Toxic Dinoflagellates - American Chemical Society

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T o x i c D i n o f l a g e l l a t e s : T a x o n o m i c and B i o g e o g r a p h i c A s p e c t s w i t h E m p h a s i s on

Protogonyaulax

F. J. R. TAYLOR

Downloaded by CORNELL UNIV on August 1, 2016 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch008

Departments of Oceanography and Botany, University of British Columbia, Vancouver, B.C., V6T 1W5 Canada This paper provides a brief summary of taxonomic developments since 1979 and also some features of the global distribution of toxic species. The continued recognition of Protogonyaulax as distinct from Gonyaulax , with some species referred to Gessnerium, seems most reasonable at present. The growing difficulty of distinguishing between P. catenella from P. tamarensis in areas where they co-exist is discussed, including electrophoretic data, principally from British Columbia isolates. In B.C. they are usually separated distributionally, but intermediate forms occur in intermediate localities. In Japan they may be temporally separated.The allocation of Gymnodinium breve to Ptychodiscus depends on the presence of a pellicle. Although not seen with TEM i t can be seen with light microscopy. Geographic distribution is closely linked to taxonomy for, although some toxin producers appear to be endemic in a restricted sense, closely similar forms occur elsewhere (e.g. p. brevis) or the same species may be known by different names in different regions ( Gyrodinium aureolum ?). P. tamarensis appears to be very widely distributed, with both Arctic and tropical forms which must differ significantly in physiology. Some tropical benthic species which produce toxins also occur in temperate regions where they should be tested for toxicity, e.g. Prorocentrum lima. Finally, i t is noted that many of the toxin producers (all of which are photosynthetic) are close relatives and suggestions are made for the testing of other related taxa. Several papers dealt with toxic dinoflagellate taxonomy at the Second International Conference on Toxic Dinoflagellate Blooms (_l)and this paper summarises developments since then. A paper by Steidinger ( 2 ) , which appeared a f t e r the ACS symposium from which the present papers a r i s e , also discusses this topic and this paper has been revised to reduce duplication. Her paper should be consulted for greater i l l u s 0097-6156/84/0262-0077$06.25/0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


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t r a t i o n of the species i n question and further h i s t o r i c a l review. In addition to taxonomy the present contribution presents some biogeographical data,since this aspect has been neglected and i s intimatel y l i n k e d to questions of species i d e n t i t y , gene pools, and biochemi c a l and p h y s i o l o g i c a l v a r i a b i l i t y within s i m i l a r morphotypes. Before dealing with the taxa i n question, some general points may a s s i s t the non-specialist i n following recent developments, f o r d i n o f l a g e l l a t e taxonomy i s currently i n a state of f l u x which must be annoying and seem capricious to those who simply require a stable name with which to relate t h e i r biochemical data. F i r s t l y , much o f this has been necessitated by the c r i t i c a l re-examination of species which, because of t h e i r association with red tides or toxin production, were often described inadequately or improperly by non-taxonomi s t s . Secondly, because of t h e i r importance these species are being subject to much closer scrutiny than others and, as usual, the more that i s known, the less simple the picture becomes (discovery of intermediates or biochemical v a r i a b i l i t y within s i m i l a r morphotypes, for example). Because d i n o f l a g e l l a t e s have been named by both botani s t s and zoologists, each of whom follows a s l i g h t l y d i f f e r e n t set of nou&nclatural rules (International Codes), complications a r i s e which can be resolved eventually. Although sexuality i s known i n d i n o f l a g e l l a t e s ^ i t i s c r y p t i c , infrequent, unknown i n many, and reproductive i s o l a t i o n under natural conditions i s very d i f f i c u l t to assess at present. This means that c l a s s i c a l species concepts are d i f f i c u l t , i f not impossible to apply to such organisms. Genera are subjective constructs, following objective guidelines, which a s s i s t i n species i d e n t i f i c a t i o n s and the recognition of species groups. The degree of difference necessary for the recognition of genera i s usually set by precedent within each group. For example, the presence or absence of chloroplasts i s a generic c r i t e r i o n i n euglenoids, but has not been used as such i n d i n o f l a g e l l a t e s (except i n Pheoppolykrikos, rejected by most recent taxonomists). I t i s evident that those working with f o s s i l d i n o f l a g e l l a t e cysts accept much smaller differences i n generic d i s t i n c t i o n s than those studying l i v i n g forms and a current aim i s to b r i n g them closer together. F i n a l l y , natural populations are mixtures of genotypes and multiple i s o l a t e s from the same population at the same time should not be expected to be s i m i l ar i n a l l respects. D i f f e r i n g environmental conditions, both short and long term, w i l l favour p a r t i c u l a r genotypes within the populations i n accordance with basic p r i n c i p l e s of population genetics. Pro to gonyaulax Three species of this genus have been linked to natural p a r a l y t i c s h e l l f i s h poisoning (PSP) events: tamarensis (which includes excavata, attempts to distinguish them by morphological or other means having been abandoned by former proponents; 3 ) , P^. acatenella and c a t e n e l l a . In addition there are f i v e other species ( see J2, 4^ plus the form described as Gonyaulax kutnerae Balech - see below) which have not been shown to produce toxins so f a r . A l l these were formerly attributed to the genus Gonyaulax, but they d i f f e r i n so many respects (epithecal plate pattern, hypothecal pattern, a p i c a l pore, degree of g i r d l e displacement, cyst type), that t h e i r generic d i s t i n c t i o n cannot be disputed. Both Taylor (4)and Loeblich & Loebl-


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Taxonomic and Biogeographic Aspects of Toxic Dinoflagellates

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ich (_5) independantly came to this conclusion at the same time but, unfortunately, offered d i f f e r e n t solutions. The l a t t e r authors e n l arged the d e f i n i t i o n of the genus Gessnerium (see below) to include a l l the "tamarensis group, minimising variations i n the f i r s t a p i c a l plate. Taylor created a genus especially for them, omitting those species i n which the f i r s t a p i c a l plate homologue (designated Is i n the Taylor homology system, or l u i n E v i t t s modification of it)does not usually make contact with the a p i c a l pore complex (APC). A typographical error i n the generic description of Protogonyaulax ( i t should read " V/VI contacting Z") was corrected l a t e r (6). At that time,the cyst of the type of Gessnerium was unknown. It was subsequently found to be similar to that of Protogonyaulax (7), as i s that of Pyrophacus horologium (8). Consequently, the d i s t i n c t i o n between Protogonyaulax and Gessnerium (not as emended by Loeblich & Loeblich) rests s o l e l y on the contact, or lack of i t , of the f i r s t a p i c a l plate. Species referable to Gessnerium using this c r i t e r i o n , are indicated i n the next section. In ]?. f r a t e r c u l a the contact area may be small because the ventral part of the APC may be very narrow. Balech (9) has argued that this c r i t e r i o n i s not r e l i a b l e for generic d i s t i n c t i o n because i n his newly described form Gonyaulax kutnerae the contact was variable. However, the specimen he i l l u s t r a t e s , i n which the f i r s t a p i c a l i s displaced from the APC, i s c l e a r l y megacytic ( a form i n which the c e l l i s greatly enlarged, usually associated with sexuality or immediately p r i o r to division) and the growth of i n t e r calary bands may cause a temporary separation. I f such secondary e f f e c t s , known only for one or two species, are taken into account, the c r i t e r i o n i s s t i l l usable and useful i n maintaining nomenclatura l s t a b i l i t y (the genus Protogonyaulax i s now i n wide use). In a culture of P_. tamarensis , Taylor observed c e l l s with s t r i k i n g l y aberrant tabulations, involving a l l the primary plate series (cingulars and s u l c a l s were not examined but are assumed to be more conservative) , but the majority of c e l l s were of the normal type and this i s the condition on which the diagnosis must be based (10).


f

In this connection i t should be noted that an insistence that there should be no overlap whatever, would r e s u l t i n the "sinking" of the genus Triadinium ( = Goniodoma, Heteraulacus) as well, because i t i s b a s i c a l l y s i m i l a r to Gessnerium ( see Figure 1) and d i f f e r s only i n the plates which are included i n the sulcus, a feature which i s not always c l e a r l y evident due to intergradation. S i m i l a r l y , the athecate genera Amphidinium, Gyrodinium and Katodinium would a l l have to be sunk into Gymnodinium because of species which exhibit borderl i n e features (see Gyrodinium aureolum below). The morphological d i s t i n c t i o n between species of Protogonyaulax i s also becoming increasingly d i f f i c u l t . A l l share the same basic plate pattern. Their features were summarised and i l l u s t r a t e d by Taylor (10). Subsequently,Balech & De Mendiolana (11) described I\ peruviana (as a Gonyaulax), distinguished c h i e f l y by i t s curving f i r s t a p i c a l plate (which, however, resembles that of P. phoneus and some c e l l s of P. dimorpha). Later Balech (9) added Gonyaulax kutnerae , a large form which he compared to G_. brevisulcaturn Dangeard but which, i f contact between the f i r s t a p i c a l plate and the APC i s the usual condition (see above), should be transferred to Protogonyaulax, and Gessnerium i f not.


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A

B

C

D

F i g u r e 1. E p i - and h y p o t h e c a l t a b u l a t i o n p a t t e r n s . A. T r i a d i n i u m . B. Gessnerium, P y r o d i n i u m . C. P r o t o g o n y a u l a x . D. Gonyaulax (spinifera).

Each of these species has morphological ideosyncrasies which distinguish i t (e.g. the angular anterior s u l c a l plate, s u l c a l l i s t s and large size of P_. cohorticula; the narrow ventral projection of the APC of P. f r a t e r c u l a ; a very narrow s i x t h precingular i n a population from New Zealand which i s currently under study), but continued observations are revealing the existence of populations with morphotypes which f a l l between "species" that were previously readily distinguishable. Unlike the genera discussed above, species are b e l ieved to be r e a l b i o l o g i c a l units which should be discrete morphol o g i c a l l y and/or reproductively. Because the Zoological Code does not regulate names below the rank of species or subspecies, zoologists working with dinoflagellates tend to create new species names for forms which d i f f e r only minimally, whereas botanists can use varietas * forma for i n f r a s p e c i f i c variants that d i f f e r i n small genetic or phenotypic ways respectively. To one using the botanical system i t appears that several members of the "Protogonyaulax complex" do not deserve separation at the species l e v e l , although further information i s required i n most instances. A case i n point, which we are investigatingi i s the d i s t i n c t i o n of P_. catenella from P_. tamarensis, the two species which have been distinguished for the longest period. When f i r s t described P_. catene l l a appeared to be c l e a r l y d i f f e r e n t because of the plate arrangement on the epitheca, as well as c e l l shape and chain formation. However, a f t e r the r e a l i s a t i o n that Lebour had erred i n the o r i g i n a l description of P_. tamarensis, drawing the epithecal pattern i n optic reversal, and the correction i s made (10), the d i s t i n c t i o n becomes largely a matter of shape. Populations can be found which can be readily attributed to P_. catenella (chains of 8 c e l l s or more, c e l l s wider than long with angularly flattened apex and antapex, APC shaped l i k e a lamb chop) or P_. tamarensis ( c e l l s single or i n p a i r s , longer than wide, more e l l i p t i c a l APC). When brought into culture and grown under i d e n t i c a l conditions the d i s t i n c t i o n often becomes more d i f f i c u l t to make, chain length becoming shorter or no more than pairs i n some s t r a i n s , and c e l l s changing shape. I f they simply became i n d i s a n c



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tinguishable we would conclude that they were phenotypic variants r e s u l t i n g from environmental influences belonging to the same morphospecies and this would be a case i n which forma might be an appropriate l e v e l of d i s t i n c t i o n . Unfortunately i t i s not so simple. Some strains maintain a catenella shape even when not i n chains. Some make short chains. Some become isodiametric. One of our strains (NEPCC 254 from Nelson Island, B r i t i s h Columbia) was'^amarensoid" when f i r s t i s o l a t e d (12),but now occurs as small, flattened c e l l s (resembling Gaarder's Goniodoma depressum ) which could be taken for P.. catenella. A ventral pore i s absent, which i s usually the case with P_. catenella, whereas i t i s of variable occurrence (although constant within a s t r a i n ) i n P_. tamarensis. An i l l u s t r a t i o n of the problems involved can be seen i n the scanning electron micrographs of Postek & Cox (13), reproduced by Steidinger (2), of a culture sent to them from Washington state as P_. catenella, but i n which the c e l l s are shown to be longer than wide, with smooth contours ( l i k e the variety globosa of _P. tamarensis), and possessing a ventral pore. I f one were not aware of the p o t e n t i a l for change i n culture i t might be assumed that the culture was misidentified, but this i s not clear. The posterior attachment pore on the posterior s u l c a l plate i s found only i n the anterior individuals i n pairs or chains and appears to be induced by the apical pore of the c e l l posterior to i t . The shape of the posterior s u l c a l plate and the p o s i t i o n of the pore, i f present, has been used as an a i d i n distinguishing these species and P_. f r a t ercula (14). However, the shape used for the l a t t e r does not agree with the o r i g i n a l description by Balech (15) and c e l l s with the poste r i o r s u l c a l form associated with "catenelloid" c e l l s i n Japan has been seen i n "tamarensoid" c e l l s from Massachusetts (material of D.M. Anderson). These problems are seen most c l e a r l y i n B r i t i s h Columbia and Japan, where both types co-occur (see d i s t r i b u t i o n a l descriptions below), as well as populations which are d i f f i c u l t to assign because of intermediate features. In an attempt to resolve this problem A l l a n D. Cembella, working with the author, has investigated the enzyme compositions of 18 cultured i s o l a t e s of tamarensis and P_. catenella, or intermediate morphotypes, using enzyme electrophoresis. The majority of these i s o lates were from B r i t i s h Columbia or adjacent Washington state waters, but i s o l a t e s from England, Portugal, the Bay of Fundy (eastern Canada) and New Zealand were available for more remote comparisons. A l l cultures were grown under i d e n t i c a l conditions i n enriched natural seawater (NSP-7 medium) and harvested i n late exponential phase for application to v e r t i c a l slab, polyacrylamide gels (PAG). Comparison with starch gel electrophoresis confirmed the superiority of PAG electrophoresis for investigating the biochemical taxonomy of Protogonyaulax, both i n terms of number of bands and resolution (Figure 2). To date we have examined the following enzymes, divided into two functional groups: 1) the pyridine-linked dehydrogenases - alanine dehydrogenase (ADH), glutamate dehydrogenase (GDH), glucoses-phosphate dehydrogenase (G6PDH), i s o c i t r a t e dehydrogenase (IDH), malate dehydrogenase (MDH), malic enzyme (ME), and succinate dehydrogenase (SDH); and 2) the non-specific hydrolases - ou- and/a- acetylesterases (oC-,/a- EST), propionylesterases (PES) , butyrylesterases (BES) , and acid phosphatases (AcPH). An analysis of the banding patterns (16), while providing evid-



S E A F O O D TOXINS

F i g u r e 2. Isozymer banding p a t t e r n i n PAG of P r o t o g o n y a u l a x t a m a r e n s i s morphotypes s t a i n e d f o r m a l i c enzyme (ME/NADPdependent malate dehydrogenase).400-406: E n g l i s h Bay i s o l a t e s ( " i n d i c a t e s c l o n a l ) . 255: Lummi I s l a n d (Washington S t a t e ) .



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ence for considerable, stable biochemical v a r i a b i l i t y between both clonal and non-clonal i s o l a t e s , and multiple isolates from the same place and time (English Bay, Vancouver,, i s o l a t e s 400 to 407, see F i g ure 2), has not supported a simple separation of P_. catenella from P_. tamarensis, although differences do exist (Figure 3). The presence of such a high degree of electrophoretic polymorphism, even for r e l a t i v e l y conservative enzyme systems, such as MDH (NAD - dependent) and ME (NADP - dependent), within a r e s t r i c t e d geographical area, i s strong circumstantial evidence that Protogonyaulax populations are multiclonal,even when morphologically indistinguishable. In a similar study of five freshwater Peridinium species Hayhome & P f i e s t e r (17) did find a correlation between banding patterns and morphological characters, but the l a t t e r (presence or absence of an APC, form of the f i r s t a p i c a l p l a t e , etc.) were more gross than the d i s t i n c t i o n s between the tamarensis and catenella morphotypes. Previous attempts to use other biochemical c r i t e r i a to distinguish Protogonyaulax species, such as luminescence or toxin content, have f a i l e d to find consistent correlations with morphotype (3,18). The d i s t r i b u t i o n of the tamarensis and catenella morphotypes i s i n t e r e s t i n g , both on global (Figure 4) and l o c a l (Figure 5) scales Z.* catenella occurs on the west coast of North America from southern C a l i f o r n i a (La J o l l a - F.Haxo and pers.obs.) to south eastern Alaska (19). Its type l o c a l i t y i s San Francisco Bay. It i s absent south of 32°N,but reappears i n southern Chile (20 and pers.obs.). Although i t has been assumed to be the source of PSP i n the Gulf of Alaska, this has not been confirmed and i s o l a t e s from cysts c o l l e c t e d from several locations i n this region were of the tamarensis morphotype (21 and pers. obs.). It i s possible that both may occur, but i t should be noted that A r c t i c records so far are r e s t r i c t e d to P_. tamarensis (see below). catenella has not been recorded throughout most of the A t l a n t i c Ocean, but has produced four PSP outbreaks o f f the west coast of South A f r i c a (22 and pers. obs.). The only other region where P_. catenella has been recorded i s the east coast of Japan (14, 23). Blooms generally occur when the temperature i s close to 20 and the form occurs i n both estuarine and open coast l o c a l i t i e s , including one chain seen i n a sample 440km away from the B r i t i s h Columbia coast (pers.obs.). The _P. tamarensis morphotype i s found over a much wider area, including both A r c t i c (24,25) and t r o p i c a l (26,27) l o c a l i t i e s , the l a t t e r only i n the western t r o p i c a l A t l a n t i c Ocean (Venezuela; northeast of the Amazon mouth, beyond the Guiana Current; and possible Puerto Rico, although some of the l a t t e r records appear to be of other related forms). In the North A t l a n t i c i t i s known from estuarine l o c a l i t i e s or shallow embayments from Long Island to the A r c t i c , being p a r t i c u l a r l y common i n the Gulf of Maine, Bay of Fundy and Gulf of St.Lawrence i n the west, and from Portugal north to Norway and the A r c t i c near Spitsbergen (25). In the South A t l a n t i c i t i s known from the central coast of Argentina (28),with an i d e n t i f i c a t i o n attributed to Braarud ot material from blooms i n Walvis Bay, S.W. A f r i c a (29). In the P a c i f i c Ocean i t occurs i n B r i t i s h Columbia (10), some coasta l locations i n the Gulf of Alaska (pers. Obs. and f i g s , i n 21), Unimak Is. i n the Aleutians (unpubl. obs., 1965), and bays i n north eastern Japan (14,30 ) . A population from the northeast coast of North Island, New Zealand, which appeared a f t e r f i s h and s h e l l f i s h k i l l s , resembled P_. tamarensis, but had an unusually narrow sixth precingtf


SEAFOOD TOXINS

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Intarm. CatansHa

Tamarensis

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Toxic Dinoflagellates - American Chemical Society

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