ultrastructure of the quadriflagellate zoospores of the filamentous green algaechaetophora...

Bot. Mat. Tokyo 102 : 533-546, 1989 The Botanical Magazine, Tokyo �9 by The Botanical Society of Japan 1989

Ultrastructure of the Quadriflagellate Zoospores

of the Fi lamentous Green Algae Chaetophora

incrassata and Pseudoschizomeris caudata

(Chaetophorales, Chlorophyceae) with E m p h a s i s

on the Flagellar Apparatus

SHIN WATANABE* AND GARY L. FLOYD**

*Department of Biology, Faculty of Education, Toyama University, Toyama 930 ; **Department of Botany, Ohio State University,

1735 Nell Avenue, Columbus, Ohio 43210, U.S.A.

Quadriflagellate zoospores of Chaetophora incrassata and Pseudoschizomeris caudata have similar features including an appressed membrane between the pyrenoid matrix and the starch sheath, and identical flagellar apparatuses. Components of the flagellar apparatus include: directly opposed upper basal bodies, lower basal bodies in the clockwise absolute orientation, a grooved distal fiber, peripheral and terminal fibers between adjacent basal bodies, proximal fibers connecting the lower basal bodies to the X-membered rootlets, two- and X-membered rootlets associated with electron-dense components, and at least one rhizoplast. The X-membered rootlets are comprised of five mierotubules in C. incrassata and four or five in P. caudata. These features of the flagellar apparatus suggest that the two algae are closely related, and together with Stigeoclonium, Uronema, Draparnaldia and Fritschiella, form a natural group, the Chaetophoraceae, Chaetophorales (sensu Mattox and Stewart).

Key words : Chaetophora - - Chaetophorales Chlorophyceae Flagellar apparatus - - Green algae - - Pseudoschizomeris.

The assemblage of filamentous green algae containing a single nucleus and a parietal chloroplast in each cell has been traditionally assigned either to the Ulotri- chales or the Chaetophorales. Fritsch (1935) evaluated the heterotrichous feature as good enough to distinguish the two orders, and circumscribed the Ulotrichales as having unbranched filaments and the Chaetophorales as having branched ones that are often comprised of both erect and prostrate portions: Such taxonomic appraisal of thallus morphology at the rank of order is followed by Bourrelly (1966), and Bold and Wynne (1985). However, Smith (1950), Printz (1964) and Fott (1971) treated it in

Abbreviations : B, basal body ; B1-B4, basal body #1-#4 ; CV, contractile vacuole ; DF, distal fiber; LB, lower basal body; N, nucleus; PF, peripheral fiber; Py, pyrenoid; PS, proximal sheath; PxF, proximal fiber; R2, two-membered rootlet; Rh, Rhizoplast; RX, X-membered rootlet ; S, starch ; TF, terminal fiber ; UB, upper basal body.

534 S. WATANABE AND G.L. FLOYD

different ways. Smith (1950) included heterotrichous forms in both the Ulotrichaceae and Chaetophoraceae, together with the Coleochaetaceae and Trentepohliaceae, of the Ulotrichineae, a suborder of the Ulotrichales. Printz (1964) included many families of different thallus morphology, such as the Ulvaceae, Ulothrichaceae, Trentepohliaceae, and Coleochaetaceae, in the Chaetophorales. Fort (1971) established the Ulotrichales to accomodate five suborders, including the Ulotrichineae and Chaetophorineae. In these traditional approaches it is apparent that the branched or unbranched habit has been generally accepted as one of the key taxonomic characters, though this feature has been used at the various ranks.

Recently the cytological features of cell division and especially of the flagellar apparatus have been used in elucidating phylogenetic relationships of green algae (for review see Mattox and Stewart, 1984; O'Kelly and Floyd, 1984a). In Utothrix, a representative genus of the Ulotrichales, two types of basal body arrangement have been reported. In the swarmers of U. zonata (Weber et Mohr) Kiitz. (Sluiman et al., 1980), U. mucosa Thuret (Lokhorst and Star, 1986) and U. flacca var. roscoffensis Berger-Perrot (Berger-Perrot et al., 1986), the basal bodies are offset in the counter- clockwise absolute orientation. However, in the zoospores of U. bel/~ae Mattox et Bold the upper basal body pair is directly opposed and the lower pair is shifted in the clockwise absolute orientation, and several associated components are dissimilar from those of U. zonata (Floyd et al., 1980). Since the basal body arrangement has been proposed as one of important characters at the rank of class, U. zonata, U. mucosa and U. flacca var. roscoffensis are classified in the Ulvophyceae and U. bel/~ae is transferred into the genus Uronema of the Chlorophyceae (Mattox and Stewart, 1984 ; O'Kelly and Floyd, 1984a).

Ultrastuctural features of the flagellar apparatus identical to that of Uronema have been reported in heterotrichous members, including Stigeoclonium sp. (Manton, 1964), Fritschiella tuberosa Iyeng. (Melkonian, 1975 ; Moestrup, 1978), and Draparnal- dia glomerata (Vauch.) Agardh (Bakker and Lokhorst, 1984). The taxonomic prob- lem we encounter is in which category of the Chlorophyceae these species should be classified. As most of these algae have been traditionally included in the Chaetophor- ales, it is important to determine whether or not the zoospore of Chaetophora, the representative genus of the order, has the same ultrastructure as the zoospores of the species listed above. In this paper the flagellar apparatus of Chaetophora incrassata (Hudson) Hazen is described for cytological circumscription of the Chaetophoraceae and for clarification of the features of the Chaetophorales.

Pseudoschizomeris caudata Deason et Bold was originally described as a member of the Ulotricaeeae, Ulotrichales. This species forms unbranched filaments which, in relatively old cultures, become pluriseriate, similar to the thallus of the genus Schizome~is, Schizomeridaceae (Deason and Bold, 1960). By thallus morphology P. caudata has a possible relationship with either Ulothrix of the Ulvophyceae or Uronema of the Chlorophyceae. We also discuss the phylogenetie affinity of P. caudata on the basis of cytological features.

Chaetophora and Pseudoschizomeris Zoospores 535

M a t e r i a l s and M e t h o d s

Chaetophora incrassata (~ 1289) and Pseudschizomeris caudata (# 1271) were obtained from the Culture Collection of Algae at the University of Texas at Austin

(Starr and Zeikus, 1987). Cultures were maintained at 21-24~ under 36 ] l E . m 2.s 1

on a 12 : 12 h l ight /dark photo-regime on agar plates containing 9 : 1 medium that

contained 9 parts of Bold's basal medium (Deason and Bold, 1960) and 1 part of soil

extract. Zoospore formation and release were promoted by transferring actively

growing vegetative cells to fresh l iquid medium over a new agar plate. Zoospores

were collected by a pipette at the beginning of the light period on the following day.

For fixation of cells, 5% glutaraldehyde (GA) in a mixture of the medium and 0.1

Fig. 1. C. incrassata. Longitudinal section of zoospore. Nucleus is below contractile vacuole in anterior part of cell. Scale bar= 1.0 z m. Fig. 2. P. caudata. Pyrenoid matrix is surrounded by appressed membrane (arrows) and starch sheath. Scale bar= 0.5 ~m. Fig. 3. C. incrassata. Longitudinal section of two-layered eyespot. Strand of rootlet microtubules (arrow) runs between plasmalemma and chloroplast envelope near eyespot. Scale bar=0.2/~m.


M sodium cacodylate buffer (pH 7.2) was added to the zoospore suspension to make a final concentration of 1% GA, for 1 h at room temperature. Cells were collected onto a Millipore membrane filter (0.8/lm pore size) and embedded in 1.5% agar. After washing with buffer, cells were post-fixed in 1% OsQ in buffer for 1 h at 4~ followed by en bloc staining with 1% aqueous uranyl acetate overnight at 4~ Dehydration was carried out in an acetone series of 2 5 ~ 50%, 750, 95%, and 100% . Samples were embedded in Epon-Araldite and cured at 60~ for 48 h. Sections were cut on an MT-1 ultramierotome and stained in uranyl acetate for 5 min and in lead citrate for 3 min. Examinations were made with a JEOL-200CX electron microscope.

R e s u l t s

Because the ultrastructural features of the zoospores of C. incrassata and P. caudata are nearly identical, only representative photographs of one or the other are shown rather than repeating the same images of both. Major components of the flagellar apparatus are diagrammed in Fig. 39 (see also Floyd et al., 1980 ; Bakker and Lokhorst, 1984). The zoospores are ellipsoidal to rain-drop shape in the actively swimming mode (Fig. 1), and become spherical upon quiescence. They are naked in the physiological sense but they have a thin mucous-like material as the outer-most covering (not shown). A large contractile vacuole is just beneath the flagellar apparatus, the nucleus is located in the anterior third of the cell, and the chloroplast is cup-shaped with a pyrenoid (Fig. 1). The pyrenoid matrix is surrounded by a t ightly appressed membrane between the matrix and starch sheath, but not penetrated by thylakoid membranes (Fig. 2). A single bilayered eyespot (Fig. 3) is located in the median part of the cell. Mitochondrial profiles are seen throughout the cell.

The basal bodies (numbered according to Floyd et al., 1980) are inserted in a cruciate papilla at the anterior end of the cell. The basal bodies of the upper pair (#1, 2) are directly opposed (Figs. 7-10). The lower pair (#3, 4) is one-half of a basal body diameter lower than the upper pair, without overlap, and in the clockwise absolute orientation (Figs. 8 10, 18).

The upper basal bodies are connected to each other by a distal fiber (Figs. 4, 12- 15). The distal fiber has a thin central region resulting in a grooved appearance (Figs. 12-15, 32) and appears weakly striated (Figs. 4, 5). Adiacent basal bodies, i.e., each upper to each lower, are connected by striated peripheral fibers (terminology after Floyd et al., 1980) (Figs. 4-7). The distal fiber contacts the peripheral fibers (Figs. 18-20, 32), and both types of fibers are connected to the same microtubular triplets of

Figs. 4 11. C. incrassata. Oblique cross section of consecutive series of anterior end of zoospore, viewed from top of cell. Fig. 4. Note striated distal fiber. Scale bar=200 nm for Figs. 4 ll. Figs. 4-7. Adjacent basal bodies are interconnected by peripheral fibers. Figs. 7, 8. Two-membered rootlets are connected to basal bodies by non-striated fibers (arrows). Figs. 8-10. Basal bodies #1 and #2 of upper pair are directly opposed, and basal bodies #3 and #4 of lower pair are in clockwise absolute orientation. Fig. 11. Proximal fiber with prominent striations is between basal body #3 and X-membered rootlet,

538 S. WATANABE AND G.L. FLoYD

Figs. 12-15. C. incrassata. Consecutive series of longitudinal, oblique sections of upper basal bodies and cross section of lower basal body, including distal fiber with grooved region (arrows). Note proximal sheaths of basal bodies. Scale bar-200 nm for Figs. 12-15.

the upper basal body (Figs. 20, 21). Basal bodies #3 and #4 are connected to the X-membered rootlets by proximal fibers with prominent striations (Figs. 11, 22, 23, 27, 28). Adjacent basal bodies are also connected by terminal fibers located beneath the connecting point of the proximal fibers to the basal body (Figs. 18, 19, 24-26). Two striations were seen in C. incrassata terminal fibers (Figs. 18, 19, 24 26), while no prominent striations were observed in P. caudata (not shown).

The four microtubular rootlets are arranged in a cruciate pattern, including two-membered rootlets between basal bodies #1 and #4, and #2 and #3 (Figs. 6-11), and X-membered rootlets between basal bodies #2 and #4, and #1 and #3 (Figs. 5-11). The proximal ends of both two- and X-membered rootlets are connected to the distal fiber (Figs. 18, 19). Each two-membered rootlet is associated with five layers of electron- dense components in C. incrassata (Figs. 29-32), and three components in P. caudata

(Figs. 33, 34). The two-membered rootlets are connected to the basal bodies by non-striated fibers (Figs. 7, 8, 19, 20, 31). In C. incrassata the X-membered rootlet is comprised of five (4/1) microtubules (Figs. 22 24), while in P. caudata it is comprised of four (3/1) or five (4/1) microtubules (Figs. 35, 36). The X-membered rootlets are associated with electron-dense components which vary in number in the two species


Figs. 16-21. C. inccassata. Consecutive series of cross sections of one upper basal body viewed from proximal to distal region, and of longitudinal sections of one lower basal body. Fig. 16. Note electron-dense material (arrowhead) in basal body adiacent to cartwheel structure. Scale bar = 200 nm for Figs. 16-21. Figs. 16, 17. Note proximal sheaths on posterior triplets of basal bodies. Distal fiber is not connected to peripheral fibers. Figs. 18, 19. Upper basal body is connected to lower basal body by striated terminal fibers. Two- and X-membered rootlets are connected to distal fiber. Arrows indicate connection of distal fiber with peripheral fibers. Figs. 19, 20. Two-membered rootlet is connected to upper basal body by non-striated fiber (arrows). Fig. 20. Note electron-dense material (small arrowhead) in basal body. Figs. 20, 21. Distal and peripheral fibers are connected to same triplets of basal body (large arrowheads).


Figs. 22-28. C. incrassata. Selected oblique sections in consecutive series of four basal bodies. Figs. 22, 23. X-membered rootlet (4/1) is associated with several electron- dense components (brackets). Lower basal body is connected to X-membered rootlet by proximal fiber. Scale bar 200 nm for Figs. 22 28. Fig. 24. Upper and lower basal bodies in adjacent position are connected by striated terminal fiber. Figs. 25, 26. Second complementary pair of upper and lower basal bodies connected by striated terminal fibers. Figs. 27, 28. Lower basal body is connected to X-membered rootlet (arrowheads) by proximal fiber. Proximal fibers in Figs. 23, 27 are more distal than terminal fibers in Figs. 24-26.

Chaetophora and Pseudoschizome~is Zoospores 541

(Figs. 22, 23, 35, 36). Some basal body triplets are coated by proximal sheaths (Figs. 12-20, 25, 26). An

electron-dense material is contained in each basal body adjacent to the cartwheel structure (terminology after Ringo, 1967). In C. incrassata a rhizoplast was observed to descend posteriorly from a proximal sheath on one of the lower basal bodies (Fig. 37), and in P. caudata one extends toward the nucleus (Fig. 38). Although it was not seen, the rhizoplast may be present beneath each lower basal body (Fig. 39).

D i s c u s s i o n

Stewart et al. (1973) have demonstrated that among fourty-two species of the algae traditionally classified in the Ulotrichales, U1vales and Chaetophorales, Chaeto- phora incrassata, Draparnaldia plumosa, Fritschiella tuberosa and seven species of Stigeoclonium possess the pyrenoid matrix surrounded by the appressed membrane. The present observations have reconfirmed the pyrenoid feature of Chaetophora incrassata and demonstrated that Pseudoschizomeris caudata has the same type. The pyrenoid with the appressed membrane appears to be a consistent feature for Chaeto- phora incrassata and its allied species.

As in D~'aparnaldia glomerata (Bakker and Lokhorst, 1984), the distal fiber of C. incrassata and P. caudata is differentiated into a median groove and two striated sub-components. The morphology of the distal fiber in D. glomerata is similar to that of Bryopsis (Melkonian, 1981) and Derbesia (Roberts et al., 1981), which, unlike D. glomerata, are marine algae and have been classified in the Ulvophyceae (O'Kelly and Floyd, 1984b). Bakker and Lokhorst (1984) pointed out that one should not overstate the use of the distal fiber alone as a feature reflecting phylogeny. As seen in the following examples, however, the morphology of the distal fiber seems to be uniform for certain groups and could be useful in considering the phylogeny of green algae when other cytological features are also taken into account. The distal fiber type present in Chlamydomonas reinhardtii Dang. (Ringo, 1967) is known for many biflagellate motile cells of Chlamydomonadales, Chlorococcales (Watanabe and Floyd, 1989) and Chlorosarcinales (Melkonian, 1978) whose basal bodies are in the clockwise absolute orientation. An elaboration of the Chlamydomonas-type distal fiber into a ribbed structure is found in motile cells of Pediastrum (Wilcox and Floyd, 1988), Hydrodictyon (Hawkins and Leedale, 1971), Sorastrum (Marchant, 1974), some species of Neochloris (Watanabe and Floyd, in press), Tetraedron, Chloroteraedron (Watanabe et al., 1988), and some species of Characium (unpublished data), all having directly opposed basal bodies, a continuous SMAC (striated microtubule-associated component, Floyd et al., 1980) on the two-membered rootlets and partial caps on the proximal ends of the basal bodies. In gametes of two species of Sphaeroplea (Cs and Robinson, 1981 ; Buchheim and Hoffman, 1986), a prominent apical cone is present on the distal fiber. In gametes of Atractomorpha (Hoffman, 1984), a genus closely related to Sphaeroplea, however, the apical cone is absent. In the algae mentioned above, the specific structure of the distal fiber appears in accordance with other cytological


Fig. 39. Three-dimensional dia- gram presenting flagellar apparatus of C. incrassata, drawn by compiling electron micrographs. Arrow indicates non-striated fiber. Proximal sheath and electron-dense component associated with two- and X- membered rootlets are omitted.

features tha t are consistent wi th the taxonomic groups. The groove like tha t observed

in D. glomerata, C. incrassata, and P. caudata is to be expected in other filamentous

genera having the same general features of the flagellar apparatus.

The peripheral fibers seen in C. incrassata and P. caudata are like those in U.

belkae and D. glomerata, and may be present in Stigeoclonium sp. and F. tuberosa though they were not described. In U. belkae and D. qlomerata, the proximal fibers

connect the two pairs of adjacent basal bodies #4 and #2, and #3 and #1, extending

beneath the X-membered rootlets (Floyd et al., 1980, Fig. 4 ; Bakker and Lokhorst,

1984, Fig. 5), while, in C. incrassata and P. caudata, the proximal fibers connect each

lower basal body to an X-membered rootlet as in Stigeoclonium sp. (Manton, 1964, Fig.

20).

In C. incrassata and P. caudata, the terminal fibers connect each pair of adjacent

basal bodies at their proximal ends. Striations were seen in the fibers of C. incrassata, bu t not observed in P. caudata. Although there are no reports of terminal fibers in the

other algae, this fiber may have been overlooked for U. belkae (see Fig. 6A in F loyd

Figs. 29-32. C. incrassata. Figs. 29-31. Selected cross sections of two-membered rootlet in consecutive series. Figs, 29, 30. Five electron-dence components (a-e) are associated with two-membered rootlet. Scale bar = 100 nm for Figs. 29 32. Fig. 31. Arrowhead indicates connection of two-membered rootlet to distal fiber, and arrows indicate connection to basal body by non-striated fibers. Fig. 32, Longitudinal section of two-membered rootlet region. Electron-dense layers (a-e) associated with rootlet, cf. Figs. 29, 30. Note oblique section of distal fiber with grooved region (arrow) and two peripheral fibers.

Figs. 33-36. P. caudata. Figs. 33, 34. Selected cross sections in consecutive series of two-membered rootlet associated with three electron-dense components (a-c), and connected to basal body. Scale bar=100nm for Figs. 33 36. Figs. 35, 36. X- membered rootlet comprised of four (3/1) and five (4/1) microtubules, associated with electromdense components.

Fig. 37. C. incrassata. Rhizoplast descends posteriorly from proximal sheath. Scale bar = 100 nm.

Fig. 38. P. caudata. Rhizoplast extends toward the nucleus. Scale bar= 200 nm.

544 S. WATANABE AND G.L. FT, OYD

et al., 1980) and for D. glomerata (see Figs. 15D and 16B in Bakker and Lokhorst, 1984). A SMAC runs from each two-membered rootlet near its proximal end and is connected to a lower basal body in U. belkae (Floyd et al., 1980) and D. glomerata (Bakker and Lokhorst, 1984). In C. incrassata and P. caudata, each two-membered rootlet is connected to a lower basal body by a non-striated fiber. However; we cannot rule out fixation quality for the lack of striation.

To our knowledge, in biflagellate motile cells of the Chlorophyceae (sensu Mattox and Stewart 1984), the two-membered rootlet is always associated with a single layer of electron-dense component, but no associated material is present near t h e X- membered rootlet. In quadriflagellate motile cells of C. incrassata and P. caudata, and Stigeoclonium sp. (Manton, 1964), U. belkae (Floyd et al., 1980) and D. glomerata (Bakker and Lokhorst, 1984), several electron-dense layers are present near the both types of rootlet, varying in number and structure according to the species and position in the flagellar apparatus. In zoospores of Chaetopeltis (O'Kelly and Floyd, 1984a), Planophila and Hormotilopsis (unpublished data), whose upper and lower basal bodies are directly opposed, the rootlets are associated with electron-dense materials. These species are considered to be more primitive than the ehaetophoralean algae by virtue of the arrangement of basal bodies and the possession of body scales. In Hafniomonas, a unicellular species resembling Pyramimonas of the prasinophyte in possession of four basal bodies inserted in an apical groove, the rootlets also have associated electron- dense materials (Ettl and Moestrup, 1980). The presence of electron-dense material of various construction around the two types of the microtubular rootlet, therefore, may be a primitive feature.

The rhizoplast has not been observed in zoospores of chaetophoralean algae to date (Manton, 1964; Melkonian, 1975; Floyd et al., 1980; Bakker and Lokhorst, 1984). However, in C. incrassata and P. caudata, very thin and short rhizoplasts were found.

�9 It appears likely that with proper fixation, rhizoplasts could be demonstrated in the other chaetophoralean algae as well.

In summarizing the cytological features discussed above, the following components are present in the green algal group that includes C. incrassata and allied species : the pyrenoid is not traversed by thylakoids, but surrounded by an appressed membrane ; two pairs of basal bodies include the directly opposed upper basal bodies and the lower basal bodies arranged in the clockwise orientation; the distal fiber linking between the upper basal bodies is comprised of the median groove and two striated sub-components; adjacent basal bodies are connected by peripheral fibers at their anterior flanks and by the terminal fibers at their posterior ; the proximal fibers link between adjacent basal bodies, or between lower basal bodies and the X- membered rootlet ; electron-dense materials are associated with both types of rootlets ; and a rhizoplast is sometimes observed.

Taxonomically P. caudata was originally described as a member of the Ulotri- ehaceae, Ulotrichales, though this species has a pluriseriate stage (Deason and Bold, 1960). The present study on the features of the pyrenoid and flagellar apparatus confirms that P. caudata closely resembles C. incrassata rather than the genus

Chaeto2hora and Pseudoschizomeris Zoospores 545

Schizomeris ( B i r k b e c k et al., 1974; F l o y d a n d Hoops , 1980). Toge the r w i t h P .

caudata, the a lga l g roup r ep resen ted b y C. incrassata cou ld be r ega rded as na tu ra l ,

regardless of the d i v e r s i t y in the t h a l l u s morpho logy . M a t t o x a n d S t e w a r t (1984)

p roposed to d i v i d e the Chae topho ra l e s in to th ree famil ies , the Chae tophoraceae ,

A p h a n o c h a e t a c e a e and Sch izomer idaceae , and we be l i eve t h a t the cha rac te r i s t i c s seen

in C. incrassata are d i agnos t i c of the Chae tophoraceae . The cy to log ica l c i rcumscr ip -

t i on of t he order Chae tophora l e s is, however , inconclus ive , s ince d a t a on the o the r two

fami l i e s is insuff ic ient a t th i s t ime.

Th is research was s u p p o r t e d b y the Min i s t ry of E d u c a t i o n , Science a n d Cul ture ,

J a p a n , to S W and b y N S F grants BSR-8319539 and BSR-8219825 to G L F .

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Accepted September 22, 1989 Received April 26, 1989

ultrastructure of the quadriflagellate zoospores of the filamentous green algaechaetophora...

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