in certain green algae than the cruciate microtubular root system.

2. Materials and methods

Fertile thalli of Enteromorpha linza (L.)J.Ag. were collected on the rocky flats at the West Cliff of the island of Helgoland during spring tides in April 1977. The fertile thalii were individually placed in plastic petri dishes in a moist but not wet condition. After return to the laboratory they were stored overnight in the petri dishes in a cool chamber (10°C). The next morning the thalli were flooded with fresh seawater and placed at a northern light window. Within minutes quadriflagellate swarmers were discharged which exhibited negative phototactic behaviour. The zoospores became immotile within minutes and fixation was therefore carried out during backward swimming of zoospores away from the light. Fixation was performed by adding an equal amount of zoospore suspension to 2% glutaraldehyde (in 0.25M sucrose adjusted to pH 8.2 with NaOH). Cells were fixed for 30min at room temperature, washed three times with seawater and postfixed for 15 min in 1% OsO4 (prepared as a 2% solution in 0.25M sucrose and mixed with equal amounts of seawater; pH adjusted to 8.2) at 4°C. After three washes in seawater they were further processed as previously described (Melkonian, 1975). Sec- tions were examined with a Siemens Elmis- kop 102.

3. Experimental sect ion

Since the recent proposal of Stewart and Mattox (1978) that the Ulvales may represent a third class of green algae C Ulvaphyceae") besides the Charophyceae and Chlorophyceae sensu Stewart and Mattox, a detailed study on the flagellar apparatus of zoospores of Ulva lactuca (Melkonian, 1979) has given evidence that the flagellar apparatus of Ulva is peculiar in that besides a "normal" cruciate microtubular root system (4-2-4-2) a second type of cruciate fibrous root system is present.

This is more internally located and contains 4 cross-striated fibres which run parallel to the microtubular roots. These fibres have an overall appearance of rhizoplasts known from a few green algae (summary by Moestrup, 1978 and Robenek and Melkonian, 1979) and have prompted a critical reevaluation of published data on fibrous structures associated with basal bodies of green algae (see Discussion).

In an attempt to relate the observations made in Ulva lactuca to other members of the Ulvales a comparative study on zoosperes of Enterornorpha linza was undertaken. The results of this study indicate that root systems in beth genera are identical. In zoospores of E. linza a cruciate microtubular root system of the 4-2-4-2-type is present (Figs. 5-7). The four-stranded root shows changes typical of root tubule configurations which have been described in detail for zoospores of Chloro- sarcinopsis (Melkonian, 1977, 1978) (Figs. 6, 7). Closely associated with both types of microtubular roots is underlying electron dense material (Figs. 1, 2, 5, 6), which is more prominent below the two-stranded root (Figs. 2, 5). In longitudinal section this underlying material in the two-stranded roots contains a cross-striated fibre (32 nm repeat; Fig. 9). In addition to this system another system of striated fibres is present, this being more internally located (Fig. 1, triangles and Figs. 2, 5, 7). Four striated fibres with 150-160 nm periodicities run from the proximal end of a basal body (shown in Fig. 8 for female gametes of Ulva lactuca, but identical in zoospores of E. linza) towards the plasmalemma, where they terminate about 2/~m away from the respective basal body. Figure 5 reveals cross sections through the two microtubular root types r~ and rn (two-stranded root and four-stranded root in a 3 over 1 configuration with associated underlying electron dense material) and through two of the four internally located striated fibres (triangles).

As in zoosperes of Ulva lactuca the basal bodies in zoosperes of E. linza are closed at their

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proximal end by a '~brminal cap" (Melkonian, 1979) (Fig. 3; zoospores of E. linza; Fig. 4; female gametes of Ulva lactuca).

Summarizing these results, it can be stated that zoospores of Enteromorpha linza exhibit an identical flagellar root system to zoospores of Ulva lactuca. It is suggested that this type of complex flagellar root system characterizes motile cells in the Ulvales and thus certain critical genera like Trichosarcina and Pseu- dendoclonium (Mattex and Stewart, 1973) should now be investigated to see if similar systems are present.

4. D i s c u s s i o n

In this review two principal types of fibrous structures associated with basal bodies of green algae are distinguished: (1) Connecting fibres; and (2) Fibrous roots.

4.1. Connecting fibres

Within the flagellar apparatus connecting fibres link different basal bodies with each other. They have not been found in some uniflagellate green algae exhibiting a second non-functional basal body (e.g., Pedinomonas (Ettl and Manton, 1964) and Monomastix (Manton, 1967)) and have also not been seen in some biflagellate spermatozoids (e.g., Golenkinia (Moestrup, 1972) and D/- chotomosiphon (Moestrup and Hoffman, 1975)). Even when connecting fibres are absent the flagellar apparatus is nevertheless physically united into a structural unit, a prerequisite for its coordinate functioning. This is especially well documented in shadow- cast preparations of an isolated flagellar apparatus (e.g., in spermatozoids of D/- chotomosiphon; Moestrup and Hoffman, 1975).

In the following discussion algae producing biflagellate or quadriflagellate motile cells are treated separately because they generally display different kinds of connecting fibres,

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which can only seldom be related (e.g., Chlamydomonas and Polytornella; Brown et al., 1976). Unfoi'tunately no detailed study exists on those few green algae producing both biflagellate and quadriflagellate swarmers. Preliminary evidence obtained from quadriflagellate zoospores and biflagellate gametes of Ulva lactuca indicates that both types of motile cells have two connecting fibres with apparently identical substructure, one difference being that in the zoospores the fibres are attached to the newly formed basal bodies of different basal body pairs (Mel- konian, 1979; Melkonian, unpublished observations).

4.1.1. Connecting fibres of the Chlamy- domonad-type in biflagellate cells

In green algae producing biflagellate motile cells the available information indicates that at least two different systems of connecting fibres are present. The first is for the purpose of this review named Chlamydomonad-type (Group I connecting fibres). This type occurs in several green algae and Table 1 lists those species in which published data indicate its presence. In some species only one of the two kinds of connecting fibres, the distal striated fibre, was observed and in a few species even the striation pattern of this fibre was poorly resolved. The general discussion is therefore mainly based on the results obtained in Chlarnydornonas reinhardi (Ringo, 1967) and zoospores of Chlorosarcinopsis (Melkonian, 1977, 1978).

The system of connecting fibres consists of a single distal striated connecting fibre and two proximal striated connecting fibres. A third

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TABLE 1

Chlamydomonad- type of connecting fibres in green algae producing biflagellate motile cells

Organism References

Chlamydomonas reinhardi

Chlamydomonas moewusii Chlamydomonas concordia Chlam ydomonas spp.

(several species) Chlam ydomonas eugametos Polytoma papillatum Chlorogonium mantonii Dunaliella primolecta Dunaliella bioculata Asteromonas gracilis

Nephroselmis olivacea ( Heteromastix angulata )a

Volvox carteri ? Tetraspora lubrica Nautococcas mammilatus Tetracystis excentrica Chlorosarcinopsis spp.

(7 species) Pediastrum boryanum Hydrodictyon rt, ticulatum ?

Dictyosphaeria cavernosa

Ringo (1967); Johnson and Porter (1968); Cavalier-Smith (1974); Goodenough and Weiss (1978)

Triemer and Brown (1975) Green et al. (1978)

Ettl (1966); Ettl (1976) Nakamura et al. (1978) Gaffal (1977) Ettl (1965) Eyden (1975) Marano (1976) Peterfi and Manton (1968);

Floyd (1978)

Mcestrup and Ettl (in press) Olson and Kochert (1970) Lembi and Walne (1969) Deason and Schnepf (1977) Arnott and Brown (1967) Melkonian (1977);

Melkonian (1978) Marchant (1979) Marchant and Pickett-Heaps

(1972) Hori and Enomoto (1978)

a Nephroselmis olivacea has a unique flagellar root system including three different microtubular roots.

type of fibre, named median proximal connecting fibre has only been found in some species of Chlorosarcinopsis (Melkonian, 1978) and it is at present unknown whether it is also characteristic of the Chlamydomonad-type.

The striation pattern of the distal connecting fibre in horizontal longitudinal section is bilaterally symmetrical consisting of three

Fig. 1. Cross section through the apical p a r t of a Enteromorpha l inza zoospore. A cruciform papi l la wi th 4 roots (arrows) is visible. Below the 4 roots 4 additional fibrous roots (system II; t r iangles) can be seen. x75000.

Fig. 2. Slightly oblique section through the apical p a r t of an Enteremorpha zoospore. 4 roots (arrows) and three additional fibrous roots (system If; triangles) are depicted, x75 000.

Fig. 3. Median longitudinal section through a basal body of E. l inza zoospore. Arrow points to " t e rmina l cap". x75000.

Fig. 4. S imi la r section through a basa l body of female gametes of Ulva lactuca. ×75000.

differently spaced pairs of electron-dense lines. Fine filaments running the length of the fibre were mentioned by Ringo (1967); their diameter has been determined in Chloro- sarcinopsis zoospores to be about 7 nm. The filaments in Chlamydomonas appear to have the same diameter as calculated from the published micrographs of Goodenough and Weiss (1978). The distal fibre attaches to the distal part of the two basal bodies below the transitional region. Observations on zoospores of Chlorosarcinopsis indicate that contact is made with the C-tubules of two different basal body triplets (Melkonian, 1978).

The functional role of the distal striated fibre is not well understood. It has formerly been assumed that it maintains the orientation of the flagellar bases with respect to each other and resists the opposing forces which the two flagella exert at their bases as they beat in opposite directions (Ringo, 1967). In the naked zoospores of some Chloro- sarcinopsis species however the distal fibre changes shape during flagellar beat, since different basal body orientations occur during forward or backward movement (Melkonian, 1978). Even in algal species, like Chlamy- dornonas, in which a cell wall might prevent autonomous changes of basal body orientation and changes in shape of the distal fibre, recent information suggests a more active function for the distal fibre. For example, Triemer and Brown (1975) observed that in motile zygotes of Chlarnydornonas rnoewusii, the flagella of the---gamete are motionless. While the internal structure of the axonemes in these cells ap-

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peared normal, the distal fibre was separated from beth basal bodies of the motionless pair of flagella thus perhaps preventing initiation of flagellar beat. Furthermore, studies on the isolated and reactivated flagellar apparatus of Chlamydomonas reinhardii (Hyams and Borisy, 1975, 1978) have led to the conclusion that the movement of the individual flagella is coordinated by the distal fibre. It remains to be determined whether this possible coordination is achieved by a contractile or a slid- ing mechanism.

The two proximal striated connecting fibres have been less well studied, though it is known that the striation pattern seems to be similar to that in the distal fibre. In zoospores of Chlorosarcinopsis (Melkonian, 1978) one electron-dense cross band of the proximal fibre is connected to a C-tubule of one basal body triplet and a second connection is established through electron dense material with a second basal body triplet. In zoospores of Chloro- sarcinopsis all 9 triplets of one basal body are linked to different connecting fibres, two triplets to the distal striated fibre, two triplets each to the two proximal striated fibres and three triplets to the median proximal connecting fibre (Melkonian, 1978, Figs. 49-51).

In two organisms with a typical Chlamy- domonad-type cell organization and flagellar apparatus, connecting fibres lacking striations have been described: Thus, in vegetative cells of Volvox carted Olson and Kochert (1970) observed non-striated "distal and proximal kinetosome bridges" linking basal bodies situated parallel to one another, and in zoo-

Fig. 5. Cross section through both root types (r~ and r~) of E. linza zoospore, r~ exhibits two root microtubules, r~ 4 microtubules (3 over 1 arrangement). Cross sections through two fibrous roots (system II; triangles) are also seen. x80 000.

Figs. 6 and 7. Cross sections through proximal and distal parts of the 4-stranded root of E. linza zoospore. Figure 6 proximal part (3 over 1 configuration), Fig. 7 distal part (2 besides 2 configuration). B = Basal body. x80000.

Fig. 8. Longitudinal section through a fibrous root (system II) of female gametes of Ulva lactuca revealing striation pattern (small arrows), b = basal body, ms = mating type structure, x75 000.

Fig. 9. Longitudinal section through a fibrous root (system I) of E. linza zoospore revealing striation pattern (small arrows), x 112 500.

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spores of Hydrodictyon reticulatum (Marchant and Pickett-Heaps, 1972) basal bodies were lying opposite to each other forming an angle of 180 ° and again a non-striated connecting fibre was described. However, in both these organisms connecting fibres were only studied in "vertical" longitudinal sections. It is thus possible that both Volvox and Hydrodictyon also have Chlamydomonad-type connecting fibres since in zoospores of some Chloro- sarcinopsis species where the basal bodies preferentially lie at an angle of 180 ° striations are indistinct when the distal fibre is cut in "vertical" longitudinal section, but when cut in '%orizontal" longitudinal section the striation pattern is always clearly visible irrespec- tive of the flagellar orientation (Melkonian, 1978, Figs. 12, 15, 24).

A very interesting deviation is found in Gloeomonas sirnulans (Schnepf et al., 1976). In mature cells of this organism the two flagellar bases are about 3-5/~m apart and the flagellar apparatus seems to be modified. Each of the four microtubular roots is distally associated with electron dense fibres (fibrous roots). Proximally these fibres leave the root microtubules and two fibres from each side converge and connect with the other two fibres from the opposite side forming a "connecting fibre". This connecting fibre is therefore not directly linked to the basal bodies but only indirectly through the microtubular roots. At least two of the four fibres are striated with a repetitive unit of 26nm (measured from the published micrographs). Secondary cytoplasmic microtubules seem to be organized at this connecting fibre. The repetitive unit and the organization of cytoplasmic microtubules strongly favour the assumption that in Gloeomonas microtubular root associated fibres (see below) function as connecting fibres. It can be speculated that during evolution Chlamydomonad-type connecting fibres have been lost and replaced.

4.1.2. Connecting fibres of the Bryopsis-type in biflagellate cells

This system is provisionally named Bry- opsis-type (Group H connecting fibres) and

much less is known about these fibres than about the Chlamydomonad-type and at present four genera are only tentatively included: Bryopsis, Microtharnnion, Ulva and Enterornorpha. The Bryopsis-type differs from the Chlamydomonad-type in several respects:

(1) The principal connecting fibres are non- striated in any plane of section. In most cases there are two principal connecting fibres each arising from a different basal body but touching each other midway between the basal bodies and so providing a connection between them.

(2) Each principal connecting fibre is directly or indirectly associated with an electron dense plate which closes the proximal end of each basal body at least temporarily (the "terminal cap" or "flagellar platform", see below).

(3) A second type of connecting fibre is only rarely found (in male gametes of Bryopsis maxima (Hori, 1977)).

Three of the four above mentioned genera are sufficiently different in the detailed structure of their connecting fibres to deserve separate discussion.

In Microthamnion kuetzingianum the published data indicate the presence of only one principal connecting fibre (Watson and Arnott, 1973; Watson, 1975). The presence of two fibres closely touching each other, which in most sections would appear as a single fibre however cannot be excluded. In Microtham- nion the electron dense plate has been named "flagellar platform" (Watson, 1975) and it is associated with the connecting fibre through a microtubular root. Watson (1975) has further demonstrated that the connecting fibre changes shape and the flagellar platform position during autonomous changes of basal body orientation. During forward swimming of the cell, the flagellar platform lies oblique to the zoospore axis and closes the proximal end of the adjacent basal body. The situation in the biflagellate gametes of Ulva has not yet been fully worked out, but two principal connecting fibres link the two basal bodies in a way described above for the Bryopsis-type. An

electron dense plate, named "terminal cap" (Melkonian, 1979) closes the proximal end of the basal body and is linked to the connecting fibre in a similar way as in Microtham- nion. In male gametes of Bryopsis (Hori, 1977) there are again two principal connecting fibres as described above. The electron dense plate is continuous with each of the connecting fibres and closes the proximal end of each basal body. The two touching connecting fibres in male gametes of Bryopsis lyngbyei are shown in Fig. 12 and part of one connecting fibre with associated electron dense plate in Fig. 13.

The function of Bryopsis-type connecting fibres and associated electron dense plates is unknown, but it is probably significant that in the four genera with non-striated connecting fibres prominent striated roots composed of a bundle of filaments (system II, see below) are present and linked to the electron dense plates either directly (e.g., Microtharnnion (Watson, 1975) and Ulva (see Fig. 8)) or possibly through a microtubular root (male gametes of Bryopsis lyngbyei; unpublished observations). It can thus be hypothesized that these roots may perform a similar function as the distal striated fibre in Chlamydomonad-type cells in coordinating and initiating flagellar beat. The electron dense plate may help to establish a separate ionic compartment (the interior of the basal body) during forward movement of the cell or prevent loss of ribosomes, which are especially abundant in these genera inside the basal body (e.g., Figs. 3, 4).

It is probable, when more green algal biflagellate motile cells are studied, that variations in detailed structure of connecting fibres will be found and that more basic types of connecting fibres will have to be distinguished.

4.1.3. Connecting fibres in quadriflagellate cells

In green algae producing quadriflagellate motile cells, variation in types and dispositions of connecting fibres is far greater than in biflagellate cells. Nearly every species

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which has been thoroughly studied exhibits its unique system of connecting fibres. The discussion below attempts to compare the different systems found in the ten studied genera.

(i) Pyramimonas and Tetraselmis. In the quadriflagellate prasinophyte Pyramimonas basal bodies are arranged as two pairs in a diamond shape. A complex system of three different connecting fibre types is present (Norris and Pearson, 1975). One prominent striated fibre (named "synistosome" by Norris and Pearson, 1975) connects the two basal bodies of different basal body pairs, which are closer together. This fibre is present in Pyr- amimonas parkeae (Norris and Pearson, 1975) and Pyramimonas orientalis (Moestrup and Thomsen, 1974). A second non-striated fibrillar band connects three different basal bodies in P. parkeae (Norris and Pearson, 1975). A third system of branching fibrils Cpericentriolar fibrils", Norris and Pearson, 1975) connects adjacent basal bodies and also links with the striated connecting fibre (Nor- ris and Pearson, 1975; Moestrup and Thorn- sen, 1974). A similar but somewhat modified system exists in the other quadriflagellate genus of the Prasinophyceae sensu Christen- sen, Tetraselmis (=Platymonas). In Tetra- selmis the two basal body pairs form a "zig- zag" row (e.g., Manton and Parke, 1965). Different basal body pairs are again connected by a prominent fibre, which is however non- striated but similar in appearance to that in Pyramirnonas. In their proximal parts the four basal bodies seem to be further connected by a system of thin fibrils and possibly two cross-striated fibres linking basal bodies i and 3 and 2 and 4 (Melkonian, unpublished observations).

(ii) PolytomeUa, Cylindrocapsa, Carteria- Group/. The quadriflagellate alga to be most likely related to Chlamydomonas is Poly- tornella agilis. One pair of basal bodies exhibits the Chlamydomonad-type of connecting fibres, and apparently even a median proximal fibre (e.g., Brown et al., 1976, Fig. 7); the second pair is connected only by a proximal

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non-striated fibre. Only opposite basal bodies are connected by fibres. The system of connecting fibres in zoospores of Cylindrocapsa geminella is similar to that in Polytomella, a slightly anteriorly positioned flagellar pair showing the Chlamydomonad-type of connecting fibres (Hoffman, 1976). In addition, however, adjacent basal bodies are linked by fibrillar connecting fibres (composed of filaments of 6-8 nm diameter). Four proximal striated fibres also probably link adjacent basal bodies. A similar system to that in Cyl- indrocapsa occurs in some Carteria species (Carteria Group I according to Lembi, 1975). In contrast to the scheme presented by Lembi (Text--Fig. C in Lembi, 1975) own observations on Carteria obtusa have indicated that the four basal bodies are not arranged in a square, but forming a diamond-shape, two opposite basal bodies being more close together than the other two (Fig. 10). This tendency towards a diamond-arrangement is however only slight compared to Cylindro- capsa. Adjacent basal bodies in Carteria Group I are linked by fibrillar fibres and proximal striated fibres as in Cylindrocapsa (Lembi, 1975; Melkonian, M., unpublished observations; e.g., Fig. 11). In contrast to Cylin- drocapsa zoospores, a distal striated fibre connecting opposite basal bodies seems to be absent in Carteria which therefore has no Chlamydomonad-type system of connecting fibres.

(iii) Carteria-Group II. Other species of Carteria (Carteria-Group II; Lembi, 1975)

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have a totally different system of connecting fibres in which similar to the situation in the biflagellate Gloeornonas fibrous roots provide basal body connections. A system of eight non- striated fibres and two striated fibres (originally connecting fibres ?) attach the fibrous roots to their respective basal bodies.

(iv) Ulva, Enteromorpha. In Ulva and Enterornorpha (Melkonian, 1979 and this paper) only two non-striated connecting fibres connect adjacent basal bodies of different basal body pairs and no other connecting fibres occur. The system of the quadriflagellate zoospores is otherwise identical to that of the biflagellate gametes of Ulva (see above).

(v) Stigeoclonium, Fritschiella. In Stigeo- clonium (Manton, 1964) and Fritschiella (Melkonian, 1975) zoospores information on connecting fibres is insufficient, but it can be seen that adjacent basal bodies are linked by non-striated connecting fibres, which differ from those of Cylindrocapsa zoospores and Carteria Group I because they cannot be resolved into fine filaments. At least two proximal striated connecting fibres linking adjacent basal bodies occur in zoospores of Fritschiella (Melkonian, 1975). It is not known if opposite basal bodies are connected by a special fibre in FritschieUa zoospores.

(vi) Schizorneris. In Schizorneris zoospores it has been reported that adjacent basal bodies are connected "by a cross-banded structure similar to the distal fibre in Chlarnydornonas" and opposite basal bodies are said not to be connected by special fibres (Birkbeck et al.,

Fig. 10. Cross section through anterior part of vegetative cells of Carteria obtusa. 4 basal bodies (b) in diamond-shape arrangement. Curved arrows point to proximal parts of 4 flagellar roots, x75000.

Fig. 11. Nearly longitudinal section through basal body of Carteria obtusa showing connecting fibre linking two basal bodies. Note electron dense material attached to the basal body (arrow) in the region where the connecting fibre attaches to the basal body. x67500.

Fig. 12. Vertical longitudinal section through connecting fibres in male gametes of Bryopsis lyngbyei. Two connecting fibres touch each other and appear as a single fibre; b = basal body. x67 500.

Figs. 13 and 14. Longitudinal section through one of the mierotubular roots in male gametes of Bryopsis lyngbyei. Curved arrows indicate path of one of the root microtubules along a striated fibre (system II; arrow) and beyond the fibre (Fig. 14) to terminate near the connecting fibre (cf). b = basal body; L = lipid droplet, x67500.

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1974). At their proximal ends adjacent basal bodies are connected by proximal striated fibres. On the basis of this Birkbeck et al. (1974) suggest that the flagellar apparatus of Schizorneris zoospores has affinities to that of the Oedogoniales, which have a conspicuous ring of cross-striated fibrillar material connecting the basal bodies (e.g., Hoffman and Manton, 1963; Pickett-Heaps, 1970).

Nevertheless both the Stigeoclonium/Frit- schieUa-system and the Schizorneris-system are in need of reinvestigation using serial sections.

With only the limited amount of information available on connecting fibres in quadriflagellate cells of green algae, it is only possible to speculate about the functional significance of the various structures. If one assumes that in analogy to the situation in biflagellate cells flagellar coordination is related to the presence of some cross-banded structure being either a connecting fibre or a striated fibrous root, then the following account in the quadriflagellate cells can be made: In Pyrarnimonas flagellar coordination could be provided by the prominent cross- striated connecting fibre, while in Tetraselrnis this function has to be performed by the two massive fibrous roots Crhizoplasts") as was already suggested by Salisbury and Floyd (1978; see below). Polytomella and Cylin- drocapsa zoospores have Chalmydomonad- type connecting fibres for flagellar coordination (at least in one flagellar pair). In the Ulvales (Ulva, Enteromorpha) again fibrous roots (system II) could be responsible for flagellar coordination, while the system of Carteria Group I species cannot at present be inter- preted in functional terms. Lembi (1975a) has found a striated fibrous root (system II) in one of the species of Carteria Group I but not in the other species of this group.

4.2. Fibrous roots

Fibrous roots can be defined as striated fibrous structures originating at basal bodies and terminating somewhere else in the cell.

They are therefore distinguished from connecting fibres, which originate at one basal body and provide a link between different basal bodies. Transitions occur in those organisms in which fibrous roots have been transformed to function as "connecting fibres" (e.g., in Gloeornonas and Carteria Group II species, see above). These fibres in a strict sense are neither connecting fibres nor fibrous roots.

A list of fibrous roots in green algae has recently been provided by Moestrup (1978). From this it is seen that fibrous roots are confined to the Chlorophyceae sensu Stewart and Mattox and the Prasinophyceae sensu Christensen, but are absent in the Charophy- ceae sensu Stewart and Mattox and also in the Bryophyta and the higher plants. Stewart and Mattox (1978) have speculated that fibrous roots (system II) of the Prasinophyceae were transformed during evolution to become the MLS-structures of the Charophyceae and higher plants (see also phylogenetic conclusions). Moestrup (1978) listed the striation periodicity of fibrous roots, but no at tempt was made to compare the different fibrous roots in other ways. Recently it has been shown that the fibrous roots Crhizoplasts") of Tetraselmis are contractile (Salisbury and Floyd, 1978) and that a single root exhibits different striation pat terns along its length which can be related to its contractile function (Melkonian, 1979; Rebenek and Melkonian, 1979). A new terminology of fibrous roots based on structural properties of the roots is therefore needed and such is at tempted in this paper. It is also apparent that the term rhizoplast, which was originally used by light microscopists, relates to different structures as seen in the electronmicroscope, some of them possibly not even being fibrous roots. It is therefore proposed that the term rhizoplast should not be used for a fibrous root without referring to its detailed structure.

At present two main types of fibrous roots can be distinguished, microtubular root associated f ibres(sys tem D and fibrous roots composed of a bundle of filaments (system II).

4.2.1. Microtubular root associated fibres (system I)

These fibres are int imately associated and physically linked to cruciate microtubular roots and therefore run parallel to the root microtubules. They overly and/or underly the root microtubules and in the lat ter case usu- ally are embedded in electron dense material. This electron dense material probably func- tions as a microtubule organizing centre for secondary cytoskeletal microtubules (e.g., Brown et al., 1976; Stearns et al., 1976). The fibres are always cross-striated with striation pat terns in the range of 25-35nm. No filaments running the length of these fibres have been observed. When the fibres are detached from the root microtubules, for example during isolation of the flagellar apparatus in Dunaliella, their striation pat tern changes (Hyams and Chasey, 1974). The spacing and numbers of cross-bars in a fibre differs between various green algae, but this may be due to different fixation procedures, insufficient resolution, or different functional states of the fibre. Termination of the fibres at basal bodies has not been studied in most species, but in Chlorosarcinopsis zoospores it has been shown that the fibre underlying four-stranded roots probably connects with two basal body triplets, while the fibre overlying the two-stranded root links to the basal body possibly only through one of the root microtubules (Melkonian, 1978). It is not known if microtubular root-associated fibres are contractile and their function is thus unknown. The fibre overlying the two stranded root in gametes of Chlamydomonas reinhardi makes direct contact with the mating type structure (Goodenough and Weiss, 1978) and the association may have a morphogenetic function or the fibre may function in signal transmission during mating structure activa- tion (Goodenough and Weiss, 1978). A similar association between a microtubular root associated fibre and a mating type structure is present in gametes of Ulva lactuca (Mel- konian, M., in preparation). The association of some fibres with electron dense material

97

organizing secondary cytoskeletal microtubules might also be functionally important.

In Table 2 are listed those species of green algae in which microtubular root associated fibres (system I) have been found. Information is also given on numbers and dispositions of the fibres in a cell. These data suggest that most if not all green algae with cruciate microtubular root systems will exhibit some kind of associated fibrous roots if carefully studied. Care is necessary since fibrous roots can be very thin (e.g., 20 nm in Chlamy- domonas reinhardi; Goodenough and Weiss, 1978) and the fine striations can only be seen in extremely thin sections. Unfor tunate ly cross sections through microtubular roots often do not reveal the presence of root associated fibres owing to associated abundant electron dense material. It is thus proposed that negative stain preparations of isolated flagellar apparatus should be used whenever possible to see these fibres more clearly.

Numbers and dispositions of root associated fibres seem to be of some taxonomic significance (see Phylogenetic conclusions).

4.2.2. Fibrous roots composed of a bundle of filaments (system H)

These fibrous roots consist of a bundle of fine filaments (diameter range of individual filaments 5-10 nm, in most cases 8 nm) inter- rupted by cross-striations with a repetitive unit greater than 80 nm. They are with one exception (the "at tachment fibres" in Tetraselmis cordiformis; Melkonian, 1979) not closely associated with microtubular roots. They originate at one to four different basal bodies and in the lat ter case they branch. System II fibres take different paths within the cell. The fibres often run towards the nucleus and in these cases they can be related to the light microscopic image of a rhizoplast. Rhizoplasts have been described by light microscopists in a number of green algae as fibrous strands connecting basal bodies of motile algae with the nucleus (e.g., Entz, 1918; Hartmann, 1921; Zimmermann, 1921; Kater, 1929; Hovasse, 1943). In some cases (e.g., Tetraselmis) the

98

TABLE 2

Microtubular root associated fibrous roots (system I) in green algae

Organism No. of flagella No. and arrangement of fibrous roots References

Chlamydomonas reinhardi 2 1 continuous fibre overlying 2-stranded Goodenough and Weiss (1978) roots

2 4, associated with both root types 2 1, associated with 3-stranded root

Dunaliella primolecta Nephroselrnis olivacea

(Heteromastix angulata ) Chlorosarcinopsis dissociata

Hyams and Chasey (1974) Moestrup and Ettl , (in press)

Pedinom onas minor 1(2) Oedogonium cardiacum 30-120

(approx.)

2 2 (1 continuous?), overlying 2-stranded Melkonian (1978) roots

Stigeoclonium sp. 4 2, underlying 2-stranded roots Manton (1964) Fritschiella tuberosa 4 2, under lying 2-stranded roots Melkonian (1975) Cylindrocapsa geminella 4 at least 4, over- and under lying

two of the four roots Hoffman (1976) Urospora penicilliformis 4 4 associated with microtubular roots? Kris t iansen (1974) Ulva lactuca 2, 4 at least 2, under lying 2-stranded roots Micalef and Gayral (1972);

Melkonian (1979) Enteromorpha linza 4 at least 2, underlying 2-stranded roots Present paper Polytomella agilis 4 1 continuous and 2 separate fibres over-

lying the four 2-stranded roots 1, underlying 3-stranded root many, underlying 3-stranded roots

Brown et al. (1976) Ettl and Manton (1964) Hoffman and Manton (1962);

Hoffman and Manton (1963); Hoffman (1970); Pickett- Heaps (1971, 1972)

light microscopic observations were verified by later electronmicroscopic work (Manton and Parke, 1965), but in others the observations could not be confirmed and Watson and Arnott (1973) have suggested tha t some rhizoplasts described by light microscopists were merely strands of endoplasmic reticulum extending between the region of the flagellar apparatus and the nucleus. Later EM-studies have shown that fibrous roots consisting of cross-striated filament bundles approach and terminate at the nucleus in some species (e.g., the four fibres in zoospores of Urospora penicilliformis; Kristiansen, 1974), and that in others they attach to the nuclear envelope and then run towards the plasmalemma to terminate there, for example in Tetraselmis (Manton and Parke, 1965; Robenek and Mel- konian, 1979). In yet other species they run in a superficial position parallel to the microtubular roots below the plasmalemma (e.g., Ulva lactuca (Melkonian, 1979) and Entero-

morpha linza, this paper). Only the four fibres of Urospora would conform to the term rhizoplast as originally proposed, though the fibrous roots of Urospora and Ulva are very similar in their detailed structure. It can thus be seen that the term rhizoplast is not unequivocally defined and should either be omitted or only used in a restricted sense for those system II fibres which attach at the nucleus.

In Table 3 are listed those species of green algae containing fibrous roots composed of a bundle of filaments (system II fibres) together with numbers per cell and path of the fibres within the cell. Moestrup (1978) has suggested that the periodicity of cross-banding in fibrous roots may be phylogenetically significant, but recent work with Tetraselmis (Salisbury and Floyd, 1978) has shown that the fibres are contractile and respond to changes in external calcium concentration by contracting or expanding. In Tetraselmis cordiformis,

TABLE 3

System II fibrous roots (fibrous roots with filaments) in green algae

99

Organism No. of flagella No. and arrangement of fibrous roots References

Nephroseimis olivacea 2 1, passes nucleus, microbody and Moestrup and Ettl (in press) (Heteromaetix angulata) terminates at chloroplast

Monostroma grevillei 2 at least 1, parallel to root micretuhules (approx. 200 nm apart)

2, 4 4, parallel to root microtubules (approx. 200 nm apart)

Ulva lactuca

Enteromorpha linza 4

Urospora peniciUiformis 4 Microthamnion kuetzingia- 2

n ~ m

Carteria radiosa 4 Pyrarnimonas spp. 4

(5 species)

Prasiola stipitata 2 Tetraselrnis spp.

(5 species) 4

Prasinocladus marinus 4

Heteromastix rotunda 2

Scourfwldia caeca 2

Pseudoscourfieldia marina 2

Monornastix sp. 1(2)

4, parallel to root microtubules (approx. 200 nm apart)

4, approaching nucleus 1, approaching nucleus, special

arrangement at ER at least 2, approaching nucleus 1 (or 2?.), passes micrebody and

terminates near nucleus

1 (or 2?.), approaching the nucleus 2, attaching to nucleus and

terminating at plasmalernma

2, attaching to nucleus and terminating at plasmalemma

1, passing nucleus, terminates at chloroplast



1, passing nucleus, termination? special striation?

Jonsson and Chesnoy (1974); Moestrup (1978)

Micalef and Gayral (1972); Melkonian (1979 and unpublished (gmnete~))

present paper Kristiansen (1974) Watson and Arnott (1973);

Watson (1975) Lembi (1975b) Manton (1966, 1968); Moestrup

and Thomsen (1974); Norris and Pearson (1975)

Friedmann and Manton (1960) Manton and Parke (1965);

McLachlan and Parke (1967); Parke and Manton (1967); Salisbury and Floyd (1978); Melkonian (1979); Robenek and Melkonian (1979)

Parke and Manton (1965)

Manton et al. (1965)

Manton (1975)

Manton (1975)

Manton (1967)

fixation carried out in a natural ionic environment (Melkonian, 1979) resulted in an image in which the striation pattern changed from the proximal regions of a single fibrous root to the distal regions (from a 105nm repeat to a 220 nm repeat). Evidence has been presented that fixation carried out in a solution lacking some ionic constituents leads to an equally spaced fibrous root, which is therefore a fixation artifact (Robenek and Melkonian, 1979). Taking all these facts into consideration, it is suggested that striation patterns in fibrous roots (system H)

should not be used for taxonomic purposes and the difference in spacing observed for example between Microthamnion zoospores (80nm repeat; Watson, 1975) and some Tetraselmis species (approx. 500 nm repeat; Manton a n d Parke, 1965) might merely be a reflection of different functional stages. The function of ~ystem 1I fibrous roots might be related to the coordination and initiation of the flagellar beat (suggested by Salisbury and Floyd, 1978) and in most cases where these fibres are present, it is noteworthy that striated connecting fibres are absent from the flagellar ap-

100

paratus (discussion see above). Furthermore, these fibres are involved in the formation of spindle microtubules during mitosis in Tetra- selmis subcordiforrnis (Stewart et al., 1974) and Nephroselmis olivacea (=Heteromastix angulata) (Mattox and Stewart, 1977). In addition they might be associated with microtubule organizing centres, since secondary cytoplasmic microtubules are sometimes found originating at these fibres.

Finally, mention should be given to the description of a very unusual fibrous component in male gametes of the siphonous green alga Bryopsis maxima (Hori, 1977). Two of the four flagellar roots were described as being cross-striated at their anterior end and microtubular at their distal end, i.e., the root is heterogeneous along its length. A different conclusion is, however, drawn from a reexamination of the root system in male gametes of another Bryopsis species (B. lyngbyei) in which a fibrous root of system II connects at least one of the two basal bodies with a microtubular root (Fig. 14). The microtubular root, however, does not terminate at the a t tachment point of this fibre but runs alongside the fibre (Fig. 13) towards the cell apex and terminates in a similar way to the other microtubular roots below the connecting fibre (curved arrows in Fig. 14). The system in Bryopsis male gametes is therefore only unique in that the fibrous root does not attach to a cell organelle (nucleus, mitochondria, microbody, chloroplast) or to the plasmalemma but to a microtubular root instead.

4.3. Phylogenetic conclusions

The presence of two lines of evolution within the green algae seems to be fairly well established (Stewart and Mattox, 1975; Moes- trup, 1978) and the ancestors of both the Chlorophyceae and the Charophyceae sensu Stewart and Mattox were possibly scaly green monads, descendants of which are placed in the Prasinophyceae sensu Christen- sen. Most members of the Prasinophyceae are characterized by a cruciate microtubular root

system (Moestrup, 1978; Melkonian, 1979) and in addition have fibrous roots of system II Crhizoplasts"). The present phylogenetic discussion concentrates on the problem how the MLS-structure of the Charophyceae and higher plants might have evolved in a scaly flagellate with a cruciate microtubular root system. Stewart and Mattox (1978) suggest that fibrous roots have been transformed to MLS-structures, while Moestrup (1978) be- lieves that a transformation of the cruciate microtubular root system took place in the evolution of the MLS-structure. Moestrup and Ettl (in press) have presented evidence in favour of the lat ter hypothesis in a study on Nephroselmis olivacea (= Heteromastix angulata), in which one of the three microtubular roots is apparently multilayered. While the present paper on fibrous structures in green algae cannot give evidence in favour of one of the hypotheses, some phylogenetic conclusions might be drawn.

Because of their nearly universal occurrence in the Prasinophyceae, the presence of fibrous roots (system II) may be regarded as a primitive character. The presence of this type of fibrous root in the Ulvales is possibly of phylogenetic significance, because the Ulvales have a peculiar cytokinetic mechanism and some swarmers have scales on their surface (review by Melkonian, 1979). Other members of the Chlorophyceae with these fibrous roots have long been regarded as occupying rather isolated systematic positions (e.g., Prasiola, Urospora, Microthamnion, etc.). Relationships between these genera at first sight seem to be unlikely, but it is for example quite interesting that the similarities in the flagellar apparatus between Bryopsis and Microthamnion are matched by the presence of the unusual carotenoid siphonaxanthin in Microtharnnion as well as in the siphonous green algae (Weber and Czygan, 1972). A further conclusion is that Carteria is possibly not a close relative to Chlarnydornonas because system II fibres were found at least in one Carteria species and Carteria has no Chlamydomonad-type connecting fibres. Such

Chlamydomonad-type connecting fibres, however, occur in quadriflagellate cells and in fact Polytomella appears to be a later offshoot of Chlamydomonas, in which the flagellar root system has doubled and a Chlamydomonad- type system of connecting fibres persisted in the original basal body pair (Brown et al., 1976). The green algae producing biflagellate cells with Chlamydomonad-type connecting fibres seem to constitute a natural group. A further evidence for this conclusion is that system I fibres in these algae preferentially overly the two-stranded microtubular roots. A puzzling exception from this is the prasinophyte Nephroselmis olivacea which contains Chlamydomonad-type connecting fibres but even no cruciate flagellar root system (Moes- trup and Ettl, in press). It should however be mentioned that Manton et al. (1965) studying the probably identical Heterornastix angulata reported the presence of a two-stranded root which has not been found by Moestrup and Ettl (in press). This discrepancy should be clarified. Nephroselmis olivacea is regarded by Moestrup and Ettl (in press) as a descendant of a Pyrarnimonas-like cell. The presence of Chlamydomonad-type connecting fibres might then indicate that the synistosome of Pyr- amimonas is homologous to the distal connecting fibre of Chlamydomonad-type cells. This hypothesis would further imply that quadriflagellate species of green algae were probably ancestors for most green algae producing biflagellate cells.

These few comments indicate the potential usefulness of fibrous structures associated with basal bodies of green algae for phylogenetic considerations in the Chlorophyceae and Prasinophyceae, but some objections to this should also be made. With respect to the enormous variability of fibrous structures, the number of thoroughly studied species is far too small for detailed phylogenetic conclusions. Furthermore one has to imagine that special modifications in fibrous structures have evolved in response to functional special- izations, e.g., with respect to gamete fusion or unusual basal body dispositions. These peculi-

101

arities should be taken into consideration when drawing phylogenetic conclusions.

Acknowledgements

Part of the study was made at the Biologi- sche Anstalt Helgoland during several weeks in 1976 and 1977 and I would express my thanks to the Director for giving me the opportunity to use the facilities of the Biolo- gische Anstalt. Technical help during various stages of the work by Mrs. B. Berns, Mrs. I. Wachholz and Mrs. E. Manshard is gratefully acknowledged.

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Note added in proof

Two m o r e a r t i c l e s c o n t a i n i n g r e l e v a n t in - f o r m a t i o n on f ib rous s t r u c t u r e s a s soc i a t ed w i t h

104

basal bodies of green algae have appeared since this paper was written: In Chlamydomonas moewusii K.R. Katz and R.J. McLean (1979) demonstrated the presence of two system II fibres for the first time in the genus Chlamy-

domonas (J. Cell Sci. 39, 373-382) and R. Birchem and G. Kochert (1979) presented micrographs indicating that sperm cells of Volvox carted contained a distal striated connecting fibre (Phycologia 18, 409-419).

ultrastructural aspects of basal body associated fibrous structures in green algae: a critical...

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