P1. Syst. Evol. 130, 265--292 (1978) Plunt |ystemutits und Egnlutiun © by Springer-Verlag 1978

Botanisches Insti tut , Westf~lisehe Wilhelms-Universit&t, M/inster

Structure and Significance of Crueiate Flagellar Root Systems in Green Algae: Comparative Investigations in Species

of Chlorosarcinopsis (Chlorosarcinales) 1

By

Michael Melkonian ~, MUnster

(Received March 10, 1978)

Key Words: Chlorophyceae, Chlorosarcinopsis, C. pseudominor, C. gela- tinosa, C. auxotrophica, C. minor, C. dissociata.--Comparative ultrastructure, zoospores, flagellar root system, function, taxonomy.

Abstract: The ultrastructure of biflagellate zoospores in seven species of the green alga Chlorosarcinopsis (Chlorosarcinales) was studied, particu- larly the cruciate flagellar root system, and a comparative approach was attempted. The seven species constitute four groups on the basis of pyrenoid ultrastructure, presence or absence of a cell envelope covering zoospores, and details of the flagellar root system. Variable structures inehide root tu- bule number (4-2-4-2 or 5-2-5-2), association of electron dense inateriM with root tubules and root tubule configurations. Otherwise the root system is similar in all species. Zoospores which lack a cell envelope (three species) show characteristic rcorientation of basM bodies with flagellar beat. This results in different images of some basal body associated structures (e.g. distal striated fiber). A fixed basal body orientation was noted in those species where zoospores are covered by a cell envelope. Structural contact of root tubules from root type I I (4- or 5-stranded root) with the outer chloro- plast membrane in the region of the eyespot was demonstrated for M1 species. On the basis of some new observations a generM description of the crueiate flagellar root system in biflagellate green algae is given and a new nomen- clature is offered. Taxonomic implications and functional aspects of erueiate flagellar root systems in biflagellate green algae are discussed.

Phyle t ic implicat ions of flagellar root systems have been suggested

as ear ly as 1965 (MA~TO~ 1965). Dur ing the past few years studies on

I First contribution of a series. 2 This work was carried out at the Inst i tut fur Allgemeine Botanik,

Universit/it Hamburg.

18"

0378-2697/78/0130/0265/$ 05.60

266 M. MELKONIAN: Cruciate Flagcllar Root Systems in Green Algae

mitosis, eytokinesis and flagellar root systems led to a reclassification of the green algae (STmvA~aT & MATWOX 1975, PICKET~-HEAPS 1975a). Most green algal genera were placed in the "Chlorophyceae" and a few were plaeed in the "Charophyceae". Besides other characteristics the "Chlorophyceae" show a cruciate flagellar root system with four micro- tubular roots. There is however a large discrepancy between the few thoroughly studied genera of the Chlorophyceae sensu ST~WAnT & MAT- • OX and the large majority of genera possibly belonging to this group. Furthermore it is not known if the cruciate flagellar root system can be used with equal success for phylogenctic considerations within the Chlorophyceae as it has been used to differentiate these from the Charo- phyceae.

The reasons for this uncertainty are manifold. There are no sound investigations into the function of root systems, there is a confusing nomenclature and no generally agreed strategy to study flagellar root systems. Even with regard to the investigated species there is a con- siderable lack of comparable data. For example it has only recently been established that one of the best known species Chlamydomonas reinhardi is characterized by a bilateral symmetric root system (4-2-4-2) including the presence of a flagellar root-associated striated fiber (Mv.L- KONIAN 1977, GOODEXO~GH & WEISS 1978).

Figs. 1-8. Different aspects of zoospore ultrastructure in Chlorosarcinopsis pseudominor. Fig. 1. Longitudinal section through the zoospore. Note posterior position of the nucleus (N). × 7,500. Fig. 2. Transverse section through the apical part of the cell revealing two basal bodies (b) and four roots. Bilateral symmetric arrangement of roots and two root types (rl and rz) can be distinguished. Small arrows indicate secondary eytoskeletal microtubules. × 60,000. Fig. 3. Median longitudinal section through apical part of the zoospore, basal bodies and distal striated fiber (d/) visible. Large arrows indicate electron dense material which forms part of the median proximal fiber. Small arrow pointing to tubule 1 of root type I. × 60,000. Fig. 4. Tangential section through the eyespot-region. Root type II in a 2 besides 2 configuration is present (large arrows). The eyespot lies between both parts of the root. Star shaped tufts of the cell envelope can be seen covering the cell (small arrow). × 27,000. Fig. 5. Cross section through the distal parts of root type I. Electron dense material overlying the root tu- bules represents the root associated striated fiber. One secondary cyto- skeletal microtubule is also present. × 60,000. Fig. 6. Distal cross section through root type II, revealing 3 + l-configuration, small arrow points to secondary rnicrotuhule. × 60,000. Fig. 7. Pyrenoid of C. pseudominor. Notice continuous starch sheath and tubular thylakoids in the pyrenoid matrix. Small dots in the lumen of the tubules represent fused thylakoid membranes. × 22,500. Fig. 8. Cross section through eyespot. Root tubules of root type II can be seen adjacent to the outer chloroplast membrane

(large arrows). × 60,000

Figs. 1-8

268 M. MEL~CONIAN: Cruciate Flagellar Root Systems in Green Algae

This au thor believes t h a t a prerequis i te for using crucia te f iagellar root sys tems in t a x o n o m y and phy logeny of green algae is de ta i led knowledge of the i r degree of va r i a t ion in small sys temat ic categories and the i r funct ional significance. I n this series of papers i t is in tended to use selected species and genera of green algae to approach this goal. The series will ma in ly deal wi th var ia t ions of f lagel lar roo t sys tems

a) wi th in different species of one algal genus, b) wi th in one algal s t ra in according to different env i ronmenta l

factors including inhibi tors , c) wi th re la t ion to sexual d i f ferent ia t ion of algal gametes , and d) wi th re la t ion to the occurrence of different numbers of f lagel la

wi th in one s train. I n this pape r f lagel lar roo t sys tems of zoospores in seven species of

the green alga Chlorosarcinopsis were compared and the i r degree of va r i ab i l i t y was de te rmined . This genus was used because i ts t a x o n o m y seems well s tud ied (Gl~oovEl~ &BOLD 1969), all species are avai lab le th rough cul ture collections and because no record of the fine s t ruc ture of f lagel lar root sys tems has been made in the order Chlorosarcinales yet .

Materials and Methods

Chlorosareinopsis minor HERNDON (Utex 949), U. dissociata HERNDON (Utex 948), C. gelatinosa CH~TANACHA~ et BOLD (Utex 1180), U. pseudo- minor GROOVER et BOLD (Utex 1702), C. auxotrophica G~OOVER et BOLD syn. Chlorosphaera consociata KLEBS (Utex 722) were all obtained from the Culture Collection of Algae at the Universi ty of Texas, Austin. Culture con- ditions of the algae and zoospore induction was the same as described for U. minuta and C. spec. by MELKO~IAN (1977). In some species zoospores were not produced in large enough amounts and the technique of zoospore collection was modified: Vegetative cell packets were centrifuged to a pellet and a concentrated solution was pipet ted into a petri dish containing fresh culture medium in such a way tha t only a small area was covered by the

Figs. 9-12. Different aspects of zoospore ul trastructure in Uhlorosarcinopsis gelatinosa. Fig. 9. Longitudinal section through the zoospore, nucleus (N) in apical position and dictyosom (d) characterist ically arranged below the nucleus. × 13,000. Fig. 10. Dried preparat ion of zoospore killed in osmie aeed vapour and shadowed. Cell shape pear-like. × 4,500. Fig. 10. a Zoo- spore killed in glutaraldehyde and photographed with phase contrast optics. Note difference in cell shape between Fig. 10 and 10 a. Small arrow indicates position of the eyespot. × 1,700. Fig. 11. Oblique section through the apical par t of the cell. Two basal bodies with transit ional regions, a proximal s t r ia ted fiber (p]) and all four roots (rl, r~) visible. × 60,000. Fig. 12. Median longitudinal section through the apieM par t of the cell. Note unelevated basal body orientation, special structure of the distal s tr iated fiber (d[) and

the two tubules of root type I (rl). × 60,000

4 4 ¸̧

4 t " ~

Figs. 9-12

270 M. !t¢[ELKONIAN: Cruciate Flagellar Root Systems in Green Algae

vegetative cells. At the beginning of the first or second light phase zoospores were collected at the meniscus of the induction medium where they tended to concentrate. Fixation and further processing of the samples have been described in earlier papers (MELKONIAN 1975, MELKONIA~r 1977). Light microscopy was carried out using a Zeiss Standard microscope fitted with phase contrast optics. For purposes of photography cells were fixed in buf- fered glutaraldehyde. For whole mounts, a drop of zoospore solution was placed on a mowital/earbon-eoated grid. This was brought into a petri dish saturated with osmie acid vapour. After 30 seconds the grid was removed from the dish, the solution allowed to dry in a stream of sterile air and the precipitated salt washed away with several washes of distilled water. The grid was then shadoweast with either chromium or platinum.

Results

For comparative aspects the previously published results on zoo-

spores of C. minuta and C. spec. (MELKONIAN 1977) are included.

Zoospore Size and Shape. Size and shape of zoospores between different species varies to a greater extent. Some k ind of var ia t ion also occurs wi thin a given strain. F ive species (C. minuta, C. spec., C. pseudo- minor, C. auxotrophica, C. minor) more or less conform to a general shape which could be described as ovoidal to slightly elongate (Fig. 1), in C. minor the posterior par t is often pointed (Fig. 24). The size is variable bu t usual ly wi thin 3.5-5 ~m and 6-10 ~zm with C. minor being slightly larger (5-7 ~m and 10-14 Ixm).

Two species show elongate to fusiform zoospores (C. gelatinosa, Fig. 9 and 10 inset and C. dissociata, Fig. 34). I n C. ffelatinosa zoospores

Figs. 13-23. x 60,000. Fig. 13. Cross section through the distal striated fiber, details of how the fiber connects to both basal bodies (b) are revealed. Fig. 14. Cross section through proximal part of root type I. Both tubules lying near the proximal striated fiber (arrow). Interconncctions of root tubules and the prominent link of tubule I with the fiber can be seen. Root type II is cut oblique (triangle). Fig. 15. Horizontal longitudinal section through the distal striated fiber. From absence of the two basal bodies in this section it is concluded that this cell has unelevated basal bodies. Stria- tion pattern of the fiber clearly visible. Fig. 16 and Fig. 17. Cross sections through different parts of root type I. In distal sections (Fig. 17) a greater number of secondary cytoskeletal mierotubules is present and the root tubules can only be distinguished through serial sections (small arrows). Fig'. 18-22. Cross sections through different parts of root type II. In prox- imal sections (Fig. 18) root tubule 1 underlies root tubule 5 (arrow). The next sections represent more distal sections. Root tubule 1 changes its path and successively underlies tubule 4 (Fig. 19), tubule 3 (Fig. 20) and tubule 2 (Fig. 21). Small arrow in Fig. 20 points to a secondary mierotubule, which is probably organized at electron dense material underlying the root tubules. Fig. 22 shows tubule 1 associated with tubule 2. Tubule 1 has a prominent link (arrow), which is also present in the eyespot region (Fig. 23, arrow).

Fig. 23 shows l besides 1 configuration of root tubules.

15

Figs. 13-23

272 M. MnLKO~IA~: Crueiate Flagellar Root Systems in Green Algae

were usually 3-4 times longer than broad (Fig. 9). Dramatic changes in zoospore size and shape were noticed when the cells were fixed with Os04 and shadowcast (Fig. 10). The posterior part of the cell rounds up and the total cell length corresponds to only one third of that of living cells or glutaraldehyde fixed cells (Fig. 10 inset). This effect is especially noticeable with regard to the relative length of the flagella to the cell body (compare Fig. 10 and inset). Rounding up of the zoospores occurs also in rive when a cover slip is applied to swimming zoospores. Within a few seconds the cell becomes sphericM (measuring about 4 × 4 ~zm) but apparently is still motile. Zoospores of C. dissociata are also elongate and pointed at the posterior end but are larger than those of C. gelatinosa (6 × 14 ~m). No apparent changes in size and shape occurs in this species when the cells were treated with Os04 and shadowcast.

(~ell Envelope. Four of the investigated species (C. minuta, C. spec., C. pseudominor, C. auxotrophica) produce zoospores which when swim- mini are covered by a delicate cell envelope. The detailed structure of the envelope is the same in each species and can best be seen in tangential sections through zoospores (Fig. 4). Star shaped tufts composed of five small fibrils constitute one single layer overlying the plasmMemma

Figs. 24-33. Different aspects of zoospore ultrastructure in Chlorosarcinopsis minor. Fig. 24. Median longitudinal section through the zoospore. Note anterior position of the nucleus (N). Small arrow depicts distal striated fiber with unMevated basal bodies. × 7,500. Fig. 25. Vertical longitudinal section through the distal striated fiber. Striation pattern can be evaluated. Note angle between the basal bodies and different shape of the distal fiber as compared to Fig. 24. X 60,000. Fig. 26. Longitudinal section through proximal parts of root type I. Tubules 1 from opposing roots are seen in longitudinal section (small arrows). They end pointed near the distal striated fiber (d/) and very close together. Curved arrow points to underlying electron dense material, x 60,000. Fig. 27. Cross section through apical part of the cell showing both root types in cross section (arrows). I~oot type I I shows 3 + l-configuration with tubule 1 already separated from tubule 2. Under- lying electron dense material is more distinct in root type I. X 60,000. Fig. 28. Cross section through distal part of root type I. Note asymmetric position of overlying striated fiber (arrow). × 60,000. Fig. 29. Details of pyrenoid ultrastructure. Only single thylakoids enter the pyrenoid matrix (arrow). × 27,000. Fig. 30. Cross section through proximal part of root type II. Tubules 2, 3 and 4 underly the distal striated fiber (d/) and tubules 2 and 3 are linked to the fiber (arrow). × 60,000. Fig. 31. More distal cross section through root type II. The electron dense plate underlying tubule 2 and 3 and linked to these tubules can be seen (arrow). × 60,000. Fig. 32. 3 besides l-configuration of root tubules from root type II. × 60,000. Fig. 33. Root tubules from root type II near the cross-sectioned eyespot. Please note cross-links of tubules 2, 3 and 4 with the outer chloroplast

membrane (small arrows), x 60,000

Figs. 24-33

274 M. MELKO~IAZ¢: Cruciate Flagellar Root Systems in Green Algae

(Fig. 5, 6, 8). The flagellar surface distal to the t ransi t ional region of the basal body is not covered by the envelope (Fig. 3). This envelope is a consistent feature of zoospores in the above named four Chlorosarcinopsis species, while it is no t present in the other three species, where the p lasmalemma is the only cell boundary of the zoospores.

In terna l Organization. Some species show different pa t te rns in the a r rangement of the major cell organelles as revealed by longi tudinal median or near ly median sections through zoospores (Fig. 1, 9, 24, 34). I n four species (C. minuta, C. spec., U. pseudominor, C. auxotrophica)

Figs. 34-47. Different aspects of zoospore ultrastructure in Chlorosarcinopsis dissociata. Fig. 34. Longitudinal section through the zoospore. Nucleus is located anterior (N) and associated with a dictyosom. The chloroplast fills the posterior part of the cell and a lobe containing the eyespot (e) extends into the anterior part of the cell. Note irregular distribution of starch (s) around the pyrenoid (P). × 9,000. Fig. 35. Transverse section through the apical part of the cell, two basal bodies (b) and four roots (rl, r2) are shown. Bilateral symmetric arrangement of root types is evident. Small arrow depicts fine striations from the root associated striated fiber. × 60,000. Fig. 36. Cross section through proximal part of root type I. The root is connected to the proximal striated fiber, x 60,000. Fig. 37. Cross section through distal part of root type I. Note striated fiber overlying root tubules as electron dense material and prominent link of tubule I (arrow). × 60,000. Fig. 38. A similar section as in Fig. 37. Striated fiber (large arrows) is con- nected to the root tubules and underlying dense material is finely striated in this section (small arrows). × 120,000. Fig. 39. Section through the apieal part of the cell revealing both root types in cross section. A 4 over 1-configura- tion is present in root type I I (tubule 1 underlies tubule 3). Note underlying electron dense material associated with both root types (small arrows). × 60,000. Fig. 40. Cross section through proximal part of root type II . Tubules 2, 3, 4 and 5 lying below the distM striated fiber (d/) and through links seem to be connected to this fiber. Small arrow points to tubule 1 which underlies tubule 5 in this proximal section. × 60,000. Fig. 41. A slighter distal section as in Fig. 40. Tubule 1 is now lying between tubule 4 and 5. × 60,000. Figs. 42 and 43. Median longitudinal sections through apical parts of zoospores depicting different basal body orientations. In Fig. 42 basal bodies are elevated and the distal striated fiber is short and thick. Please note presence of a few secondary microtubules above the flagellar apparatus in an apical papilla (small arrow). Fig. 43 shows unelevated basal bodies and the distal fiber although cut oblique assumes a different shape as in Fig. 42. × 39,000. Fig. 44. Cross section through root type II . Electron dense plate assoicated with tubule 2 and 3 can be seen (small arrow). × 60,000. Fig. 45. A more distal section as in Fig. 44. Tubule 1 underlies tubule 3.

Note presence of underlying electron dense material. × 60,000 Fig. 46. Distal cross seetion through root type II . Root tubules in a 4 besides I-configuration. Note electron dense material associated with tubule 1 (arrow). × 60,000. Fig. 47. Cross section through the eyespot. Root tubule 1

is linked to the outer chloroplast membrane (arrow). × 60,000

Figs. 34--47

276 M. M]~LKONIA~: Crueia~e Flagellar Root Systems in Green Algae

the position of the nucleus in the cell is quite variable differing between anterior, central and posterior (Fig. 1) in one population of zoospores. Correspondingly other cell organelles including the chloroplast and dictyosomes show similar variations. In the other three species cell organelles are fixed in certain positions. The nucleus is located in the anterior part of the cell below the two contractile vacuoles (Fig. 9, 24, 34). In close association with the nuclear envelope a dictyosom occupies a position in the center of the cell. The chloroplast including the pyrenoid always constitute the posterior part of the cell, while one or more lobes of the chloroplast extend into the anterior part of the cell. The most anterior lobe usually contains the eyespot (Fig. 34). More variable is the distribution of mitochondria, lipid droplets and vacuoles.

Pyrenoid Ultrastructure. Each zoospore contains one prominent pyrenoid. Three types of pyrenoids were recorded in the investigated species.

In five species (C. minuta, C. spec., C. pseudominor, C. auxotrophica, C. gelatinosa) the pyrenoid is surrounded by a continuous starch sheath which is traversed by a few thylakoids entering the matrix. Usually two thylakoids enter the matrix at one time. They give rise to tubular channels, which when cross sectioned show an electron dense spot in the lumen (Fig. 7). In longitudinal sections through thylakoid tubules these spots are rod-like, They are formed from fusion of two adjacent thylakoid membranes. Stroma material is excluded from the tubules.

In C. minor (Fig. 29) the starch sheath is traversed by many lamellar structures. These lamellae consist of only one thylakoid which is appar- ently not modified. Stroma-hke material is again excluded.

In C. dissociata (Fig. 34) the massive starch sheath, which is dis- continuous oyez larger areas, is traversed by few thylakoids. Two thylakoids enter the matrix at one place and form large irregular areas inside the matrix. These areas probably include stroma-like material. Thylakoid membranes do not fuse in this pyrenoid type.

Basal Body Orientation and Distal Striated Fiber. Median longi- tudinal sections through zoospores in the plane of the flagella reveal both basal bodies and the respective angle between them. This angle was determined in a greater number of such sections for each species. Zoospores, which are covered by a cell envelope showed a rather constant basal body orientation with an angle of 90-110 ° (Fig. 3). If the flagella are extended backward, the bending of the flagella mainly occurs near their bases distal to the transitional region of the basal bodies. In species without an envelope basal body orientation seems to differ to a greater extent and is related to the position of the flagella. If the flagella are orientated laterally or backwards, the basal bodies project horizontally

Fig. 48. Cross section th rough prox imal pa r t of root type I in zoospores of 6'hlorosarc~nopsis minuta. R o o t tubule 1 is associated wi th the over lying electron dense plate, which af ter mak ing contac t wi th tubule 1 arches around and links to the distal s t r ia ted fiber. Tubules 1 and 2 are in te rconnec ted and tubule 1 is fur ther connected to a basal body t r iple t . In this section t e rmina t ion of a root tubu le (presumably tubule 2) f rom the o ther root t ype (II) is as well documen ted (large arrow). I n more proximM par ts the electron dense plate of this root t ype (small arrow) dissociates f rom the root

tubu le and approaches the basal body (b). × 120,000 Fig. 49. Cross section th rough a basM body in zoospores of Ghlorosarcinopsis spee. Dista l a t t a c h m e n t points of the med ian proximal fiber are seen as three electron dense areas associated wi th three basal body tr iplets (trian- gles). The proximal s t r ia ted fiber (pf) a t taches to a C-tubule of one t r iplet , while it is Mso connected to a C-tubule of the ad jacen t t r ip le t v ia electron dense mater ia l . The electron dense pla te of root t ype I I ex tends below the distal s t r ia ted fiber (d[) (small short arrow). The pla te seems to be connected v ia electron dense mater ia l to two tr iplets (long small arrow). × 120,000 Fig. 50. Cross section th rough a basal body in zoospores of G. spec. more p rox imal as in Fig. 49. P rox ima l s t r ia ted fiber (large arrow) and l ink be tween C-tubule of one t r iplet and distM s t r ia ted fiber visible (small arrow). × 120,000 Fig. 51. Ano the r cross section th rough a basal body in zoospores of G. spec. This shows t e rmina t ion of a root tubule f rom root t ype I I near the dis tal s t r ia ted fiber and detai ls of the connect ion be tween the electron dense pla te and two basal body t r ip le ts (small arrow). Large arrow probab ly depicts under ly ing electron dense mater ia l , d[ = distM s t r ia ted fiber. × 120,000

278 M. MEL~:ONIA~ :

in opposite directions (Fig. 12, 24, 43). If the flagella are extended forward, the basal bodies constitute an angle of varying degree (in most eases greater than 90°; Fig. 25, 42).

The basal bodies are joined near their distal ends by a distal striated connecting fiber (Fig. 3 d]). The striation pattern is visible in horizontal longitudinal sections (Fig. 15) or in vertical longitudinal sections (Fig. 3, 25), but not in cross sections (Fig. 13) through the fiber. The striation is bilateral symmetric and obviously the same in each species. A central dark line, sometimes visible as two closely adjacent dark lines, with a light line and another pair of dark lines at either side is the usual appearance. Fine filaments running the length of the fiber and con- necting the dark lines can be seen in horizontal longitudinal sections (Fig. 15).

Differences were observed in appearance of the distal striated fiber in those species showing different basal body orientations. In unelevated basal bodies (flagella posteriolaterMly, Fig. 12, 43) the fiber is thin and arches to form a bridge with a horizontal midregion and nearly vertical lateral regions connecting the fiber to the basal bodies. In elevated basal bodies (flagella projecting forward) the fiber is thicker and shorter and resembles distal fibers of zoospores with an envelope (compare Fig. 42 with Fig. 3).

The distal striated fiber is probably connected to two triplets of the respective basal body via C-tubules (Fig. 50, small arrow).

The variable basal body orientation observed in three species also affects the external shape of the anterior part of the cell. In unelevated basal bodies zoospores show a neck-like region posterior to the flagellar apparatus (Fig. 12), this is not observed in elevated basal bodies (Fig. 42). In the latter case however a prominent apical papilla is visible (Fig. 42), which again disappears in unelevated basal bodies (Fig. 43).

Proximal Fibers. Two proximal connecting fibers link the basal bodies at their proximal ends (Fig. 11, 14, 36, 49, 50). Striations are observed in longitudinal sections through this fiber (Fig. 11, 50). Stria- tion pattern seems to be the same in different species, while it is not known, if this fiber changes length and thickness with different basal body orientations. The fibers are connected to the basal body at its outside edge at either side. In appropriate cross-sections through basal bodies a proximal fiber seems to link with one triplet, two dark lines being connected to a C-tubule (Fig. 49, 50).

If a basal body pair is cut in a median longitudinal section (Fig. 3) electron dense material can be seen at the proximal ends of those basal body triplets which are faced to the posterior part of the cell (Fig. 3, large arrows). This material seems to be interconnected and results in a third type of connecting fiber (median proximal connecting fiber).

Cruciate Flagellar Root Systems in Green Algae 279

This fiber is not striated in any plane of section, its presence has con- vincingly been shown only in species with a fixed basal bedy orientation. When a basal body is cross-sectioned in this region (Fig. 49, 50) the fiber underlies three triplets and more distally seems to split up into three parts (Fig. 49, triangles).

Flagellar Root System. The Flagellar Root System in all inves- tigated species is cruciate with two alternating root types. One root (root type I, r 1 in Fig. 2, 11, 12, 35) is always two-stranded consisting of two microtubules along its length. The other root (root-type II, r 2 in Fig. 2, l l , 35) is four- or five-stranded with characteristic root tubule configurations along its length. Presence of two root types is indicated by transverse sections through the apical part of the cell (Fig. 2, 35) and proved in cross sections through adjacent roots (Fig. 27, 39). The orientation of the four roots with respect to the basal bodies is bilateral symmetric, the two roots of type I arranged in a straight line, the two roots of type II displaced against each other (Fig. 2, 35).

Some new informations on details of both root types were obtained and are used to introduce a new nomenclature of root tubule numbering.

Root Type I. The two tubules are numbered according to their position with respect to the adjacent basal body. In proximal cross sections through this root type one tubule has a prominent link to a basal body triplet (Fig. 14, 36, 48). This tubule is named tubule 1, the other tubule 2. The proximal termination of the two tubules was inves- tigated and it was found that tubule 2 terminates earlier, leaving only tubule 1 (Fig. 3, small arrow). The termination of tubule 1 was shown in a favorable longitudinal section through the proximal parts of such tubules from opposing roots (Figs. 26, small arrows). Both tubules 1 end pointed with their tips touching a faint dark line on either side at exactly the same position. This faint dark line has obvious connections to the distal striated fiber (Fig. 26 d/). An electron dense fiber overlies the two tubules for a considerable part of their length (Fig. 5, 28, 37, 38, 39). In proximal cross sections it is broader and constitutes the electron dense plate. This plate associates with tubule 1 (Fig. 48) and links with the distal striated fiber (Fig. 48). I t is about 15 nm thick. In distal sections this fiber preferent!y overlies tubule 2 (Fig. 27, 28) while still more distally it is more centrally located (Fig. 37, 38). In horizontal longitudinal section through this fiber it sometimes appeared that a fine striation was present, however only in C. dissociata a repet- itive unit of 23-25 nm (center to center) could be measured in three different sections.

R o o t T y p e I I . In this root type the tubules (either four or five) change their positions relative to one another in different parts of the cell, they exhibit different configurations. Through a greater number

19 PI. Syst. Evo1., Vol. 130, No. 3--4

2 8 0 M. MELKOI',IIAlq- :

of serial sections it has been possible to trace the pa th of every individual tubule, which could consequently be numbered. In more proximal cross sections through this root the tubules lie in two rows in a 3 over 1 (3 + 1) or 4 over 1 (4 + 1)-configuration. The tubule of the second row was named tubule 1. The other tubules from the first row were numbered according to their position to the adjacent basal body. I f the basal body is located at the left side of the root, the numbering begins on the left side of the row with tubule 2.

The proximal termination of root tubules was studied. Each root approaches the region of the distal striated fiber and makes contact with it (Fig. 30, small arrow, Fig. 40). Termination of root tubules are seen in longitudinal sections through proximal parts of the root (Fig. 48, 51). In Fig. 48 the tubule (large arrow) terminates with a pointed tip near the distal fiber, in Fig. 51 another tubule ends with a blunted tip in a similar region. In both sections tubules from the upper row are pictured, while it is impossible to designate a definite number to them. The termination of root tubule 1 is unclear. I t may be tha t it terminates earlier, which results in a 3 + 0-configuration.

Changes of root tubule configuration are mainly the result of the changing pa th of root tubule 1 along the length of the root. In the most proximal sections tubule 1 underlies tubule 4 (or 5 in the 5-stranded root) (Fig. 18 arrow, Fig. 30, 40 arrow). More distally tubule 1 moves with respect to the other tubules in such a way tha t it underlies suc- cessively tubule 3 (or 4) (Fig. 19, 44), tubule 2 (or 3) (Fig. 20, 45) and also in the 5-stranded root tubule 2 (Fig. 21). No exception has been found in any species from these configurational changes. More distally the dif- ferent species show differences in root tubule configurations (see below).

t o o t type I I is also associated with an electron dense plate at its most proximal part (Fig. 31, 44). This plate extends not into a fiber as in root type I. I t occupies a position between the root tubules and the adjacent basal body. In proximal cross sections it is associated with tubule 1 lying in the same plane (Fig. 3i, but best seen in Fig. 9 of MELKONIAS 1977) aS this tubule. In more distal parts the plate underlies preferently tubules 2 and 3 and is connected with these tubules by small thin links (Fig. 31, 44, 48 small arrow). The plate is also connected to the basal body. In Fig. 5l the plate links with the C-tubule of one and the B-tubule of the next basal body triplet (small arrow). The plate extends into a region below the distal striated fiber (Fig. 49, small short arrow) but whether it is continuous with the electron dense plate of root type I is not known, although both structures lie in the same plane (see Fig. 48) and approach each other. Striations were not observed.

C o m p a r a t i v e A s p e c t s of t h e F l a g e l l a r l~oot S y s t e m s . In those species where zoospores are covered by a cell envelope (C.

Cruciate Flagellar l~oot Systems in Green Algae 28i

minute, C. spec., C. pseudominor, C. auxotrophica) a 4-2-4-2-system is present. No electron dense material underlies the root tubules (Fig. 5, 6) and the root tubule configuration in distal parts of root type I I is the following: Tubule 1 associates with tubule 2, while tubule 2 dissociates from tubule 3, which results in a 2 besides 2 configuration (Fig. 4, large arrows), the eyespot lying between the two pairs (Fig. 4, 8). In C. minor again a 4-2-4-2-sys tem exists but both root types are associated with underlying electron dense material (Fig. 26 curved arrow, Fig. 28, 30). Tubule 1 in distal cross sections through root type I I is not associated with tubule 2 but leaves the other three tubules (3 besides 1 configura- tion; Fig. 32). This configuration is still seen in the region of the eyespot, which is located between both parts of the root (Fig. 33). In C. gelatinosa a 5-2-5-2-sys tem can be seen, root type I I is associated with underlying electron dense material (Fig. 18, 20, 21). Root tubule configuration in distal parts of root type I I is 4 besides 1, which is maintained in the eyespot region (Fig. 23). In C. dissociate again a 5-2-5-2-system was observed, both root types are associated with electron dense material (Fig. 38, 39, 40, 41, 45). Configuration of root tubules near the eyespot is 4 besides 1 (Fig. 46, 47). A difference exists to C. gelatinosa because in about 25% of the sections a 4-2-4-2-sys tem was observed. I t has been shown by serial sections tha t a root is either a 4- or a 5-stranded root along its length. Comparison between cross sections of a 4- and 5-stranded root in this species led to the conclusion that the 5th tubule in the 5-stranded root is added in the upper row at the distal end with respect to the basal body.

Secondary Cytoskeletal Microtubules. In addition to the flagellar root microtubules secondary cytoskeletal microtubules are present in different numbers according to different species. In those species where the zoospores are covered by a cell envelope only three or four secondary microtubules were seen in several cross-sections through the central par t of the zoospore. In zoospores without a cell envelope they are more numerous, the highest numbers were recorded in C. gela- tinosa. In this species the number of tubules varies between 12 and 23 in 20 randomly chosen sections. The presence of a larger number of secondary microtubules correlates well with the presence of electron dense materiM underlying the root tubules. In such species cytoskeletal tubules may be seen originating at the sides of this electron dense ma- terial (see Fig. 20 arrow). If no serial sections were taken these tubules could easily be counted as root tubules. In serial sections however only those tubules which are consistently present in different sections, show definite positions (configurations) and link with other tubules or asso- ciated structures prove to be root tubules. Secondary tlibules usually diverge at an angle of about 60 ° from the electron dense material and

19"

282 M. ME:SKONIAN :

are therefore not seen in adjacent serial sections. I t should be mentioned that although secondary microtubules are frequently seen in distal sections through root type I (Fig. 17), their termination in proximal sections at the electron dense material was never observed. I t is notice- able that some of these secondary tubules are visible above the flagellar apparatus in median longitudinal sections (Fig. 42, arrow) where they seem to stabilize an apical papilla.

Summarizing the results obtained from comparative ultrastrncture the zoospores of the seven species constitute four different groups:

Group I (C. minuta, 6'. spec., C. pseudominor, C. auxotrophica) 1. Presence of cell envelope, variable position of cell organelles,

fixed basal body orientation, few secondary cytoskeletal microtubules. 2. Pyrenoid with tubular thylakoids, rod-like inclusions and con-

tinuous starch sheath. 3. 4-2-4-2 crueiate flagellar root system, no electron dense material

underlying root tubules, distal configuration of root tubules from root type II 2 @ 2 (2 besides 2).

Group II (C. minor) 1. Zoospores without cell envelope, fixed position of cell organelles,

variable basal body orientations, larger number of secondary cytoskeletal microtubules.

2. Many single lamellar thylakoids traverse pyrenoid matrix, several starch plates present.

3. 4-2-4-2 eruciate flagellar root system, electron dense material underlies root tubules, distal configuration of root tubules from root type II 3 besides 1.

Group I|1 (C. gelatinosa) 1. As in Group II. 2. As in Group I. 3. 5-2-5-2 crueiate flagellar root system, electron dense material

underlies root tubules, distal configuration of root tubules from root type II 4 besides 1.

Group 1¥ (C. dissociata) 1. As in Group II. 2. Few pairs of thylakoids traverse pyrenoid matrix forming irregular

stroma-like areas, several irregular starch plates. 3. As in Group I I I with a tendency towards a 4-2-4-2 root system

resembling that of Group II.

Discussion

General Aspects of Zoospores. The presence of a cell envelope covering zoospores in four Chlorosarcinopsis-speeies was surprising

Crueiate Flagellar Root Systems in Green Algae 283

because it is somehow in contrast to the definition of the genus Chloro- sarcinopsis as given by HEI~NDON (1958). Chlorosarcinopsis produces zoospores of the protosiphon type (STA~I~ 1955), becoming spherical at quiescence. This has usually been interpreted as reflecting zoospores only covered by the plasma]emma. Gl~oovEI~ and BOLD (1969) studied zoosporogenesis in C. minor and C. dissociata and found no cell wall covering the developing zoospores. I t is proposed from the present study that the term protosiphon type should include those zoospores which show a delicate cell envelope not preventing rounding up at quiescence.

This is also substantiated by the fact that the cell wall covering zoospores in the related Tetracystis (Bl~ow~ & BOLD 1964) is very different from the cell envelope of Chlorosarcinopsi8 zoospores.

The nature of the fibrillar material of the envelope is not known, but its ultrastructure is similar to the extraee]]ular polysaccharide of Porphyridium (I~AMIYS 1972). Similar tufts are also overlying the outer investment of Cylindrocapsa zoospores (HoFFMAN 1976) and might be present as wall layer 7 in Chlamydomonas (t~O~ERTS & a]. 1972). The envelope therefore might consist of slime-like material.

In spite of the delicate nature of the envelope a certain rigidity is proposed. Reasons for this assumption is presence of only a few addi- tional cytoskeletal microtubules in those species and fixed basal body orientation. That cytoplasmic microtubules form a cytoskelet in wall- less cells is well known (e.g. Bl~ow~ & BolrcI~ 1973). The presence of larger numbers of secondary microtubules in C. minor which otherwise conforms to a similar shape as the species with a cell envelope supports the above made assumption. The cytoskeletal role of the secondary mierotubules is also stressed in zoospores of C. gelatinosa. By applying an external pressure (cover slip) or fixing the cells in osmie acid (which does not preserve cytoplasmic mierotubules) the cell shape drastically changes. That this effect is not observed in zoospores of C. dissociata probably reflects the ability of this species to develop an internal cyto- skelet by a characteristic arrangement of the major cell organelles (nucleus and chloroplast; compare Fig. 9 with Fig. 34).

Frequent and autonomous changing of basal body orientations were observed in zoospores of the green alga Microthamnion (WATso~ 1975). That this is not an isolated example was shown in this study. In one genus both situations, either fixed basal body orientations and changing basal body orientations occur in different species. WATsox (1975) discussed basal body orientations with respect to lack of a cell wall in Microthamnion zoospores. He suggested that in those species lacking basal body movements the cell wall prevents these movements. I t is significant in this respect that only species without a cell envelope

284 M. MELIiiONIAN :

exhibit basal body movements. I t cannot be decided if the cell envelope plays a direct physical role preventing basal body movements or if its presence resulted in co-evolution of a fixed basal body orientation. Organisms with fixed basal body orientations include the walled zoo- spores of Tetracystis (ARxoTT & BROWN 1967) and the vegetative cells of Chlamydomonas (RINGO 1967).

The distal fiber changes length and thickness with basal body reorientation. This was also observed in Microthamnion zoospores (WATso~ 1975), whereas it has otherwise been pointed out that the fiber holds the basal bodies in a fixed position and resists the opposing forces which the two flagella exert at their bases as they beat in opposite directions (RI~GO 1967). While according to WATSOX (1975) the fiber in Microthamnion is not striated in any plane of section, it is striated in the Chlorosarcinopsis zoospores. When in unelevated basal bodies the fiber is cut vertical and longitudinal (Fig. 12) the striation pattern is difficult to resolve, in the respective horizontal longitudinal sections however the striation pattern is clearly visible (Fig. 15). Although it is difficult to conclude from such horizontal sections through the fiber if the basal bodies are elevated or unelevated, it appears that they are unelevated in Fig. 15. The striation pattern is then the same in unele- ra ted and elevated basal body orientations. This cannot be explained if the fiber simply contracts or expands. I t is highly hypothetical, but instead some kind of sliding mechanism might exist. From the above mentioned it is clear that some basal body associated structures show changes in morphology when they exert their function. From the taxo- nomic point of view some further problems should be mentioned. On reacting to the added fixative the zoospores might not be fixed randomly, but preferently in a definite flagellar orientation. Furthermore this reaction might be different in different species. Even when the cells are fixed in their random flagellar orientations one has to take into account that some time phases of flagellar beat are longer than others (e.g. the motionless phase) and certain orientations might not be present very often.

A third type of connecting fiber is described in this paper. I t is suggested that this fiber is present in more biflagellate green cells, but has been overlooked. In several green algae (e.g. Chlamydomonas, l~I~Go 1967, Fig. 13) its termination at the basal body, visible as electron dense material, was photographed. In the somatic cells of Volvox carteri (OLsox & KOCtIERT 1970) a "proximal kinetosome bridge" was described (Fig. 1, 2 in OLSOer & KOC~E~T 1970) which was not striated and might correspond to the fiber described in this paper.

Flagellar Root System. I:)IGN:ETT-HEAI~S (1975b) has given a general description of basal body associated structures in gametes and

Crueiate Flagellar Root Systems in Green Algae 285

zoospores of green algae with respect to the two main types: Cruciate systems and MLS-systems. The crueiate system was named "Chlamydo- monad-Type" and the description was mainly based on the results obtained by gl>-Oo (1967) which have recently been modified in several respects (MxLxO~IA~ 1977, GOODE~OIrGK & WEISS 1978). In this paper some new observations are added and a new nomenclature with regard to the numbering of root tubules is introduced.

It has become obvious that in most if not all biflagellate green algae which correspond to the erueiate system of flagellar roots, two root types are present. In Chlamydomona8 this was suggested (MELxO- NIA~- 1977) and has recently been proved (GooI)XXOUGH & WEISS 1978). Chloro~arcinopsis zoospores and Chlamydomonas show a ~ - 2 4 - 2 or 5-2-5-2 erueiate flagellar root system. In the following some genera are listed in which the available ul t ras tmctural data suggest identical systems, although some were described as 4 4 - 4 4 - s y s t e m s and others were not studied in detail: Tetraspora (L~M~I & WAL~E 1969), Volvox (OLso~ & KOCHEaT 1970), Dunaliella (HYAMS &CHAsm ~ 1974, EYDE~ 1975), Chaetomorpha (MAxTo~ & al. 1955; 3-2-3-2-system), Sorastrum (MA~cgA~T 1974~a), Pediastrum (M~cHA~T 1974b), Eudorina (HoBBS 1971) and the more specialized male gametes of Golenkinia (Mo~s~R~P 1972; 3-1-3-1-system) and Sphaeroplea (MoEs~auP 1975). Excluded from this list are zoospores of Microthamnion and gametes of Bryopsis and Dichotomosiphon because although they might show two root types other details of basal body associated structures are different from Chlamydomonas or Chlorosarcinop.~is (WATsO~ 1975, Mo~sT~ur& HOFFM~ 1975, HOXI 1977, and own unpublished results).

R o o t T y p e I. This root type is always two-stranded (exception the specialized gamete of Golenkinia, Mo~sTx~P 1972). From studies of quadriflagellate zoospores of ~ritschidla (MELEOmA~ 1975), it was suggested tha t root type I would always be associated with a striated fiber. In biflagellate green algae this was recently shown for Chlamydo- monas (Goo~)~OUaH & Wxlss 1978) and in this paper for Chlorosarci- nopsis. I t is interesting tha t the striation pat tern seems to be identical in Chlamydomonas and Chloroaarcinopsis with a center to center repeat of 23-25 nm. This fiber is difficult to resolve and might have been over- looked in other biflagellate organisms.

G o o n x ~ o u ~ & Wxlss (1978) introduced a numbering of the tubules in root type I. The tubule which in distal sections underlies the striated fiber was named tubule 1. In this paper a different numbering is pro- posed. Reasons for this are a) the tubule named tubule 2 by GOODE~OUOH & W~ISS is in the proximal par t of the root physically linked to the striated fiber, b) this tubule terminates later than the other and c) has a prominent link with a basal body triplet. This tubule which might be

286 M. M]~L~:ONIAN :

functionally more important is therefore designated tubule 1, the other tubule tubule 2.

Termination of tubule 1 was shown in this s tudy to be complex, the tips of two tubules from opposing roots are connected to a faint electron dense sheet which is in an unknown way linked to the distal striated fiber. From horizontal longitudinal sections through proximal parts of this root GOOD~NO~G~ & W~ISS (1978) concluded that tubules 1 of both roots terminate at some distance from each other.

R o o t T y p e I I . This root type is characterized by larger numbers of root tubules and some kind of variation in tubule numbers seems to occur, The presence of an electron dense plate in this root type has so far only been established in the seven Chlorosarcinopsi8 species studied, but some micrographs of Chlamydomonas (e.g. RI~CGo 1967, Fig. 22) indicate a more general occurrence of this structure. This plate is not easily visible in those species where secondary cytoskeletal microtubules are abundant, because electron dense material underlying the root tu- bules obscures its fine structure. Since this plate extends distally not as far the striated fiber of root type I, it might not have been noticed in other organisms. The detailed structure of this plate, especially its links with root tubules was observed best in those species of Chlorosarcinopsis without underlying electron dense material. I t should be investigated if this plate is also striated when cut in horizontal longitudinal section and if it is continuous with the electron dense plate (striated fiber) of root type I.

During the course of this s tudy it was possible to individually number the tubules of root type I I . All tubules from the upper row (tubules 2-4[5]) seem to terminate at the distal striated fiber. I t is an intrinsic character of this root type that root tubules change their positions with regard to each other in different cell parts. Such con- figurational changes were observed in a larger number of algae (MA~TO~ 1964, RII~GO 1967, BII~KBECK & al. 1974, MELKONIAX 1975, BROWlV &; al. 1976, HOFF~A~¢ 1976, M~r~KONIA~ 1977). The path of tubule 1 from an initially present 3 over 1 configuration to 4 single tubules lying besides one another was already suggested by RtNGo (1967). Details and pres- ence of this pa th in all seven Chlorosarcinopsis species was shown in this study. Furthermore differences in distal configurations were pIesent between different species and could be used for taxonomic purposes (see below). The tubules are obviously held in their respective position through cross-links, which also distinguish them from secondary cyto- skeletal microtubules.

Secondary Cytoskeletal Mierotubules. The termination of secondary cytoskeletal microtubules at electron dense material associated with flagellar roots seems to be well established (e.g. BROWN & al. 1976,

Crueiate Flagellar Root Systems in Green Algae 287

STEAa~VS & al. 1976). I t has been one result of this study that the number of cytoskeletal microtubules can be correlated with absence or presence of electron dense material underlying the root tubules of both root types. Possible initiation of eytoskeletal mierotubules was often observed at the outer edges of electron dense material from root type II , while the electron dense material underlying root type I was not seen to be intimately associated with cytoskcletal tubules. In distal cross sections through this root type however abundant cytoskeletal microtubules were seen lying near the root. Several explanations can be offered: Either the cytoskeletal tubules diverge from the dense material at an angle much greater than 60 ° or the tubules are not organized randomly along the length of the dense material, but rather at a specific point which could be missed in cross sections. I t might also be that they are organized somewhere else near a basal body or in the most apical region of the cell. That tubules can be found above the flagellar apparatus in an apical papilla was observed in several algal species (WcJEK & CI~E- L U ~ 1975, GRIrBER & I~OSaRIO 1977) and also in Chlorosarcinopsis. I t is not clear where these secondary tubules originate.

Taxonomic Considerations. G~oov~R & BOLD (1969) in their exten- sive study on taxonomy of 15 Chlorosarcinopsis species separated these species into 7 groups according to the presence or absence of secondary carotenoids (measured as color changes of agar grown cul- tures), morphological characteristics of vegetative cell packets and macroscopic attributes of the plant mass on agar cultures. Their group 1 with four species is believed to be the most distinct species-group in that they don' t synthesize secondary earotenoids upon ageing and exhibit vitamin Blz requirements or are enhanced in the presence of vitamin B12. The two species of this group, which were investigated in this paper, U. minuta and C. auxotrophica are not distinguishable with respect to their zoospore ultrastrueture. U. spec. which was isolated by this author in 1974 belongs to the same group because it remains green on agar cultures for longer than two months and then dies and is vita- min B12 dependent. Its ultrastructural aspects of the zoospore are identical to C. minuta (MELxO~IAX 1977). Only C. ~)seudominor was placed in another species-group by G~OOVE~ & BOLD (1969). Its color changes on agar were described as: "Three month old cultures on 1 N BBMV agar are green on yellow green." With respect to C. minuta they state: "On 1 N BBMV agar it is green at 1 month but yellow green at two months." The differences in color changes do not seem to be distinc- tive enough to qualify separation in two clear cut species-groups. C. pseudominor is however separated from the other 4 (or 5 including C. spec.) species by presence of Hormotila-like stages in older agar cultures and no vitamin B!2 requirement. The ultrastructure of the zoospores

288 M. MELKOI~IAlq :

shows its close affinites to group 1-species. On this basis it cannot be distinguished from C. minuta and C. auxotrophica and should therefore be placed into group I (terminology of this author) with the above n a m d three species.

The other three species studied in this paper C. minor, C. gelatinosa and C. dissociata also belong to three different groups in the scheme of GgoovEg & BoLD (1969).

Ultrastructural characteristics therefore fit quite well into the taxonomic scheme of different species of Chlorosarcinopsis as based on comparative morphological and physiological data and seem to clarify the taxonomic position of ill-defined species (e.g.C. pseudominor).

The taxonomic significance of crueiate flagellar root systems in green algae is still uncertain. There are only two reports on comparative ultrastructure of these systems within one green algal genus. BIRKBECK (1976) studied flagellar root variations in Chlamydomonas~ but since this genus has recently undergone a revision of the root system (MELxO~IA~ 1977, GOODE~OUG~ & W]~ISS 1978) and since his results were only presented as an abstract, a detailed discussion is difficult. In one species a 5-2-5-2-system was reported and its detailed structure would be interesting. The only other comparative study is that of LEMBI (1975) on 5 species of the quadriflagellate Carteria. This work has raised doubts on the significance of root systems for taxonomic and phyletie purposes because two very distinct root types occur within this genus. Since however other ultrastructural details also differ between the respective species, it is more probable that they belong to different genera. In genera where large numbers of species are assembled (e.g. Carteria and Chlamydomonas) detailed investigations including ultrastructural work, might lead to some separations into different genera.

Summarizing the taxonomic implications of cruciate flagellar root systems in green algae producing biflagellate zooids the present state of knowledge indicates the following:

a) A naturally constructed genus should be characterized by essen- tially the same flagellar root system in each species belonging to this genus:

b) Between different species of one genus small variations of flagellar root system structure may occur. These differences together with other ultrastructural details of zooids can be used to separate species in certain groups. These species-groups should conform with other schemes based on morphological and physiological data.

c) With regard to the cruciate flagellar root system of biflagellate cells such small variations include presence or absence of electron dense material (as microtnbule nucleating sites) underlying root microtubules and variations of tubule number and configuration in root type II.

Cruciate Flagellar Root Systems in Green Algae 289

Functional Considerations. One serious limitation in using flagellar root systems for taxonomic purposes is the lack of information on their function. I t is not known whether this structure changes in morphology during flagellar function as do some basal body associated structures.

Previously two main functions of cruciate root systems have been considered (RINGO 1967). They might serve in distributing stresses caused by the flagellar movement and they may function as an addi- tional cytoskelet. Their cytoskeletal role seems not to be very significant. When a cell assumes an elongate shape additional secondary micro- tubules are formed which constitute the main cytoskelet. In the ovoid zoospores of Chlorosarcinopsis, either a cell envelope is present or again additional microtubules are formed as in C. minor. Furthermore the distribution of root tubules in cross sections through the cell does not reveal their cytoskeletal function.

The function of distributing stresses is more obvious. The detailed structure of flagellar root systems however seems to be far too complex to account for this single function. I t cannot be explained for example why there should be definite tubule numbers and configurations in the respective roots. The complex association of root tubules to basal bodies and their associated structures again points to some other function.

Recently several reports were made on structural connections be- tween flage]lar root microtubules and cell organelles and functional explanations were offered (MELKO~IA~ 1975, WATSO~ 1975, LARGE & OLSO~ 1977, KBISTIANS]{~ &WAL~E 1977, B]~LL 1978, MYL~{S & a]. 1978, GOODEI~OUGI{ • WEISS 1978). In all these cases flagellar root microtubules may function in determining the exact positions of cell organclles or associated structures with respect to the flage]lar apparatus. A more direct role of root microtubules in the function of these organelles or associated structures was also suggested. One example may be detailed. Mierotubules lying near an eyespot in green algal zooids were frequently observed and photographed (e.g. MANTO~ 1964, ETTL & M ~ T O ~ 1964, HOBBS 1971) although their identi ty with flagellar root microtubules was only established later (MELKOI~IA~ 1975, WATSO~ 1975). I t was shown in this s tudy tha t all species of Chlorosarcinopsis exhibit an eyespot-flage]lar root microtubule association. Other un- published observations suggest tha t this situation is of widespread occurrence in different orders of green algae. In all studied species it is root type I I being associated and physically linked with the outer chloroplast membrane. I t is now generally believed tha t the eyespot in green algae acts as a shading device (HAUPT 1966, SCt{LETz 1976), shading a photoreceptor which is thought of being localized in the plasma- lemma overlying the eyespot (WAL~E & A~OTT 1967, AI~NOTT & BROWN

290 M. MELKONIAN :

1967, SC~T.ETZ 1976). Ano the r possible site for the pho to recep to r might be the chloroplas t membrane . Through roo t t y p e I I the chloroplas t mem- brane over ly ing the eyespo t is d i rec t ly connected to the d is ta l s t r i a t ed f iber and to some basa l body t r iplets . Some eonformat iona l changes in the chloroplas t m e m b r a n e might lead to changes in those pro te ins l inking the root tubules to the membrane . Since A T P a s e a c t i v i t y has r ecen t ly been found in basa l bodies and some associa ted s t ruc tures (A~DEnSO~ 1977), changes in the chloroplas t m e m b r a n e could u l t i m a t e l y resul t in a different f lagel lar beat . This is ve ry hypo the t i ca l and fu r the r s tudies are needed to inves t iga te this p rob lem and to reveal the functio- na l significance of crueia te f lagel lar roo t sys tems in green algae.

The author would like to thank Mrs. E. MA~rS~IA~D and Mrs. Ct~. ADA~I for careful technical assistance.

Note Added in Proof

M o ~ s T ~ P (Bio Sys tems 10, 117--144; 1978) has r ecen t ly given a comprehens ive review on f lagel lar root sys tems in green a lgae inclu- ding some original work on those s t ra ins of Chlamydomonas reinhardi

which R I ~ a o (1967) used, where he found a 4 - 2 - 4 - 2 f lagel lar root sys tem.

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Crueia te F lage l l a r R o o t Sys tems in Green Algae 291

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Address of the author: MIOI~AEL MELKO~IAN, Botanisehes Inst i tut , Westf/~lische Wilhelrns-Universitat, Schlol3garten 3, 1)-4400 MOnster, Federal Republic of Germany.