comparative ultrastructure of the zoospores of nine species ofneochloris (chlorophyta)

P1. Syst. Evol. 168, 195-219 (1989) Plant

Systematics and

Evolution © by Spfinger-Verlag 1989

Comparative ultrastructure of the zoospores of nine species of Neochloris ( Chlorophyta)

SHIN WATANABE and GARY L. FLOYD

Received November 8, 1988

Key words: Algae, Chlorophyta, Chlorophyceae, Pleurastrophyceae, Hydrodictyon, Neo- chloris, Pediastrum, Sphaeroplea.- Ultrastructure, flagellar apparatus.

Abstract: Nine species of Neochloris can be divided into three groups on the basis of comparative ultrastructure of the flagellar apparatus, the cell wall and the pyrenoid of zoospores. In Group I, N. wimmeri and N. minuta, zoospores are thin-walled, pyrenoids are penetrated by stromal channels, and the basal bodies are in the clockwise absolute orientation and connected by the distal and two proximal fibers. In Group II, N. aquatica, N. vigenis, N. terrestris, N. pyenoidosa, and N. pseudostigmatica, zoospores are naked or covered by fuzzy material, pyrenoids are covered by a continuous starch sheath or invaginated by cytoplasmic channels, basal bodies are directly opposed, the distal fiber is dif- ferentiated into a ribbed structure at the central region, a striated microtubule-associated component (SMAC) is continuous between opposite two-membered rootlets and connected to the ribbed structure, proximal ends of basal bodies are covered by partial caps, each two-membered rootlet and a basal body are connected by a striated fiber to the X-membered rootlet associated with the opposite basal body, and the basal bodies, when oriented at wide angles, are joined at their proximal ends by core extensions. In Group III, iV. pseudoalveolaris and N. cohaerens, zoospores are naked, pyrenoids are traversed by parallel thylakoids, basal bodies are in the counterclockwise absolute orientation and overlapped, and each X-membered rootlet is connected to the end of the opposite basal body by a terminal cap. It is suggested that the genus Chlorococcopsis gen. nov. be erected for the Group I species. Group II, which includes the type species, N. aquatica, should be preserved as Neochloris. The group appears to be closely related to the coenobial genera Pediastrum, Hydrodictyon, and Sorastrum, and to have affinities with the coenocytic genera Sphaeroplea and Atractomorpha as well. It is also suggested that the genus Parietochloris gen. nov. be erected in the Pleurastrophyceae for the species of Group III.

The unicellular green algae that possess non-motile vegetative cells and produce swarmers have been traditionally classified in the Chlorococcales and Chlorosar- cinales (BoLo & WYNNE 1985). These two orders are usually considered intermediate between algae forming motile, unicellular vegetative cells and those forming non- motile, multicellular thalli. Ultrastructural studies of cell division and the flagellar apparatus have been the primary basis for the recently proposed phylogenetic relationships of green algae (for current reviews see MATTOX & STEWART 1984,

196 S. WATANABE & G. L. FLOYD:

O'KELLY & FLOYD 1984 a). Previous studies have demonstrated that various types of flagellar apparatuses exist in biflagellate zoospores and gametes of the Chlo- rococcales and Chlorosarcinales. Chlorokybus atmophyticus GEITLER, an alga with the sarcinoid habit, has zoospores which have a multi-layered structure in the flagellar apparatus, and is thus recognized as a member of the Charophyceae (Ro- ~ERS & al. 1980). Zoospores of several genera have basal bodies in the clockwise absolute orientation, including Golenkinia rninutissima IYENGAR & BOLD (MOES- TRUe 1972), Chlorosarcinopsis spp. (MELKONIAN 1977, 1978), Desmotetra stigmatica (DEASON) DEASON & FLOYD (DEASON & FLOYD 1987), Chlorococcum minutum STARR (GROMOV & GAVRILOVA 1985), Chlorococcum hypnosporum STARR, Tetra- cystis aeria BROWN & BOLD, Pseudotetracystis terrestris ARNESON, Spongiochloris spongiosa STARR, Protosiphon botryoides (KuTz.) KLEBS (WATANABE & FLOYD 1989 a), Ascochloris multinucleata BOLD & MACENTEE and Urnella terrestris (PLAY- FAIR) WATANABE (WATANABE & FLOYD 1989b). These algae are thought to be allied with Chlamydomonas. WILCOX & FLOYD (1988) have demonstrated that gametes of Pediastrum duplex MEYEN possess directly opposed basal bodies and that the flagellar apparatus is very similar to that of Hydrodictyon gametes and zoospores (HAWKINS & LEEDALE 1971, MARCHANT & PICKETT-HEAPS 1972a, b). In addition, directly opposed basal bodies have been found in sperm of the coenocytic, filamentous genus Sphaeroplea (CACERES & ROBINSON 1981, BUCHHEIM & HOFFMAN 1986) and the unicellular genus Atractomorpha (HOFFMAN 1984). Basal bodies in Friedmannia israeliensis CHANTANACHAT & BOLD overlap and are in the counterclockwise absolute orientation (MELKONIAN & BERNS 1983). Other organisms with this feature include Trebouxia and Pseudotrebouxia (MELKONIAN & BERNS 1983), Chlorosarcina spp. (DEASON & FLOYD 1987), Myrmecia spp. (DEA- SON 1987, WATANABE unpubl.), and Neochloris alveolaris BOLD (GRoMOV & GAV- RILOVA 1987).

STARR (1955) described the genus Neochloris with the type species of N. aquatica, placing it in the Chlorococcales. Vegetative cells are spherical and have a parietal chloroplast with pyrenoids. Zoospores are without walls and have two isokont flagella. Although this genus resembles Chlorococcum, the naked zoospores have been emphasized as a critical character to distinguish between the two genera. The following additional species have been described for Neochloris: N. gelatinosa, N. terrestris (HERNDON 1958), N. fusispora, N. minuta, N. pyrenoidosa (ARCE & BOLD 1958), N. alveolaris (BOLD 1958), N. pseudoalveolaris (DEASON & BOLD 1960), N. oleoabundans (CHANTANACHAT & BOLD 1962), N. pseudostigmatica (BISCHOFF & BOLD 1963), N. cohaerens (GRooVZR & BOLD 1969), N. texensis, N. conjuncta, N. vigenis (ARCHIBALD 1973), and N. bilobata (VINATZER 1975). ARCHIBALD & BOLD (1970) transferred Chlorococcum wimmeri (RABENHORST) STARR to Neochloris on the basis of morphological features of the zoospores. WAa'ANABE (1983) synony- mized two Neochloris species; N. fusispora with Pseudotetracystis terrestris and N. bilobata with N. alveolaris. For the present paper we ultrastructurally examined zoospores of nine of the 14 species. We describe the flagellar apparatuses in detail, and include other features such as the type of pyrenoid and cell wall. By comparing these ultrastructural characteristics with those of other green algae we propose phylogenetic relationships and offer specific taxonomic treatments for these nine species.

Ultrastructure of zoospores of Neochloris 197

° ~

o

© ;>

;>

~ o

~ c

2

-~

e.,,..

~.~

~ 2

o

~.~ ~ :~ ~_ .~.~

.~ ~ . =

~.o

~o~

©

t ~

t

o ~

o

I

o

©

<

~ - ~ ~.~ ~.~ .

+ +

~ o o o o

I l I I J

0

z ~ 0 ~ ~

~ .~ ~

C~ ¢'q

I

Cq

oqp

I

. ~

~4

t¢'3 t"¢3

I

i

©

> 0 0


Materials and methods

Neochloris aquatica (#138), N. cohaerens (#1707), N. minuta (#776), N. pseudoalveolaris (#975), N. pseudostigmatica (#1249), N. pyrenoidosa (#777), N. terrestris (#947), N. vigenis (#1981), and N. wimmeri (#113) were obtained from the Culture Collection of Algae at the University of Texas at Austin (STARR & ZZI~=US 1987). Cultures were maintained at 2 1 - 24 °C under 35 gE. m-2 . s-1 on a 12:12 L/D photo regime, for one to three weeks on 9 : 1 (STARe, & ZZIKUS 1987) or 3N 9 : 1 (Nitrogen increased to 3 × normal 9 : 1) agar plates (1.5% agar). Zoospore release was promoted by flooding the plates with fresh media, with collection at the beginning of the light period on the following day.

For transmission electron microscopy, 5% glutaraldehyde (GA) in the medium or a mixture of the medium and 0.1 M sodium cacodylate buffer (pH 7.2) was added to the cell suspension to make a final concentration of 1% GA. Material was fixed for 1 h at room temperature. Cells were collected by mild centrifugation, pipetted onto a Millipore membrane filter (0.4 or 0.8 gm pore size) and embedded in 1.5% agar. After washing with liquid medium, post-fixation was in 1% OsO4 in medium or cacodylate buffer for 1 h at 4 °C, followed by en bloc staining with 1% aqueous uranyl acetate overnight at 4 °C. Dehydration was carried out in an acetone series of 25, 50, 75, 95, and 100%. Samples were embedded in Epon-Araldite, and cured at 60 °C for 48 h. Sections were cut on an MT-1 ultramicrotome and stained in uranyl acetate for 5 min and in lead citrate for 3 min. Specimens were examined on an Hitachi H-300, JEOL-200CX or Zeiss EM-10 CA with goniometer.

Results

The species investigated are grouped into three categories; Group I includes N. wimmeri and N. minuta, Group II includes N. aquatica, N. terrestris, N. pyrenoidosa, N. vigenis, and N. pseudostigmatica, and Group III includes N. pseudoalveolaris and N. cohaerens. Characteristics of each group are summarized in Table 1. Selected, representative micrographs are used for showing common features of each group rather than showing all features for every species.

Abbreviations used in all figures: ABB accesory basal body, CH chloroplast, CS continuous SMAC, DF distal fiber, G Golgi body, M mitochondrial profile, N nucleus, Pa papilla, PF proximal fiber, PS proximal sheath, Py pyrenoid, R 2 two-membered rootlet, Rh rhizoplast, RS ribbed structure, R J( X-membered rootlet, SF striated fiber

Figs. 1 - 8. Neochloris spp . - Figs. 1 and 2. N. minuta.- Fig. 1. Longitudinal section of zoospore. Note thin cell wall (arrowhead). Bar: 1 gin. - Fig. 2. Pyrenoid covered by several starch segments, and penetrated by tubular channels. Bar: 1 gm.-Figs. 3 - 8. N. wimmeri .- Fig. 3. Pyrenoid covered by nearly continuous starch sheath and penetrated by tubular channels. Bar: 0.5 gm. - Fig. 4. Longitudinal section of basal bodies. Note prominent papilla at anterior end of zoospore. Basal bodies are connected by distal fiber. The ratio of lengths of distal to proximal cylinders in the transition region is about 2 : 1 (arrow). Bar: 0.5 ~tm.-Fig. 5. Oblique section showing a proximal fiber and three microtubules with SMAC (arrow) in the two-membered rootlet position. Bar: 200 nm for Figs. 5 - 8. - Fig. 6. Cross section of basal body showing connections to distal fiber and the two- and X- membered rootlets. Note fibrous material (arrowhead) extending from accessory basal body to distal fiber. - Figs. 7 and 8. Two consecutive sections of SMAC (arrows). It is continuous between two-membered rootlets and connected to the distal fiber. Note fibrous material on the distal fiber (arrowheads)

200 S. WATANABE • G. L. FLOYD:

Group I. Zoospores are covered by a thin cell wall comprised of a single layer (Fig. 1). The nucleus is anterior and mitochondrial profiles are found throughout the cell. The single chloroplast extends the entire cell length in N. minuta, while it covers the median to posterior part of the cell in N. wimmer i (not shown). In N. minuta the pyrenoid is surrounded by several discontinuous starch segments (Fig. 2), and in N. w immer i it is covered by a nearly continuous starch sheath (Fig. 3). The pyrenoid matrix in both species is penetrated by tubular channels of thylakoid membranes (Figs. 2 and 3).

The angle between basal bodies is relatively fixed between 130- 150 °. A prominent papilla is present (Fig. 4). The basal bodies are in the clockwise absolute orientation and connected by the distal and two proximal fibers (Figs. 4, 5, and 9 - 13). In N. wimmeri , the rootlets also are slightly offset in a clockwise orientation (Fig. 11). Each two-membered rootlet is joined by a third microtubule in the region of the basal body (Figs. 14 and 15) and is associated with a SMAC (striated microtubule-associated component, FLOYD & al. 1980), although the striation is obscure. The SMAC is continuous between the distal ends of opposite rootlets and is connected to the distal fiber (Figs. 7 and 8). The X-membered rootlets consist of four microtubules in a 3 over 1 configuration. However, one relatively short microtubule is added to make a 4 over 1 arrangement near the basal body (Figs. 16 and 17). Both the two- and X-membered rootlets are connected to the basal bodies and to the distal fiber (Fig. 6). In N. minuta, the rootlet in the two-membered position consists of only one microtubule (Fig. 18), and the SMAC is absent. The X-membered rootlet consists of either one or two microtubules connected to the basal body directly or by electron-dense material (Figs. 19 and 20). In N. w immer i

each basal body is connected to a mitochondrion by a rhizoplast (not shown), while in N. minuta each basal body is in direct contact with a mitochondrion, the chloroplast or the nucleus (Figs. 18-21) . In N. wimmeri , accessory basal bodies are

Figs. 9 - 22. Neochloris spp. Bar: 200 n m . - Figs. 9 - 17. N. wimmeri. - Figs. 9 - 13. Five consecutive cross sections of anterior end of cell.- Fig. 9. Note striated distal fiber.

- Figs. 10- 13. Basal bodies are in clockwise absolute orientation.- Fig. 11. Two-membered rootlets are slightly offset in a clockwise orientation. Note accessory basal body connected to the two-membered rootlet (arrow). - Fig. 12. Accessory basal bodies connected to the two-membered rootlet (arrow) and primary basal body (arrowhead).- Fig. 13. Pri- mary basal bodies are connected by two proximal fibers (small arrowheads). Accessory basal body is connected to the primary basal body (large arrowhead).- Figs. 14 and 15. Two consecutive cross sections of two-membered rootlet. Arrows indicate SMAC. - Fig. 14. Two microtubules near the basal body. - Fig. 15. An additional microtubule is present. - Figs. 16 and 17. Two selected sections from consecutive series of cross sections of an X-membered rootlet. - Fig. 16. Four microtubules away from the basal body region.- Fig. 17. Near basal body, microtubules increase to five.- Figs. 18- 22. N. minuta.

- Fig. 18. Note cross section of single microtubule (arrowhead) in the two-membered rootlet position, connected to the distal fiber and basal body. Basal body is in contact with nucleus.- Figs. 19 and 20. Cross sections of X-membered rootlets.- Fig. 19. Rootlet with single microtubule (arrowhead). Basal body is in contact with chloroplast. - Fig. 20. Rootlet with two microtubules (arrowhead). - Fig. 21. Cross section of basal body in contact with a mitochondrial profile. - Fig. 22. Note absence of proximal cylinder in transition region (arrow)


present which connect to the two-membered rootlets, the primary basal body and extensions from the distal fiber (Figs. 6 and 11 - 13). Accessory basal bodies are not present in N. minuta. The transition region between basal body and flagellum measures 9 5 - 110 nm in length in N. wimmeri and 8 5 - 100 nm in N. minuta, and the ratio of the length of distal to proximal cylinders of the transition region is approximately 2:1 in N. wimmeri (Fig. 4). The proximal cylinder is absent in N. minuta (Fig. 22).

Group II. A cell wall is absent from all species of Group II (see Fig. 23), however, in N. pseudostigmatica, N. pyrenoidosa and N. vigenis, a fuzzy coating is present on the surface of the cell body and flagella (Fig. 24). The nucleus is usually situated anteriorly. In N. aquatica and N. vigenis mitochondrial profiles are present under the basal bodies, while in the other species they are variously located. The single chloroplast covers the posterior half to three quarters of the cell. The pyrenoid is covered by a continuous starch sheath and the matrix is uninterrupted (Fig. 25) except in N. terrestris, where the pyrenoid is invaginated by cytoplasmic channels (Fig. 26). These channels are membrane-lined. A mitochondrion is positioned at, and appears to extend into, the invaginations (Fig. 26).

In all cases the two basal bodies are directly opposed without displacement (Figs. 3 3 - 36). In side view, the basal bodies show various angles between them (see Table 1). When the basal bodies are at wide angles, their proximal ends are connected by the basal body cores (Fig. 30), however, at narrow angles they are not connected (Fig. 31). The central region of the distal fiber differentiates into a ribbed structure (terminology after WiLcox & FLOYD 1988) (Figs. 32 and 37). The latter is connected to the SMAC on the two-membered rootlets (Figs. 28-30) . The SMAC is continuous between the opposite two-membered rootlets (Fig. 42). In N. aquatica and N. vigenis, the two- and X-membered rootlets consist of two and four microtubules respectively. In N. terrestris, a single microtubule is present in the two-membered rootlet position (Fig. 43), and three, four or five microtubules comprise the X-membered rootlets (not shown). Opposite X-membered rootlets of some cells had the same number ofmicrotubules, while others had unequal numbers. In N. pseudostigmatica, four, five or six microtubules were present in the two- membered rootlet position (Figs. 4 4 - 46). In most cells the opposite rootlets have the same microtubule number, however, unequal numbers were also found (not

Figs. 23 - 31. Neochloris spp. - Fig. 23. N. aquatica. Longitudinal section of cell in a plane including basal bodies. Bar: 1 ~tm. - Fig. 24. N. pseudostigmatica. Oblique section of anterior end of cell including flagellum. Note deposition of fuzzy material at the surface of cell body and flagellum (arrowheads). Bar: 200 rim.- Fig. 25. N. vigenis. Pyrenoid covered by continuous starch sheath. Bar: 0.5 ~tm.-Fig. 26. N. terrestris. Pyrenoid invaginated by cytoplasmic channels. Note mitochondrial profile situated at the channel. Bar: 0.5 ~tm.-Figs. 27-30. N. vigenis. Four consecutive longitudinal sections of basal bodies.-Fig. 27. Note striated fiber. Arrowhead indicates SMAC. Bar: 200nm for Figs. 27- 31.- Figs. 28 and 29. Note partial cap (large arrowheads) covering proximal end of basal body.- Figs. 28- 30. Note continuous SMAC (small arrowheads) connecting to the ribbed structure (arrow) at different positions. - Fig. 30. The ratio of distal to proximal cylinder length in transition region (large straight arrows) is about 2.5 : 1. Basal body cores are connected (curved arrow) when basal bodies are at wide angles. - Fig. 31. N. terrestris. Longitudinal section of basal bodies oriented at narrow angle, with cores not connected

Ultrastructure of zoospores of Neochloris

ii~ ~~ ...... , , ~ ~,i~ii~i i~

~ ~̧i~'~̧~̧ ¸ ¸~ ~'~ ~"~'~ ~iii!i~~

#,

~r

U

203

~ii~i~ ~ S I ! ~


shown). In N. pyrenoidosa the two-membered rootlet is sometimes associated with four or five extra microtubules, in a somewhat loose arrangement (Figs. 4 7 - 49). The SMAC covers only two or three of the microtubules in the two-membered rootlet posit ion in N. pseudostigmatica (Figs. 4 4 - 4 6 ) and N. pyrenoidosa (Figs. 47 - 49). The X-membered rootlets have the more typical four microtubules in these two species (Fig. 50). Both the two- and X-membered rootlets are connected to the basal bodies (Figs. 3 8 - 4 1 ) . Several cytoplasmic microtubules emanate f rom the rootlets. Extensive bands of microtubules like those involved in coenobium for- mat ion in Pediastrum (MARCHANT 1979) were not observed.

A partial cap covers the proximal end of each basal body at the edge where the X-membered rootlet terminates (Figs. 28, 29, and 3 4 - 3 8 ) . The two-membered rootlet and the basal body are connected by a striated fiber to the X-membered rootlet of the opposite basal body (Figs. 27, 3 4 - 3 6 , 38, and 50). In N. pyrenoidosa and N. pseudostigmatica proximal sheath material is present on some triplets (Fig. 52). A rhizoplast extends to the nucleus in N. terrestris, N. pyrenoidosa, and N. pseudostigmatica (Figs. 51 and 52). A rhizoplast was not found in N. aquatica, but one of the two basal bodies is directly appressed to a mi tochondr ion (not shown). The ratio of lengths of the distal to proximal cylinders in the transition region is approximately 2 - 2.5 : 1 in N. aquatica and N. vigenis (Fig. 30), while it is about 1.5 : 1 in N. pseudostigmatica (Fig. 51). The transit ion region in these species ranges between 1 1 5 - 160 nm in length. The proximal cylinder is absent in N. terrestris (Fig. 53). The transit ion region in N. pyrenoidosa is reduced, i.e., no cylinder is present, but "dots" of electron-dense material are present (Fig. 54). An addit ional electron-dense region is located between one of the opposing dot pairs (Fig. 54), which corresponds to the electron-dense, central par t of the stellate pat tern (Figs. 5 5 - 57). No accessory basal bodies are present in any Group II species.

Group IIL Zoospores of Group III have neither a wall nor fuzzy material (Fig. 58). The nucleus is situated anteriorly in N. pseudoalveolaris (Fig. 58) and is nearly median in N. cohaerens (not shown). The chloroplast covers one side of the cell and extends posteriorly. One to three pyrenoids are surrounded by scattered starch segments and traversed by several, parallel thylakoids (Figs. 58 and 59).

The two basal bodies assume various angles to each other (Table 1) and are in a counterclockwise absolute orientat ion with considerable overlapping (Figs. 64 - 66 and 71 - 73). They are connected by a distal fiber (Figs. 68 - 73) possessing

Figs. 32 - 42. Neochloris spp . - Figs. 32 - 41. Bar: 200 n m . - Figs. 32 - 41. N. vigenis. - Figs. 32 - 36. Consecutive cross sections of anterior end of cell. - Fig 32. Note continuous SMAC (bracket) running obliquely to ribbed structure (arrow) of the distal fi- be r . - Figs. 33 - 36. The basal bodies are directly opposed.- Fig. 33. Note connection of distal fiber to basal bodies. - Figs. 34 - 36. X-membered rootlets terminating at partial caps (arrowheads). The terminal region of a two-membered rootlet is connected to X-membered rootlet of opposite basal body by a striated fiber. - Figs. 37 - 41. Consecutive cross sections of basal body from proximal to distal end. - Fig. 37. Note ribbed structure, striated fiber (arrow) and partial cap (arrowhead). - Fig. 38. The X-membered rootlet is connected to basal body were partial cap (arrowhead) covers triplet. The striated fiber (arrow) extends toward X-membered rootlet of second basal body. - Figs. 39-41. Note connections between basal body, rootlets and distal fiber. - Fig. 42. N. pseudostigmatica. The SMAC (arrow) is continuous between two-membered rootlets. Bar: 400 nm


a prominent central cross striation (Figs. 60 -63) . The distal fiber lies very close to the anterior end of the cell. No papilla is present. The triplets of each basal body are aligned closely together in the overlap region (Figs. 71 - 73). In N. pseudoalveolaris two microtubules occupy the two-membered rootlet position, while in N. cohaerens the number is one (Fig. 74) or two (not shown). SMACs were not observed on these rootlet microtubules. In both species the X-membered rootlets are comprised of four microtubules in the 3 over 1 configuration (Fig. 74). Each X-membered rootlet is directly connected to one basal body (Figs. 64 and 69), and to the second basal body via a plate-like terminal cap (Fig. 61). The X-membered rootlet terminates at the distal fiber (Fig. 69). A spherical, electron-dense mass is situated between the proximal end of the basal body and the terminal cap (Figs. 60, 61, 65, and 66). A single, bifurcate rhizoplast extends from a basal body to the nucleus through amorphous, rather electron-sparse underlying material (Figs. 67, 70, and 71). Accessory basal bodies were not observed. The transition region ranges between 1 7 0 - 2 0 0 n m in length in N. pseudoalveolaris and 1 2 5 - 1 3 5 n m in N. cohaerens. The ratio of the length of the distal to proximal cylinders in the transition region is 1 : 1 in N. pseudoalveolaris (Fig. 60) and 2:1 in N. cohaerens (not shown).

Discussion

Structural and functional aspects. T h e n u c l e a r c o n d i t i o n in v e g e t a t i v e ce l l s . Mature vegetative cells of Group I and Group III are uninucleate, while those of Group II are multinucleate (see ARCHIBALD 1973). In the uninucleate species, each nuclear division is followed by cytokinesis (i.e., successive bipartition). Many species of green algae, like those in Group I, have uninucleate vegetative cells and the clockwise basal body orientation in their zoospores, including ChIorococcum, Te- tracystis (WATANABE ~ FLO'VD 1989 a), Chlorosarcinopsis (MELKONIAN 1978) and Desmotetra (DEASON & FLOYD 1987). In addition, the following genera have mul-

Figs. 43 - 57. Neochloris spp. - Figs. 43 - 50, 51 and 52, 53, and 54-57. Bars: 200 nm. - Fig. 43. N. terrestris. Single microtubule with SMAC (arrow) in the two-membered rootlet position. - Figs. 44 - 46. N. pseudostigmatica. Four, five and six microtubules in the two- membered rootlet position. The SMAC (arrows) overlies three microtubules in each. - Figs. 47 - 49. N. pyrenoidosa. Selected cross sections from a consecutive series of the two- membered rootlet position, moving away from origin. - Fig. 47. Two microtubules with SMAC and three additional microtubules (arrow). - Fig. 48. There are now four additional microtubules (arrow) with SMAC overlying one of the four . - Fig. 49. Additional microtubule is seen on opposite side of previous four microtubules (arrow). - Fig. 50. N. vigenis. Cross section of X-membered rootlet in 3 over 1 configuration. The striated fiber (arrow) extends from this rootlet. - Figs. 51 and 52. N. pseudostigmatica. - Fig. 51. Anterior portion of cell, including bifurcate rhizoplast extending toward nucleus. Ratio of cylinders in the transition region is about 1.5 : 1 (arrow). - Fig. 52. Rhizoplast extends from the proximal sheath on the basal body. - Fig. 53. N. terrestris. Note absence of proximal cylinder in transition region (arrow). - Figs. 54- 57. N. pyrenoidosa. - Fig. 54. Oblique longitudinal section of transition region. Cylinders are seen as "dots" (arrows). Note electron-dense region in proximal portion of transition region (arrowhead). Three arrows from distal to proximal positions correspond to Figs. 55 to 57, consecutively. - Figs. 55 - 57. Three consecutive cross sections of transition region proceeding proximally. Electron-dense, central part (arrowhead) of stellate pattern in Fig. 56 corresponds to electron-dense region in Fig. 54


7


tinucleate vegetative cells and clockwise absolute orientation: Spongiochloris, Pro- tosiphon, Ascochloris, and Urnella (WATANABE & FLOYD 1989 a, b).

Group II species are multinucleate and have directly opposed basal bodies. Other genera with these same two features include Pediastrum (WILCOX & FLOYD 1988), Hydrodictyon (HAWKINS & LEEDALE 1971, MARCHANT & PICKETT-HEAPS 1972 a), Atractomorpha (HOFFMAN 1984), Sphaeroplea (CACERES & ROBINSON 1980, BUCHHEIM & HOFFMANN 1986), Tetraedron, Chlorotetraedron (WATANABE & al. 1988), and Characium (some species) (WATANABE & FLOYD, unpubl.). In Group III, N. pseudoalveolaris and N. cohaerens, are uninucleate. The same is true for all other pleurastrophycean algae examined to date including Friedmannia, Trebouxia, Pseudotrebouxia (MEL~ONIAN & BERNS 1983), Myrrnecia (DEASON 1987, WATAN- ABE unpubl.), and Chlorosarcina (OEASON & FLOYD 1987).

P y r e n o i d . There are four types of pyrenoid structure in the nine species of Neochloris. Type 1: the pyrenoid matrix is penetrated by tubular channels of chloroplast thylakoids. This type is present in Group I, and resembles that of some Chlorococcum spp. (BROWN & MCLEAN 1969). Type 2: the pyrenoid matrix lacks invaginations and is apparently covered by a continuous starch sheath. This type is seen in four species of Group II and has also been observed in other multinucleate algae with directly opposed basal bodies and the chaetophoralean alga Stigeo- elonium spec. (MANTON 1964), Fritschiella tuberosa IYENGAR (MELKONIAN 1975), Ulothrix belkae MATTOX & BOLD (FLOYD & al. 1980), Draparnaldia glomerata (VAucH.) AGARDH (BAKKER & LOKHORST 1984), Chaetophora incrassata (Hubs.) HAZEN and Pseudoschizomeris caudata DEASON & BOLD (WATANABE unpubl.). Type 3: the pyrenoid matrix is invaginated by cytoplasmic channels. This type is found in one species of Group II, and is also found in various other taxa, including Hafniomonas reticulata Ea'a-L & MoEsxRw' (1980), Hormotilopsis spp. (FLOYD unpubl.), Chaetopeltis spec. (FLOYD & O'KELLV unpubl.), Cylindrocapsa geminella WOLLE (HOFFMAN 1976), Oedogonium cardiacum WITXR. (HOFFMAN 1968), Oedo- cladium carolinianum BEANEY & HOFFMAN (MARKOWITZ & HOFFMAN 1974), Bul- bochaete hiloensis (NORDST.) TIFFANY (REXALLACK & BUXLEe, 1970), Sphaeroplea annutina (RoTH) AGARDH (CACERES & ROBINSON 1980), Ankyra spec. (SWALE & BELCHER 1971), A. starii LEE & BOLD, and Centrosphaera spec. (WATANABE & FLOYD unpubl.). Type 4: the pyrenoid is traversed by several, parallel thylakoids,

Figs. 58 - 67. NeochIoris spp. - Fig. 58. N. pseudoaIveolaris. Longitudinal section of zoospore. Bar: 1 ~tm. - Fig. 59. N. cohaerens. Two pyrenoids traversed by parallel thylakoids. Bar: 0.5 ~tm. - Figs. 60- 67. N. pseudoalveolaris. Bar: 200 nm. - Figs. 60 and 61. Two consecutive longitudinal sections of basal body. X-membered rootlet is connected to opposite basal body by plate-shaped terminal cap (arrowhead in Fig. 61). Spherical, electron-dense mass (small arrows) lies between terminal cap and proximal end of basal body. Ratio of lengths of cylinders in transition region is about 1 : 1 (large arrow in Fig. 60). - Figs. 62- 67. Six consecutive sections of anterior end of cell. - Fig. 62. Note striated distal fiber. - Fig. 63. Note edges of distal fiber and connections to basal body. - Figs. 64- 66. Basal bodies are in the counterclockwise absolute orientation with overlap.- Fig. 64. Note connection of X-membered rootlet to basal body (arrow).- Fig. 65. Connection of X-membered rootlet to terminal cap (arrowhead).- Fig. 66. Note spherical electron-dense masses (arrows) at proximal ends of basal bodies. - Fig. 67. Rhizoplast (arrowhead) extends downward through amorphous material (arrow) beneath one basal body


i i i ~ i ~ ¸ . . . . . . . .


Figs. 68 - 74. Neochloris spp. Bar: 200 nm. - Figs. 68 - 73. N. pseudoalveolaris. Selected sections from a consecutive series of cross sections of both basal bodies. - Fig. 68. Basal body connected to distal fiber.- Fig. 69. The X-membered rootlet is connected to basal body (arrow) and terminates at distal fiber. - Figs. 70 and 71.Rhizoplast extends from posterior proximal sheaths to nucleus through amorphous material (arrows). - Figs. 72 and 73. Basal bodies are overlapped and appressed (arrowheads).- Fig. 74. N. cohaerens. Note one (arrow) and four (arrowhead) microtubules in the two- and X-membered rootlet positions, respectively

and surrounded by scattered starch segments, as seen in Group III. This same or very similar type has been reported for genera of several classes, including Des- motetra stigmatica (Chlorophyceae, DEASON & FLOYD 1987), Stichococcus chlor- anthus KRUG. (Charophyceae, PICKETT-HEAPS 1975), and Pleurastrum spec. (Pleu- rastrophyceae, MOLNAR & al. 1975).

Z o o s p o r e c o v e r i n g . NTARR (1955) described the zoospores of Neochloris as naked. This description was based on whether zoospores retained their original, ellipsoidal shapes or became rounded upon quiescence. The former was called the Chlamydomonas-type and the latter the Protosiphon-type (STARR 1955). Although only two types had been recognized in coccoid green algae, MELKONIAN (1978)


discovered a third type of zoospore in some species of Chlorosarcinopsis (= Neo- chlorosarcina, WATANABE 1983), with the aid of the electron microscope. The third type possesses a thin cell wall but also becomes rounded after the swimming period. In addition to N. wimmeri and N. minuta of Group I, WATANABE & FLOYD (1989 b) report this type of zoospore for Ascochloris multinucleata and Urnella terrestris. Zoospores of A. multinucleata were first described as becoming rounded upon quiescence (BOLD & MACENTEE 1974), while those of U. terrestris were observed to retain the original form when they ceased moving (WATANABE 1981). The thin cell wall is so delicate that it does not prohibit the zoospore from becoming round, however, the time it takes to do so may depend on physiological conditions. The Chlamydomonas-type zoospores of Chlorococcum hypnosporum and Tetracystis aeria possess a thick, multi-layered cell wall (WATANABE & FLOYD 1989 a).

In two species of Group II, the zoospores are naked, while in three species, a mucous, fuzzy coating is present on the plasma membrane. This fuzzy coating resembles the surface coat of Dunaliella (OLIVEIRA & al. 1980, EYDEN 1975, MEL- KONIAN & PREISIG 1984a, CHARDARD 1987, WATANABE & FLOYD 1989a). In Dunaliella the basal bodies are found in a limited range of angles, whereas in the three species of Group II they can assume a wide variety of angles, evidently not restricted by the external material. Although the surface coat of these two genera appears similar to one another under the electron microscope, their structure is nonetheless distinct.

Zoospores of all pleurastrophycean members, including the Group III Neochloris species have neither a cell wall nor surface coat.

C o n n e c t i n g f ibe rs b e t w e e n basa l bodies . The distal fiber of Group I is similar to most swarmers having the clockwise absolute orientation. The variation of the sawhorse shape with beam-like extensions also has been reported in Chlo- rogonium elongatum DANG. (HooPs & WITMAN 1985), Chlorococcum hypnosporum, Spongiochloris spongiosa, Protosiphon botryoides, Pseudotetraeystis terrestris (WA- TANABE & FLOYD 1989 a). However, the extension from the distal fiber to accessory basal bodies in N. wimmeri is unusual.

The algae with directly opposed basal bodies have additional structural vari- ations of considerable interest. In Sphaeroplea an apical cone projects from the distal fiber (CACERES & ROBINSON 1981, BUCHHEIM & HOFFMAN 1986), whereas in A tractomorpha the distal fiber is without an apical cone (HOFFMAN 1984). BUCH- HEIM & HOFFMAN (1986) concluded that the apical cone represents a modification of the central portion of the distal fiber of Atraetomorpha gametes. The distal fiber in Group II is elaborated into a ribbed structure across the central region. This structure was first clearly demonstrated by WILCOX & FLOYD (1988)in Pediastrum duplex gametes, and it is also present in Hydrodictyon (HAWKINS & LEEDALE 1971: P1. 3 B). In cross section, the ribbed structure is psi-shaped and connected to the SMAC. The distal fiber with ribbed structure is obviously different from the distal fiber of Atraclomorpha or Sphaeroplea.

The basal bodies of Group I are connected by two proximal fibers. In Group II, there are two striated fibers that are connected to the X-membered rootlet near one basal body and at the point where the two-membered rootlet terminates at the second basal body.

In Group III, the morphology of the distal fiber, the absence of proximal or


striated fibers, and the interconnection of basal bodies at the overlapped portion are identical to those of other pleurastrophycean algae.

M i c r o t u b u l a r roo t l e t s . The microtubule number in the rootlets is quite variable from species to species in Neochloris, and is not consistent according to groups. A single microtubule is present in the two membered rootlet position in N. minuta and N. terrestris, as seen in Golenkinia minutissima gametes (MOESTaUP 1972) and Bracteacoccus spec. zoospores (FLOYD & WATANABE, unpubl.). In the two-membered rootlet position of N. cohaerens, one or two microtubules descend from the basal body. In N. wimmeri, a third microtubule is added to the two- membered rootlet near the basal body. In N. pseudostigmatica, four, five or six microtubules occupy the two-membered position, while in N. pyrenoidosa, cytoplasmic, non-rootlet microtubules are in close proximity. Although these additional microtubules vary in number and formation in the latter three species, only two or three of the microtubules are covered by the SMAC. It should be noted that the SMAC is typically associated with two microtubules, and only in Group II is it broad enough to cover a third microtubule.

In N. wimmeri, an additional microtubule joins the X-membered rootlet near the basal body as in the case of the two-membered rootlet. Different numbers of microtubules constitute the two X-membered rootlets in a single cell of N. terrestris and N. minuta. A similar situation has been noted in Pseudotetracystis terrestris (WATANABE 84 FLOYD 1989 a), Bracteacoccus spec. (FLOYD 84 WATANABE, unpubl.) Pediastrum duplex (WILCOX & FLOYD 1988), and Friedmannia israelensis (MEL- KONIAN 84 BERNS 1983). The number of microtubules in the X-rootlets ranges between three and six in these organisms, while in Atractomorpha echinata (HOFF- MAN 1984) and Sphaeroplea robusta (BucHHEIM 84 HOFFMAN 1986) microtubule numbers in multistranded rootlets range from eight to eleven. Although variation in the number of microtubules of three or more microtubules is found elsewhere (see MOESTRUP 1978), the large number of eight to eleven seems restricted to the last two species.

SMAC. In the majority of swarmers having basal bodies in the clockwise absolute orientation, the two-membered rootlet has a SMAC, except for Ascochloris multinucleata and Urnella terrestris in which the rootlets are greatly reduced in length (WATANABE 84 FLOYD 1989 b). Likewise, the single microtubule in the two- membered rootlet position ofN. minuta does not appear to have a SMAC. Although the SMAC usually terminates at the distal fiber, it is sometimes continuous between the two anterior ends of opposite two-membered rootlets, as seen in N. wimmeri. This also has been reported in Spermatozopsis similis PREISIG & MELKONIAN (MEL- KONIAN 84 PREISIG 1984 b) and Dunaliella lateralis (WATANABE 84 FLOYD 1989 a), and Chlamydomonas reinhardtii DANG. (RINGO 1967: Fig 13). Since many algae presumably related to these latter four species do not have a continuous SMAC (e.g., Chlorococcurn, Tetracystis, Spongiochloris), this structural variation seems to be secondary. The continuous SMAC in Group II species is, however, consistently present in their fiagellar apparatuses, and identical to that of Pediastrum (WILCOX 84 FLOYD 1988). The SMAC of Group II which is connected to the ribbed structure of the distal fiber, is obviously different from the continuous SMAC of most other algae. The SMAC was not observed in Group Ill, nor in any other pleurastrophycean algae (MELKONIAN 84 BERNS 1983, DEASON & FLOYD 1987).

P a r t i a l cap and t e r m i n a l cap. In Groups I and II, the X-membered rootlets


associated with each basal body do not also connect with the other basal body. However, in Group lII the X-membered rootlet of one basal body is connected to the second basal body by the terminal cap. This structure has been reported in the Ulotrichales and Ulvales of the Ulvophyceae (O'KELLY & FLOYD 1984 b). The partial cap that covers the proximal end of each basal body in Group II is present in Pediastrum (WILCOX & FLOYD 1988) and Hydrodictyon (MARCHANT • PICKETT- HEAPS 1972 b). This structure resembles the terminal cap in its location. However, these two structures are apparently not the same, because the general morphology is different, and because the partial cap of one basal body is not connected directly to the X-membered rootlet of the other basal body as in the case of the terminal cap.

R h i z o p l a s t ( s ) . Generally a rhizoplast extends from the basal body region to the nucleus in most green algal swarmers. It extends to a mitochondrion in some species, including Dunaliella salina (DUNAL) TEOD. (MELKONIAN & PREISIG 1984 a), D. lateralis, Chlorococcum hypnosporum, Tetracystis aeria (WATANABE & FLOYD 1989 a), Bracteacoccus spec. (FLOYD & WATANABE unpubl.) and Brachiomonas submarina (WATANABE & al. 1989). In Desmotetra stigmatica one rhizoplast con- nects to the chloroplast and a second may connect to a mitochondrion and to the nucleus (DEASOY & FLOYD 1987). In N. minuta, the basal bodies are directly connected to the mitochondrion, the chloroplast or the nucleus.

To date, rhizoplasts have not been reported for algae whose basal bodies are directly opposed, and no rhizoplast between the basal body and an organelle (or nucleus) was found in N. vigenis of Group II. However, in N. terrestris, N. pyrenoidosa and N. pseudostigmatica, a rhizoplast extends to the nucleus, and in N. aquatica one of the two basal bodies is directly connected to a mitochondrion.

As in Group III, a single rhizoplast extending to the nucleus has been reported for the members of the Pleurastrophyceae, including Friedmannia israelensis (MEL- KONIAN & BERNS 1983), and Chlorosarcina spp. (DEASON & FLOYD 1987). In Microthamnion kuetzingianum NX~ELI (WATSON & ARNOTT 1973), tWO rhizoplasts descend from the basal bodies and fuse together into a single strand. In N. alveolaris the basal body is connected by electron-dense material (probable rhizoplast) to the nucleus (GRoMOV & GAVRILOVA 1987). The single connection between the basal body and the nucleus appears to be a representative feature of the Pleurastrophyceae.

T r a n s i t i o n reg ion . The length of the transition region and the ratio of the distal to proximal cylinders vary from species to species in Neochloris. In most green algae the cylinders are continuous, and sometimes associated with "dots" (MELKONIAN 1984). The reduced nature of the transition region in N. pyrenoidosa, including the septum-like electron-dense region and "dots" located both distally and proximally, is unusual. In N. minuta and N. terrestris the transition region lacks the proximal cylinder. This absence might be a secondary reduction, as proposed for Heterochlamydomonas (FLOYD & al. 1989). MELKONIAN (1984) noticed a certain tendency in the length of the transition region and the ratio of lengths of cylinders in green algae. A range of 100 - 160 nm in length and a ratio of 2 - 3 : 1 are often encountered in the Chlorophyceae, however, exceptions such as Sorastrum spinulosum NXGELI in which the ratio is 1 : 1 (MARCHANT 1974) are known. BUCH- HEIM & HOFEMAN (1986) have reported that in Sphaeroplea and Atractomorpha the transition region measures 9 0 - 110 nm in length and the ratio is 1 : 1. In Pe- diastrum the ratio is also 1 : 1 (MARCHANT 1979: Fig. 8, WILCOX & FLOYD 1988).


Although there are some exceptions, the ratio of 2 - 3 : 1 is often observed in algae whose basal bodies are in the clockwise orientation, and the ratio of 1 : 1 is frequent in those whose basal bodies are directly opposed.

Basal b o d y core c o n n e c t i o n . In Group II, when the basal bodies are oriented at wide angles, their proximal ends are connected by cores, but they are not connected when at narrow angles. This characteristic was noted for Hydro- dictyon (MARCHANT & PICKETT-HEAPS 1972 a) and Pediastrum (WILCOX & FLOYD 1988). Likewise, the core connections may be present in Sphaeroplea annulina (CACERES & ROBINSON 1981 : Fig. 12) and A tractomorpha echinata (HOFFMAN 1984: Fig. 36).

A c c e s s o r y basa l bodies . Accessory basal bodies are connected to the distal fiber, rootlets and basal bodies in N. wirnmeri. In C. reinhardtii the accessory basal bodies are connected to the basal body and rootlets (GouLD 1975, WEISS 1984), and in some chlorococcalean members they are placed along the two-membered rootlets (WATANABE • FLOYD 1989 a). WATANABE & al. (1989) have proposed that since the accessory basal bodies are in contact with the two-membered rootlets in Brachiomonas, they might remain tethered to these rootlets during cell division, insuring semi-conservative replication of the basal body pairs in daughter cells. The role of the connection of the distal fiber extension to the accessory basal bodies in N. wimmeri is unknown.

The distribution of accessory basal bodies is somewhat restricted in the Chlo- rophyceae. They are present in bifiagellate motile cells with basal bodies in the clockwise absolute orientation, including Chlamydomonas (RINGO 1967), Chloro- gonium (HooPs & WITMAN 1985), Dunaliella (MELKONIAN & PREISIG 1984 a, WA- TANABE & FLOYD 1989a), Brachiomonas (WATANABE & al. 1989), Desrnotetra (DEASON & FLOYD 1987), Chlorococcum and Tetracystis (WATANABE & FLOYD 1989 a), Ascochloris and Urnella (WATANABE & FLOYD 1989 b), Dysmorphodoccus, Pteromonas (WATANABE unpubl.), and N. wirnmeri. In the Chlamydomonadales, accessory basal bodies function as centrioles during mitosis, however, in the Chlo- rococcales their role remains to be determined. It is interesting that accessory basal bodies have not been reported in algae with directly opposed or counterclockwise basal bodies.

Phylogenetic and taxonomic aspects. Basal body arrangement has been considered important in understanding algal phylogeny. It has been proposed that algae having clockwise absolute orientation be included in the Chlorophyceae and those having counterclockwise absolute orientation in the Pleurastrophyceae and Ulvo- phyceae (MATTOX & STEWART 1984, O'KELLY & FLOYD 1984a). In the present study, the clockwise absolute orientation, and vegetative cell morphology of Group I species indicate that they should be assigned to the Chlorophyceae. In addition they resemble the genus Chlorococcum. However, it is recommended that these species be included in the new genus Chlorococcopsis, primarily because the zoospores have thin cell walls that are very different from the thick ones of Chloro- coccum. Chlorococcopsis resembles Ascochloris in being unicellular, however, they are distinguished by following features; in Ascochloris mature cells are multinucleate and the flagellar apparatus possesses a plate-like structure (WATANABE & FLOYD 1989b), while in Chlorococcopsis mature cells are uninucleate and the flagellar apparatus lacks the plate-like structure.

Even though the growth habits are different, the flagellar orientation of the


Group II algae with directly opposed basal bodies and Sphaeroplea indicates a phylogenetic relationship. The coenobial genera Pediastrum, Hycrodictyon and Sorastrum also should be considered phylogenetically related. In addition to the directly opposed basal bodies, these algae have several shared characteristics including: multinucleate mature cells, the absence of accessory basal bodies in zoospores, and pyrenoids covered by a continuous starch sheath or invaginated by cytoplasmic channels. Some of these characteristics are encountered in algae whose basal body orientations are other than directly opposed. However, for the algae with the directly opposed basal bodies, these characteristics are consistently present. On the basis of these combined features, algae with directly opposed basal bodies should be taxonomically separate from those having the basal bodies in the clockwise absolute orientation.

Since the type species of the genus Neochloris, N. aquatica is included in Group II, the generic name should be preserved for the species of this group. Since the flagellar apparatus of Neochloris has the combined features of the ribbed strucure, the continuous SMAC and the partial caps, this genus is probably more closely related to hydrodictyacean genera than to Sphaeroplea. The flagellar apparatus of the latter genus lacks these structures and possesses large numbers of microtubules in the X-membered rootlet position. WATANABE & al. (1988) have demonstrated that the zoospores of the unicellular algae, Tetraedron bitridens BECK and Chlo- rotetraedron polymorphum MACENTEE, BOLD & ARCHIBALD have the same kind of ultrastructural features in the flagellar apparatus as those of Neochloris and hydrodictyacean genera. However, these unicellular genera are distinguished from the coenobial genera, because in zoospores of the latter, microtubules involved in colony formation are present, while in zoospores of the former such microtubules are absent.

It is evident that Group III should be classified in the Pleurastrophyceae. In this class, features such as growth habit of vegetative cells, chloroplast morphology, and presence or absence of a pyrenoid are useful for generic level taxonomy. There are two genera Trebouxia and Myrmecia, whose vegetative cells are solitary, as in the Group III species. However, Group III should be included in a new genus Parietochloris, because Trebouxia has an axile chloroplast and Myrmecia lacks pyrenoids, while Parietochloris possesses a parietal chloroplast with pyrenoids. This new genus resembles the filamentous genus Pleurastrum in chloroplast and pyrenoid morphology. It is interesting to note that Pleurastrum tends to become solitary when cultured axenically (MATTOX & STEWART 1984).

Diagnoses

Chlorococcopsis WATANABE & FLOYD, gen. nov.

Cellulae vegetativae solitariae, sphaericae. Paries cellulae laevis. Chloroplastus pa- rietalis pyrenoidibus. Reproductio asexualis per aplanosporas et zoosporas. Zoo- sporae parietibus cellularum tenuibus in uno strato, duobus flagellis aequalibus, interdum sphaericae ubi quiescentes. Corpora basalia zoosporarum in absoluta dispositione helicte.

Genus speciebus sequentibus: Chlorococcopsis wimmeri (RABENHORST) WATANABE • FLOYD, comb. nova (Ge-

neris typus). Basionym: Chlorococcum wimmeri RABENHORST (1868) emend. STARR (1953); Neochloris wimmeri ARCHIBALD & BOLD (1970).


Chlorococcopsis minuta (ARcE & BOLD) WATANABE & FLOYD, comb. nova. Ba- sionym: Neochloris minuta ARCE & BOLD (1958).

Vegetative cells solitary, spherical. Cell wall smooth. Chloroplast parietal with pyrenoids. Asexual reproduction by means of aplanospores and zoospores. Zoo- spores with a thin cell wall comprised of a single layer, with two flagella of equal length, may become spherical upon quiescence. Basal bodies of zoospores arranged in clockwise absolute orientation.

This genus includes following species: Chlorococcopsis wimmeri (RABENHORST) WATANABE & FLOYD and Chloroeoccopsis minuta (ARcE & BOLD) WATANABE ~; FLOYD.

ParietocMoris WATANABE & FLOYD, gen. nov.

Cellulae vegetativae solitariae, sphaericae. Paries cellulae laevis. Chloroplastus pa- rietalis pyrenoidibus. Reproductio asexualis per aplanosporas et zoosporas. Zoo- sporae sine parietibus cellularum, duobus flagellis aequalibus. Corpora basalia zoosporarum imbricata, in absoluta dispositione antihelicte.

Genus speciebus sequentibus: Parietochloris Mveolaris (BOLD) WATANABE & FLOYD, comb. nova (Generis ty-

pus). Basionym: Neochloris alveolaris BOLD (1958). Parietochloris pseudoalveolaris (DEASON & BOLD) WATANABE • FLOYD, comb.

nova. Basionym: Neochloris pseudoalveolaris DEASON & BOLD (1960). Parietockloris cohaerens (GROOVER & BOLD) WATANABE ~g FLOYD, comb. nova.

Basionym: Neochloris cohaerens GROOVER & BOLD (1969). Vegetative cells solitary, spherical. Cell wall smooth. Chloroplast parietal with

pyrenoids. Asexual reproduction by means of aplanospores and zoospores. Zoo- spores without cell wall, with two flagella of equal length. Basal bodies of zoospores overlapped and arranged in counterclockwise absolute orientation.

This genus includes following species: Parietochloris alveolaris (BOLD) WATAN- ABE • FLOYD, P. pseudoalveolaris (DEAsON & BOLD) WATANABE & FLOYD, and P. cohaerens (GROOVER & BOLD) WATANABE & FLOYD.

Support of the National Science Foundation is gratefully acknowledged. We thank Drs KEN STEWART and TEMD DEASON for helpful comments. We also thank Dr LEE WILCOX for many constructive suggestions and technical assistance, and Dr ToP STUESSY for assistance with the Latin diagnosis.

References

ARCE, G., BOLD, U. C., 1958: Some Chlorophyceae from Cuban soils. - Amer. J. Bot. 45: 492- 503.

ARCHIBALD, P. A., 1973: The genus Neochloris STARR (Chlorophyceae, Chlorococcales). - Phycologia 12: 187- 193.

-BOLD, H.C.,1970:Reclasgificationofthreeunicellulargreenalgae. - Phytomorphology 20: 383- 389.

BAKKER, M. E., LOKHORST, G. M., 1984: Ultrastructure of Draparnaldia glomerata (Chae- tophorales, Chlorophyceae). 1. The flagellar apparatus of the zoospore. - Nordic J. Bot. 4:261-273.

BISCHOFF, H. W., BOLD, U. C., 1963: Phycological studies 4. Some soil algae from Enchanted Rock and related algal species. - Univ. of Texas Pub1. No. 6318: 32-36.


BOLD, H. C., 1958: Three new chlorophycean algae. - Amer. J. Bot. 45: 737-743 . - MACENTE~, F. J., 1974: Phycological notes. 3. Two new saccate unicellular Chloro-

phyceae. - J. Phycol. 10: 189-193. - WYNNE, M. J., 1985: Introduction to the algae. - Englewood Cliffs: Prentice-Hall. BROWN, R. M., Jr., MCLEAN, R. J., 1969: New taxonomic criteria in classification of

Chlorococcum species. 2. Pyrenoid fine structure. - J. Phycol. 5 : 1 1 4 - 1 1 8 . BUCHHEIM, M. A., HOFFMAN, L. R., 1986: Ultrastructure of male gametes of Sphaeroplea

robusta. - J. Phycol. 22: 176-185. CACERES, E. J., ROBINSON, D. G., 1980: Ultrastructural studies on Sphaeroplea annulina

(Chlorophyceae). Vegetative structure and mitosis. - J. Phycol. 1 6 : 3 1 3 - 320. 1981: Ultrastructural studies on Sphaeroplea annulina (Chlorophyceae). 2. Sper-

matogenesis and male gamete structure. - J. Phycol. 17:173 - 180. CHANTANACHAT, S., BOLD, H. C., 1962: Phycological studies 2. Some algae from arid soils.

- Univ. Texas Publ. No. 6212: 1 - 75. CHARDARD, R., 1987: L'infrastructure du plasmalemme de Dunaliella biocuIata (algae verte)

mise en evidence d'un cell-coat; essai de localisation des charges negatives. - Cryp- togamie, Algologie 8:173 - 189.

DEASON, T. R., 1987: An ultrastructural comparison of two coccoid green algae. - J. Phycol. 23 (Suppl.): 14.

- BOLD, H. C., 1960: Phycological studies 1. Exploratory studies of Texas soil algae. - Univ. Texas Publ. No. 6022: 2 8 - 3 0 .

-- FLOYD, G. L., 1987. Comparative ultrastructure of three species of Chlorosarcina (Chlo- rosarcinaceae, Chlorophyta). - J. Phycol. 23: 187-195.

ETTL, H., MOESTRUP, O., 1980: Light and electron microscopical studies on Hafniomonas gen. nov. (Chlorophyceae, Volvocales), a genus resembling Pyramimonas (Prasinophy- ceae). - P1. Syst. Evol. 135: 177-210.

EYDEN, B. P., 1975: Light and electron microscope study of Dunaliellaprimolecta BUTCHER (Volvocida). - J. Protozool. 22: 336-344.

FLOYD, G. L., WATANABE, S., WILCOX, L. W., 1989: Structure and flagellar beat in Het- erochlamydomonas inaequalis, a motile, unicellular, green alga with unequal flagella and parallel basal bodies. - Cryptogamic Botany (in press).

- HooPs, H. J., SWANSON, J. A., 1980: Fine structure of the zoospore of Ulothrix belkae with emphasis on the flagellar apparatus. - Protoplasma 104: 17 -31 .

GOULD, R. R., 1975: The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation and partial characterization. - J. Cell Biol. 65: 6 5 - 7 4 .

GROMOV, B. V., GAVRILOVA, O. V., 1985: Ultrastructural patterns of the flagellar apparatus of Chlorococcum minutum (Chlorophyceae) zoospore. - Cytology 27: 940-943 , (in Russian).

1987: Ultrastructural specificity of zoospores of a unicellular green alga Neochloris aIveolaris. - Cytology 29: 637-641 , (in Russian).

GROOVER, R. D., BOLD, H. C., 1969: Phycological studies 8. The taxonomy and comparative physiology of the Chlorosarcinales and certain edaphic algae. - Univ. of Texas PuN. No. 6907: pp. 4 5 - 4 7 .

HAWKINS, A. F., LEEDALE, G. F., 1971: Zoospore structure and colony formation in Pediastrum spp. and Hydrodictyon reticulatum (L.) LAGERHEIM. -- Ann. Bot. 35: 201-211 .

HERNDON, W. R., 1958: Some new species of chlorococcalean algae. - Amer. J. Bot. 45: 308 - 323.

HOFFMAN, L. e . , 1968: Observations on the fine structure of Oedogonium. 5. Evidence for the de novo formation of pyrenoids in zoospores of Oe. cardiacum. - J. Phycol. 4: 212-218 .

- 1976: Fine structure of Cylindrocapsa zoospores. - Protoplasma 87:191 -219 . - 1984: Male gametes ofAtractomorpha echinata HOFFMAN (Chlorophyceae). - J. Phycol.

20:573 - 584.

218 S. WATANABE & G. L. FLoYo:

Hooes, H. J., WITMAN, G. B., 1985: Basal bodies and associated structures are not required for normal flagellar motion or phototaxis in the green alga, Chlorogonium elongatum.

- J. Cell Biol. 100: 297-309. MANTON, I., 1964: Observations of the fine structure of the zoospore and young germling

o f Stigeoclonium. - J. Exp. Bot. 15: 399-411. MARCHANT, H. J., 1974: Mitosis, cytokinesis and colony formation in the green alga

Sorastrum. - J. Phycol. 10: 107-120. - 1979: Microtubular determination of cell shape during colony formation by the alga

Pediastrum. - Protoplasma 98:1 - 14. - PICKETT-HEAPS, J. D., 1972a: Ultrastructure and differentiation of Hydrodictyon re-

ticulatum. 3. Formation of the vegetative daughter net. - Austral. J. Biol. Sci. 25: 265 - 278.

1972b: Ultrastructure and differentiation of Hydrodictyon reticulatum. 4. Conju- gation of gametes and the development of zygospores and azygospores. - Austral. J. Biol. Sci. 25: 279-291.

MARKOWITZ, M. M., HOFFMAN, L. R., 1974: Chloroplast inclusions in zoospores of Oedo- cladium. - J. Phycol. 10: 308-315.

MATTOX, K. R., STEWART, K. D., 1984: Classification of the green algae: a concept based on comparative cytology. - In IRVINE, D. E. G., JOHN, D. M. (Eds.): The systematics of the green algae, pp. 2 9 - 72. - London: Academic Press.

MELKONIAN, M., 1975: The fine structure of the zoospores of Fritschiella tuberosa [YENG. (Chaetophorineae, Chlorophyceae) with special reference to the flagellar apparatus. - Protoplasma 86:391 - 404.

- 1977: The flagellar root system of zoospores of the green alga Chlorosarcinopsis (Chlo- rosarcinales) as compared with Chlamydomonas (Volvocales). - P1. Syst. Evol. 128: 79 - 88.

- 1978: Structure and significance of cruciate flagellar root systems in green algae: Com- parative investigations in species of Chlorosarcinopsis (Chlorosarcinales). - P1. Syst. Evol. 130: 265-292.

- 1984: Flagellar apparatus ultrastructure in relation to green algal classification. - In IRVmE, D. E. G., JOHN, D. M. (Eds.): The systematics of the green algae, pp. 73 - 120. - London: Academic Press.

- BERNS, B., 1983: Zoospore ultrastructure in the green alga Friedmannia israelensis: an absolute configuration analysis. - Protoplasma 114: 67-84.

- PREIS~, H. R., 1984a: An ultrastructural comparison between Spermatozopsis and DunalieIla (Chlorophyceae). - P1. Syst. Evol. 146:31-46.

1984 b: Ultrastructure of the flagellar apparatus in the green flagellate Spermatozopsis similis (Chlorophyceae). - P1. Syst. Evol. 146: 145- 162.

MOESTRUP, O., 1972: Observation on the fine structure of spermatozoids and vegetative cells of the green alga Golenkinia. - Brit. Phycol. J. 7: 169- 183.

- 1978: On the phylogenetic validity of the flagellar apparatus in green algae and other chlorophyll a and b containing plants. - BioSystems 10 :117- 144.

MOLNAR, K. E., STEWART, K. D., MATTOX, K. R., 1975: Cell division in the filamentous Pleurastrum and its comparison with the unicellular Platymonas (Chlorophyceae). - J. Phycol. 11: 287-296.

O'KELLY, C. J., FLOYD, G. L., 1984 a: Flagellar apparatus absolute orientation and the phylogeny of the green algae. - BioSystems 16: 227-251.

1984 b: Correlations among patterns of sporangial structure and development, life histories, and ultrastructural features in the Ulvophyceae. - In IRVINE, D. E. G., JOHN, D. M. (Eds.): The systematics of the green algae, pp. 121 - 156. - London: Academic Press.


OLIVEIRA, L., BISALPUTRA, T., ANTIA, N. J., 1980: Ultrastructural observation of the surface coat of Dunaliella tertiolecta from staining with cationic dyes and enzyme treatment. - New Phytol. 85: 385-392.

PICKETT-HEAPS, J. D.,1975: Green algae. - Sunderland, Mass.: Sinauer. RETALLACK, B., BUTLER, R. D., 1970: The development and structure of pyrenoids in

Bulbochaete hiloensis. - J. Cell Sci. 6: 229-241 . RINOO, D. L., 1967: Flagellar motion and fine structure of the flagellar apparatus in

Chlamydomonas. - J. Cell Biol. 33: 5 4 3 - 571. ROGERS, C. E., MATTOX, K. R., STEWART, K. D., 1980: The zoospore of Chlorokybus

atmophyticus, a charophyte with sarcinoid growth habit. - Amer. J. Bot. 67:774 - 783. STARR, R. C., 1953: Further studies in the genus Chlorococcum MENEOI4~NI. -- Lloydia

16: 142- 148. - 1955: A comparative study of Chlorococcum MENEOnINI and other spherical, zoospore-

producing genera of the Chlorococeales. - Indiana Univ. Publ. Sci. Ser. No. 20:1 - 111. - ZEI~ZUS, J. A., 1987: U T E X - the culture collection of algae at the University of Texas

at Austin. - J. Phycol. 23 (Suppl.): 1 - 4 7 . SWALE, E. M., BELCHER, J. H., 1971: Investigation of a species of Ankyra FOTT by light

and electron microscopy. - Brit. Phycol. J. 6: 4 1 - 50. VINATZER, G., 1975: Neue Bodenalgen aus den Dolomiten. - P1. Syst. Evol. 123:213 -235 . WATANABE, S., 1981: Observations on Urnella terrestris PLAYFAIR (Chlorophyceae, Chlo-

rococcales) in culture. - Phycologia 20: 1 2 - 15. - 1983: New and interesting green algae from soils of some Asian and Oceanian regions.

- Arch. Protistenk. 127: 223-270 . - FLOYD, G. L., 1989 a: Variation in the ultrastructure of the biflagellate motile cells of

six unicellular genera of the Chlamydomonadales and Chlorococcales (Chlorophyceae), with emphasis on the flagellar apparatus. - Amer. J. Bot. 76: 307-317.

1989 b: Ultrastructure of the zoospore of the coenocytic algae Ascochloris and Urnella (Chlorophyceae), with emphasis on the flagellar apparatus. - Brit. Phycol. J. 24: 143 - 152.

WILCOX, L. W., 1988: Ultrastructure of the zoospores and vegetative cells of Te- traedron and Chlorotetraedron (Chlorophyceae). - J. Phycol. 24: 490-495 .

- TSUCI-ITMOTO, K. FLOYD, G. L., 1989: Light and electron microscopy of Braehiomonas submarina BOnLIN (Chlamydomonadales, Chlorophyeeae). - Phycologia 28: 188-196.

WATSON, M. W., ARNOTT, H. J., 1973: Ultrastructural morphology of Microthamnion zoospores. - J. Phycol. 9 :15 - 29.

WEISS, R. L., 1984: Ultrastructure of the flagellar roots in Chlamydomonas gametes. - J. Cell Sci. 67: 133- 143.

WILCOX, L. W., FLOYD, G. L., 1988: Ultrastructure of the gamete of Pediastrum duplex (Chlorophyceae). - J. Phycol. 24: 140-146.

Addresses of the authors: G. L. FLOYD, Department of Botany, The Ohio State Uni- versity, 1735 Neil Avenue, Columbus, OH 43210, U .S .A . - S. WATANABE, Department of Biology, Faculty of Education, Toyama University, Toyama, Japan.

comparative ultrastructure of the zoospores of nine species ofneochloris (chlorophyta)

Documents