British Phycological Journal

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The taxonomy of Prymnesium (Prymnesiophyceae) including a description of a new cosmopolitan species, P. Patellifera sp. nov., and further observations on P. parvum N. carter J.C. Green , D.J. Hibberd & R.N. Pienaar To cite this article: J.C. Green , D.J. Hibberd & R.N. Pienaar (1982) The taxonomy of Prymnesium (Prymnesiophyceae) including a description of a new cosmopolitan species, P. Patellifera sp. nov., and further observations on P. parvum N. carter, British Phycological Journal, 17:4, 363-382, DOI: 10.1080/00071618200650381 To link to this article: http://dx.doi.org/10.1080/00071618200650381

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Br. phycoL J. 17:363-382 1 December 1982

THE TAXONOMY OF PRYMNESIUM (PRYMNESIOPHYCEAE) INCLUDING A DESCRIPTION OF A NEW COSMOPOLITAN S P E C I E S , P. P A T E L L I F E R A SP. N O V . , A N D F U R T H E R O B S E R V A T I O N S O N P. P A R V U M N. C A R T E R By J. C. GREEN Marine Biological Association of the U.K., Citadel Hill, Plymouth, Devon PL1 2PB

D. J. HIBBERD Institute of Terrestrial Ecology, Culture Centre for Algae and Protozoa, 36 Storey's Way, Cambridge CB3 0DT a n d R. N . PIENAAR Dept. of Botany, University of Natal, Pietermaritzbarg 3200, Natal, South Africa Isolates of Pryrnnesium from the Pacific coast of North America, Europe and South Africa have been examined using both light and electron microscopy. Some have been identified as P. parvum N. Carter whilst others have been shown to represent a new species, P. patellifera sp. nov. Both species have two layers of body scales which in P. parvurn have a pattern of radial ridges on the proximal face but concentrically arranged fibrils on the distal face; in P. patellifera the scales of both layers have a pattern of radial ridges on both faces. In addition, in P. patellifera the scales of the outer layer have a tall upright rim, whilst in P. parvum the scales of the inner layer have a markedly inflexed rim, an observation not previously recorded. A systematic revision of Prymnesium is given and the taxonomic status of the genus Chrysochromulina in relation to Prymnesium is discussed. The genus Prymnesium C o n r a d has been o f interest for several decades because its representatives are widely d i s t r i b u t e d a n d have been identified as the causative agents o f extensive fish mortalities in b r a c k i s h w a t e r f r o m n o r t h e r n E u r o p e to the M i d d l e East, p a r t i c u l a r l y Israel. T h e toxic m e c h a n i s m a n d the n a t u r e o f the toxin have consequently been the subjects o f intense s t u d y (for a recent review, see Collins, 1978) a n d the fine structure o f the species concerned, identified as P. parvum N. Carter, has been e x a m i n e d in detail b y M a n t o n & L e e d a l e (1963) a n d M a n t o n (1964a,b, 1966, 1968). The latter studies have y i e l d e d m u c h inf o r m a t i o n o n scale p r o d u c t i o n , h a p t o n e m a structure a n d cell division a n d thus P. parvum is one o f the best k n o w n species in the P r y m n e s i o p h y c e a e . N o t w i t h s t a n d i n g this interest in P. parvum, the t a x o n o m y o f the genus in general h a d been a l m o s t c o m p l e t e l y neglected. A l t h o u g h only f o u r species have been described, their t a x o n o m y is confused owing to their similar a p p e a r a n c e 363 0007-1617/82/040363+20

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© 1982 British Phycological Society

364

J. C. GREEN, D. J. HIBBERD AND R. N. PIENAAR

i n the light microscope a n d the fact t h a t o n l y one h a d b e e n studied u s i n g electron microscopy. Recently we have been investigating a n u m b e r o f isolates of Prymnesium f r o m western N o r t h America, South Africa a n d Britain using b o t h light a n d electron microscopy. These studies resulted in the recognition o f a second scale type in P. parvum a n d the description o f P . patellifera sp. nov., also with two distinct scale types. These new o b s e r v a t i o n s m a d e necessary a n assessment o f the t a x o n o m y o f the genus as a whole a n d the basis of the separation f r o m it o f the genus Chrysochromulina Lackey with which it shares a n u m b e r o f morphological a n d structural features. MATERIALS AND METHODS ALGAL STRAINS USED The following isolates have been used in this investigation:

Prymnesium patellifera sp. nov. (described below) Strain JG/Sll : collected by F. J. R. Taylor from Sequin Bay, Washington, August, 1969. Isolated 1970 by R. Waters and sent as strain no. HD2. Strain JG/S12: collected by F. J. R. Taylor from English Bay, British Columbia, 1970. Isolated by R. Waters and sent as culture no. H47. Strain X28: Isolated by R. N. Pienaar from Old Town Lagoon, Friday Harbor, San Juan Island, U.S.A., October 1974. Strain CCAP 946]4: Isolated by D. J. Hibberd from The Fleet, Dorset, 1976, as DH189.

P. parvum N. Carter Strain JG/S10: collected from English Bay, British Columbia in 1969 by K. Barrett. Isolated 1970 by R. Waters and sent as culture no. HA2B. Strain Plymouth No. 94 (=CCAP 946/lb): isolated by R. W. Butcher from a sea-water sample from Northey Island, R. Blackwater, Essex in 1952. Found to be toxic to fish when tested by D. Ballantine in 1955 (unpublished result). Strain RNP/E2: isolated by R. N. Pienaar from the Swartkops Estuary, Port Elizabeth, South Africa in 1976. Strain CCAP 946/ld: isolated by K. Reich in 1953 from fish-ponds in Israel. Strain CCAP 946/3: isolated by P. A. Holdway in 1976 from Horsey Mere, Norfolk. MICROSCOPY For the electron microscopy of whole cells, the material was dried on to formvar or collodion coated grids and shadowed either with gold/palladium (Fig. 42) or chromium (Figs 18-20, 37-39). For sectioning, several different fixation procedures were used. (a) Cells were fixed for 1 h in 2.5 ~ glutaraldehyde by adding 1 ml of 25 ~o electron microscopy (EM) grade stock solution to 9'0 ml of culture. After centrifuging the material was rinsed three times in medium followed by 2 ~ OsO~ in medium for 2 h. The material was then washed in medium, dehydrated through a graded ethanol series and embedded in Spurr's low viscosity resin (Figs 21, 27). (b) Cells were concentrated by centrifuging and fixed in 5 ~ glutaraldehyde in 0-1 M sodium cacodylate, pH 7-0, with 0.25 Msucrose. After rinsing in buffer with and without added sucrose, they were post-fixed for 1 h in 2 ~o OsO4 in 0.1 M sodium cacodylate, pH 7-0, washed in buffer and dehydrated through a graded series of alcohols. The material was embedded in Epon (Figs 22, 25, 26). (c) The cells were fixed in 2 ~ OsO4 in 0"1 M sodium cacodylate buffer pH 7"0, for 20 min followed by washing in buffer, dehydration in a graded ethanol series and embedding in Epon (Figs 23, 24, 40, 41). Sections were cut using LKB and Reichert ultramicrotomes, double-stained with uranyl acetate and lead citrate and examined using Philips 300 (Plymouth), AEI 801 (Cambridge) and Jeol 100CX (Cambridge and Pietermaritzburg) electron microscopes.

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Living cells were photographed at Plymouth using a Reichert Zetopan microscope fitted with bright field, Anoptral contrast and interference contrast optical systems and electronic flash. Cultures were maintained in a variety of media including Erd-Schreiber, a 1 : 1 mixture of Erd-Schreiber and Provasoli's ASP2 (Provasoli, McLaughlin & Droop, 1957) and Provasoli's sea-water enrichment medium (Provasoli, 1968). In order to obtain cysts, material was grown in media of salinities 10, 20, 35, 50 and 70%0. The high salinities were obtained by evaporating sea-water (35%0) and the low salinities by the dilution of sea-water using double glass-distilled water before the addition of the enrichment.

PRYMNESIUM PATELLIFERA SP. N O V . Synonym:

Prymnesium aft. saltans sensu Pienaar, R. N. & Kleizen, H. G. i n Proceedings of the Electron Microscopy Society of S. Africa, 6 : 5 5 (1976).

DIAGNOSIS Cellulae natantes 6-12 tzm longae, 3.5-8.0 tzm latae, plerumque subsphericae vel cylindricae, taro leviter flexae vel pyriformes, postice rotundatae vel raro contractae, antice obliquetruncatae. Flagella duo, 10-14.5/zm longa, aequalia vel subaequalia, heterodynamica, ad apices attenuata; haptonema 3.0-5.0 t~m longum, flexile sed spiram non formans; flagella atque haptonema e fovea superficie truncatae subantice exorientia. Cellulae placide natantes, circum axem longitudinalem volventes, polo antico ira helicem delineante; flagella polum posticum versus deflexa; cellulae rare recessi natantes. Squamae corporis 0.364).37 tzm x 0-250'27/~m magnae, duas formas in duobus stratis praebentes; squamae strati interioris marginibus angustis infiexis, umbonibus centralibus, fibrillis radiantibus in superficiebus utrisque; squamae strati exterioris similes sed marginibus verticalibus, comparate altis. Chloroplasti duo, laterales atque parietales, flavo-virentes vel olivacei, lobati vel dissecti, omnes pyrenoide immersa. Nucleus inter chloroplastos centralis; corpusculum Golgianum parabasale. Materia penaria in vacuola postica condita, probaliter "chrysolaminarin" ("leucosin"). Corpuscula mucigera peripherica praesentia atque plerumque aliquot globuli substantiae lipoidis suppetentes. Cystae ovoideae, 9-3-10.8/~m x 6.0-6.4/~m, poro simplici subantico, 2.75-3.00/zm diametro; paries e stratis squamarum compositus cum materia silicea in superficiebus extrorsis squamarum extimarum deposita. Holotypus: Figs 18-21.

i

Swimming cells 6-12 ~m long x 3.5-8-0 ~m broad; mostly sub-spherical to elongate with more or less parallel sides and rounded posterior end; sometimes bent or pyriform or with a tapered posterior end; anterior end obliquely truncate. Two equal or sub-equal distally tapered heterodynamic flagella, 10-14"5/~m in length and a short, 3"0-5-0/~m, flexible noncoiling haptonema arising sub-apically from a groove or depression in the truncate face. Swimming motion smooth, the cell rotating about its longitudinal axis and the anterior (flagellar) pole describing a helix about the path of travel; flagella directed posteriorly. Body scales 0-36--0-37tzm x 0.25-0.27/zm, of two types in two layers: scales of the inner layer with a narrow inflexed rim, a central thickening on the distal face, and a radial fibrillar pattern on both faces; scales of the outer layer similar but with relatively tall upright rims. Chloroplasts two, lateral and parietal, yellow-green to olive, lobed or dissected, each with an immersed pyrenoid. Nucleus central between the chloroplasts, Golgi body parabasal. Reserve metabolite probably chrysolaminarin 0eucosin) accumulated in a posterior vacuole. Peripheral muciferous bodies and usually several lipoidol globules present. Cysts ovoid, 9.3-10.8/~m x6-0-6.4/zm, with a simple sub-anterior pore, the latter 2.753.00/~m in diameter; wall composed of layers of scales with electron-dense siliceous material deposited on the distal surfaces of the outermost scales. Holotype: Figs 18-21. LtGHT MICROSCOVV (Figs 1-17) The cells of P. patellifera are 6-12/~m long a n d 3.5-8-0/~m b r o a d a l t h o u g h within this range their shape can vary between m a r k e d l y elongate to a l m o s t spherical (Figs 1-12). Elongate cells were seen m o s t frequently, these being

366

J. C. GREEN, D. J. HIBBERD A N D R. N. PIENAAR

iii

J FIGS 1-12. Light microscopy of Prymnesium patellifera; live cells showing variation in size and shape. Note the two flagella and the short haptonema arising sub-anteriorly. Some cells contain lipoidal globules (1) and chrysolaminarin (ch). Figs 1-5. Strain X28. FIGS 6-9. Strain CCAP 946/4. FIGS 10-12. Strain S12. Electronic flash, interference contrast. All × 2,000.

Taxonomy of Pryrnnesiurn

367

pyriform, ovoid or with more or less parallel sides (the whole cell sometimes slightly bent) or tapered to an acute posterior end. The anterior end is always obliquely truncate with the appendages arising sub-terminally from a depression or pit (Figs 4, 7, 8, 10, etc.). The cells are not compressed, or perhaps only slightly so, with a more or less circular profile in polar view. They do not show noticeable metaboly although in older cultures (approximately 1 month or more) the cells become more variable in shape, some appearing very irregular in form. The appendages include two equal or sub-equal flagella, 10.0-14.5 tzm in length (i.e. approximately 1.5xcell length), each with a terminal hair-point which is easily detected in flattened cells by light microscopy (Figs 16, 17) and a short flexible haptonema (3-5 tzm) which has never been observed to coil. When the cell is swimming the flagella beat rapidly so that it is difficult to determine their movements precisely, but it appears that the more posteriorly inserted flagellum is directed posteriorly often close to the cell-body and beats with an undulating motion, whilst the other flagellum passes over the most anterior part of the cell, backwards along the side opposite to that on which the appendages are inserted and beats with a violent flicking movement. When the cell has come to rest both flagella may beat in a similar way, though here also the anteriorly inserted flagellum may beat more rapidly. The posterior flagellum may eventually become almost stationary and be held against the cell either anterio-posteriorly or obliquely so that it is partly wrapped around the cell. During normal swimming the haptonema is directed forwards and the cell proceeds smoothly, spinning about its longitudinal axis but with the anterior end describing a helix about the path of travel. Occasionally cells were seen swimming with the posterior (non-flagellar) pole directed forwards and in such cells the flagella adopted the trailing position shown in Fig. 6. The erratic movements described for P. saltans by Massart (1920) and Conrad (1926, 1941) were never seen by us. After a time the cells often come to rest attached to the surface of either the slide or coverslip by the tip of the haptonema. They may then revolve about the point of attachment or simply vibrate rapidly. As the cell begins to compress and deform under the coverslip, movements gradually become slower. The most conspicuous cell contents are the two large yellow-green to olive chloroplasts which are situated laterally and parietally. They are often deeply lobed or dissected (Figs 13-15) and each contains an immersed pyrenoid which cannot easily be seen except in flattening cells under phase-contrast illumination (Fig. 16). The nucleus lies between the chloroplasts (see below), but again it is identifiable with certainty in the light microscope only in compressed cells. There are always a number of large round bodies within the ceil, often situated centrally, which from their refractivity and staining reaction with OsO~, appear to have a high lipid content. A large number of smaller somewhat refractile bodies may often, though not always, be detected at the periphery (Figs 15, 17). These may be the structures identified in other strains of Prymnesium by previous workers as muciferous bodies. The posterior part of the cell is occupied by a large vesicle containing the reserve metabolite, probably chrysolaminarin (leucosin). Although this vesicle often causes the posterior part of the cell to bulge, it has never been seen to be extruded as reported by Carter (1937) in P. parvum. The posterior part of the

368

J. c. GREEN, D. J. HIBBERD AND R. N. PIENAAR

Fits 13-17. Light microscopy o f P . patellifera; compressed live cells to show the organelles. Note the lobed chloraplasts, muciferous bodies (rob), immersed pyrenoids (py), the position of the nucleus (n) and the hair-tips of the flagella. Figs 13-15. Strain X28, interference contrast. Fig. 16. Strain Sll, Anoptral contrast. Fig. 17. Strain S12, Anoptral contrast. All x 2,000. cell m a y sometimes also contain a number of small refractile bodies, of unknown nature, which undergo violent Brownian movement. They are usually found in cells which are beginning to flatten under the cover-slip and they may, therefore, be a pathological response to this stress. A m o e b o i d cells have not been seen in cultures grown under our conditions. Cysts have been observed by one of us (RNP) in strains X28, Sl 1 and $12 grown in Provasoli ES medium with a salinity of 35~oo and strain X28 also produced cysts in medium of 20~o salinity. Observed under the light microscope, the cysts are ovoid, 9.3-10.8/zm x 6.0-6.4/Lm, with a simple sub-anterior pore (2-75-3.00/~m diameter) and are similar to those previously described for P. sultans (Conrad, 1941) and P. parvum and P. minutum (Carter, 1937). ELECTRON-MICROSCOPY(Figs 18-27) Whole-mounted shadow-cast cells clearly show the two sub-equal flagella with their long attenuated tips and the short haptonema (Fig. 18). However, the cell shape is often lost in such dried preparations. At higher magnifications it can be seen that the cell is surrounded by scales shed from the cell-body and these are of the type commonly found in other members of the Prymnesiophyceae with a pattern of radial ridges on both the distal and proximal sides. In addition, each scale has a central protuberance on the distal side which may be a single more or less rounded swelling, an elongate hump, a double hump, or two distinct and separate protuberances (Figs 19, 20; see also Pienaar & Kleizen,

Taxonomy of Prymnesium

FIGS 18-21. Electron microscopy of P. patellifera. Fig. 18. Dried shadow-cast cell, strain X28. x 5,000. Figs 19, 20. Shadow-cast body scales, some displaying the proximal face with radial fibrils extending to the extreme periphery and others showing the distal face with the central thickening. Some scales (a) have small rims whilst others (b) have collapsed raised rims. Fig. 19. Strain S11. Fig. 20. Strain CCAP 946/4. Both x 70,000. Fig. 21. Section through the periphery of a cell showing the scale layers, the tall-rimmed scales outermost. For further details see text, p. 370. Strain X28. x 66,000.

369

370

J. C. GREEN, D. J. HIBBERD AND R. N. PIENAAR

1976, fig. 4). Many of the scales have tall upright rims which collapse in direct preparations (Fig. 19) and sections show that these form an outer layer on the cell, the scales of the proximal (under) layer having smaller, inflexed rims (Figs 21, 22, 27). Beneath the scales the plasmalemma carries a layer of lightly staining material (Figs 21-23), probably a glycocalyx. Thick sections show that the tallrimmed scales of the distal layer have, in the angle between the rim and the distal

FIGS 22-24. Electron microscopy of P. patellifera. Fig. 22. Thick section through the scale layers showing the disposition of the scale types. Strain CCAP 946/4. x 100,000. Fig. 23. Vertical section through a single scale of the distal layer showing especially the superficial material (arrow). Strain CCAP 946]4. x 100,000. Fig. 24. Glancing section through the scale layers, the superficial material of the scales of the distal layer arrowed. Strain CCAP 946/4. x 100,000.

Taxonomy of Prymnesium

371

face of the plate, material which appears amorphous after fixation (Figs 21-23). Similar material has been reported by Pienaar (1978) in another Prymnesiumlike organism but in this case it is organized into distinct, radially arranged flanges. The tall-rimmed scales also have coarse material distributed over the distal face often towards the periphery of the scale (Figs 23, 24, arrows). There was no indication of any fibrils arranged concentrically on the distal face of either scale type. The internal structure of the cell resembles that of P. parvum as described by Manton & Leedale (1963) and Manton (1964a,b, 1966). Longitudinal sections (Fig. 25) clearly show the sub-apical insertion of the two flagella with the hapto-

FIG. 25. Electron microscopy of P. patellifera. Median longitudinal section showing the disposition of the organelles and particularly the sub-terminal insertion of the appendages. Strain CCAP 946/4. x 15,000.

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Taxonomy of Prymnesium

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nema between them. Transverse sections of the latter show that it has seven microtubules in the free part. There are generally several chloroplast profiles as would be expected from the extreme lobing seen with the light microscope and the nucleus is situated more or less centrally in the cell with the usual confluence of the outer membrane of the nuclear envelope with the outer membrane of the chloroplast endoplasmic reticulum (ER). Each chloroplast has a more or less immersed pyrenoid (Fig. 26) penetrated or traversed by a single two-thylakoid lamella. The Golgi body is situated in the anterior part of the cell between the nucleus and the flagellar basal bodies and the central parts of several adjacent cisternae are often dilated in the way now known to be characteristic of the Prymnesiophyceae. The Golgi vesicles may also contain nascent scales. The cell always contains many vacuoles, often concentrated in the posterior part which may be connected with the formation of reserve metabolite. It may be noted here, as recorded by Manton (1964a, 1968) in P. parvum, that there is a complicated system of microtubular flagellar roots; these will be the subject of a further paper. A cross-banded ftagellar root is lacking. Electron microscopy of the cysts has shown that they possess a wall composed of layers of scales [cf. benthic cells of Cricosphaera carterae (Braarud et Fagerl.) Braarud; Paddock, 1968] with electron-dense siliceous material (detected by Kevex analysis) deposited on the distal face of the outermost scales (Pienaar, 1980, 1981). Full details of the structure and chemical composition will be published elsewhere (Pienaar, in preparation). F U R T H E R OBSERVATIONS ON P R Y M N E S I U M P A R V U M N. C A R T E R Among the isolates investigated were several which differed in their scale structure from those now named as P. patellifera. Some of the scales were identical to those described by Manton & Leedale (1963) for P. parvum, but a second scale type with a broad inftexed rim was also present, apparently indicating a second new species. However, an examination of some of the micrographs in other publications, e.g. fig. 8 in Manton (1964a) and pl. 140 in Manton (1964b), showed fragments of scales possibly of this second type. Through the courtesy of Professor Manton we were able to examine all the micrographs relating to her work on P. parvum (Manton, 1964a,b,c, 1966, 1968; Manton & Leedale, 1963), all of which was carried out using a strain sent from Israel in 1961 by E. I. Friedmann but which is no longer available in culture. (This strain also existed in the Cambridge Culture Collection as Nos 946/la and 946/lc). In every case it was confirmed that scales with a broad strongly inftexed rim were present in addition to those already known (Figs 37-42), making it necessary to redescribe P. parvum to include the new observations. In an effcrt to determine whether it was possible to distinguish between P. patellifera, P. parvum and the type species, P. saltans Conrad (see p. 377) at the light microscope level, detailed observations were made on all available strains of the two former species. It was found that there was considerable overlap between P. parvum and P. patellifera and a selection of micrographs representing some of the forms of P. parvum encountered are shown in Figs 28-36 (of. Figs 1-12).

374

J. C. GREEN, D. J. HIBBERD AND R. N. PIENAAR

FIGS 28-36. Light microscopy of P. parvum; live cells showing the range of variation in size and shape: lipoidal globules (1), chrysolaminarin (ch), muciferous bodies (mb) and one cell (Fig. 33) with a basal "rust-red body" (rb) of unknown nature. Figs 28-30. Strain Plymouth No. 94. Figs 31-35. Strain S10. Figs 34-36. Strain CCAP 946/ld. Electronic flash, bright field (Fig. 28) and interference contrast (Figs 29-36). All × 2,000. Cysts of P. parvum have b e e n f o u n d i n cultures of isolates S10 a n d E2 g r o w n i n salinities o f 20 a n d 35%o t h o u g h they were more a b u n d a n t at the higher salinity. T h e y were identical in form a n d size to those of P. patellifera. REVISED DESCRIPTIONOF P. PAR VUM N. CARTER Swimming cells 8"0-11"0(-15"0) ~m long, 4-0-6.0(-10.0) ~m broad: sub-spherical to elongate, not or only slightly compressed; posterior end rounded or tapered; anterior end obliquely truncate. Two equal or sub-equal typically heterodynamic flagella, 12'0-15.0(-20)t~m long, and a short, 3.0-5.0 ~m, flexible non-coiling haptonema arising sub-apically from a groove or depression in the truncate face. Swimming motion smoothly forward, the cell spinning about its longitudinal axis and the anterior (flagellar) pole describing a helical path around the path of travel. Body scales of two types in two layers: scales of the outer layer 0.30-0.43 ~m × 0-23-0.30 vm with narrow inflexed rims, a pattern of radiating fibrils on the proximal face and concentrically arranged fibrils, sometimes occupying only the central area, on the distal face; scales of the

Taxonomy of Prymnesium

375

inner layer 0.29-0.36 tzm x 0-26--0-32tzm with surface patterning similar to above but with a wide rim strongly inflexed over the distal face. Chloroplasts two, lateral and parietal, yellow-green to olive, lobed or dissected each with an immersed pyrenoid. Nucleus central between the chloroplasts, Golgi body parabasal. A single contractile vacuole sometimes present. Reserve metabolite probably chrysolaminarin (leucosin) accumulated in a posterior vacuole. Peripheral muciferous bodies and usually several lipoidal globules present. Cysts ovoid, 9.3 tzm x 6-0-6.2 t~m, with a simple sub-anterior pore, the latter 2-75-3.00 p.m in diameter; wall composed of layers of scales with electron-dense siliceous material deposited on the distal face of the scales. The flagellar action is usually h e t e r o d y n a m i c in actively s w i m m i n g cells with the anteriorly inserted flagellum beating with a rapid, almost "flicking" movement. However, in cells at rest, the flagellar action m a y be h o m o d y n a m i c , the

FIGS 37-39. Electron microscopy of P. parvum. Fig. 37. Shadow-cast cell. x 5,000. Figs 38, 39. Body-scales from a shadow-cast preparation showing the pattern of radial fibrils on the proximal face and concentric fibrils on the distal face. Fig. 39. Scales with broad inflexed rims (arrows). Strain CCAP 946/3. Both x 70,000.

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Taxonomy of Prymnesium

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beat being slower and more regular with the amplitude of the waves increasing towards the distal end of the flagellum. The pyrenoid is always difficult to see with the light microscope and is usually only detectable in flattened cells. Electron microscopy of sections shows that it is traversed or penetrated by a single lamella consisting of a pair of thylakoids. In cells from older cultures lipoidal globules may accumulate obscuring the other organelles. In some cells granular accretions of carotenoid material can be observed as dark rust-red bodies (Fig. 33). DISCUSSION TAXONOMYOF PR YMNESIUM Prymaesium Massart ex Conrad in Archivfi~r Protistenkunde, 56:219 (1926) Syn. Prymnesium Massart in Annales de la Socidtd beige de Microscopie, 27: 79 (1900); in Bulletin de l'Acaddmie royale de Belgique, Classe des Sciences, Ser. 5, 6:133 (1920) nom. nud. P. saltans Massart ex Conrad in Archivfiir Protistenkunde, 56:219 (1926) Syn. P. saltans Massart in Annales de la Socidtd beige de Microscopie, 2 7 : 7 9 (1900); in Bulletin de l'Acad~mie royale de Belgique, Classe des Sciences, Set. 5, 6:133 (1920) nora. nud. The name of the type species of the genus, P. saltans, first appears as a nomen nudum in a list of flagellates found by Massart (1900), in a brackish tidal ditch in Belgium. Massart later described the swimming movements of the cells in some detail and provided labelled diagrams of "Prymnesium saltans, n.g., n.s." (Massart, 1920, fig. 24) but it was not until 1926 that a description of the species was given by Conrad. This description was based solely on Massart's observations, Conrad not having seen P. saltanshimself at that time. Although Massart is generally accepted as the authority for the genus Prymnesium and the species P. saltans, the names cannot be considered as validly published by Massart in 1920 according to Art. 42 of the International Code of Botanical Nomenclature (I.C.B.N.; Steflev et al., 1978) since he provided only illustrations and no description; prior to 1 January, 1908, Massart's figures would have validated the names (I.C.B.N. Arts 42, 44). Thus, Conrad (1926) must be recognized as the authority for both the genus Prymnesium and for P. saltans, having validated them by providing a combined generic and specific description (I.C.B.N. Art. 42). Conrad (1941) eventually gave a more complete description of P. saltans based on material he had collected in the region of Lilloo and this appears to be the last published record in which the species has been identified with certainty. According to Massart (1920) and Conrad (1941) the cells of P. sultans vary in shape from sub-ovoid or ellipsoidal to sub-cylindrical and are not compressed. The recorded size range is 11-18/~m × 5.0-7.5/~m. The cells bear two equal flagella approximately twice the cell length and a haptonema ("un troisi~me fouet, extrSmement court"; Conrad, 1941), 2-5/~m long according to Massart and 5/~m long according to Conrad. The cells contain two parietal yellow-gold chloroplasts, a single posterior nucleus, a single anterior contractile vacuole

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and a mass ofleucosin (chrysolaminarin) posteriorly. Other droplets are scattered through the cytoplasm and many muciferous bodies occur at the periphery. Conrad also describes rhizopodial, amoeboid and palmelloid stages and ovoid cysts, 9-16/~m x 8.0-14/~m with a short neck, a thick rugose brownish wall and a plug in the shape of an inverted saucer. The unusual pattern of swimming of P. saltans is stressed by both Massart and Conrad. The cells do not swim in a smooth path but after swimming forwards for several cell lengths skip backwards before swimming forwards once again. Repetition of these movements produces the characteristically jerky pattern of swimming described by Massart as a "suite de gambades". One further apparently characteristic feature connected with this is the "rippling" beat of the flagella, not specifically mentioned but clearly illustrated by both Massart and Conrad. We have assembled and made a detailed comparison of all the available data for P. saltans with those for the various species described subsequently and from this have concluded that at the level of the light microscope there are no significant morphological differences between P. saltans, P. parvum, P. minutum and the species newly described here. Although P. saltans is larger than the remaining species it overlaps with all except P. minutum at the lower end of its size range. Its peculiar manner of swimming thus represents the only clear distinguishing feature. Since this character has not been described since Conrad's paper of 1941, however, it is now not always taken into consideration. Heynig (1978) for example, refers to P. saltans forms which swim smoothly. Some material did show jumping movements, however, and this, together with the fact that cells are illustrated with the forwardly extending rippling flagellar beat otherwise seen only in the drawings ofP. saltans by Conrad (1941) and Massart (1920), suggests that Heynig may indeed have had authentic material ofP. saltans. The taxonomic enigma of this species can clearly only be resolved by EM examination of a Prymnesium which swims in the way described for P. saltans. In the meantime we prefer to continue to recognize P. saltans as a separate species distinguished by its swimming pattern. P. parvum N. Carter, Archivfiir Protistenkunde, 90:40 (1937) Carter distinguished P. parvum from P. saltans by its slightly smaller size, relatively long "small flagellum" (haptonema) the absence of a contractile vacuole and the swimming action, described as straight ahead and lacking the jerky movements of P. saltans. The material described by Conrad (1941) as P. saltans was also distinguished by him from P. parvum mainly by its different pattern of swimming. The first electron micrographs of Prymnesium were published by Manton & Leedale (1963) using material from Israel identified as P. parvum. The information obtained in this investigation provided a useful identification marker since it was shown that the cells were covered by elliptical scales, each 0.3 × 0.25 ~m (given as 1-0 x 0.75/zm in the text) with a narrow raised rim, a pattern of radial fibres on the proximal face and concentrically arranged fibrils on the distal face. Our material also has scales with this structure but, in addition, an under-layer of body-scales has been demonstrated, each with a strongly inflexed rim (see p. 373). Although it can never be ascertained whether the species described by

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Carter had scales resembling those described by Manton & Leedale (1963) and ourselves, it cannot be separated on morphological characters visible in the light microscope and must now be considered to be characterized by its scale features. P. minutum N. Carter, Archivfiir Protistenkunde, 90:43 (1937) Carter distinguished this species from P. parvum only by its smaller size (6.0-8-5/~m x 3.5 tzm) and the colour of its chloroplasts which were greenishyellow rather than brownish-yellow as in P. parvum. The cysts were also smaller than those ofP. parvum with an almost smooth wall, lacking the fine dimples of P. parvum. If, as is probable, P. parvum is as variable in size as P. patellifera, then P. minutum may be merely a small form of P. parvum since it otherwise resembled this species and always occurred with it (Carter, 1937). Once again, however, this can be determined only by EM examination of material closely resembling P. minutum. P. patellifera Green, Hibberd et Pienaar (1982), herein This species, newly described here, has been distinguished from P. saltans mainly because it does not swim in the way considered to be so characteristic of this species and because it is smaller than P. saltans except at the maximum of its size range in culture. As already mentioned the significance of these differences can be determined only by further study of material, preferably in culture, identical with P. saltans. It is clearly distinguished from P. parvum in having a radial fibrillar pattern on both surfaces of all its scales and, in addition, the scales of the outer layer possess very obvious vertical rims and the distal face shows a pronounced thickening in the centre. An examination of the two micrographs published by Valkanov (1964) suggests that the organism responsible for a sudden mass fish mortality in the Varna Lakes, Bulgaria in 1959 may not have been P. parvum, as was reported at the time, but P. patellifera since the single scale shown in fig. 5 of Valkanov's paper appears to have a distinct raised rim and a central protuberance which Valkanov himself noted; these features are also detectable on close examination in the scales of his fig. 4. P. czosnowskii Starmach, Chrysophyta I. Chrysophyceae Ztotowiciowie. In Flora stodokowodnapolski (Starmach, K., Ed.), Tom 5, 155. Warszawa (1968) Syn. P. minutum Czosnowski, Poznahskie Towarzystwo Przyjaci6l Nauk, Prace Komisji Biol. Ogicznej, 11:368 (1948). non P. minutum Carter, Archiv fiir Protistenkunde, 90:43 (1937). Czosnowski distinguished this species by the shape of the cells which are either globular or, more commonly, wider than long. They are also very small, 6-5/~m long x 7-5/zm wide, and are therefore similar in size to P. minutum Carter. Starmach (1968) provided a new name for this species since P. minutum Czosnowski, 1948 is a later homonym ofP. minutum Carter, 1937. Until further information becomes available it is impossible to say whether this is a distinct species or simply a small form of another.

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UNIDENTIFIED SPECIES

Liebert & Deems (1920) published a description of an organism responsible for a fish-kill in the Netherlands which they were unable to identify but which from the description and drawings is certainly a species of Prymnesium. From the information provided, however, it is impossible to decide whether it corresponds more closely to P. saltans or P. parvum, or indeed to P. patellifera. It is significant, in view of the uncertainty still surrounding the recognition of P. saltans as a separate species, that Conrad (1941) identified this species as P. saltans whereas Valkanov (1964) identified it as P. parvum. This discussion emphasizes the need for EM examination of more material of Prymnesium since it is clearly only by a combination of EM methods with careful light microscope observations that the taxonomic problems in the genus can be solved. Cultures provide the best material for electron microscopy and are not difficult to establish since species of Prymnesium are easy to grow. It should be stressed, however, that shadowcast preparations may be made from natural populations of plankton containing Prymnesium and the wild material described by Heynig (1978), for example, would have been ideal for this purpose. Material may also be fixed for sectioning without recourse to cultures when a species occurs in bloom proportions. Finally, it should also be mentioned that we have found that good shadowcast preparations of scales may be made from cells sedimented with Lugol's iodine. Material may thus be preserved by this method until it can be prepared and examined in an EM laboratory. DISTINCTIONBETWEENPR YMNESIUM CONRAD, 1926 ANt) CHR YSOCHROMULINA LACKEY, 1930 The discovery that two species of Prymnesium produce more than one type of scale re-opens the question of the taxonomic distinction between Prymnesium and Chrysochromulina, as indicated by Pienaar & Norris (1979) after the preliminary report by Pienaar & Kleizen (1976) on the species we have named P. patellifera. Until now, Prymnesium has been regarded as having a short noncoiling haptonema and only one type of scale, this concept of the genus being based solely on the description of P. parvum by Manton & Leedale (1963). Chrysochromulina species usually have two or more very elaborate types of scale and a long, often coiling haptonema. However, the type species of Chrysochromulina, C. parva Lackey, is described as having only one type of body-scale (Parke, L u n d & Manton, 1962) while on the other hand, some species, e.g. C. spinifera (Fournier) Pienaar et Norris and C. parkeae Green et Leadbeater, have short non-coiling haptonemata. Indeed, C. spinifera was originally described by Fournier (1971) under a new genus, Chrysocampanula Fournier, but was transferred to Chrysochromulina by Pienaar & Norris (1979) on the basis of an ultrastructural study. In our opinion, the possession of either a very long coiling haptonema or of highly elaborated non-mineralized scales is sufficient to distinguish Chrysochromulina from Prymnesium, species of the latter genus always having simple plate scales and a short non-coiling haptonema. In addition, the normally homodynamic flagellar action of Chrysochromulina spp. with the appendages borne posteriorly during rapid swimming contrasts with the

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totally different swimming action in Prymnesium where they are borne anteriorly and beat heterodynamically. The appendages of species of Chrysochromulina are also inserted in a polar position with no obvious depression, in contrast to Prymnesiurn in which they arise sub-apically from a groove or depression on the obliquely truncate anterior end. Furthermore, examination of the published micrographs of scales of species of Chrysochromulina suggests that they are all more robustly constructed than those of Prymnesium, being formed from coarser fibrils. This taxonomic decision, based on morphological differences, is supported by other, less easily defined differences between the two genera. Firstly, the general robustness and longevity ofPrymnesium spp. both in cultures and during observation in the light microscope contrasts markedly with the delicacy of all Chrysochromulina species. This is probably connected with the fact that Chrysochromulina species are mostly oceanic or, in the case of the few freshwater forms, lacustrine, whereas Prymnesium spp. are apparently found only in brackish waters, often limited in area and of very variable salinity, and never in the euplankton of the sea or lakes. Finally, it should also be mentioned that whereas cysts appear to be the alternative stage in the life history of Prymnesium, they have never been described for any species of Chrysochromulina. ACKNOWLEDGEMENTS We are particularly grateful to Professor I. Manton, F.R.S., for access to her unpublished data and for the micrograph reproduced here as Fig. 42. We also wish to thank D r F. J. R. Taylor and Mrs R. Waters for three of the strains used in this investigation and Miss P. A. Course for invaluable technical assistance. D r F. K. Kupicha kindly translated the diagnosis into Latin and criticized the manuscript. The thanks of R.N.P. are also due to the Directors of the Friday Harbor Laboratories and the Marine Biological Association's Laboratory, Plymouth, for accommodation and facilities during 1974-75, the Council for Scientific and Industrial Research (South Africa) for financial support and D r R. E. Norris for valuable discussions and comments. REFERENCES

CARTER,N., 1937. New or interesting algae from brackish water. Arch. Protistenk., 90: 1-68. COLLINS, M., 1978. Algal toxins. Microbiol. Rev., 42: 725-746. Cor~,Ao, W., 1926. Recherches sur les Flagellates de nos eaux saumatres. 2 e Partie: Chrysomonadines. Arch. Protistenk., 56: 167-231. CONRAD, W., 1941. Notes protistologiques. XXI. Sur les Chrysomonadines /l trois fouets. Aperqu synoptique. Bull. Mus. r. Hist. nat. Belg., 17: 1-16. Czosr~owsKi, J., 1948. Materiali do flory wiciowcow Polski. Pr. Kom. mat.-przyr., Poznaft, 11 : 363-402. FOtmNIER, R. O., 1971. Chrysocampanula spinifera gen. et sp. nov., a new marine haptophyte from the Bay of Chaleur, Quebec. Phycologia, 10" 89-92. HEYN~G, H., 1978. Prymnesiurn saltans Massart (Chrysophyceae) in Gewassern des Bezirks Halle (DDR). Arch. Protistenk., 120: 222-228. LIEBERT, F. & DEERNS, W. M., 1920. Onderzoek naar de oorzaak van een vischsterfte in den polder Workumer-Nieuwland, nabij Workum. Verh. Rapp. Rijksinst. VisschOnderz., 1 : 81-93. MAtCrON, I., 1964a. Further observations on the fine structure of the haptonema in Prymnesium parvum. Arch. MikrobioL, 49: 315-330. MANTON, I., 1964b. Observations with the electron microscope on the division cycle in the flagellate Pryrnnesium parvum Carter. Jl R. microsc. Soc., 83: 317-325. MANTON, I., 1964C. The possible significance of some details of flagellar bases in plants. Jl R. microsc. Soc., 82: 279-285. MArCrON, I., 1966. Observations on scale production in Prymnesium parvum. J. Cell ScL, 1" 375-380.

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MANTON,l., 1968. Further observations on the microanatomy of the haptonema in Chrysochromulina chiton and Prymnesium parvum. Protoplasma, 66: 35-53. MANTON, I. & LEEDALE,G. F., 1963. Observations on the fine structure of Prymnesium parvum Carter. Arch. Mikrobiol., 45: 285-303. MASSART, J., 1900. Liste des flagellates observ6s aux environs de Coxyde et de Nieuport. Ann. Soc. beige Microsc., 27: 75-83. MASSART,J., 1920. Recherches sur les organismes inf6rieurs. VIII. Sur la motilit6 des flagellates. Bull. Acad. r. Belg. CL Sci. Sdr. 5, 6: 116-141. PADDOCK,T. B. B., 1968. A possible aid to survival of the marine coccolithophorid Cricosphaera and similar organisms. Br. phycoL Bull., 3: 519-523. PAgKE, M., Luha), J. W. G. & MANTON,I., 1962. Observations on the biology and fine structure of the type species of Chrysochromulina (C. parva Lackey) in the English Lake District. Arch. Mikrobiol., 42: 333-352. PIENAAR,R. N., 1978. Preliminary observations on the microanatomy of an unusual member of the Prymnesiophyceae. Proc. Electron Microscopy Soc. S. Africa, 8: 77-78. PmNA~at, R. N., 1980. Observations on the structure and composition of the cyst of Prymnesium (Prymnesiophyceae). Proc. Electron Microscopy Soc. S. Africa, 10: 73-74. PmNAAR, R. N., 1981. Ultrastructural studies on the cysts of Prymnesium (Prymnesiophyceae). Phycologia, 20: 112. P~NAA~, R. N. & KLVJZEN,H. G., 1976. A comparative study of two species of the toxic alga Prymnesium. Proc. Electron Microscopy Soc. S. Africa, 6: 55-56. PIENAAR,R. N. & NORRIS,R. E., 1979. The ultrastructure of the flagellate Chrysochromulina spinifera (Fournier) comb. nov. (Prymnesiophyceae) with special reference to scale production. Phycologia, 18: 99-108. PROVASOU, L., 1968. Media and prospects for cultivation of marine algae. In Culture and Collection of Algae. (Watabe, A. & Hattori, A., Eds.), 63-75. Japanese Society of Plant Physiology. PROVASOLI, L., McLAUGnLIN, J. J. A. & DROOP, M. R., 1957. The development of artificial media for marine algae. Arch. MikrobioL, 25: 392--428. STAI~LEV,F. A. et al. (Eds), 1978. International Code of Botanical Nomenclature Adopted by the Twelfth International Botanical Congress, Leningrad, July 1975. Bohn, Scheltema & Holkema, Utrecht. STARMACU, K., 1968. Chrysophyta I. Chrysophyceae Zlotowiciowie. In Flora slodokowodna polski (Starmach, K., Ed.), Tom 5. Warszawa. VALKANOV, A., 1964. Untersuchungen fiber Prymnesium parvum Carter und seine toxische Einwirkung auf die Wasserorganismen. Kieler Meeresforsch., 20: 65-81.

(Accepted 15 March 1982)