P1. Syst. Evol. 146, 3 1 ~ 6 (1984) Plunt §ystemuti(s nnd Euolution @ by Npril~ger-Verlag 1984

An Ultrastructural Comparison Between SpermatozopMs and Dunaliella ( Chlorophyceae)

By

Michael Melkonian and Hans Rudolf Preisig

(Received September 21, 1983)

Key Words: Chlorophyceae, Spermatozop,s~is, Dunaliella, D. salina.--Green flagellates, ultrastrueture, taxonomy, systematics.

Abstract: The ultrastructure of the type species of the genus Dunaliella, D. salina, has been reinvestigated in an attempt to clarify the relationships between Dunaliella and Spermatozopsis. Dunaliella .salina differs in the following ultrastruetural characters from Spe~wtatozopsis (as exemplified by S. similis PRE~Sm et MF~LKOXIAX): presence of a distinctive surface coat covering the plasmalemma; presence of a prominent pyrenoid (with pairs of thylakoids partially entering the pyrenoid matrix); dietyosomes parabasal; endoplasmic retieulum closely underlying the plasmalemma around most of the ceil; contractile vacuoles absent; cell form ovoid to elongated and not spirally twisted; mitochondrial profiles near the fl~gellar apparatus. Differences in the ultrastructure of the flagellar apparatus: basal body angle more or less fixed; distal connecting fibre cross-striated; system I I fibre (rhizoplast) present, associated with mitoehondrial profile; system I fibre underlying two-stranded microtubular root; mating structure present. These ultrastruetural differences justify distinction between the two taxa at generic level. The problematical status of "freshwater" species of Dunaliella is briefly discussed.

Dunaliella TEODORESCO is a biflagellate green alga of usually ellipsoid to cylindrical cell shape which lacks a distinct cell wall and divides by longitudinal fission. The type species was originally described by DUNAL (1838) under the name Haematococcus 8alinus and later redescribed under the new generic name Dunaliella, as D. salina by TEODORESCO (1905). Dunaliella species occur in ext remely saline habi ta ts and have been extensively used in biochemical and physiological studies of osmotic adapta t ion to saline environments (e.g. BE~-AMoTz & AVRON 1978, Baowx & BOgOWITZKA 1979).

Of the 30 species of Dunaliella so far described 24 occur in saline

32 M. M~.:LKOM~N & H. R. Pm~:ISI~:

habitats (TEoDOI%ESCO 1905, 1906, NICOLAI ~5 BAAS BECKING 1935, LERCHE 1937, BUTCHER 1959 a, 1959 b, MASYUK 1969, 1971, 1973 a, 1973 b, 1973 e, ~¥[ASYUK ~5 ]~ADCNENKO 1973) and only 6 are known from freshwater (PASCHER & JAHODa 1928, PASCHER 1932, SKVORTZOV 1968, MASYUK 1971, 1973 a). Although typical species of Dunaliella (e.g.D. salina) are readily distinguished from Spermatozopsis by light microscopy because of their different cell shape and presence of a pyrenoid, some Dunaliella species have been described as typically asymmetric ( e .g .D . asymmetrica MASYUK, MASYUK ] 971). In addition, three freshwater species have been described lacking a pyrenoid (MAsYVK 1973 a). That some confusion exists about the separation of Spermatozopsis from Dunaliella is also evident from the work of KALINA (1965) who described a new species of Spermatozopsis, S. acidophila, which differs from the type S. exsultans in tha t it lacks the typical spirally twisted cell shape of S. exsultans and contains a pyrenoid which is absent in S. exsultans. Because of these characters MASYUK (1971) transferred S. acidophila to Dunaliella as D. acidophila (KALINA) MASYUK, but other authors still use the name Spermatozopsis acidophila for this organism (ALBERTAXO & al. 1981).

In an a t t empt to clarify the situation, an ul trastructural comparison between Spermatozopsis and Dunaliella is made in this study. We have collected known data on the ul trastructure of various species of Dunaliella and, in addition, reinvestigated the ul trastructure of the type species of Dunaliella, D. salina, with emphasis on features now believed to be of prime taxonomic significance (disposition of cell organelles, fiagellar apparatus). We compare our observations on D. salina with published data on the ul trastructure of species of Dunaliella (ALBERTANO & al. 1981, ANGHEL & al. 1980, BA~oN-MaRANO & IZARD 1968, BERKALOFF 1966, EYDEN 1975, HOSHAW & MALUr 1981, HYAMS & CHASEY 1974, MA~ANO 1976, OLIVEIRA & al. 1980, PETEaFI & MANTON 1968, TaEZZI & al. 1964, VLA~IMmOVA 1978, WERZ & KELLNER 1970), and relate this to the ul trastructure of Spermatozopsis as exemplified by the recently studied Spermatozopsis similis (PREISIG & MELKONIAN 1984, MELKONIAN & PREISlG 1984). We demonstrate tha t Dunaliella and Spermatozopsis are clearly separate genera and tha t the present taxonomic status of some of the freshwater species of Dunaliella must be regarded as uncertain.

Material and Methods

Dunaliella salina (DuNAL) TEODORESCO was obtained from the "Sammlung von Algenkulturen", Pflanzenphysiologisches Institut, GSttingen (strain 19-3, SCHL0SSE~ 1982). The organism was cultured in Erd-Schreiber (ES) seawater medium (MELKO~IA~ 1981) at 15 °C in a 14/10 h light-dark cycle and illuminated with 3 0001x light intensity from fluorescent lamps (Osram L 40 W/30-1 warm white). Cells were fixed for electron microscopy according to the following procedure: prefixation with glutaraldehyde (1~ in ES-medium, pHS.2) for

Comparative EM of Spermatozopsis and Dunaliella 33

10 see at 4 °C immediat.ely followed by addition of 1~o OsO 4 (in 50~o ES-medium, pH8.2) to the glutaraldehyde-eontaining cell suspension. The fixation was extended for another 10 rain at 4 °C. Sample preparation for thin section electron microscopy was standard (MEI~KO~IAN 1975).

Results

The general ul trastructure of Dunaliella salina has been previously investigated (TREzzI & al. 1964, VLADIMmOVA 1978) and only those ultrastructura] features tha t are either new or particularly relevant for the ul trastructural comparison with Spermatozopsis will be emphasized here.

Dunaliella salina is not naked in the sense that it is only limited by the plasmalemma, but instead the plasmalemma is surrounded by a distinct cell coat. The cell coat can be visualized in the light microscope with Indian ink (not illustrated) whereas in thin sections this coat is seen as irregular electron dense material covering the plasmalemma (Figs. 2, 3, 6-9).

A longitudinal section through a cell of D. salina is shown in Fig. 1 revealing distribution of major cell organelles and ellipsoid shape of the cell. Dictyosomes (probably 2-3) are exclusively found in a parabasal position at the anterior end of the cell (Figs. l, 2). They are typically intercalated between the a.nterior end of the nucleus and the basal bodies. The forming face of the dictyosomes lies towards the plasmalemma and is associated with a prominent strand of endoplasmie reticulum (Fig. 2). The endoplasmie reticulum typically underlies the plasmalemma over most parts of the cell (Figs. 1-3). In addition, a strand of EI{ links the nuclear envelope with the basal parts of the flagellar apparatus (part of this ER-system can be seen in Fig. 6). The distribution of mitochondrial profiles has not been studied in detail, but it is probably significant to note tha t mitoehondrial profiles have always been seen in proximity of the basal bodies (Figs. 2.6) and also between the chloroplast and the subplasmalemmal EI~ (Fig. 3). Contractile vacuoles are not developed. The nucleus occupies a more or less central position in the anterior half of the cell and contains a single prominent nucleolus (Fig. 1). Lipid droplets and numerous vacuoles representing characteristic features of our cells are located centrally near the nucleus (Fig. 1). The chloroplast is cup-shaped and has several anterior lobes and a prominent centrally located pyrenoid (Figs. 1, 3). The pyrenoid is penetrated by pairs of thylakoids, their lumen being slightly swollen, perhaps a fixation artifact (Fig. 3). The thylakoids entering the pyrenoid matr ix are short and never traverse the pyrenoid (Fig. 3). Eyespots were only rarely seen in our material and consisted of a single layer of lipid globules located in an anterior lobe of the chloroplast. On several

3 PI. Syst. Evo]., Vol. 146, No. 1--2

34 M. M~:tKONIA~ & H. R. PREISm:

occasions we observed an onion-shaped protrusion of the p lasmMemma near the basal bodies (Fig. 5). Inside this protrusion an electron-dense layer is directly a t tached to the p lasmalemma and additional fibrillar material is present. We suspect tha t the protrusion is a mat ing structure (partially act ivated?) similar to the mat ing structure found in Chlamydomonas reinhardtii (GooDENOUGH & WEISS 1975). The presumpt ive mat ing structure is associated with a two-stranded microtubular flagellar root (Fig. 5).

The two flagella of D. salina are equal in length and their transit ion region is of the type found in other Chlorophyceae (Chlorophyceae-type sensu MELKOMAX 1984). The basal bodies are interconnected by a prominent distal connecting fibre which is distinctly cross-striated in a bilateral fashion (Fig. 4) and, in addition, there are also two smaller str iated proximal connecting fibres. The flagellar root system is complex and its absolute configuration has not been determined. As in most other Chlorophyceae there is a sys tem of 4-2-4-2 eruciate microtubular roots and these are associated with system I fibres (Fig. 9). Although this association is not very distinct for the four-stranded root it is p robably present, whereas the two-stranded microtubular root is clearly underlain by a prominent system I fibre (Figs. 8, 9). The striation pa t t e rn of this fibre has a periodicity between 28 and 32 nm. In addition to system I fibres, the flagellar appara tus of D. salina also contains at least one conspicuous system I I fibre (rhizoplast; Figs. 6, 7). This system I I fibre extends from the basal body towards the nuclear membrane and is closely associated with mitoehondrial profiles over pa r t of its length (Fig. 6), whereas ER-profiles do not lie immediate ly adjacent. The striation pa t t e rn of the system I I fibre has a periodicity of about 80 - 90 nm.

Discussion

Surface Coat. Dunaliella has often been described as a naked equivalent of Chlamydomonas (PAscI~ER & JAHODA 1928, FRITSCI~ 1935, FOTT 1971). Most u! t ras t ructura l studies on Dunaliella species have

Figs. 1-3. Sections through cells of Dunaliella salina.--Fig. 1. Longitudinal section through a cell of Dunaliella salina revealing distribution of major cell organelles. N: nucleus, d: dictyosome, L: lipid droplet, v: vacuole, el: chloroplast, p: pyrenoid, s: starch grain. Bar = 5#m.--Fig. 2. Anterior end of a cell in longitudinal section, m: mitoehondrial profile, d: dietyosome, ER: endoplasmic reticulum at the forming face of the dictyosome. Bar = 1 # m . ~ i g . 3. Section through the posterior part of a cell. p: pyrenoid, s: starch grain surrounding the pyrenoid, m: mitochondrial branch, cl: chloroplast, ER: subplasmalemmal endoplasmic reticulum, small arrows: eelI surface coat, large arrows: thylakoid

pairs inside the pyrenoid matrix. Bar = 1 #m

Comparative EM of Sper'matozopo'i~' and DunalieIla 35

J I

3* Figs. 1-3

36 M. M~:LKONIAN & al.: Comparative EM of Spermatozopsis and Dunaliella

failed to recognize a surface coat outside the p lasmalemma and it was therefore concluded tha t Dunaliella is only covered by the p lasmalemma (TuEzzT & al. 1964, HYAMS & CHaSEY 1974, VLAHMmOVa 1978, ANOHEL & al. 1980). The wall-like s tructure reported for D. salina by TOMASELLO & al. (1980) is disregarded here since the material of their investigation was certainly different from Dunaliella spp. and more likely belonged to Tetraselmis ( = Platymonas). On the other hand, light microscopic studies on living cells of Dunaliella spp. have clearly shown a surface coat (thick mucilaginous sheath) using Indian ink preparat ions (HAMBURGER 1905, PRI~TZ 1927, LERCnE 1937). These observations were verified in a recent u l t ras t ruetural analysis of the surface coat of D. tertiolecta (OLrVEma & al. 1980). The lat ter authors demonst ra ted tha t cells of D. tertiolecta displayed a distinct surface coat when t rea ted with ru thenium red and alcian blue. This coat was also affected by proteolytic enzymes and neuraminidase (OHvEmA & al. 1980) and because of these results the authors suggested tha t the surface coat of Dunaliella is glycoproteid in nature with some additional neuraminic acid residues present. I t is clear tha t dehydrat ion and other prepara t ive steps for thin section electron microscopy reduce the thick surface coat of Dunaliella to nothing more than a thin fuzz. This fuzz, however, is always recognizable in ordinarily stained thin sections and apparent ly has been overlooked by most previous investigators. EYDEX (1975), however, noted its presence in D. primolecta and the surface coat can also be seen in other published mierographs ofDunaliella spp. (e.g. Figs. 37 and 39 of PETERFr & MANTON 1968, Figs. 5 and 11 ofTI~EZZI & al. 1964, Figs. 2 e and f of MA~ANO 1976). In the type species D. salina a surface coat has been visualized by both light and electron microscopy in this study. In

Figs. 4-9. Sections through the flagellar apparatus of Dunaliella salina. Fig. 4. Oblique section through both basal bodies revealing distal striated connecting fibre (small arrows: bilateral pattern of cross-striations), b: basal bodies. Bar = 0.5/~m.--Fig. 5. Section through the presumptive mating structure (ms). 2: cross-section through two-stranded mierotubular flagellar root. Bar = 0.5 ~m.--Fig. 6. Longitudinal section through a system I I fibre (rhizoplast) originating at a basal body (b) and terminating near the nuclear surface (N: nucleus). The path of the fibre is indicated by small arrows, m: mitochondrial profiles. Bar = 0.5/~m.- Fig. 7. Another slightly oblique section through a system I I fibre revealing striation pattern (arrows), b: basal body. Bar = 0.5 #m.--Fig. 8. Cross-section through two-stranded mierotubular flagellar root with prominent underlying electron dense material (large arrow) and an inconspicuous small plate-like structure overlying the root mierotubules (small arrow), b: basal body. Bar = 0.25 #m.--Fig. 9. Vertical longitudinal section through a system I fibre underlying the two-stranded microtubular root. Small arrows indicate the cross striation pattern. Large arrow: longitudinal section

through root microtubule, b: basal body. Bar = 0.25 gm

Figs. 4-9

38 M. M,,:LKOX~A?,~ & H. R. PR~:~sm:

conelusion, all species of Dunaliella tha t have been investigated by light and electron microscopy have been shown to possess a distinctive surface coat, which therefore should be regarded as a generic characteristic of Dunaliella. Regarding the absence of a surface coat in "fi 'eshwater" species of Dunaliella see below.

Disposition of Major Cell Organelles. In Dunaliella salina the 2-3 dietyosomes occupy a characteristic parabasal position. This disposition of dictyosomes is apparent ly characteristic for the genus Dunaliella (D. viridis: A>'GHEL & al. 1980; D. primolecta: EYDEN 1975; D. tertiolecta: HOSHAW & MALUF 1981; D. bioculata: MARANO 1976; D. salina: TREZZ~ & al. 1964, VLADI~mOVA 1978; D. sp.: PETERFI & MANTON 1968). The forming face of the dictyosome is always located towards the plasmalemma and a system of ER is close to the forming face of the dictyosome. Mitochondrial profiles are found both n e a r t h e flagellar apparatus and in a more peripheral position between the chloroplast and the plasmalemma in D. salina (this study). A similar distribution of mitoehondrial profiles is evident from published micrographs of other Dunaliella species [around the nucleus and near the basal bodies in D. viridis (ANG~EL & al. 1980) and D. primolecta (EYDEN 1975), around the nucleus and near dietyosomes in D. salina (TREzZI & al. 1964), near the flagellar apparatus in D. sp. (PET~RFI & MANTON 1968)]. The mitoehondrial profiles of Dunaliella form a reticulum (e.g. EYDEN 1975) and at least one anterior lobe is close to the flagellar apparatus.

All marine species of Dunaliella so far investigated lack contractile vacuoles. In fact the usual position of contractile vacuoles in Chlamydomonas-type green algae is occupied in Dunaliella spp. by the prominent dictyosomes.

Another characteristic feature of Dunaliella is the presence of a large centrally located pyrenoid. The pyrenoid is apparent ly of the same type in all species of Dunaliella which have been investigated with the electron microscope, with a starch shell composed of a greater number of individual starch grains and pairs of thylakoids entering but not traversing the pyrenoid matrix. This pyrenoid type can be found in D. bioculata (MARANO 1976), D. salina (TREzZI & al. 1964, VLAHMmOVA 1978, this study), D. sp. (PETERFI & MANTON 1968), D. primolecta (EYDEN 1975), D. tertiolecta (HOSHAW & MALUF 1981) and D. viridis (ANtHEr, & al. 1980).

Cell Shape. The cell shape of the type species ofDunaliella, D. salina, is ellipsoid to cylindrical (TEoDOgESCO 1905, HAMBUeGER 1905, LERCHE 1937). Most of the marine species of Dunaliella have a similar cell shape, but sometimes with a tapering posterior end (LERcI-IE 1937, RUINER 1938, MASYUK 1973 a, EYDEN 1975, M_4~ANO 1976, ANGHEL & al. 1980) and sometimes the cell body is f lattened (D. peircei, NICOLAI & BAAS

Comparative EM of Spermatozopsis and Dunaliella 39

BECKING 1935). As already observed by TEODORESCO (1905) and HAS~BUR(~E~. (1905) the cell shape of Dunaliella salina appears to be stable under a given set of environmental conditions, but with changing salt concentrations the cell shape can change drastically. This ability to change shape has been incorporated by MASYt-~ (1971) in a new circumscription of the genus Dunaliella to include some typically asymmetric forms, such as D. turcomanica and D. aaymmetrica. Unfortunate ly the ul trastructure of these asymmetric species has not been investigated yet.

Flagellar Apparatus. The flagellar apparatus of Dunaliella has so far only been examined in a preliminary way (HYAMS & CHASEY 1974, EYDEX 1975). The two flagella are of equal length (HYAsiS & CHASEY 1974) and the flagellar transition region is of a type common in the Chlorophyceae sensu STEWART & MATTOX (HYAMS ~; CHASEY 1974, EYDEX 1975, MARAXO 1976, PETERF~ & MA~TO~ 1968; review by MELKONIAN 1984). The angle formed between the two flagellar bases is usually 90-120 ° and does not seem to vary much (EYDEX 1975, ANGttEL & al. 1980, MARa~0 1976, PETEaF~ & MA~TO~ 1968, HYAs~S & CHASEY 1974). The two basal bodies are interconnected by a prominent distal striated connecting fibre of the Chlamydomonas-type [review in MELKOX~IAX 1980; reported for D. bioculata (MARANO 1976), D. sp. (PETERFI & MANTON 1968), D. viridis (ANOHEL & al. 1980), D. primolecta (EYI)EN 1975, HYAMS & CHASE¥ ] 974), D. tertiolecta (HosHAw & MALUr 1981) and D. salina (this study)]. The flagellar root system is cruciate and of the X-2-X-2 type [4-2-4-2 system in D. primolecta (HYAsrs & C~As~¥ 1974, EYDEN 1975) and in D. salina (this study)].

The presence of system I fibres in the flagellar apparatus of Dunaliella spp. was first reported by HYAS~S & CHASEY (1974) from negative stain preparations of disrupted cells ofD. primolecta. There, the four system I fibres were closely associated with the four microtubular flagellar roots over their proximal 200-300nm. EYDEN (1975) also observed fibrous material associated with all four microtubular roots but he could not detect cross-striations in his material (i.e. system I fibres). We have demonstrated the presence of system I fibres probably associated with all four microtubular roots in D. salina and, in addition, we have demonstrated for the first time that the prominent electron- dense material underlying the two-stranded microtubular roots is in fact a system I fibre with a typical striation pat tern (28-32 nm periodicity; see MELKO~AN 1980 for a review). System I fibres underlying two- stranded roots have so far not been demonstrated in Chlamydomonas- type green algae, which usually have system I fibres overlying two- stranded microtubular roots (MELKO~AN 1980). On the other hand, system I fibres underlying two-stranded microtubular roots are common

40 M. MELKONIAN ~; H. P~. PREISIC:

in the Ulvophyceae sensu STEWART & MATTOX, but also occur in some Chlorophyceae sensu STEWART & MATTOX (some Chaetophoralean-type organisms: MANTON 1964, MELKONIAN 1975, FLOYD • al. 1980 and in the Oedogoniales: ttOFFMAN 1970). The significance of these varying positions of system I fibres is unknown but,obviously of taxonomic importance.

In addition to the system I fibres, D. salina also possesses a prominent system II fibre (rhizoplast). This feature has apparently been overlooked in most previous ultrastructural investigations of Dunaliella species, though in D. primolecta EYDE~ ~ (1975) observed a cross-striated fibril arising from the basal bodies consisting of filaments and traversed by cross-bars with 80-90nm periodicity. However, in the schematic drawing of D. primolecta provided by EYDEN (1975) the path of the system II fibre is almost certainly wrong since there the system II fibre runs in a superficial position below the plasmalemma, whereas in D. 8alina it clearly approaches the nucleus like a typical rhizoplast. I t is interesting to note tha t the present demonstration of a rhizoplast in Dunaliella salina is another example where very exact early observations by light microscopists (HAMBURGER 1905) have been verified later by electron microscopy. The close association of the system II fibre in Dunaliella with mitoehondrial profiles is unusual since system II fibres are usually linked to other cell organelles (nucleus, chloroplast, microbody, and ER).

The plasmamembrane specialization and protrusion found in D. salina is probably a mating structure, because its fine structure, location and association with two-stranded microtubular fiagellar roots is very similar to what has been reported in Chlamydomonas and other green algae. In addition, LER.CWE (1937) has studied sexual reproduction in DunaIiella species in detail and observed sexual fusion of gametes initiated by fusion of apical plasmic protrusions. She also demonstrated tha t thick-walled cysts are always zygotes while asexual resting cysts are lacking in Dunaliella. Since we have quite frequently observed thick- walled cysts in our cultures of D. salina it is possible tha t our strain is a sexually reproducing clone. This is quite interesting because LERC~E (1937) has reported tha t D. salina is heterothallic, in contrast to most other DunaIielIa species.

"Freshwater" Species of DunMieIIa. Six freshwater species of Dunaliella have been described (PASCHER & JAgODA 1928, PASCH~R 1932, SKVO~TZOV 1968, MASYUK 1971, 1.973 a), but all of them appear to be extremely rare. Although interpretations on these species are still almost exclusively based on the incomplete original descriptions, there is sufficient evidence to suggest that at least some of these species are clearly different from D. salina and other marine species of Dunaliella.

D. cordata PASCHER et JAHODA (PAscnE~ & JAHODA 1928) has a well

Comparative EM of Spermatozopsis and Dunaliella 41

developed apical flagellar groove and short and rather thick flagella (an indication for the presence of flagellar scales?) suggesting prasinophycean relationships. Because of the presence of a flagellar depression this species has subsequently been removed from Dunaliella to the new genus Papenfu.s~iomonas, as P. cordata (PaSCHEg et JAHODA) DESIKACHARY (DESIKACHAI~Y 1971).

D. lateralis PASCHER et JAHODA (PAsCHER ~5 JAHODA 1928) is characterized by a distinctly lateral position of the pyrenoid, again a feature not found in any of the marine Dunaliella species. Classification of this species within Dunaliella must be viewed with some reservations.

D. paupera PASCHER (PAscHER 1932), D. flagellata SKvoaTzov (SKVORTZOV 1968) and D. obliqua (PAscHER) MASYUK (1937a) [ = Apiochloria obliqua PASCn~:~ (1930)J should almost certainly be excluded from Dunaliella since they lack a pyrenoid which is such a characteristic feature of the marine Dunaliella species. Presence or absence of a pyrenoid is usually regarded as a character of generic significance in green flagellates (cf. Chloromona8 which has been separated from Chlamydomonas because of the lack of a pyrenoid; ETTL 1970). D. paupera is also distinctive by the lack of an eyespot (present in all other Dunaliella species) and both D. paupera and D. obliqua are unusual regarding cell division, often producing unequal daughter cells (PASenEa 1930, 1932).

The sixth freshwater species, D. acidophila (KALINA) MAS~UK, has originally been described as Spermatozopsis acidophila from highly acid habitats (KALTNA 1965; see also discussion of this work by ETTI~ 1965, MASYUK 1971 and PREISm & MEL~:O~IAN 1984). This species has recently been studied in a preliminary way with the electron microscope (A~BERTANO & al. 1981). These authors still use the name Spermatozopsis acidophila which is untenable because the organism lacks the typical spirally twisted cell shape of this genus and contains a pyrenoid. In addition, the ultrastruetural analysis reveals profound differences to Spermatozopsi8 simili8 (as described by PREISm & MELKON~A~ 1984 and MELKOXIA~ & PI~EIS~G 1984): D. acidophila is presumably covered by a surface coat (ALBF~RTASO & al. 1981: "The periphery of the cell is delimited by a thin electron opaque layer beneath which the plasmalemma can be seen"). Further characteristics of this species include the two flagella of equal length, the dictyosome and mitoehondrial profiles near the flagellar apparatus, ER extending around the cell periphery, and paired thylakoids penetrating into the pyrenoid matrix as in the marine Dunaliella species. Interestingly, D. acidophila has also been shown to be moderately salt tolerant (ALBEI~TAXO & al. 1981). Although most. ultrastruetural features described by ALBERTANO & al. (1981) conform to those of the marine

42 M. MELKONIAN ~5 H. R. PREISI~:

species of Dunaliella, a final designation must depend on further characterization of the cell surface coat of D. acidophila and particularly on the analysis of the flagellar apparatus, which has not been studied by ALBERTANO & al. (1981).

Apart from the distinctive features discussed above, all the freshwater species of Dunaliella also differ from the marine species by the presence of contractile vacuoles (MASYUK 1972, 1973 a) and, except for D. acidophila (see above), it appears tha t the freshwater species lack the thick mucilaginous coat which is characteristic for the marine species of Dunaliella.

Comparison Between Dunaliella and Spermatozopsis. Bearing the reservations for the freshwater species in mind, Dunaliella is ultrastructurally a well characterized and fairly homogeneous green algal genus. An ultrastructural comparison between Dunaliella and Spermatozopsis (as exemplified by S. simili8 studied by PREISlG & MELKONIAlXT 1984 and MELKO~IA~- & PREISlG 1984) on the other hand reveals major differences between these two genera (summarized in Table 1). Spermatozopsis can be easily distinguished from Dunaliella in the light microscope by its characteristic spirally twisted cell shape, probably a consequence of the special disposition of secondary cytoskeletal mierotubules, and the absence of a pyrenoid. Although some Dunaliella species may show an asymmetric cell shape none is known to be spirally twisted (MAsYUK 1973 a). Sioermatozopsi~ also lacks the prominent cell surface coat which is characteristic for the marine species of Dunaliella. Disposition of most major cell organelles is also different between the two genera (Table 1). In addition, some peculiar differenees are observed in the detailed ultrastrueture of the flagellar apparatus between the two genera (summarized in Table 1).

The taxonomic affinities of both genera to each other and to other green flagellates still remain to be elucidated, though it is safe to say tha t both genera exhibit similarities and differences when compared to Chlamydomonas. On the basis of its ultrastructure Dunaliella cannot be regarded simply as a "naked" Chlamydomonas (major differences in the disposition of dictyosomes, mitochondrial profiles and system I fibres), though it must be mentioned that only few of the hundreds of Chlamydomonas species described (ETTL 1976) have been studied in any detail regarding their ultrastructure. Dunaliella appears to be more closely related to Asteromonas AI~TARI than to other green flagellates (PETERFI & MANTON !968, FLOYD 1978): Similarities include overall cell shape, presence of a surface coat, similar events occurring during mitosis and eytokinesis, similar flagellar apparatus [including distal striated connecting fibre, electron-dense material underlying root microtubules (system I fibres ?), 4-2-4-2 cruciate flagellar root system, and a system II

Table 1.

Comparative EM of Spermatozopsis and Dunaliella 43

Differences between Spermatozopsis (reference S. similis) and Dunaliella (reference D. 8alina)

Spermatozopsia Dunaliella

Cell Shape spirally twisted, sickle ovoid to shaped cylindrical

(sometimes asymmetric)

Cell Surface absent present Coat (mucilaginous)

Dictyosomes 1, perinuelear, posterior 2~, parabasal end of nucleus

Contractile 2 absent Vacuoles

Pyrenoid absent present, pairs of thylakoids enter matrix

Mitochondrion 1, compact, posterior to reticulum, nucleus anterior lobe

Flagellar distal connecting fibre not distal connecting fibre apparatus cross-striated cross-striated

Basal Body Angle variable fixed System I Fibre overlies underlies

microtubular root mierotubular root

System II Fibre absent present (rhizoplast)

fibre (FLOYD 1978 and pers. commun.)], dietyosomes in the anterior part of the cell associated with the nucleus (though more symmetrically located around the anterior portion of the nucleus; PETEa~ & MaNTON 1968) and presence of thick-walled cysts. Differences between Dunaliella and Aateromonas mainly occur in pyrenoid structure and external cell shape. The pyrenoid of Asteromona8 is t raversed by channels containing nuclear extensions, whereas the external cell shape of Aateromona8 is characteristic by the presence of three to six longitudinal ridges running lengthwise down the cell body. Interestingly, both genera also use the same mechanism for osmoregulation, accumulating large amounts of intraeellular glycerol in response to saline conditions (BEN-AMoTz & GI~UNWALD 1981). Apparent ly the same enzymes are involved in metabolic production and degradation of glycerol in the two genera (BEN-AMoTZ & GRUXWALD 1981).

In conclusion, the marine green flagellate Dunaliella is ul t rastruetural ly a well circumscribed genus. Spermatozopsi.s (as exemplified by S..simil i8 PRErS~O et MELKON~aX) differs in several

44 M. MELKONIAN ~5 H. P~. PREISrG:

d i s t i nc t f ea tu res f rom the t y p e species of Dunaliella a n d is the re fore c lear ly a genus in i ts own r ight . On the o the r hand , Spermatozopsi8 acidophila KALINA [ = Dunaliella acidophila (KALINA) MASYUK] is no t a S~oermatozopsis, more l ike ly a Dunaliella or even a s e p a r a t e genus. Spermatozopsis a n d Dunaliella u l t r a s t r u e t u r a l l y e xh ib i t s imi la r i t i es and di f ferences to species of Chlamydomonas a n d canno t a t p r e sen t be v iewed s i m p l y as a " n a k e d " c o u n t e r p a r t of Chlamydomonas. Most of t he desc r ibed "freshwater" species of Dunaliella p r o b a b l y do no t be long to th is genus. The genus mos t closely r e l a t ed to Dunaliella a ppe a r s to be Asteromonas which differs f rom Dunaliella in some c ha ra c t e r s (py reno id s t ruc tu re , d i spos i t ion of d i c tyosomes ) re f lec t ing i ts a n c e s t r y f rom sca ly green f lagel la tes .

We would like to thank Mrs. B. BEI~N8 for considerable help during some stages of this study.

R e f e r e n c e s

ALBERTANO, P., PINTO, G., SANTISI, S., TADDEI, R., 1981: Spermatozopsis acidophila KAI,~NA (Chlorophyta, Volvocales), a little known alga from highly acidic environments. - - Giorn. Bot. Ital. 115, 65--76.

ANGHEL, I., BREZEA~U, A., To~A, N., SilAGEANU, V., 1980: Studiu electronomieroscopie privind ul trastructura Mgei verzi Dunaliella viridis TEoD. - - Stud. si Cereet. Biol., Ser. Biol. Veget. 32, 159--162.

BARON-M~aRANO, F. LE, IZARD, C., 1968: Observations d'anomalies ultrastructurales dans la descendance d'Mgues trait~es par l'acrol6ine. - - C.R. Acad. Sci. Paris 267, 2137--2139.

BaN-AMOTZ, A., AwoN, M., 1978: On the mechanism of osmoregulation in Dunaliella. - - In CAPI~AN, 8. R., GINZBUt~G, M., (Eds.): Energetics and Structure of Halophilic Microorganisms, 529--541. - - A m s t e r d a m : Elsevier.

- - GnUNWALD, T., 1981: Osmoregulation in the halotolerant alga Asteromonas gracilis. - - P1. Physiol. 67, 613--616.

B~:RKALOFF, C., 1966: Observations sur l 'organisation infrastructurale d 'une Volvocale. - - C.R. Acad. Sci. Paris 262, 1232--1234.

BROWN, A. D., BOROWITZKA, L. g., 1979: Halotolerance of Dunaliella. - - In LEWaNDOWSKY, M., HUTNER, S. H., (Eds.): Biochemistry and Physiology of Protozoa 1 (2nd edit.), 139--190. - - New York: Academic Press.

BUTCHER, I~. W., 1959 a: An introductory account of the smaller algae of British coastal waters. I. Introduction and Chlorophyceae. Fishery Invest. London, Ser. 4, 1--74.

- - 1959 b: An undescribed species of Dunaliella from the Cambridge collection of a l g a e . - Hydrobiologia 12,249 250.

DESlKACHARY, T. V., 1971: Notes on Volvocales II . - - Phycologia 10, 429~430. DUNAL, F., 1838: ExtrMt d 'un m6moire sur les algues qui colorent en rouge

certaines eaux des marais salans m6diterran6ens. - - Ann. Sci. Nat. (Bot.), Paris, Ser. 2, 9, 172--175.

ETTL, H., 1965: Untersuchungen an Flagellaten. - - 0st . Bot. Z. 112, 701 745. - - 1970: Die Gattung Chloromonas GOBI emend. WILLE (Chlamydomonas und die

ngchstverwandten Gattungen, I). - - Nova Hedwigia, Beih. 34, 1--283. - - 1976: Die Gattung Chlamydomonas EH~E~-BERG (Chlamydomonas und die

ngchstverwandten Gattungen, II). - - Nova Hedwigia, Beih. 49, 1--1122.

Comparative EM of Spermatozopsis and Dunaliella 45

EY,),,:x, B. P , 1975: Light and electron microscope study ofDunaliella primolecta BUTC~:~ (Vo l vo c i d a ) . - J. Protozool. 22, 336--344.

FLOYD, G. L., 1978: Mitosis and cytokinesis in Asteromona.s gracilis, a wall-less green monad. J. Phycol. 14, 440--445. HooPs, H. J., SWANSOX, J. A., 1980: Fine structure of the zoospore of Ulothrix belkae with emphasis on the flagellar apparatus. - - Pro~oplasma 104, 17--31.

FOTT, B., 1971: Algenkunde. (Ynd edit.) - - J e n a : Fischer. FRTTSCH, F. E , 1935: The Structure and Reproduction of Algae, vol. 1 . . -

Cambridge: University Press. GOODE~;OUCH, U. W., WEISS, R. L., 1975: Gametic differentiation in

Chlamydomona.s reinhardtii. III . Cell walt lysis and microfilament-associated mating structure activation in wild-type and mutant strains. - - J. Cell. Biol. 67,623 637.

HAMbUrGER, C., 1905: Zur Kenntnis der Dunaliella .salina und einer AmSbe aus Salinenwasser yon C a g l i a r i . - Arch. Protistenk. 6, 111--130.

HOFF~L~N, L. R., 1970: Observations on the fine structure ofOedogonium VI. The striated component of the compound flagellar "roots" of O. cardiacum. - - Can. J. Bot. 48, 189--196.

HosHaw, R. W., MALUF, L. Y., 1981: Ultrastrueture of the green flagellate Dv, naliella tertiolecta ( Chlorophyceae, Volvocales) with comparative notes on three other species. - - Phycologia 20, 199 206.

HYaMS, J., CHas~;Y, D., 1974: Aspects of the flagellar apparatus and associated microtubules in a marine alga. - - Exp. Cell Res. 84, 381--387.

KALes'a, T., 1965: Zur Morphologie und Taxonomic der Gattung Spermatozop.si.~ KogsemKOW (Volvocales). Spe~'matozopsis acidophila sp. n. - - Preslia 37, 9---12.

LJ~mcul~, W., 1937: Untersuchungen fiber Entwieklung und Fortpflanzung in der Gattung Dunaliella. - - Arch. Protistenk. 88,236--268.

MANTO.X', I., 1964: Observations on the fine structure of the zoospore and young germling of S t i g e o c l o n i u m . - J. Exp. Bot. 15, 399--411.

MaRaxo, F., 1976: Etude ultrastrueturale de la division chez Dunaliella. - - J. Mierose. Biol. Cell. 25, 279--282.

MasYuK, N. P., 1969: A new species of the genus Dunaliella TEOD. - - Ukr. Bot. Zh. 26, 87--90.

- - 1971: New species of Dunaliella with asymmetric cells. - - U k r . Bot. ~. 28, 148--152.

- - 1972: On phylogeny and taxonomy of the genus Dunaliella TEOD. - - Ukr. Bot. ~. 29, 744--749. 1973 a: Morfologija, sistemetika, ekologija, geografiSeskoe rasprostranenie roda Dunaliella TEOD. - - "Naukova Dumka", pp. 1--244, Kiev.

- - 1973 b: New taxons from the genus Dunaliella TEOD., part 1. - - Ukr. Bot. Z. 3 0 , 175--183.

- - 1973 e: New taxons from the genus Dunaliella TEOD., part 2. - - Ukr. Bot. Z. 3 0 , 345--354.

- - RaDCX~:NKO, M. I., 1973: New taxons from the genus Dunaliella TEOD., part 3. Ukr. Bot. Z. 30, 4 6 8 ~ 7 1 .

MFLKON~AN, M., 1975: The fine structure of the zoospores ofFritschiella tuberosa IY~xG. (Chaetophorineae, Chlorophyceae) with special reference to the flagellar apparatus. - - Protoplasma 86, 391--404, 1980: Ultrastructural aspects of basal body associated fibrous structures in green algae: a critical review. BioSystems 12, 85--104.

- - 1981: Structure and significance of cruciate flagellar root systems in green algae: female gametes of Bryopsis lyngbyei (Bryopsidales). - - Helgol. Meeresunters. 34, 355--369.

46 M. M~'I~KONIAN & al.: Comparative EM of Spermatozopsis and DunalieUa

MELKOR~AN, M., 1984: Flagellar apparatus ultrastrueture in relation to green algal c l a s s i f i ca t ion . - In IRVINTI~ :, D. E. G., JOHN, D. M., (Eds.): The Systematies of the Green Algae. - - London: Academic Press. (In press.) PREISIG, H. R., 1984 b : Ultrastructtlre of the flagellar apparatus in the green flagellate Spermatozopsis similis. - - P1. Syst. Evol. (in press).

N~COLA~, E , BAAS B1~CKINC, L. G. M., 1935: Einige Notizen fiber Salzflagellaten. Arch. Protistenk. 85, 319--328.

OLIVEII~A, L., BISALPUTRA, ~I~., ANTIA~ N. J.~ 1980: Ultrastruetural observation of the surface coat of Dunaliella tertiplecta from staining with cationic dyes and enzyme treatments. - - New Phytol. 85, 385--392.

1)ASCHEH, A., 1930: Neue Volvocalen (Polyblepharidinen--Chlamy- d o m o n a d i n e n ) . - Arch. Protistenk. 69, ]03--146. 1932: Zur Kenntnis der einzelligen Volvocalen. - - Arch. Protistenk. 76, 1 - - 82. JAHO DA, R., 1928: Neue Po]yblepharidinen und Chlamydomonadinen aus den Almtfimpeln um Lunz. - - Arch. Protistenk. 61,239 281.

PI~TJ,]RFI, L. S., MANTON, I., 1968: Observations with the electron microscope on Asteromonas gracilis AHTARI emend. [Stephanoptera gracilis (ARTARI) WISL.], with some comparative observations on Dunaliella sp. - - Br. Phycol. Bull. 3, 423--440.

Pm,:rs~G: H. R., M~:LKONIAN: M., 1984: A light and electron microscopical study of the green flagellate Spermatozopsi8 similis sp. nov. - - P1. Syst. Evol. 146. 57--74.

PRINTZ, H., 1927: Chlorophyceae. In E~GLER, A., PRANTL, K , (Eds.): Die 5,Tatfirlichen Pflanzenfamilien 3 (2rid edit.), 1--463. Leipzig: Engelmann.

RUINEN, J., 1938: Notizen fiber Salzflagellaten. II . Uber die Verbreitung der Salzflagellaten. - - Arch. Protistenk. 90~ 210 258.

SCHL0SSnR, U. G., 1982: Sammlung von Algenkulturen. - - Bet. Deutsch. But. Ges. 95, 181--276.

SKVORTZ0V, B. V., 1968: New genera of green flagellatae recorded in N.-O. China. - - Revue Algol., N. S. 9, 121--130.

T~co~oHEsco, E. C, 1905: Organisation et d6veloppement du Dunaliella, nouveau genre de Volvoeac6e-Polyblepharid6e. - - But. Centralbl., Beih. 18, 215-- 232.

- - 1906: Observations morphologiques et biologiques sur le genre Dunaliella. Revue G6n. But. 18, 353--371,409--427.

TOMASELLO, D., PAL~,~mm, R. M., Lo GIUDICE, L.: SO~TINO, M., 1980: Ulteriori ricerche sulla ul t ras t rut tura di Dunaliella salina TEOD. Riv. Biol. N o r m . Patol. 6, 165--170.

TREZZl, F., GALLI, M. G., BELL~N4 E., 1964: L~infrastruttura di Dunaliella salina. - - Giorn. But. Ital. 71, 127--136.

VLADIMIROW, M. G., 1978: Ultrastruetural organization of the cell of Dunaliella salina and functional changes of it in relation to light intensity and temperature. Fiziol. Rust. 25, 443--449. (Engl.)

WE~z, G., KELLNER, G., 1970: Die Struktur des Golgi-Apparates bei gefrier- ge£tzten Dunaliella-Zellen. - - Protoplasma 69, 351 364.

Addresses of the authors: MICHAEL MELKONIAN, Botanisches Inst i tut , Westf/~lisehe Wilhelms-Universit~t, Sehlossgarten 3, D-4400 Mfinster: Federal P~epublie of Germany. - - H~Ns RU~OL~ PRn~sm, Ins t i tu t ffir Systematische Botanik der Universit~t Zfirich, Zollikerstrasse 107, CH-8008 Zfirich, Switzerland.