MOLECULAR ANALYSIS OF PORPHYRA 517

dophyta) in North America. I. Biogeographic trends in mor- Waaland, J. R., Dickson, L. G. & Duffield, E. C. S. 1990. Con- phology. Phjcologia 23:383-96. chospore production and seasonal occurrence of some Por- Sogin, M. L. & Edman, J. C. 1989. A self-splicing intron in the phjra species (Bangiales, Rhodophyta) in Washington State. small subunit rRNA gene of Pneumocjstis carinzz. Nurleir Acids Hjdrobiologia 204/205:453-9. Res. 17:5349-59. Wang, J-C. & Chiang, Y-M. 1977. Notes on the marine algae of Sogin, M. L., Gunderson, J. H., Elwood, H. J., Alonso, R. A. & Taiwan. 11. The genus of Porphp. Taiwania 22:105-12. Peattie, D. A. 1989. Phylogenetic meaning of the Kingdom White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification concept: an unusual ribosomal RNA from Giardia lamblia. and direct sequencing of fungal ribosomal RNA genes for Sciencp (Wash. D.C.)243:15-7. phylogenetics. In Innis, M. A., Gelfand, D. H., Sninsky, J. J. Tokida, J. 1935. Phycological observations. 11. On the structure & White, T. J. [Eds.] PCR Protocols: A Guide to Mpthods and ofPorphjra onoi Ueda. Trans. Sapporo ‘Vat. Hist. Sac. 14: 11 1-4. Applications. Academic Press, San Diego, pp. 3 15-22. Van de Peer, Y., Neefs, J-M. & De Wachter, R. 1990. Small Wilcox, L. W., Lewis, L. A., Fuerst, P. A. & Floyd, G. L. 1992. ribosomal subunit RNA sequences, evolutionary relation- Group I introns within the nuclear-encoded small-subunit ships among different life forms, and mitochondria1 origins. rRNA gene of three green algae. Mol. Biol. EL~o~.9:1103-18. J. Mol. Errol. 30:463-76. Woese, C. R. & Olsen, G. J. 1986. Archaebacterial phylogeny: van Oppen, M. J. H., Olsen, J. L. & Stam, W. T. 1993. Evidence perspectives on the Urkingdoms. Sjst. Appl. Microbial. 7: 16 1- for independent acquisition ofgroup I introns in green algae. 77. Mol. Biol. Evol. (in press).

J. Phycol. 29, 517-528 (1993)

SCRIPPSIELLA VELELLAE SP. NOV. (PERIDINIALES) AND GLOEODINIUM VISCUM SP. NOV. (PHYTODINIALES), SYMBIONTS OF TWO HYDROZOANS (CNIDARIA)’.’

Anastazia T. Banaszak, Roberto Iglesias-Prieto, and Robert K. TrenchS Department of Biological Sciences, University of California at Santa Barbara, Santa Barbara, California 93106

ABSTRACT dophyta, Bacillariophyta, Chlorophyta, Prasinophy- The alga symbiotic with the “By the Wind Sailor” Ve- ta, Pyrrophyta). Such a wide phyletic distribution of lella velella L. (Hjdrozoa, Chondrophora) collected off symbionts and hosts is consistent with the concept the coast of Galijornia, and the alga symbzotic with the of a polyphyletic origin of microalgal-invertebrate hydrocoral Millepora dichotoma Forskal (Hydrotoa, symbiosis (Trench 1992). Symbioses between dino- Milleporina) from the Gulf of Aqaba, were isolated and flagellates (Pyrrophyta) and invertebrates dominate brought into unialgal, axenic culture. Based on laght mi- the marine environment (Trench 1987a, 1992), are croscopj and ultrastructural analyses by transmission and essential to the high primary productivity of coral scanning electron microscopy, the algal population from reefs (Muscatine 1980), and are sensitive indicators V. velella is described as Scrippsiella velellae sp. nov. of ecological perturbations in such shallow water (Peridiniales). The algal population from the dark morph ecosystems (Iglesias-Prieto et al. 1992). The concept of M. dichotoma is described as Gloeodinium viscum of a monophyletic origin of dinoflagellate-inverte- sp. nov. (Phytodiniales). These are the first descriptions of brate symbioses was founded on the premise that sjmbiotic belongzng to the orders Perzdinia- symbiotic dinoflagellates are restricted to the genus les and Phqtodiniales. When viewed with descriptions of . This concept is giving way to one of symbiotic dinoflagellates belonging to the Prorocentrales polyphyletic origins of dinoflagellate-invertebrate and Gymnodinzales, the evidence currentlq points to seven symbioses (Trench 1992) that currently embrace genera in four orders of dtnojagellates havzng represen- several species of Symbiodinium (Blank and Trench tataves that haw entered a symbiotic mode of existence. 1985, Trench and Blank 1987, Blank and Huss 1989, Rowan and Powers 1992), two species of Amphidi- Key index words: Dinophyceae; dizwsity; Gloeodini- nium (,,. L. Taylor 1974, Trench and Winsor 1987), um viscum sp. nov.; Pyrrophyta; Scrippsiella velellae two species of Gymnodinium (Lee 1980, Spero 1987), sp. nov.; symbiosis Aureodinium hindler and Hemleben 1980). and ,I Prorocentruin (famasu 1988). A wide phyletic range of invertebrates harbors The dinoflagellate symbiont of the “By the Wind endosymbiotic microalgae (used here to include Sailor” Velplla velplla L. was originally named zoo- cyanobacteria) representing S~eraldivisions (Rho- xanthella (Brandt 1881, 1882) or Philozoon sifhonoph- oruvn (Geddes 1882a, b) and, subsequently, Endodi- nium chattonii (Hovasse 1922, 1923) (Blank and ’ Received 14 December 1992. Accepted 19 April 1993. We dedicate this paper to the memory of the late Professor Trench 1986)*More using v’ velella from Luigi Provasoli, a mentor and friend to R.K.T. the Mediterranean, D. L. Taylor (1971a, 1974) $Author for reprint requests. claimed that the alga in culture was amphidinioid 518 ANASTAZIA T. BANASZAK ET AL. and assigned the alga to Amphidinium [as A. chattonii cells were fixed and processed as described previously (Trench (Hovasse) D. L. Taylor]. However, this generic des- 1974, Trench and Blank 1987). The methods used for exami- ignation was disputed by Hollande and CarrP (1 974), nation of motile stages by scanning electron microscopy (SEM) who, although they studied only ‘the coccoid phase were those described by Spero (1987). hospite of the symbiont of Mediterranean velella, in V. RESULTS nonetheless felt justified in retaining the alga in En- dodinium. The alga was never described in detail by Scrippsiella velellae sp. nov. (Figs. 1- 15) Brandt, Geddes, nor Hovasse, who regarded it as a Order, Peridiniales; Family, Calciodinellaceae parasite related toBlastodinium (Hovasse 1922, 1923). Unicellularis alga status sjmbiosis cum Velella velella D. L. Taylor and Hollande and CarrP described in (Pactjica), locatus in hospite ac coccoidales (non-mobiles) detail the ultrastructure of the alga in hospite but cellulae diametro 17-18 pm intra endodermatibus cel- offered no description of the alga in culture. We lulae gonophorum et medusarum, dividens status mitosis have successfully isolated the symbiotic alga from V. in statu coccoidales in hospite et in cultura. Magna nu- velella collected off the coast of California and have cleus cum circa 90 chromosomatibus; inuolucrum nuclei maintained it in unialgal, axenic culture. We de- compositus duarum membrana cum areoles. Involucrum scribe the alga as Scrippsiella zelellae sp. nov. Light chloroplastium coinpositus triuin membrane; thjlakoidea and electron microscopic descriptions of coccoid chloroplastium maximum partern binae aggregatae; dua stages in hospite and in culture and of the dinomas- pjrenoides cum inuadens chloroplasts thjlakoides. “Ac- tigote stage in culture are provided. cumulate corpora” praesens in hospite et in cultura. Schonwald et al. (1987) described studies of “pho- Trichocjsta praesens in hospite et in cultura. to-adaptation” of photosynthesis in light- and dark- Status dinomastigota peridinoide in cultura; ualuae de- colored colonies of the hydrocoral Millepora diclio- signatus pp x 4’3a 7“ 5c 3s 5“ Op 2”; 17-20 pm in toma Forskil from the reefs off Eilat, Israel. We have longitudine; cellulae demonstratus lateralis in statu ec- isolated the algae from both types of hydrocoral and djsis. Trichocjsta p raesens. brought them into culture. While the alga associated Unicellular alga symbiotic with Velella idella L. with the light morph of M. dichotoina conformed in (Pacific), located in hospite as coccoid (non-motile) its morphology to a species of Synbiodinium (R.K.T., cells 1’7-1 8 pm in diameter, within endodermal cells unpubl.), the alga isolated from the dark morph did of the gonophores and medusae, dividing mitotically not. A description of the ultrastructure of the coc- in the coccoid state in lzospite and in culture. Large coid cells in culture is provided. We have not been nucleus with approximately 90 chromosomes; nu- able to study the dinomastigote stage in detail. clear envelope comprised of two membranes with pores. Chloroplast envelope comprised of three MATERIALS AND METHODS membranes; chloroplast thylakoids predominantly Colonies of Vrlrlln 1vlrlln L. were collected during “blooms” in groups of two; two pyrenoids with invasive chlo- in 1988 and 1991 off the coast of Santa Barbara, California. After roplast thylakoids. “Accumulation body” present in the were transferred to the laboratory, the gonozooids host and in culture. Trichocysts present in hospite were dissected from the colony, and released medusae were col- and in culture. lected, fixed, and prepared for light and electron microscopic Dinomastigote stage in culture peridinioid; thecal examination, as described later. The algal cells were isolated by blending several medusae or gonozooids in a Virtis blender, fil- plate designation pp x 4‘ 3a 7” 5c 3s 5” OP 2””; 17- tering the slurry through cheese cloth, and repeatedly washing 20 Prn in length. Cells demonstrate lateral ecdysis. by centrifugation and resuspension in sterile sea water (Trench Trichocysts present. 1971). Samples of the crude isolates were immediately fixed for The holotype was collected from Velella velella L. subsequent examination by transmission electron microscopy off the coast of California and brought into axenic (TEM). The algae were inoculated into 10 mL sterile ASP 8A culture. It is available in liquid culture at the Uni- (Blank 1987) at initial cell densities of approximately 102.mL-’. Algal cells were routinely maintained at 17” C and illuminated versity of California at Santa Barbara. with GroLux fluorescent tubes at 80 pmol quanta.m-2.s-’ (pho- The algal cells 272 hospite are intracellular in the tosynthetically active radiation [PAR]) on a 14:lO h LD photo- endodermal cells of the gonophores and the me- period. In some instances, cells were grown under blue light using dusae (Figs. 1,2). This is consistent with descriptions a plastic filter with the following characteristics: transmission by Hovasse (1922, 1923), D. L. Taylor (1971a), and maximum, 490 nm with a full width at half maximum of 41 nm. Hollande and CarrP (1974) for the symbiont of the Colonies of the light and dark morphs of iMillrporn dichotomn Mediterranean V. velella. In the coccoid state in the Forskil were collected from the reefs near the Interuniversity Marine Institute, Eilat, Israel. Tissues fixed for examination by host, the algal cells are approximately 17.60 pm (fSD TEM, after removal of the calcium carbonate skeleton, were 1.70 pm, n = 200) in diameter and divide in the provided by Dr. Tamar Berner, Bar Ilan University, Ramat-Gan, coccoid state (Fig. 1) in the host’s cells as well as in Israel. To isolate the algae, the animals’ tissues were removed culture (Fig. 3). The boundary between algae and from the carbonate skeleton by WaterPik Uohannes and Wiebe host cell cytoplasm is difficult to resolve, as a clear 1970), and the resulting slurry was treated as already described. algal cell wall is not readily discernible (cf. Hollande The cultures were maintained at 27” C and illuminated by GroLux lamps at 80 pmol quanta.m-2.s-l (PAR) on a 14:lO h LD pho- and CarrP 1974). The plasmalemma is located with- toperiod. in the coccoid cell wall in hospite and in culture and For examination by light microscopy and TEM, tissues and within the amphiesma of the dinomastigote stage in SCRIPPSIELLA AND GLOEODINIUM 519

.

r 3

FIGS. 1-7. Scrippsirlln wlrllnr sp. nov. FIG. 1. A light micrograph of a thin section through a released medusa from V. vrlella showing the algal symbionts [in one case, dividing (arrow)] in the host’s endodermal cells. e, endoderm; ec, ectoderm; m, mesoglea. Scale bar = 5 pm. FIG. 2. TEM of a section through a medusa of V. ivlella showing the algal symbionts in the endodermal cells of the . Arrows indicate the trichocysts. e, endoderm; m, mesoglea. Scale bar = 5 pm. FIG. 3. TEM of a coccoid cell that has recently undergone karyokinesis and cytokinesis. N, nucleus; nu, nucleolus. Arrows point to the “fracture” zone in the cell wall. Scale bar = 1 pm. FIG. 4. TEM of a cell ijt iiosfiitp showing the pyrenoid with invasive chloroplast thylakoids and a prominent starch sheath. Scale bar = 1 pm. FIG. 5. TEM of a coccoid cell in culture. Arrowheads indicate trichocysts; arrow indicates the cell wall. Scale bar = 1 pm. FIG. 6. Light micrograph of a cell undergoing ecdysis and cytokinesis. Arrow indicates the discarded amphiesma of the motile cell. Scale bar = 5 pm. FIG. 7. SEM of a motile cell showing details of the apical “tuft” and associated plates. The small pores (arrows) are trichocyst pores. Scale bar = 1 pm. 520 ANASTAZIA T. BANASZAK ET AL

FIGS. 8-13. Scrippsiella velellae sp. nov. Scale bars = 1 pm. FIG. 8. SEM of a motile cell showing the ventral aspect. FIG. 9. SEM of a motile cell showing the dorsal aspect. Arrows indicate trichocyst pores. FIG. 10. TEM of a motile cell that is in the process of reverting to the coccoid stage as indicated by the development of the cell wall under the amphiesma. Karyokinesis has not yet been initiated. The girdle is shown in section from the lower left to upper right hand of the photograph, with the apical tuft at the upper left. ac, the “accumulation body.” FIG. 1 1. SEM of the amphiesma after ecdysis. The epicone and hypocone separate at the anterior edge of the girdle. FIG. 12. TEM through a motile cell (in the process of becoming coccoid) showing details of the protuberances (arrows) on the thecal plates and the sutures between plates (arrowheads). The cell wall (cw) is discernible beneath the thecal plates. FIG. 13. TEM of a motile cell showing the “simple” eyespot (arrowhead) beneath the girdle. Arrows indicate trichocysts (in transverse section). SCRIPPSIELLA AND CLOEODINIUM 52 1

FIGS. 14, 15. Scrippsir//n vrlellar sp. nov. FIG. 14. ’IEM showing details of the paired thylakoid arrangement in a cell in hospitr. The plastome (chloroplast genome) is located in the thylakoid-free region (cg). Scale bar = 0.5 pm. FIG. 15. TEM of a cell cultured under blue light showing the fused chloroplast thylakoids (arrowheads). Arrows indicate trichocysts. Scale bar = 1 pm.

culture. The lobed chloroplast is predominantly pe- cies. A prominent nucleolus (Fig. 3) is present in ripheral. As is characteristic of dinoflagellates, it is most of the stages of the life history of the alga. The limited by an envelope composed of three mem- nuclear envelope is composed of a double mem- branes and possesses thylakoids usually in groups of brane with interruptions for nuclear pores. As in two (Figs. 4, 14). However, cells cultured under blue the host’s cells (Fig. l), mitosis occurs only in the light (40 pmol quanta.m-2.s-’ PAR) demonstrated coccoid state in culture (Figs. 3, 6). Trichocysts are thylakoid stacking (Fig. 15), a phenomenon wherein present (Figs. 2, 5, 13, 15) in IzospitP and in culture. severaI thylakoids become tightly appressed creating Details of the morphology of the conversion from the impression of grana. This phenomenon has been the coccoid to motile state in culture remain unclear, reported in a putative S~vibiodiniumsp. in a Zoantlzus but it appears that the algae escape from the cell sp. from shallow intertidal environments (Thinh et wall of the coccoid cell prior to developing the theca al. 1986) and in S. microadriaticuwi grown in culture and other attributes of the dinomastigote. The cell at 280 pmol quanta.m-2.s-’(PAR) (A. Mandura and wall appears to have suture points (Fig. 3) that may R. K. Trench, unpubl.). Thylakoid membranes in- facilitate the escape. A similar observation was re- vade the pyrenoids (Figs. 2, 4). We have only re- ported by Hollande and Enjumet (1953) in cultures solved with certainty two pyrenoids per cell, and of Endodinium (= Zooxantlz~lla)wutricola (= nutricula) they are attached to the chloroplast by two (or more) from the radiolarian Collosphapra. stalks. The algae, in lzospitp or in culture, possess an In the dinomastigote state in culture, the algae “accumulation body” (Fig. lo), which, based on the are peridinioid and are 18.2 pm (fSD 2.8 pm, n = cytochemical localization of acid phosphatases with- 35) in length. The epicone is obtusely triangular in it, is probably a secondary lysosome. The nucleus, with an apical tuft (Fig. 7), and the hypocone is generally centrally located in the coccoid cells and rounded. The epicone and hypocone are separated in the hypocone region in the motile cells, appears by a broad girdle (Figs. 8, 9). Transition from the to contain many chromosomes. Although we did not dinomastigote to the coccoid stage involves the for- prepare chromosome squashes or serial sections mation of the coccoid cell wall within the theca of through the nucleus to enumerate the chromosomes the dinomastigote (Figs. 6, 10). The epicone and as was done by Trench and Blank (1 987), we counted hypocone ultimately separate at the anterior aspect the chromosomes in several randomly chosen pro- of the girdle (Figs. 1 1, 12), a process referred to as files of the nucleus and estimate that there are in “lateral ecdysis” (Morrill 1984, F. J. R. Taylor 1987). excess of 90. This is consistent with the estimates of The cast-off theca stains strongly with the fluores- Fine and Loeblich (1976) for other Scrippsidla spe- cent dye Calcofluor white M2R, and consistent with 522 ANASTAZIA T. BANASZAK ET AL.

its cellulosic composition, it does not stain after As is the case in most cnidarians, the algae are treatment with cellulase (Markell et al. 1992). Mi- located in the endodermal cells of the host. In hospite, tosis in the coccoid state usually ensues soon after the algae are coccoid and oval in outline and divide ecdysis (Fig. 6), as has been reported in cultures of in the coccoid state (Fig. 16), just as they do in cul- Scrippsiella sweeneyae, S. trochoideum, and S. faerognse ture. The mean diameter of the algae in hospite is (Fine and Loeblich 1976). 1 1.03 pm (i-SD 1.4 pm, n = 76), essentially identical The thecal plate designation of S. velellae is based to that reported by Schonwald et al. (1987). In cul- on the system of Kofoid (1 907, 1909, F. J. R. Taylor ture the algae are 13.1 pm (kSD 3.2 pm, n = 40). 1987) and is characteristic of the Peridiniales. The One reason for the larger standard deviation in the thecal plates are densely covered, except at the size of the cultured algae is the large size (up to 27 trichocyst pores and the apertures for the lateral pm in diameter) that some cells attain prior to di- and longitudinal flagella, with small, regularly dis- viding. tributed blisters (Fig. 8) in scanning electron micro- As described by Bouquaheux (1971) for G. mari- graphs or protuberances (Fig. 12) in transmission num, the algal cells in culture are mucilaginous and electron micrographs. The sutures between the the- form clumps of predominantly two, but often four, cal plates appear to be formed by overlapping ex- cells within an extracellular matrix. Consistent with tensions of the plates (Fig. 12). Situated just behind this is the ultrastructural resolution of matrix ma- the sulcus, the modified chloroplast contains glob- terial in the cells (Figs. 17, 19) and outside the cells. ules of lipid (Fig. 13) between the plastid envelope High synthetic activity is also indicated by the prom- and the thylakoids, similar to the “simple” eyespot inence of Golgi cisternae (Figs. 18, 23). described in Peridi?ziu?n westii (Messer and Ben-Shaul The coccoid cell is bounded by a prominent cell 1969). wall (Figs. 18-21), which has thickened areas that are apparently points of rupture (Figs. 18, 19) and Gloeodinium viscum sp. nov. (Figs. 16-23) may allow the motile cell to escape. We have ob- served the dinomastigotes, but they were infrequent Order, Phytodiniales; Family, Gloeodiniaceae and low in number and, hence, could not be studied Unicellularis alga status synbiosis cum Millepora di- in detail. However, it is possible that Figure 19 rep- chotoma, locatus in hospite et iiztra eizdodermatibus resents a coccoid cell in the process of becoming a lzospitibus atque cellulae coccoidales se dizlidunt in statu motile cell. Our interpretation is that the plasma- coccoidali ut in cultura. In hospite, cellulae algal dia- lemma lies beneath the cell wall and that the ma- metro inter 11-13 pin, in cultura diainetro inter 11-15 terial observed external to the wall (Fig. 21) is not pm. Cellulae in cultura sunt palinelloidibus ruin inter a plasmalemma. uizuin ue1 quatruin cellulae in inucilaginis matricis. Nu- The chloroplast is peripheral and is enclosed by cleus contient circa 30 inagna chroinosoinatibus limitatus an envelope comprised of three membranes, with a involucruin singulare coinpositus duuin inembranarum groups of three closely appressed thylakoids (Fig. ruin areoles. Chloroplast inuotucra triplica membrana- 21). Each cell contains two pyrenoids with invasive rum; thylakoidae trinae aggregatae; dua pjrewoides cum thylakoid membranes (Figs. 17,20,23).The nuclear intiadens chloroplasts thjlakoides. “Accumulate corpora” envelope is comprised of two membranes, with in- praesens in hospite et in cultura. Trichocyta nullus terruptions for nuclear pores (Figs. 17-19). The obsenlatus. Status diizoinastigota ne in singulus inuesti- characteristically condensed chromosomes are large, gatet. and we estimate that there are about 30 chromo- Unicellular alga symbiotic with Millepora dichoto- somes per nucleus. A prominent nucleolus (Fig. 17) 7na Forskal (dark morph), located in hospite within is always present. An “accumulation body” (Fig. 17) hosts’ endodermal cells as coccoid cells that divide is present in hospite and in culture. Fibrous bodies in the coccoid state, as they do in culture. In the (Fig. 22), whose function in dinoflagellates remains hosts, the algal cells are 11-13 pm in diameter, in unresolved, are present. No trichocysts have been culture 11-1 5 pm. Cells in culture are palmelloid, observed. with one to four cells in a mucilaginous matrix. Nu- cleus with approximately 30 large chromosomes, DISCUSSION limited by an envelope comprised of two membranes Our description of Scrippsiella uelellae in its host with pores. Chloroplast envelope comprised of three Velella ivlella from the Pacific is in general agree- membranes; thylakoids in groups of three; two py- ment with those given for the symbiont of the Med- renoids with invasive chloroplast thylakoids. “Ac- iterranean V. velella (D. L. Taylor 1971a, as Amphi- cumulation body” present in host and in culture. No dinium clzattoizii; Hollande and Carrk 1974, as trichocysts observed. Dinomastigote stage not stud- Endodinium chattonii). The algae from the two host ied in detail. populations are different in two major ways: the cells The holotype was collected from Millepora dicho- that we have studied are larger, and they contain toma Forskil off Eilat, Israel, and brought into axe- trichocysts in hospite and in culture. These charac- nic culture. It is available in liquid culture at the teristics confirm that the cells we obtained in culture University of California at Santa Barbara. are in fact the symbionts and not a contaminant. D. SCRIPPSIELLA AND GLOEODINIUM 523

FIGS.16-19. GloPodinium itiscum sp. nov. Scale bars = 1 fim. FIG. 16. TEM of a divided cell in hoxfiztt. FIG. 17. TEM of a coccoid cell in culture. The cell is limited by a wall, beneath which is the plasmalemma (arrow). The mucilaginous material secreted by the cell is within the plasmalemma and is contiguous with vesicles presumably arising from the Golgi. The chloroplast is peripheral, with two pyrenoids with invasive thylakoid membranes. The nucleus (N) appears to contain a few large chromosomes and a prominent nucleolus (nu). An “accumulation body” (ac) is present. FIG. 18. TEM of a coccoid cell showing a suture zone (arrowhead) in the cell wall. Arrows indicate nuclear pores. FIG. 19. TEM of a coccoid cell perhaps in the process of becoming a motile cell. Arrowheads indicate the cell wall separated at the suture points. The extracellular “matrix” is shown in the background surrounding the cell. 524 ANASTAZIA T. BANASZAK ET AL.

FIGS. 20-23. Glopodiniuur viscu~~~sp. nov. FIG. 20. TEM of a coccoid cell in culture. The continuous cell wall (cw) surrounds the coccoid cell, and the plasmalemma (arrow) is beneath it. The pyrenoid (py) has invasive thylakoid membranes and a starch sheath. Scale bar = 1 pm. FIG.2 1. TEM showing details of the chloroplast with thylakoids in groups of three. The arrowhead indicates the chloroplast envelope membranes. Scale bar = 0.25 pm. FIG. 22. I’EM showing intracellular vesicles containing what is interpreted as mucilaginous material secreted by the cell. Fibrous bodies (f) of unknown function are also shown. Scale bar = 0.5 pm. FIG. 23. TEM of a coccoid cell showing details of the Golgi cisternae. Scale bar = 0.5 pm. SCRIPPSIELLA AND GLOEODILIUM 525

L. Taylor did not mention the presence of tricho- possibility that Zooxanthella, Endodinium, and Scrip@- cysts, and Hollande and Carrk categorically stated siella are synonyms, with Zooxanthella Brandt having that trichocysts were absent. The latter authors also priority. As Brandt did not describe the dinomas- stated that there were eight pyrenoids; we could only tigote stage, which is diagnostic of Scrippsiella, we resolve two. We find it justifiable to conclude that recommend that Zooxanthella also be replaced by the alga we studied is not the same as that in the Scrippsiella. Other strong reasons for eliminating Zo- Mediterranean V. velella. Hence, we have described oxanthella as a synonym of Sjmbiodinium, as proposed it as a new species. by Loeblich and Sherley (1 979) and Loeblich (1 984), D. L. Taylor (197 la) claimed to have brought the have already been given (Trench and Blank 1987). alga from the Mediterranean V. velella into culture. As a result, the family Zooxanthellaceae (Dodge Considering the cells to be amphidinioid, he trans- 1984, F. J. R. Taylor 1987) should also be elimi- ferred the species from Endodinium to Amphidiniurn, nated, as it incorporated algae that have taxonomic as A. chattonii (Hovasse) D. L. Taylor. A detailed affiliations elsewhere. For example, in their taxon- description of the motile stage of the alga in culture omy of dinoflagellates, Dodge (1984) and F. J. R. was not provided. A major characteristic on which Taylor ( 1987) classified Zooxanthella, Endodinium, and Taylor based his decision was the presence of py- Sjmbiodinium as representative of the family Zooxan- renoids with invasive thylakoids, but this feature is thellaceae, order Gymnodiniales, while Loeblich shared by several distinct dinoflagellates. Hollande (1984) placed Sjmbiodinium, and by implication, Zo- and Carri. disputed Taylor’s conclusion and retained oxanthella, in the order Zooxanthellales. Sjmbiodini- the alga in Endodinium. Our analysis of the alga from um should be classified with the Gymnodiniaceae Pacific V. velella shows that it is representative of (Trench and Blank 1987), and Zooxanthella and En- Scrippsiella (Peridiniales). The morphology of the dodiniun should be replaced by Scrippsiella (Calciodi- motile cells agrees completely with that described nellaceae, Peridiniales). for Scrippsiella (Balech 1959, Fine and Loeblich 1976, The characteristics of the freshly isolated cells and Roberts et al. 1987). isolates that we cultured from the dark morph of It is very likely, based on their marked similarity M. dichotoma are identical, indicating that the alga in hospite, that the algae in Pacific and Mediterranean in culture is not a contaminant. In the general as- V. zlelella are congeneric. Strict adherence to the pects of its morphology in culture, including the rules of priority would dictate that the generic name secretion of an extracellular mucilaginous matrix, Zooxanthella Brandt should be adopted rather than this alga is similar to those that have been described Scrippsiella Balech ex Loeblich 111 1965. This would as Gloeodinium or Hemidinium, which some authors serve no useful purpose. As neither Brandt, Ho- (Dodge 1984, F. J. R. Taylor 1987) believe to be vasse, D. L. Taylor nor Hollande and Carr; de- congeneric. However, Popovskji (1 97 1) determined scribed the motile stage of the symbiotic algae from that G. inontanurn and H. nasutum, both of which are the Mediterranean V. zlelella, we recommend that freshwater dinoflagellates, were not congeneric. use of the generic name Endodinium be discontinued Bouquaheux (1971) and F. J. R. Taylor (1976) and that Endodiniuin be replaced by Scrippsiella. Thus, described G. marinum from the plankton as palmel- the symbiont of Mediterranean V. velella would be loid cells ensheathed in an extracellular mucilagi- Scrippsiella chattoniz comb. nov., and the alga in Pa- nous matrix. The cells described by Bouquaheux cific V. velella, S. uelellae sp. nov. Given the specificity were 25-35 x 20-30 pm, whereas those described of many dinoflagellate-invertebrate symbioses by F. J. R. Taylor were about 20 pm in diameter. (Trench 1987b, 1992), it is possible that the two Both of these are significantly larger than the alga populations of V. tielella are not conspecific. that we isolated from M. dichotoma. In addition, Bou- In their discussion, Hollande and Carrk (1974) quaheux’s diagram and electron micrographs indi- emphasized the marked similarity between E. chat- cate the presence of trichocysts in G. marinum. We tonii Hovasse and Zooxanthella nutricula Brandt, the did not observe trichocysts. In no previous study of latter being the symbiont of the radiolarians Collo- the genus Gloeodinium has there been a detailed anal- zouvn and Collosphaera. In fact, they transferred the ysis of the mastigote stage. Although we did observe species Zooxanthella nutricula to Endodinium, making motile cells, their extremely low numbers and in- the combination E. nutricula (Brandt) Hollande and frequency of occurrence frustrated any attempt at Carrk. Earlier, Hovasse (1924) decided that Endodin- scanning or transmission electron microscopic anal- zuin chattonii, which he had described in 1922, was ysis. There are no detailed descriptions of the motile representative of Zooxanthella Brandt. He therefore stage of Gloeodinium, the description of the genus made the combination Z. chattonii (Hovasse) Ho- being based exclusively on the morphology of the vasse. The ultrastructure in hospite of the symbiont coccoid cells. We therefore feel justified in conclud- of Coltozoum inerme from the Sargasso Sea (Anderson ing that the symbiont is representative of Gloeodini- 1976) also shows a marked similarity to the symbiont um but that it is not G. marinum. We describe it as of V. velella. The alga from Collozouin (or Collosphae- G. uiscum sp. nov. ra) has never been cultured; therefore, the motile With the description of two new species of sym- stage is unknown. This state of affairs raises the biotic dinoflagellates provided in this paper, despite 526 ANASTAZIA T. BANASZAK ET AL.

TABLE1. Classijcation of known sjinbiotic dinojagellates (orders andfamilies afer F.J. R. Taylor 1987). The host species are given in partnthtses. *The genus Amphidinium is ambiguous and inaj not be a member of the Gq’mnodinialts (McNallj, Govind, Thofn;, and Trench, unpubl.).

Species Reference Gymnodiniales Gymnodiniaceae Gjinnodiniuni b6i (Orbulina universa) Spero 1987 Gjinnodinium zlertebralis (Maginopora vertebralis) Lee 1980 Aureodinium sp. (Globigeriizoides sacculifer) Spindler and Hemleben 1980 Sjinbiodinium goreauii (Heteractis lucida) Trench and Blank 1987 S. inicroadriaticutn (Cassiopeia xamachana) Freudenthal 1962 S. kaulagutii (Montipora verrucosa) Trench and Blank 1987 S. pilosuin (Zoanthus sociatus) Trench and Blank 1987 S. corculoruin sp. nov. (Corculum cardissa) Trench (unpubl.) S. ineandrinae sp. nov. (Meandrina meandrites) Trench (unpubl.) S. pulchroruin sp. nov. (Aiptasia pulchella) Trench (unpubl.) S. cariborum sp. nov. (Condjlactis gigantea) Trench (unpubl.) S. bermudetzse sp. nov. (Aiptasia tagetes) Trench (unpubl.) S. californium sp. nov. (Anthopleura elegantissirnu) Trench and Blank 1987 ’*Amphidinium klebsii (Ainphiscolops langerhansi) D. L. Taylor 197 1 b *A. belauense sp. nov. (Haplodiscus sp.) Trench and Winsor 1987 Peridiniales Calciodinellaceae Scrippsiella zielellae sp. nov. (Velella zfelella) Pacific Banaszak et al. 1993 S. nutricula comb. nov. (Collozoutn inerine) Banaszak et al. 1993 S. chattonii comb. nov. (V. velella) Mediterranean Banaszak et al. 1993 Phytodiniales Gloeodiniaceae Gloeodinium ziiscuin sp. nov. (Millepora dichotoma) Banaszak et al. 1993 Prorocentrales Prorocentraceae Proroceiztrutn concazlum (Ainphiscolops sp.) Yamasu 1988

the elimination of Zooxanthella and Endodinium, the algal taxa that are involved in algal-invertebrate diversity among dinoflagellates that have entered a symbioses. The concept that there is a single alga, symbiotic way of life is greatly increased, now rep- a zooxanthella, associated with various marine in- resented by seven genera of dinoflagellates in four vertebrate taxa has pervaded the literature and con- orders and four families (Table 1). The classification tinues as an underlying assumption in several phys- employed in Table 1 follows F. J. R. Taylor (1987); iological and ecological studies. there are significant disagreements between this and Schonwald et al. (1 987) explained the differences the classification by Dodge (1 984), according to in the “photo-adaptive” (photo-acclimatory) re- whom Scrippsiella is in the family Peridiniaceae and sponses of the light and dark morphs ofM. dichotoma Gloeodinium is in the order Gloeodiniales. A reclas- as a function of different densities of algae per unit sification of living and fossil dinoflagellates has re- area in the different colonies. The underlying as- cently been published (Fensome et al. 1993). sumption was that the two hydrocorals harbored the Following D. L. Taylor (1973), the capacity to same algal taxon. A more plausible explanation revert from motile to coccoid (“vegetative cyst” of would be that two distinct dinoflagellate taxa that F. J. R. Taylor 1987) states is thought to represent demonstrate different photo-acclimatory responses a “preadaptive” characteristic of the genus Sjmbio- are involved. This hypothesis is strengthened in light dinium. From our description of S. velellae and G. of their ecological distribution, where the hosts co- viscum, it is apparent that other genera of symbiotic occur in identical habitats. It is also possible that the dinoflagellates also share this character. The exis- two morphs of M. dichotovna are not conspecific. tence of a coccoid stage in symbiosis and of coccoid Assuming that symbiotic dinoflagellates are and motile stages in culture in these genera is a “zooxanthellae” whose classification has been in- different situation from that observed in symbiotic tractable, Rowan and Powers (1991a, b, 1992) iso- Amphidinium, which retain their dinomastigote mor- lated symbiotic algae from a range of invertebrates phology in hospite and in culture (D. L. Taylor 197 1 b, and without determination of their identity, con- Trench and Winsor 1987). The inferred phylogeny ducted phylogenetic analyses based on restriction of dinoflagellates based on small subunit ribosomal fragment length polymorphism (RFLP) of amplified DNA (SSU rDNA) sequence analyses suggests that genomic SSU rDNA genes and partial SSU rDNA the genus Amphidinium is distantly related to Sym- sequences. One of the major conclusions of this se- biodinium (K. McNally, N. S. Govind, P. Thome, and ries of studies was that the “diversity within the R. K. Trench, unpubl.). genus Symbiodinium is comparable to that observed The description of two new symbiotic dinoflagel- among different orders of non-symbiotic dinoflagel- late species raises at least two significant issues per- lates.’’ Our observations indicate that extreme cau- taining to the assumptions of investigators of the tion should be exercised when studying any aspect SCRIPPSIELLA AND GLOEODINIUM 527 of dinoflagellate-invertebrate symbiosis in assuming 1992. Photosynthetic response to elevated temperature in that different taxa of invertebrate hosts a11 harbor the symbiotic dinoflagellate Synbiodi~ziuwinirroadria/icu,n in Symbiodinium-likedinoflagellates. culture. Proc. ‘Vat. Acad. Sci. U.S.A. 89:10302-5. Johannes, R. E. & Wiebe, W. J. 1970. A method for determi- nation of coral tissue biomass and composition.Limnol. Ocean- We thank Patricia E. Thome for maintenance of the culture ogr. 15:822-4. collection of symbiotic dinoflagellates and Shane Anderson and Kofoid, C. A. 1907. Dinoflagellata of the San Diego region. 111. Jim McCullagh for collecting the specimens of V. zwllela. Robert Descriptions of new species. Utzizd. Calq Publ. Zool. 3:299- Gill is gratefully acknowledged for his assistance with SEM and 340. TEM techniques. Dr. Barry Tanowitz provided the Latin trans- 1909. On Prridiiiiuin steini Jergensen, with a note on the lation of the species diagnoses. Professors David J. Chapman and nomenclature of the skeleton of the Peridinidae. Arch. Pro- Paul C. Silva read an early draft of this manuscript, and their tistrnk. 16:25-47. astute and insightful comments have contributed to the enhance- Lee, J. J. 1980. Nutrition and physiology of the Foraminifera. ment of the paper. R.1-P. acknowledges a predoctoral scholarship In Levandowsky, M. & Hunter, S. H. [Eds.] Biochrinistrj and from La Universidad Nacional Aut6noma de Mexico. This study Phj.~iolog~of Protozoa, Vol. 3. Academic Press, London, pp. was supported by a grant from the Office of Naval Research (ONR 43-66. Loeblich 111, A. R. 1965. Dinoflagellate nomenclature. Taxon N00014-92-J-1131 to R.K.T.). 14:15-6. 1984. Dinoflagellate evolution. In Spector, D. L. [Ed.] Anderson, 0. R. 1976. Ultrastructure of a colonial radiolarian DiizoJ?ag~l/ates.Academic Press, New York, pp. 48 1-522. COllozOUlff inerme and a cytological detrrmination of the role Loeblich 111, A. R. & Sherley, J. L. 1979. Observations on the of its zooxanthellae. Tissue U Cell 8:195-208. theca of the motile phase of free-living and symbiotic isolates Balech, E. 1959. Two new genera of dinoflagellates from Cali- of Zooxanthalla inicroadriatica (Freudenthal) comb. nov. J. Mar. fornia. Bid. Bull. 1 16: 195-203. Biol. Assoc. U.K. 59: 195-205. Blank, R. 3. 1987. Cell architecture of the dinoflagellate Syn- Markell, D. A., Trench, R. K. & Iglesias-Prieto, R. 1992, Mac- biodiniuin sp. inhabiting the Hawaiian stony coral Montzpora romolecules associated with the cell walls of symbiotic di- 7~errucosa.Mar. Bid. (Btrl.) 94:143-55. noflagellates. Synbiosis 12: 19-31. Blank, R. J. & Huss, V. A. R. 1989. DNA divergency and spe- Messer, G.& Ben-Shaul, Y. 1969. Fine structure of Prridiniuin ciation in Sjinbiodiniuin (Dinophyceae). PI. Sjst. Eid. 163: uvstii Lemm, a freshwater dinoflagellate. J. Protozool. I6:272- 153-63. 80. Blank, R. J. & Trench, R. K. 1985. Speciation and symbiotic Morrill, L. C. 1984. Ecdysis and the location of the plasma mem- dinoflagellates. Science (Wash. D.C.) 229:656-8. brane in the dinoflagellate Heterocnpsa niei. Protoplasma 1 19: ’ 1986. Nomenclature of endosymbiotic dinoflagellates. 8-20. Taxoiz 35:286-94. Muscatine, L. 1980. Productivity of zooxanthellae.In Falkowski, Bouquaheux, F. 197 1. Glorodiniuin inarinuiit nov. sp. Peridinien P. G. (Ed.] Prirnarj Producfizitj in the Sea. Plenum Press, New dinocapsale. Arch. Protistrnk. 1 13:314-2 1. York, pp. 381-402. Brandt, K. 1881. Ueber das Zusammenleben von Algen und Popovskf, J. 1971. Some remarks to the life cycle of Gloeodinium Tieren. Bid. Crn/ralbl. 1524-7. mxz/anum Klebs and Hrmidinium nasutuin Stein (Dinophy- 1882. Ueber das Zusammenleben von Tieren und Algen. ceae). Arch. Protistenk. 1 13: 131-6. Bot. Ztg. 40:248-54. Roberts, K. R., Timpano, P. & Montegut, A. E. 1987. The apical Dodge, J. D. 1984. Dinoflagellate taxonomy. In Spector, D. L. pore fibrous complex: a new cytological feature of some di- [Ed.] Dinof7agdlnfrs. Academic Press, New York, pp. 17-42. noflagellates. Protoplasm 137:65-9. Fensome, R. A., Taylor, F. J. R., Norris, G., Sarjeant, W. A. S., Rowan, R. & Powers, D. A. 1991a. A molecular genetic classi- Wharton, D. I. & Williams, G. L. 1993. A classification of fication of zooxanthellae and the evolution of animal-algal living and fossil dinoflagellates. 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]. Ph@. 29, 528-535 (1993)

PREDICTING DESICCATION STRESS IN MICROSCOPIC ORGANISMS: THE USE OF AGAROSE BEADS TO DETERMINE EVAPORATION WITHIN AND BETWEEN INTERTIDAL MICROHABITATS’

Susan H. Brawley2 Department of Plant Biology, University of Maine, Orono, Maine 04469 and Ladd E. Johnson Maritime Studies Program, Williams College-Mystic Seaport, Mystic, Connecticut 06355

ABSTRACT species distributions within the intertidal zone on We describe an easj and inexpensiue waj to determine rocky shores (e.g. Moore and Seed 1986). Desicca- whether intertidal microhabitats remain wet during tidal tion is regarded as one of the most important de- emersion. This new technique uses agarose beads (120 pm terminants of the characteristic distribution that diameter when fully hjdrated) that shrink in a graded many organisms have within the intertidal zone (e.g. fashion as they dry. The agarose beads allow variability Baker 1909, 1910, Zaneveld 1937, Jenik and Law- in surface wetness to be gauged ouer distances of less than son 1967, Schonbeck and Norton 1978, Vermeij 1 mm. Describing this parameter of microclimate is irn- 1978, Garrity 1984, Wethey 1984, Palumbi 1985, portant in order to predict the likelihood and spatial pat- Buschmann 1990, McMahon 1990). Most studies of tern of sunliual of settled larvae, reproductiue propagules, desiccation have been done on adults; however, many and other microscopic stages in the life histories of organ- important events affecting community structure oc- isms growing in intertidal and other water-stressed envi- cur at earlier stages in the life histories of intertidal ronments. For the brown seaweed Pelvetia fastigiata u. organisms (e.g. Keough and Downes 1982, Brawley Ag.) DeToni (Fucales, Phaeophjta), the use of agarose and Johnson 199 1 and references therein). Desic- beads demonstrated that survival of zygotes during tidal cation stresses will vary throughout an organism’s emersion was highest at those sites that remain damp. life history and are likely to be more severe for Temperature alone wasfound to be an unreliable measure younger or smaller organisms due to their greater ofwetness within a single microhabitat (e.g. red algal turf). ratio of body surface area to body volume. Key index words: agarose beads; desiccation; emersion Unfortunately, the most common method previ- ously used to assess desiccation stress in adults (i.e. stress; Fucales; intertidal zone; laruae; marine ecology; Pelvetia fastigiata; Phaeophyta; propagule weight before and after emersion [e.g. Jenik and Lawson 1967, Schonbeck and Norton 1979, Garrity 19841) is unsuitable for microscopic stages of or- Considerable effort has been devoted to elucidat- ganisms because of the difficulties inherent in weigh- ing the physical and biological factors that control ing small organisms, especially in the field. Other techniques (Piche evaporimeter with 3-cm disks, Received 11 February 1993. Accepted 21 April 1993 Jenik and Lawson 1967; evaporation of water from Address for reprint requests. open vials, Garrity 1984) used to describe evapo-