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Variability and species discrimination within the Protogonyaulax tamarensis/catenells species complex… Cembella, Allan Douglas 1986

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VARIABILITY AND SPECIES DISCRIMINATION WITHIN THE PROTOGQNYAULAX TAMARENSIS/CATENELLA TOXIC RED-TIDE  SPECIES COMPLEX:  DINOFLAGELLATES By  ALLAN DOUGLAS CEMBELLA B.Sc,  Simon F r a s e r  University,  1.977,.  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES Departments o f Botany and Oceanography  We a c c e p t t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1986 © A l l a n Douglas Cembella, 1986  In p r e s e n t i n g requirements  this thesis  f o r an a d v a n c e d  of  British  it  freely available  agree for  that  Columbia,  f o r reference  the L i b r a r y  shall  and s t u d y .  I  f o r extensive  p u r p o s e s may  f u l f i l m e n t of the  degree a t the U n i v e r s i t y  I agree that  permission  scholarly  in partial  for  that  shall  Department o f  Date  1Q\  Q|J/r  y  Hag  of this  It is thesis  n o t be a l l o w e d w i t h o u t my  permission.  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  thesis  be g r a n t e d by t h e h e a d o f my  copying or p u b l i c a t i o n  f i n a n c i a l gain  further  copying of t h i s  d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s - . understood  make  Columbia  written  i  ABSTRACT  VARIABILITY AND  SPECIES DISCRIMINATION WITHIN THE  PROTOGONYAULAX  TAMARENSIS/CATENELLA SPECIES COMPLEX: TOXIC RED-TIDE DINOFLAGELLATES  A l l a n Douglas Cembella U n i v e r s i t y of B r i t i s h  Columbia  1986  Thecate  gonyaulacoid  Protogonyaulax regions  Taylor  throughout  catenella  contemporaneous  analysis  of  world.  analysis toxin  from of  the  components  microphotometry.  taxonomic  variation  within  between patterns degree  of  isolates  of  and  group.  acclimated this  geographical  genetic  to  in coastal tamarensis/  were  subjected  to  g e l e l e c t r o p h o r e s i s , an (performance)  DNA  results  estimate  These  liquid  determinations were  compared  by with  phenotypic  biochemical  methods,  and  genotypic  along  with  growth r a t e , o f f e r e d a means of d i s t i n g u i s h i n g species  polymorphism  populations.  nuclear The  were s u b s t a n t i a l l y the same. of  genus  c r i t e r i a used t o d i s c r i m i n a t e among s p e c i e s , to  linkages  this  by  high-pressure  quantitative  morphological  location,  isozymes  using  epifluorescence  establish  same  soluble  and  measurements  the  I s o l a t e s of the Protogonyaulax  chromatography  conventional  to  complex from d i v e r s e g e o g r a p h i c a l r e g i o n s , i n c l u d i n g t e n isolates  chemotaxonomic  referable  cause p a r a l y t i c s h e l l f i s h p o i s o n i n g (PSP)  the  species  dinoflagellates  Yet,  complex, The  for  which  the t h e c a l p l a t e  isozyme p a t t e r n s r e v e a l e d a h i g h  within within  and the  among  morphotypes  tamarensoid  and  morphotype,  isolates  from  elsewhere. and  conservative isolates  same  This  profiles  through  the  general  DNA  trend  analysis,  measure  varied  location  of  were was  more  supported  although  toxin  v a r i a t i o n than  markedly  in  similar by  than t o those evidence  from t o x i n  heterogeneity  isozyme d i v e r s i t y .  from  was  a  more  Protogonyaulax  t o t a l t o x i n c o n c e n t r a t i o n and  toxicity,  even  the c u l t u r e c y c l e of i n d i v i d u a l i s o l a t e s , but the t o x i n r a t i o s were  distinctive  and  forms,  dominant morphotypes w i t h i n t h i s s p e c i e s complex, were not w e l l  the  relatively  correlated  with  occasional  presence  features  presently  always  be  used  appear  to  be  smaller  of  characters  catenelloid  and  investigated.  intermediates,  the  tamarensoid  Given  cannot  r e l i a b l y d i s c r i m i n a t e between these morphospecies,  from  stable  allopatric large  At l e a s t  two  populations e x h i b i t e d morphological  and  enough genetic  indicate  d i s t r i b u t e d Protogonyaulax p o p u l a t i o n s .  reflected  species  area  may  diversity  possible  h e t e r o g e n e i t y w i t h i n p o p u l a t i o n s from a g i v e n g e o g r a p h i c a l species  of  to  descriptors.  the  sibling  level  species  and  in  that  high  as  the  morphological  to i d e n t i f y P. c a t e n e l l a and P. tamarensis  inadequate  The  The  morphological  differences  divergence. biochemical  biochemical  used  to  isolates  biochemical  suggests  the  constant.  a l s o have a r i s e n w i t h i n s y m p a t r i c a l l y  i i i  E s a r a mia  c o l p a se c o s i  e?  - Machiavelli  The phosphorescence, t h a t b l u e y Very good f o r the b r a i n . - " U l y s s e s " , James Joyce  But not on us! the o y s t e r s c r i e d , Turning a l i t t l e blue. A f t e r such k i n d n e s s , t h a t would be A d i s m a l t h i n g t o do! - "Through the Looking  G l a s s " , Lewis C a r r o l l  greeny.  iv  TABLE OF CONTENTS  ABSTRACT  i  TABLE OF CONTENTS  iv  LIST OF FIGURES  ix  LIST OF TABLES  xiii  ACKNOWLEDGEMENTS  CHAPTER I .  xvi  INTRODUCTION  A. C l a s s i f i c a t i o n B. V a r i a b i l i t y  1  and t h e S p e c i e s Problem i n D i n o f l a g e l l a t e s  and S p e c i a t i o n  7  1. P h e n o t y p i c v e r s u s Genotypic E v i d e n c e 2. Mechanisms f o r M a i n t a i n i n g V a r i a b i l i t y  1  7 and S t a b i l i t y  3. P h e n e t i c and P h y l o g e n e t i c Taxonomic Linkage  16 22  C. G e n e r a l D e s c r i p t i o n and Taxonomic P o s i t i o n o f t h e Genus Protogonyaulax D. H i s t o r i c a l the  26  and Recent P e r s p e c t i v e s on Taxonomic V a r i a n t s W i t h i n  Protogonyaulax t a m a r e n s i s / c a t e n e l l a S p e c i e s Complex  E. R a t i o n a l e f o r E x p e r i m e n t a l S t u d i e s  39 47  1. G e o g r a p h i c a l D i s t r i b u t i o n and E n v i r o n m e n t a l S i g n i f i c a n c e of Protogonyaulax spp. blooms  47  2. The Problem o f Morphotypic G r a d i e n t s and S t a b i l i t y i n Protogonyaulax 3. Research O b j e c t i v e s  54 56  CHAPTER I I .  MATERIALS AND METHODS  58  A. C u l t u r e and Maintenance  58  1. I s o l a t e O r i g i n s and Growth C o n d i t i o n s  58  2. Development and F o r m u l a t i o n o f NWSP-7 Growth Medium  59  3. I s o l a t i o n and C l o n i n g  65  4. P r e p a r a t i o n and T e s t i n g o f A x e n i c C u l t u r e s  66  B. M o r p h o l o g i c a l Examination of Protogonyaulax  Isolates  68  1. I s o l a t e I d e n t i f i c a t i o n and M o r p h o l o g i c a l C h a r a c t e r i s t i c s . . . .  68  2. Chain Length Experiments  74  C. V a r i a t i o n i n Growth Rates  75  1. D e t e r m i n a t i o n o f A c c l i m a t e d Growth Rates  75  D. DNA A n a l y s i s by E p i f l u o r e s c e n c e Microphotometry 1. N u c l e a r DNA  I  Determination  78 78  E. Method f o r P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s  79  1. E l e c t r o p h o r e s i s o f S o l u b l e Enzymes  79  a. C e l l c u l t u r e , h a r v e s t and s t o r a g e  79  b. B a c t e r i a l c o n t a m i n a t i o n  81  c. D e t e r m i n a t i o n o f t o t a l p r o t e i n  81  d. Enzyme e x t r a c t i o n  82  e. E l e c t r o p h o r e t i c s e p a r a t i o n  83  f . G e l s t a i n i n g and band s c o r i n g  84  F. T o x i n A n a l y s i s by H i g h - P r e s s u r e (Performance) Chromatography 1. HPLC A n a l y s i s o f Toxins  Liquid 86 86  a. C e l l c u l t u r e and h a r v e s t  86  b. T o x i n e x t r a c t i o n  87  c. A n a l y t i c a l method  88  CHAPTER I I I .  EXPERIMENTAL RESULTS AND  DISCUSSION  92  A. M o r p h o l o g i c a l C h a r a c t e r i s t i c s  92  1. V a r i a t i o n i n C e l l S i z e 2. V a r i a t i o n and  92  S t a b i l i t y i n C e l l Shape and  Other  C h a r a c t e r i s t i c Features  100  3. Chain Length  117  4. D i s c u s s i o n .  126  B. Growth Rates  134  1. A c c l i m a t e d Growth Rates of Protogonyaulax i s o l a t e s . . . . a. R e s u l t s  134  b. D i s c u s s i o n  144  C. Q u a n t i t a t i v e 1. R e s u l t s D.  134  N u c l e a r DNA  Content  and  Discussion  Electrophoresis  of S o l u b l e  150 150  Enzymes  164  1. G e n e r a l E l e c t r o p h o r e t i c Theory  164  2. The  Use  and 3.  of E l e c t r o p h o r e s i s  Population  i n Chemosystematics, Taxonomy,  Genetics  165  I n t e r p r e t a t i o n of E l e c t r o p h o r e t i c Data  169  4. Enzyme R e a c t i o n s : P y r i d i n e - l i n k e d Dehydrogenases  173  5. E l e c t r o p h o r e t i c P r o f i l e s of P y r i d i n e - l i n k e d Dehydrogenases..  176  6.  Isozyme-based Chemotaxonomic R e l a t i o n s h i p s  among  Protogonyaulax I s o l a t e s  191  a. P h e n e t i c c l u s t e r a n a l y s i s  191  b. P h y l o g e n e t i c  204  linkage analysis  7. D i s c u s s i o n i. ii.  The  214  dehydrogenase isozymes and  Isozyme v a r i a t i o n and  genetic  genotype identity  214 218  vii  iii.  E v o l u t i o n a r y d i v e r g e n c e and phylogeny  i v . E l e c t r o p h o r e t i c e v i d e n c e and s p e c i a t i o n E. V a r i a t i o n i n T o x i n Composition and T o x i c i t y  222 224 225  1. I n t r o d u c t i o n  225  2. T o x i n C o n c e n t r a t i o n s and P r o f i l e s  231  3. Toxin-Based Chemotaxonomic R e l a t i o n s h i p s among Protogonyaulax I s o l a t e s  260  a. P h e n e t i c c l u s t e r a n a l y s i s  260  b. A n a l y s i s o f p r i n c i p a l components  266  4. D i s c u s s i o n  CHAPTER IV.  278  GENERAL DISCUSSION AND  A. G e n o t y p i c Polymorphism  IMPLICATIONS  287  and E n v i r o n m e n t a l H e t e r o g e n e i t y  287  B. E v o l u t i o n a r y D i v e r g e n c e , Phylogeny and S p e c i a t i o n  290  C. S u g g e s t i o n s f o r F u r t h e r Research  300  CHAPTER V.  303  SUMMARY AND  CONCLUSIONS  REFERENCES  306  APPENDICES  333  APPENDIX 1  333  A. A l t e r n a t i v e Growth Media  333  1. C u l t u r e Methods  333  2. A r t i f i c i a l Seawater Enrichment  334  3. N a t u r a l Seawater Enrichment  342  4. The. E f f e c t o f T r i s on Growth  345  viii  APPENDIX II  346  A. Modifications of Electrophoretic Protocol  346  1. Alternative Gel Formulations  346  2. Electrophoretic Buffer Systems  346  3. Alternative Sample Preparation  355  4. Alternative Running Conditions  367  5. Modifications of Gel Staining Techniques  368  6. Unsuccessful Enzyme Staining  378  APPENDIX III  380  A. Alternative Schemes of Chemotaxonomic Analysis  380  1. Isozyme-based Taxonomic Relationships  380  2. Toxin-based Taxonomic Relationships  400  ix  LIST OF FIGURES  Fig.  1  Fig. 2  Fig. 3  Fig. 4  Fig. 5  Fig. 6  Fig. 7  Fig. 8  Fig. 9  G e n e r a l morphology o f t h e tamarensoid morphotype o f Protogonyaulax..  30  G e n e r a l morphology o f t h e c a t e n e l l o i d morphotype o f Protogonyaulax, showing a n t e r o - p o s t e r i o r l y compressed c e l l s i n a c h a r a c t e r i s t i c chain c o n f i g u r a t i o n  30  T h e c a l p l a t e c o n f i g u r a t i o n o f Protogonyaulax i n t h e K o f o i d n o t a t i o n system, as a p p l i e d by Fukuyo (1985)  33  H i s t o r i c a l taxonomic scheme f o r some members o f t h e Protogonyaulax t a m a r e n s i s / c a t e n e l l a s p e c i e s complex  41  G l o b a l d i s t r i b u t i o n o f some members o f t h e genus Protogonyaulax  50  Map o f t h e n o r t h e a s t P a c i f i c c o a s t Protogonyaulax i s o l a t e s  52  showing  o r i g i n of  Frequency d i s t r i b u t i o n o f a p i c a l and t r a n s a p i c a l diameters (nm) o f Protogonyaulax i s o l a t e s (n=30) A p i c a l view o f empty t h e c a o f NEPCC 403 s t a i n e d w i t h 0.1% c a l c o f l u o r showing e p i t h e c a l p l a t e s under e p i f l u o r e s c e n c e microscopy (400X)  104  A p i c a l view o f e p i t h e c a l p l a t e s o f NEPCC 516 s t a i n e d w i t h 0.1% c a l c o f l u o r showing v e n t r a l pore on t h e f i r s t a p i c a l ( l ) p l a t e under e p i f l u o r e s c e n c e microscopy (800X)  104  Photomicrograph o f NEPCC 180, a tamarensoid morphotype from Brentwood Bay, B.C., e x h i b i t i n g a rounded apex and antapex i n e q u a t o r i a l view (250X)  107  Photomicrograph o f NEPCC 181(b), i s o l a t e d as a c h a i n forming c a t e n e l l o i d morphotype from P a t r i c i a Bay, B.C., i n e q u a t o r i a l view (400X)  107  C h a r a c t e r i s t i c morphology o f t h e a p i c a l pore complex and the p o s t e r i o r s u l c a l p l a t e o f Protogonyaulax i s o l a t e s i n culture  111-113  1  Fig.  Fig.  Fig.  10  11  12  94-99  Fig.  13  A n t a p i c a l view o f h y p o t h e c a l p l a t e s from tamarensoid specimens s t a i n e d w i t h c h l o r a l h y d r a t e - i o d i n e - h y d r i o d i c a c i d , examined under p h a s e - c o n t r a s t microscopy (250X). ... 116  Fig.  14  Photomicrograph o f c h a i n - f o r m i n g P. c a t e n e l l a i n a p h y t o p l a n k t o n sample from Puget Sound, WA (250X)  116  Growth c u r v e s o f NEPCC 355 on c o n t r o l ESNW medium ( • ) , low N ( • ), low P ( • ) and low Fe ( X )  119  Fig.  15  X  Fig.  Fig.  16  17  . F i g . 18  Fig.  19  F i g . 20  F i g . 21  F i g . 22  F i g . 23  F i g . 24  F i g . 25  F i g . 26  F i g . 27  F i g . 28  F i g . 29  Histograms o f the percentage o f c e l l s p r e s e n t i n c h a i n s of v a r i o u s l e n g t h s throughout the growth c y c l e of NEPCC 355  121-124  Histogram o f a c c l i m a t e d growth r a t e s o f Protogonyaulax i s o l a t e s from B a m f i e l d and E n g l i s h Bay i n B r i t i s h Columbia, and o t h e r r e g i o n s  136  A p i c a l view o f NEPCC 516 s t a i n e d w i t h DAPI showing c r e s c e n t - s h a p e d n u c l e u s and r e d a u t o f l u o r e s c e n c e under e p i f l u o r e s c e n c e microscopy (800X)  153  E q u a t o r i a l view o f NEPCC 516 s t a i n e d w i t h DAPI showing f l u o r e s c e n t n u c l e a r bar a t the cingulum under e p i f l u o r e s c e n c e microscopy (500X)  153  L i n e a r r e g r e s s i o n a n a l y s i s o f the r e l a t i o n s h i p o f mean n u c l e a r DNA c o n t e n t t o time e l a p s e d s i n c e the o r i g i n of isolates in culture  157  N u c l e a r DNA c o n t e n t of Protogonyaulax i s o l a t e s i n r e l a t i o n t o mean c e l l volume  161  Zymograms of dehydrogenases isolates  179-182  e x t r a c t e d from Protogonyaulax  Zymogram of NAD-dependent glutamate dehydrogenase (GDH) isozymes of some Protogonyaulax i s o l a t e s from E n g l i s h Bay, B.C  187  Zymogram of NAD-dependent malate dehydrogenase isozymes o f Protogonyaulax i s o l a t e s  187  (MDH)  Zymogram o f NAD-dependent malate dehydrogenase (MDH) isozymes o f some Protogonyaulax i s o l a t e s from E n g l i s h B.C  Bay,  Zymogram o f NADP-dependent m a l i c enzyme (ME) isozymes of some Protogonyaulax i s o l a t e s from E n g l i s h Bay, B.C UPGMA l i n k a g e dendrogram i n d i c a t i n g e l e c t r o p h o r e t i c s i m i l a r i t i e s among Protogonyaulax i s o l a t e s , c o n s t r u c t e d from S j v a l u e s  190  190  194  Unsealed Prim network i n d i c a t i n g minimum spanning d i s t a n c e s between Protogonyaulax i s o l a t e s , based upon e l e c t r o p h o r e t i c p r o f i l e s of dehydrogenase isozymes  209  O p t i m i z e d m i d p o i n t r o o t e d Wagner t r e e showing p h y l o g e n e t i c r e l a t i o n h i p s between Protogonyaulax i s o l a t e s (n=20), based upon e l e c t r o p h o r e t i c p r o f i l e s o f dehydrogenase isozymes  212  xi  F i g . 30  S t r u c t u r e s and a b b r e v i a t i o n s f o r s a x i t o x i n (STX) and t h e e l e v e n other PSP t o x i n s o b t a i n e d from Protogonyaulax spp  229  F i g . 31  T o x i n c o m p o s i t i o n o f Protogonyaulax i s o l a t e s (as % t o t a l t o x i n ±1.0 s.d.; n=4) d u r i n g e x p o n e n t i a l growth phase. ... 238-249  F i g . 32  HPLC chromatogram o f PSP t o x i n s e x t r a c t e d from a tamarensoid i s o l a t e , NEPCC 255 (Lummi I s l a n d , WA)  251  HPLC chromatogram o f PSP t o x i n s e x t r a c t e d from a c a t e n e l l o i d i s o l a t e , NEPCC 529 ( F r i d a y Harbor, San Juan I s l a n d , WA)  253  HPLC chromatogram o f PSP t o x i n s e x t r a c t e d from an i s o l a t e o f i n t e r m e d i a t e morphotype, NEPCC 402 ( E n g l i s h Bay, B.C.)  255  HPLC chromatogram o f PSP t o x i n s e x t r a c t e d from a h i g h l y t o x i c tamarensoid i s o l a t e , NEPCC 545 (Bay o f Fundy, N.B.)  257  C e n t r o i d - l i n k a g e dendrogram i n d i c a t i n g E u c l i d e a n d i s t a n c e s between Protogonyaulax i s o l a t e s based upon r a t i o s o f e q u a l l y weighted t o x i n s  262  O r d i n a t i o n o f Protogonyaulax i s o l a t e s by p r i n c i p a l component a n a l y s i s based upon t h e p e r c e n t o f t o t a l t o x i n c o m p o s i t i o n r e p r e s e n t e d by each t o x i n component  273-275  Growth curve o f NEPCC 409 r e p r e s e n t e d by i n v i v o f l u o r e s c e n c e i n d i c a t i n g r e l a t i v e growth on a r t i f i c i a l seawater (ESAW) ( • ) compared w i t h n a t u r a l seawater (ESNW) ( A ) enrichments d u r i n g t h e second t r a n s f e r cycle  341  Zymogram o f enzyme e x t r a c t s o f NEPCC 355 s t a i n e d f o r NADdependent glutamate dehydrogenase (GDH) i n 0.1 M T r i s ( t r a c k s 1-5) v e r s u s 0.1 M T r i c i n e ( t r a c k s 6-10) s t a i n i n g b u f f e r , pH 8.0  351  Zymogram o f enzyme e x t r a c t s o f NEPCC 355 s t a i n e d f o r NADPdependent m a l i c enzyme (ME) comparing t h e e f f e c t o f T r i s s t a i n i n g b u f f e r w i t h T r i c i n e ' b u f f e r a t pH 8.0  351  Zymogram o f NAD-dependent GDH isozymes o f NEPCC 409 e x t r a c t e d from f r o z e n l y o p h i l i z e d v e r s u s n o n - l y o p h i l i z e d frozen c e l l s '.  354  Zymogram o f enzyme e x t r a c t s o f NEPCC 409 s t a i n e d f o r a- and 3~esterases i n 0.1 M c i t r a t e b u f f e r , pH 6.0  354  F i g . 33  F i g . 34  F i g . 35  F i g . 36  F i g . 37  F i g . 38  F i g . 39  F i g . 40  F i g . 41  F i g . 42  xii  F i g . A3  F i g . AA '  F i g . A5  F i g . A6  F i g . A7  F i g . A8  D e n s i t o m e t r i c scan of g e l s s t a i n e d f o r NAD-dependent malate dehydrogenase (MDH) e x t r a c t e d from NEPCC 529 comparing enzyme e x t r a c t i o n by m e c h a n i c a l homogenization (A3a) w i t h h i g h i n t e n s i t y u l t r a s o n i c a t i o n (A3b)  359  D e n s i t o m e t r i c scan of g e l s s t a i n e d f o r NAD-dependent malate dehydrogenase (MDH) e x t r a c t e d from NEPCC 529 showing the e f f e c t on band i n t e n s i t y of e x t r a c t c o n c e n t r a t i o n and volume a p p l i e d to g e l s 36A-366 D e n s i t o m e t r i c scan of g e l s s t a i n e d f o r NAD-dependent glutamate dehydrogenase (GDH) e x t r a c t e d from NEPCC A09 showing the e f f e c t of a l t e r n a t i v e t e t r a z o l i u m s t a i n s on band i n t e n s i t y : A5a, MTT; A5b, NBT  370  D e n s i t o m e t r i c scan of g e l s s t a i n e d f o r a l a n i n e dehydrogenase (AlaDH) e x t r a c t e d from NEPCC 25A showing the e f f e c t of s t a i n i n g b u f f e r pH on band i n t e n s i t y : A6a, pH 8.0; A6b, pH 7.0  373  D e n s i t o m e t r i c scan of g e l s s t a i n e d f o r NADP-dependent m a l i c enzyme (ME) e x t r a c t e d from NEPCC 355 showing the e f f e c t of a l t e r n a t i v e s t a i n i n g b u f f e r s on band i n t e n s i t y : A7a, 0.1 M T r i s , pH 8.0; A7b, 0.1 M T r i c i n e , pH 8.0  375  D e n s i t o m e t r i c scan of g e l s s t a i n e d f o r NAD-dependent glutamate dehydrogenase (GDH) e x t r a c t e d from NEPCC A09 showing the e f f e c t of a l t e r n a t i v e s t a i n i n g b u f f e r s on band i n t e n s i t y : A8a, 0.1 M T r i c i n e , pH 8.0; A8b, 0.1 M T r i s , pH 8.0  377  F i g . A9  S p e c i a l s i m i l a r i t i e s unweighted p a i r - g r o u p average (SUPGMA) l i n k a g e dendrogram f o r Protogonyaulax i s o l a t e s 383  F i g . 50  R e g r e s s i o n a n a l y s i s f o r the c o p h e n e t i c c o r r e l a t i o n c o e f f i c i e n t (R = coph ^? based on the SUPGMA s p e c i a l s i m i l a r i t i e s dendrogram ( F i g . A9) 387 F i v e a l t e r n a t i v e m i d p o i n t - r o o t e d d i s t a n c e Wagner t r e e s produced by the PHYSIS subprogram SWAG (TREES=5; TRIES=500), based on the Manhattan d i s t a n c e m a t r i x d e r i v e d from electrophoretic profiles 390-39A r  F i g . 51  F i g . 52  F i g . 53  F i g . 5A  a s  O p t i m i z e d Wagner t r e e showing p h y l o g e n e t i c r e l a t i o n s h i p s between Protogonyaulax i s o l a t e s (n=20), based upon e l e c t r o p h o r e t i c p r o f i l e s of dehydrogenase isozymes  398  O r d i n a t i o n of Protogonyaulax i s o l a t e s by p r i n c i p a l component a n a l y s i s based upon the a b s o l u t e concentrations o f t o x i n components per c e l l  A06-A07  O r d i n a t i o n of Protogonyaulax i s o l a t e s by p r i n c i p a l component a n a l y s i s based upon the r a t i o s of e q u a l l y weighted t o x i n s  A11-A12  xiii  LIST OF TABLES  Table 1  F o r m u l a t i o n o f NWSP-7 n a t u r a l seawater enrichment medium.  60  Table 2  A n t i b i o t i c mix f o r Protogonyaulax p u r i f i c a t i o n  67  Table 3  M o d i f i e d STP s t e r i l i t y t e s t medium f o r t h e m o n i t o r i n g of p o s s i b l e b a c t e r i a l growth i n a n t i b i o t i c - t r e a t e d Protogonyaulax c u l t u r e s  67  L o c a t i o n o f o r i g i n and m o r p h o l o g i c a l c h a r a c t e r i s t i c s o f i s o l a t e s o f t h e Protogonyaulax t a m a r e n s i s / c a t e n e l l a s p e c i e s complex  70-73  Table 5  Polyacrylamide g e l formulation  83  Table 6  S t a i n p r o t o c o l s f o r dehydrogenase  Table 7  A b b r e v i a t i o n s and names o f PSP t o x i n s from Protogonyaulax spp.; s a x i t o x i n and i t s n a t u r a l l y - o c c u r r i n g derivatives  Table 4  Table 8  isozymes  85  ANOVA comparing v a r i a n c e i n c e l l volume between Protogonyaulax i s o l a t e s grouped by l o c a t i o n o f o r i g i n , p r e s e n t morphotype i n c u l t u r e and c l o n a l i t y t h e time o f original culture i n i t i a t i o n  90  101  Table 9  ANOVA comparing v a r i a n c e i n a c c l i m a t e d growth r a t e s between Protogonyaulax i s o l a t e s grouped by l o c a t i o n o f o r i g i n , p r e s e n t morphotype i n c u l t u r e and c l o n a l i t y a t t h e time of i s o l a t i o n '. 137-138  T a b l e 10  Nonparametric Mann-Whitney U - t e s t f o r t h e s i g n i f i c a n c e of t h e d i f f e r e n c e i n mean a c c l i m a t e d growth r a t e s f o r Protogonyaulax i s o l a t e s grouped by l o c a t i o n o f o r i g i n , p r e s e n t morphotype i n c u l t u r e and c l o n a l i t y a t t h e time of i s o l a t i o n  140  Maximum growth r a t e s o f Protogonyaulax spp. i n e x p o n e n t i a l phase from b a t c h c u l t u r e experiments  141-143  Mean n u c l e a r DNA content o f Protogonyaulax i s o l a t e s by morphotype and l o c a t i o n o f o r i g i n  162  T a b l e 11  T a b l e 12  T a b l e 13  P a i r w i s e comparison o f t h e d i f f e r e n c e i n mean n u c l e a r DNA c o n t e n t between grouped Protogonyaulax i s o l a t e s . Test of s i g n i f i c a n c e i s t h e Student's t - t e s t ; t w o - t a i l e d a t  a=0.05; H : m= Q  T a b l e 14  T a b l e 15  "2  1  6  2  N u c l e a r DNA content o f v e g e t a t i v e c e l l s o f Protogonyaulax i s o l a t e s r e l a t i v e t o t h a t o f zygotes  163  S i m i l a r i t y c o e f f i c i e n t s ( S j ) o f Protogonyaulax based upon dehydrogenase band p a t t e r n s  183  isolates,  xiv  T a b l e 16  T a b l e 17  T a b l e 18  T a b l e 19  Mean s i m i l a r i t y c o e f f i c i e n t s ( S j ) o f Protogonyaulax i s o l a t e s by g e o g r a p h i c a l o r i g i n and morphotype f o r i n d i v i d u a l dehydrogenases  184  Cophenetic c o r r e l a t i o n m a t r i x c o n t r u c t e d from UPGMA dendrogram ( F i g . 27) based on dehydrogenase banding patterns  195  B i n a r y c h a r a c t e r s t a t e m a t r i x f o r dehydrogenase isozymes coded as p r e s e n c e ( l ) - a b s e n c e ( 0 ) d a t a Manhattan d i s t a n c e m a t r i x computed from t h e c h a r a c t e r m a t r i x ( T a b l e 18) showing d i s t a n c e r e l a t i o n s h i p s between Protogonyaulax i s o l a t e s (n=20)  198-203  207  T a b l e 20  PSP t o x i n composition o f Protogonyaulax i s o l a t e s expressed as t h e c o n c e n t r a t i o n o f each t o x i n ( f m o l c e l l " ^ - ) , p e r c e n t of t o t a l t o x i n composition r e p r e s e n t e d by each component (% t o t a l ) and t o x i c i t y (uMU c e l l ~ l ) , determined i n l a t e e x p o n e n t i a l growth phase by HPLC o f t o x i n e x t r a c t s i n 0.03 N a c e t i c a c i d 232-236  T a b l e 21  Amalgamation sequence f o r c e n t r o i d - l i n k a g e c l u s t e r f o r m a t i o n from u n s t a n d a r d i z e d % t o x i n d a t a  264  V a r i a b l e r a t i o s o f t o x i n components o f Protogonyaulax isolates. The maximum r a t i o o f t h e t o t a l t o x i n c o n t e n t among a l l i s o l a t e s f o r each t o x i n i s a r b i t r a r i l y g i v e n as 1.0; r e l a t i v e v a l u e s f o r o t h e r i s o l a t e s a r e expressed as a r a t i o o f t h e maximum  265  T a b l e 22  T a b l e 23  U n i v a r i a t e summary s t a t i s t i c s  f o r % t o x i n d a t a used i n  the p r i n c i p a l components a n a l y s i s  268  T a b l e 24  C o r r e l a t i o n m a t r i x c o n s t r u c t e d from % t o x i n d a t a  269  T a b l e 25  E i g e n v a l u e s g i v i n g v a r i a n c e e x p l a i n e d by each f a c t o r i n the a n a l y s i s o f p r i n c i p a l components  270  T a b l e 26  Orthogonal r o t a t e d f a c t o r loading f o r the f i r s t four p r i n c i p a l components a f t e r varimax r o t a t i o n . Loadings a r e the e i g e n v e c t o r s o f t h e c o r r e l a t i o n m a t r i x based on % t o x i n d a t a m u l t i p l i e d by t h e square r o o t s o f t h e c o r r e s p o n d i n g eigenvalues 271  T a b l e 27  E s t i m a t e d f a c t o r s c o r e s and Mahalanobis d i s t a n c e s ( c h i - s q u a r e s ) from each case t o t h e c e n t r o i d o f a l l cases f o r t h e o r i g i n a l d a t a based on % t o x i n d a t a c o m p o s i t i o n ( T a b l e 20)  276  C e l l d e n s i t y and y i e l d r a t i o s a f t e r 12 days f o l l o w i n g i n o c u l a t i o n from r e f e r e n c e c u l t u r e s m a i n t a i n e d on ESNW medium. I n i t i a l t r a n s f e r s e r i e s  335  T a b l e 28  XV  T a b l e 29  A c c l i m a t e d maximum growth r a t e (k) determined i n e x p o n e n t i a l growth phase d u r i n g t h e second t r a n s f e r series  336  T a b l e 30  A c c l i m a t e d maximum growth r a t e (k) f o r NEPCC 180 grown on v a r i o u s n u t r i e n t media, determined f l u o r o m e t r i c a l l y d u r i n g e x p o n e n t i a l phase 337-338  T a b l e 31  E u c l i d e a n d i s t a n c e m a t r i x computed from the c h a r a c t e r m a t r i x ( T a b l e 18) showing d i s t a n c e r e l a t i o n s h i p s between Protogonyaulax i s o l a t e s (n=20)  385  P h e n e t i c goodness o f f i t s t a t i s t i c s f o r d i s t a n c e Wagner t r e e s produced by the random a d d i t i o n sequence subprogram SWAG (TREES=5; TRIES=500) t o the Manhattan d i s t a n c e matrix  396  Table  32  T a b l e 33  P h e n e t i c goodness o f f i t s t a t i s t i c s f o r d i s t a n c e Wagner t r e e s produced by the random a d d i t i o n sequence subprogram SWAG (TREES=5; TRIES=500) t o the Manhattan d i s t a n c e m a t r i x a f t e r f i t t i n g t r e e s t o t h e c h a r a c t e r m a t r i x u s i n g command DIAG t o maximize parsimony 396  T a b l e 34  P h e n e t i c goodness o f f i t s t a t i s t i c s f o r t h e two optimum d i s t a n c e Wagner t r e e s i n Fig'. 51 comparing f i t t o t h e Manhattan d i s t a n c e m a t r i x a f t e r r e - o p t i m i z a t i o n t o minimize p e r c e n t s t a n d a r d d e v i a t i o n (PSDOPT) o r t h e S - s t a t i s t i c (SOPT) o f the PHYSIS command F I T  396  Orthogonal r o t a t e d f a c t o r l o a d i n g f o r the f i r s t f o u r p r i n c i p a l components a f t e r varimax r o t a t i o n . Loadings a r e the e i g e n v e c t o r s o f t h e c o r r e l a t i o n m a t r i x based on a b s o l u t e c o n c e n t r a t i o n o f t o x i n components ( T a b l e 20) m u l t i p l i e d by the square r o o t s o f t h e c o r r e s p o n d i n g eigenvalues  403  E s t i m a t e d f a c t o r s c o r e s and Mahalanobis d i s t a n c e s ( c h i - s q u a r e s ) from each case t o t h e c e n t r o i d o f a l l cases f o r the o r i g i n a l data based on a b s o l u t e c o n c e n t r a t i o n o f t o x i n components ( T a b l e 20)  404  T a b l e 35  T a b l e 36  T a b l e 37  Orthogonal r o t a t e d f a c t o r l o a d i n g f o r t h e f i r s t f i v e p r i n c i p a l components a f t e r varimax r o t a t i o n . Loadings a r e t h e e i g e n v e c t o r s o f t h e c o r r e l a t i o n m a t r i x based on n o r m a l i z e d v a r i a b l e r a t i o s o f t o x i n components ( T a b l e 22) m u l t i p l i e d by the square r o o t s o f t h e c o r r e s p o n d i n g eigenvalues 408  T a b l e 38  E s t i m a t e d f a c t o r s c o r e s and Mahalanobis d i s t a n c e s ( c h i - s q u a r e s ) from each case t o t h e c e n t r o i d o f a l l cases f o r t h e o r i g i n a l data from n o r m a l i z e d v a r i a b l e r a t i o s o f t o x i n components ( T a b l e 22)  409  ACKNOWLEDGEMENTS  The  ultimate  dissertation  rests  significantly support and  responsibility  to  and  with  P.J. H a r r i s o n and  particular, provided  my  the  and  senior  since  have c o n t r i b u t e d  wish t o acknowlege t h e  of  of  and Dr. J.D. Berger o f t h e Dept. o f Zoology.  In  advisor,  framework  British  Dr.  within  Columbia  F.J.R. which  ("Max") these  of  Taylor  has  i n q u i r i e s were  i n t e r e s t s i n t h e b i o l o g i c a l realm, which range taxonomy,  symbiosis,  eukaryotes, w i t h a p a r t i c u l a r f o c u s ( o b s e s s i o n ? ) on  have  w i t h an u n e r r i n g a b i l i t y t o pose t h e b i o l o g i c a l l y served  to  stimulate  my  own i n t e r e s t i n t h e s e  Dr. P a u l J . H a r r i s o n has been a prominent source o f encouragement e n t e r e d graduate  relating  perspective,  to  Dr.  differences has  I  for this  Depts.  thesis  coupled  questions,  I  advice  others  t i d e e c o l o g y and p h y s i o l o g y t o p h y t o p l a n k t o n  dinoflagellates,  topics.  completion.  the U n i v e r s i t y  Botany,  the e v o l u t i o n  relevant  of  His eclectic  red,  but many  undertaken  o f my t h e s i s committee, composed o f Drs. F.J.R. T a y l o r  intellectual  undertaken. from  t h e author,  i t s successful  advice  Oceanography  f o r the research  phytoplankton  J i m Berger  between  generously  s c h o o l , and has been e s p e c i a l l y h e l p f u l i n g i v i n g physiology.  has  helped  dinoflagellates  and  me  From  a protozoologist s 1  t o a p p r e c i a t e t h e l i n k s and  other p r o t i s t s .  I n a d d i t i o n , he  p e r m i t t e d t h e use o f h i s e p i f l u o r e s c e n t microphotometer f o r  the d e t e r m i n a t i o n o f n u c l e a r DNA. As Antia  a -  scientific  prospective  scientist,  I  owe  S c i e n t i s t , mentor, and f r i e n d inquiry  me t o attempt  has  never  t o do l i k e w i s e .  faltered  an enormous debt t o Dr. Naval J . - whose d e v o t i o n t o t h e p u r s u i t o f  i n h i s long c a r e e r and has i n s p i r e d  xvii  Drs.  Greg  Boyer  chemical  analysis  linkages  described  Richard  0'Grady  progam  PHYSIS  helped  of in  and and  been  this  Sullivan  have l e n t t h e i r e x p e r t i s e i n the  enabling thesis.  me I  to e s t a b l i s h the chemotaxomic am  i n d e b t e d t o Drs. Dan  P a u l G a b r i e l s o n f o r i n t r o d u c i n g me for  assistance in i t s application. with  analysis. a  John  toxins,  significantly  components has  and  the  interpretation  of  Judy Acreman, c u r a t o r of the NEP  f a i t h f u l shepherd f o r my  t o the  Brooks,  phylogenetic  Dr. Gregory Gaines data  by  principal  Culture Collection,  f a l t e r i n g c u l t u r e s d u r i n g my  frequent  absences from the l a b o r a t o r y . I  am  grateful  Sciences  de  (Quebec), final  to  la  Canada,  stages  of  Lucy of  the  has  Maurice final  the  tolerance.  thesis  des  technical  program.  of the Centre Champlain Peches and  et  des  f i n a n c i a l support  L i o n e l C o r r i v e a u was  des  Oceans i n the  particularly  i n the d r a f t i n g of s e v e r a l f i g u r e s .  has  my  Therriault  Ministere  arranging  devoted s u f f i c i e n t time and e f f o r t  manuscript  tolerated  J.-C.  Mer, for  helpful in assisting  Dr.  i n the p r e p a r a t i o n  to be j u s t i f i a b l y c o n s i d e r e d as a co-author.  frequent  l a p s e s of good humour w i t h remarkable grace  With g r a t i t u d e , i t i s t o her t h a t I r e s p e c t f u l l y d e d i c a t e  She and this  thesis. Finally, times  if  I  to  my  long-suffering  parents,  who  must have wondered many  would ever " f i n i s h s c h o o l " , I hope t h a t I can assure them t h a t  the time i s n i g h .  1  CHAPTER I  INTRODUCTION  A.  C l a s s i f i c a t i o n and  The appear  obvious to  Linnean with  of  came  individuals  Problem i n D i n o f l a g e l l a t e s  morphological,  higher  be  possessing  but  determining  whether  as  and  ecological  gaps  that  l e d almost i n t u i t i v e l y t o  e s s e n t i a l l y immutable, w h i l e  With  regarded  the  advent of e v o l u t i o n a r y  rather  subjective  the  imbued ideas,  aggregations  of  s i m i l a r d e r i v e d c h a r a c t e r s , which change i n response  pressure.  pragmatic,  organisms  as f i x e d and  significance.  to  selective  opinion  most  species  "objective"  species  to  reproductive,  separate  view  the S p e c i e s  Indeed,  nonetheless  Darwin  (1859)  intellectually  a form should  of n a t u r a l i s t s having  offered  the  unsatisfying  following  advice:  "In  be ranked as a s p e c i e s or a v a r i e t y , the  sound judgement and wide e x p e r i e n c e  seems the  o n l y guide to f o l l o w . " Later, (Haldane, 1978)  the  contributions  of  geneticists  1929;  Dobzhansky,  1950;  Carson,  introduced  fundamental that  gene  interbreed,  reproductive  the  pool  years,  a  1957;  population  in  perspective  Concept" (BSC)  has  (Mayr, 1940;  been c h a r a c t e r i z e d as  1957;  phenetic  adopted  to  the  populations  are i s o l a t e d  by the  1963).  a r e i n e x t r i c a b l y l i n k e d t o the assumptions schemes  Wright,  s e r v e as  S p e c i e s were d e f i n e d as dynamic a c t u a l i t y or p o t e n t i a l l y , and  This  biologists  Lewontin, 1974;  concept w i t h i n which p o p u l a t i o n s  units.  either  concepts  classification  recent  species  barriers.  " B i o l o g i c a l Species Species  a  and  underlying  d i s c r i m i n a t e between s p e c i e s .  In  s p e c i e s concept, e s s e n t i a l l y an e x t e n s i o n of  the  2  typological discrete  species  groupings  without  1962;  1973).  "Phylogenetic the  organisms  a  on  numerical  systematists  of  with  the b a s i s of c h a r a c t e r  which  define  similarities, and  advocated a c l a s s i f i c a t i o n system based  derived characters  systematists  views  adopted  to  been developed (Sneath  known  antithetical  (synapomorphies), served  as  to  cladists  those  of c l a s s i c a l e v o l u t i o n a r y c l a s s i f i c a t i o n  have  analysis  p u b l i c a t i o n of Hennig's (1966) c o n t r o v e r s i a l work  of shared  school  Advocates  The  Systematics",  possession  spawn  1974)  of  utilizing  i m p l y i n g e v o l u t i o n a r y r e l a t i o n s h i p s , has  Sokal,  on  approach,  methods  of  both p h e n e t i c s  of  or  to  phylogenetic  the  pheneticists.  (Mayr, 1963;  1970;  and p h y l o g e n e t i c s ,  Bock,  but  are  s e v e r e l y c r i t i c i z e d by p u r i s t s i n these a l t e r n a t i v e camps. Problems definition aspects  in of  1957;  intractable.  past  In  by  and  and  have  been  species  and  Crovello,  intense  an  populations  led  some  genotypic,  little  1974;  the  and has  consensus  Sneath  and  1966;  often personal.  operational exhibiting  geographical taxonomists  been  definition,  region. to  The  classify  emerged, as  1962;  1976  species  are  morphological  1973),  1970;  the  Wiley,  Bock,  1974)  i n the d e f i n i t i o n  and  of  "classic"  Jameson, 1977).  often  recognized  as  characters inhabiting a  l a c k of a u n i f i e d s p e c i e s concept populations  the  taxonomists)  to the r e p r i n t i n g of  Slobodchickoff,  distinctive  has  Brooks, 1981;  Yet p r o g r e s s  limited  phylogenetic  Zoology d u r i n g  Sokal, Funk and  applicable  1974), remain  p h e n e t i c i s t s (numerical  (Hennig,  often  and  Sokal,  i n i s s u e s of Systematic  1970;  (cladists)  universally  c l a s s i c a l e v o l u t i o n a r y taxonomists (Mayr, 1963;  concepts  discrete  modern  Slobodchikoff,  between  p u b l i c a t i o n s (as i n Mayr, 1976; As  a  surprisingly  Debates  phylogeneticists 1981)  Doyen  fact,  of  u n i f y i n g phenotypic,  a r t i c l e s appearing  decade.  (Sokal  development  "species",  (Mayr,  evidenced  the  which  are  has  geographically  3  disjunct  (allopatric)  indistinguishable, have  been  or  as  reproductively  separate  recognized  species.  distinguishable.  such  from  species.  Mayr  species  morphological applied since  morphologically  Alternatively, different  or  coined  g e o g r a p h i c a l v a r i e t i e s o f polymorphic  the  term  " s i b l i n g species" f o r incipient  which r e p r o d u c t i v e b a r r i e r s have a r i s e n without divergence.  extensively  species  I t o f t e n becomes i m p o s s i b l e t o s e p a r a t e  ecological  (1942)  within  but  among s y m p a t r i c a l l y d i s t r i b u t e d p o p u l a t i o n s t h a t a r e  morphologically "species"  isolated,  to  The  sibling  speciation  species  among  concept  concomitant  was o r i g i n a l l y  D r o s o p h i l a p o p u l a t i o n s , but has  been adopted f o r use w i t h i n a wide v a r i e t y o f o t h e r taxonomic groups,  including  protists  (Sonneborn,  1975;  Beam  and Himes, 1977; 1980b; 1982;  1984). The stable  problems  of  sibling  species,  variability, more  although  profound  morphospecies) plasticity conservatism  as  many  of  This  conservation  are  clearly  of  species form  within  or  populations.  I f morphological  selection  of  be  attributed  to  lower  taxonomists  by  the  eukaryotes, who  such  as  would a p p l y t h e  (typological  simultaneous  species or  morphological  changes, and t h e  and among g e o g r a p h i c a l  populations.  morphotype may mask v a r i a n t s ( c r y p t i c s p e c i e s ? ) t h a t  genotypically  the  to  i n response t o environmental  continuum  of  with  identification  confused  morphological  renditions  significance  dealing  unit  general  p a r t i c u l a r l y the d e l i n e a t i o n of  f o r taxonomists o f h i g h e r organisms, a r e even  often  of  the  dinoflagellates,  a  are  of  and  those  For  "species"  definition,  acute  for  dinoflagellates. term  species  master  characters  biochemically  exists,  rather  than  a  Frequently  discrete  gap  a  between  v a r i a n t s a r e merely c o n s i d e r e d as i m p e r f e c t  archetype and  distinct.  the  which  level  of  d e f i n e s t h e s p e c i e s , then t h e deviation  from t h e "normal"  4  required  to  taxonomist  segregate  and  significance  species  i s subject  of  i s an a r b i t r a r y d e c i s i o n o f t h e i n d i v i d u a l  to  dispute.  Decisions  made  regarding the  p a r t i c u l a r m o r p h o l o g i c a l c h a r a c t e r s a r e almost always made  without  knowledge o f , o r r e f e r e n c e t o , t h e genomic code r e q u i r e d t o produce  them.  Furthermore,  interpretation  of  simultaneous Clearly,  numerical  related,  features  multi-gene the  genotypic  morphological  distantly plate  t h e p l e i o t r o p i c e f f e c t o f many genes c o m p l i c a t e s t h e  analysis variants  in a  used  complex  and p h y l o g e n e t i c r e l a t i o n s h i p s based upon t h e  to  of  several  characters.  among d i n o f l a g e l l a t e p o p u l a t i o n s a r e more  phylogenetic separate  expressed  morphological  sense,  them  i f , f o r example, t h e t h e c a l  a r e t h e product o f a c o o r d i n a t e d  through s e v e r a l m e t a b o l i c r e a c t i o n s , than i f  s t r u c t u r a l v a r i a t i o n i s the r e s u l t of a single point-mutation.  also  true  i f the  observed  polyphenism  a l t e r n a t e morphotype due t o e n v i r o n m e n t a l The the  case  is a  arising  aggregation  influence.  individuals  and  conceived obligately  to  the  level  rather  possessing  mutually for (Mayr,  on t h e b e l i e f t h a t  e v o l u t i o n a r y u n i t c o m p r i s i n g a common gene  interfertility,  particularly  conventionally  restricted  sexual  are not  synonymous,  asexual  fundamental  from of  definitions  t h e e x p r e s s i o n o f an  f o r t h e e s t a b l i s h m e n t o f t h e BSC i s based  species  pool,  i s simply  This i s  shared  exclusive, the  but  lower  1957),  they  than  the s u b j e c t i v e  similarities.  These  are not n e c e s s a r i l y  eukaryotes.  The  BSC,  as  i s inappropriate for exclusively  self-fertilizing  organisms,  of the i n d i v i d u a l .  s i n c e t h e gene p o o l i s  This r e s t r i c t i o n  effectively  e l i m i n a t e s i t s a p p l i c a t i o n t o a huge p r o p o r t i o n o f t h e p r o t i s t s . The to the  usefulness  adequately leading  o f t h e BSC depends upon t h e a b i l i t y o f t h e i n v e s t i g a t o r  d e s c r i b e t h e common gene p o o l . proponents  of  t h e BSC,  deleted  I n f a c t , Mayr (1969), one o f references  to p o t e n t i a l l y  5  interbreeding which  populations  referred  "potentially"  mainly  to  to  his earlier  geographically  definition  (Mayr, 1940),  i s o l a t e d populations.  I f the p o t e n t i a l f o r interbreeding  laboratory  settings,  populations  which  geographical  or  other  i f breeding  hand,  co-occurring population  may  be,  the in  The word  i t open t o v a r i o u s  i s a c c e p t e d as a v a l i d  f o r membership i n t h e same s p e c i e s , but i n t e r b r e e d i n g  optimal  sensu  in  has a degree o f i m p r e c i s i o n which has l e f t  interpretations. criterion  used  i s limited  BSC i s p o o r l y a p p l i c a b l e t o n a t u r a l  reality,  reproductively  isolated  e c o l o g i c a l ( d i s t i n c t habitat preference) b a r r i e r s .  members cannot  stricto  "potential"  of  be  On t h e  i s n o t a c c e p t e d as p a r t o f t h e BSC,  t h e same morphospecies from the same  a c c e p t e d as b e l o n g i n g  unless  by  interbreeding  geographical  t o t h e same b i o l o g i c a l  i s actually  observed  or  species can  be  definitively inferred. The  recognition  o f d i s c r e t e " b i o l o g i c a l s p e c i e s " among s e x u a l l y a c t i v e  protists  i s hampered  mating.  Although  low  viability  flow  this  "species",  in  the  use  the  exceptional  occurrence  of  interspecific  o f t e n r e s u l t s i n progeny t h a t a r e i n f e r t i l e o r o f  (Sonneborn,  between  ambiguity  by  1957), albeit  i t demonstrates the p o s s i b i l i t y o f gene in  a  restricted  sense.  o f t h e word " s p e c i e s " t o a p p l y  typological  (1957)  f a v o u r e d t h e use o f t h e term "syngen" f o r organisms known t o share a  common  gene  or  similar  interfertile  and  whether  of  minimize  groups  pool,  aggregations  t o both  To  is  an  additional  Sonneborn  n o t such groups c o u l d be d i f f e r e n t i a t e d as  s e p a r a t e s p e c i e s on t h e b a s i s o f c o n v e n t i o n a l There  individuals,  problem  in  taxonomic  criteria.  the r e c o g n i t i o n of species  dinoflagellates  which  the  d i f f e r e n t taxonomists have adopted t h e s e organisms i n t o t h e  fact  botanical  that or  i s e s s e n t i a l l y a nomenclatural a r t i f a c t a r i s i n g  among  zoological  realms,  or  have  assigned  them an  from  intermediate  6  position  as  protists  (approximately primary  half)  (Taylor, are  producers,  clearly  they  are  community.  Yet  the  parasites,  or  free-living  Dinoflagellates (Taylor,  have  1976b;  animal-like accorded whereas  their  the  or  status  1964)  level,  the  example,  the  to  same  evolutionary  affinities  and  their  "plants",  (Dodge, 1984)  autotrophic  phytoplankton  obligate  heterotrophs.  w i t h the c i l i a t e s  flagellar  motility  dinoflagellates  is  are  usually  (DIVISION: PYRRHOPHYTA or DINOPHYTA),  diminished  to  the  order  level  [ORDER:  (Hyman, 1940; G r e l l ,  exhibiting  a  and  only  At t h e  Nomenclature (ICBN; Voss e t a l . , 1983)  of  dinoflagellates  1976a;  1985).  compressum  variants -  for  within  a  At  "subspecies",  which i m p l i e s  reflects  a  e.g.,  dinoflagellate  infraspecific  greater  commonly  a species,  tropical  morphotype.  probably  the  For  r e c o g n i z e the use o f t h e  "forma" o r " v a r i e t a s " , which b o t a n i s t s  var.  use  (Taylor,  or f o r m a l l y  morphological  compressed  can  1973)]  can a r i s e due t o t h e a p p l i c a t i o n o f e i t h e r  not r e g u l a t e  minor  bahamense  isolation,  group  designations  Pyrodinium  an  Code o f Z o o l o g i c a l Nomenclature (ICZN; S t o l l e t a l . ,  does  designate  zoologist  the  or  Code o f B o t a n i c a l  ICZN  infraspecific use  of  facultative  As  anomalies  International to  members  (Barnes, 1968) or D i n o f l a g e l l a t a  International the  as pigmented  s p e c i e s a r e n o n - p h o t o s y n t h e t i c symbionts,  1980a),  is  -  phylum PROTOZOA, when they a r e c o n s i d e r e d as "animals".  infraspecific the  1978;  significance  Dinoflagellida within  possible  Many d i n o f l a g e l l a t e s p e c i e s  plant-like  prominent  remaining  characteristic.  phylum  1976b; 1985).  l e v e l , the  p a r t i a l reproductive degree  of  genetic  differentiation. A species  further is  discrepancy  related  hybridization.  Among  to  the  between  the  significance  botanical attached  and z o o l o g i c a l view o f to  the p o t e n t i a l f o r  z o o l o g i s t s , e v i d e n c e o f h y b r i d i z a t i o n would  generally  7  be  considered  (Mayr,  to  1970).  Yet  reproductive  as  evolutionary  populations,  botanical present  the  major  emphasis than  species  affinities  who  facto  and  of  totally  t o our  However,  as  within  V a r i a b i l i t y and  i f ecological  intact  (Grant,  and  among  satisfactory. remain  and  1971).  populations,  i n t e r p r e t a t i o n of the BSC  little  As  long as the  studied,  the  s i g n i f i c a n c e of  u n d e r s t a n d i n g of s p e c i a t i o n  to  relies  on  breeding divergent  hybridization  i n t h i s group of  these r e l a t i o n s h i p s become b e t t e r  such u n r e s o l v e d i s s u e s w i l l prove i n c r e a s i n g l y  B.  among  on n o n - i n t e r b r e e d i n g w i t h o t h e r  z o o l o g i c a l a t t i t u d e s toward the threat  hybridization  speciation, remain  placed  This  dinoflagellates  little  organisms.  been  interbreeding  not  involving  relationships  boundaries.  is  in and  on  speciation  o f t e n de-emphasize the p o t e n t i a l f o r  criterion  has  case f o r incomplete  tend t o be more aware of v a r i a t i o n s i n  particularly  discontinuity  more  exclusion,  de  versus outbreeding species,  interfertility  establish  a  botanists,  strategies,  inbreeding  Recently,  constitute  characterized,  problematic.  Speciation  1. Phenotypic v e r s u s Genotypic E v i d e n c e  The  fossil  Mesozoic, 1980a  and  remarkable (Taylor, virtually  and b).  record  of  p o s s i b l y the P a l e o z o i c In  some  conservatism 1980b;  dinoflagellates  Evitt,  identical  to  species, of  1985). their  is  long  -  dating  from  or Precambrian p e r i o d s ( T a y l o r , it  form, A few  i s also distinguished  particularly  of  extant species  fossilized  progenitors  cyst can  by a  the 1978;  rather  morphotypes,  be r e c o g n i z e d  as  (e.g., S p i n i f e r i t e s  8  spp.)  (Taylor,  1976a).  physiological apparently 1951; in  Nevertheless,  variants  within  culture. of  among  in  size  and  involving  variants  shape.  differences  in  plate  dinoflagellate  populations  i s a l s o w e l l known (Braarud, 1945;  1976a; 1980b), both from n a t u r a l assemblages and  Morphological  cell  expressed those  and  b e l o n g i n g t o t h e same s p e c i e s  Bursa, 1963; T a y l o r ,  basis  t h e o c c u r r e n c e o f morphotypic and  may  be g e n e r a l l y r e c o g n i z e d  on t h e  F o r t h e c a t e forms, v a r i a t i o n may a l s o be  p l a t e t a b u l a t i o n and s t r u c t u r e , p a r t i c u l a r l y  fusions  or  subdivisions,  or  the  presence  of  supernumerary p l a t e s . Dinoflagellate to  make  taxonomists working w i t h f i e l d m a t e r i a l  species  decisions  "prehistory",  and  morphological  characters  and  distortion  depletion "normal"  or  of  sexual  expressed patterns,  specimens  of  the  range  population.  represent  of  a  unknown  of v a r i a t i o n i n  The d i m e n s i o n a l changes  t h a t can occur f o l l o w i n g substantial  observations and  on  fixed), could  Although a  cultures  Scrippsiella  that stable differences  plates,  within  few  nutrient  d e v i a t i o n from t h e  by t h e i r u n u s u a l appearance.  genetically  thecal  the  a  forced  An unwary o b s e r v e r o f such specimens i n n a t u r a l  1951)  suggested  morphospecies.  plate  cell.  tamarensis  trochoideum)  was  events  (1945;  (=Gonyaulax)  knowledge  within  may be m i s l e d  Braarud's  (presumably  little  upon  thecal plate patterns  vegetative  populations  certain  with  based  are often  of  Protogonyaulax  trochoidea  (=Peridinium  i n morphological  i n c l u d i n g mean c e l l be  recognized  among  s i z e and t h e shape o f clones  of  some v a r i a t i o n i n t h e s e m o r p h o l o g i c a l given  clone,  characters  t h e same characters  primary f e a t u r e s , e s p e c i a l l y t h e c a l  remained e s s e n t i a l l y unchanged i n long term c u l t u r e s .  This  was  p a r t i c u l a r l y t r u e w i t h i n t h e c l o n e which he d e s i g n a t e d as G. tamarensis  var.  excavata.  9  Among  dinoflagellates,  the  examples  of  features  ( T a y l o r , 1976a; 1980b; E v i t t ,  number  morphological  genus  variation  o f t h e a p i c a l horns s u b j e c t  changes,  varying  status,  but  contemporaneous  considered  i n s i z e , o v e r a l l shape, and e x t e r n a l 1985).  Not o n l y a r e t h e l e n g t h s and  t o e n v i r o n m e n t a l l y induced m o r p h o l o g i c a l  d i f f e r e n t morphotypes may c o e x i s t even w i t h i n a  natural  population.  continuum,  to  p r o v i d e s t h e most extreme  w i t h changes o f season, water temperature and n u t r i t i o n a l  substantially  morphological  Ceratium  be  are  validly  observed  separable  criteria,  the  terminal  would  t r a n s f e r a b l e t o another subgenus ( E v i t t ,  daughter  cells  within  a  usually  species cells  accounted  f o r by  may  above  be  attributed  to  be  due  to  sexual  dimorphism  a  previously  1976a).  Extreme  based upon m o r p h o l o g i c a l  i f considered 1985).  differences  in isolation,  Such i n t r a - c h a i n between p a r e n t and  I n some c a s e s , v a r i a t i o n  l i f e h i s t o r y stage d i f f e r e n c e s between cysts. and  A d d i t i o n a l v a r i a t i o n may be  anisogamy expressed i n o p p o s i t e  1980b).  example s e r v e s t o i l l u s t r a t e t h a t t h e b a s i s f o r v a r i a t i o n i n  dinoflagellates,  may  c e l l of a c l o n a l chain,  and gametes o r s e x u a l  mating types ( T a y l o r ,  usually  that,  a r i s i n g through m i t o t i c d i v i s i o n .  vegetative  The  extent  forming  variants  (Taylor,  occur  can  an  species  can  variation  such  between  variation  be  to  also  Intergradations,  including  alternate  stages  be d i r e c t l y determined from o b s e r v a t i o n s  in  t h e l i f e h i s t o r y , cannot  of f i e l d  specimens, but i t  be e m p i r i c a l l y demonstrable through e n v i r o n m e n t a l m a n i p u l a t i o n o f c e l l s  in  culture.  at  least  Many d i n o f l a g e l l a t e s (perhaps a l l ) have a s e x u a l  potentially  sexual,  sexuality  is  currently  sexuality  is  characterized  induced  under  conditions  but f o r t h e v a s t m a j o r i t y  unknown in  ( P f i e s t e r , 1984). dinoflagellates,  stage o r a r e  of species  (>98%),  I n t h e few cases where i t can  u s u a l l y o n l y be  o f e n v i r o n m e n t a l s t r e s s , such as n i t r o g e n  (Turpin  10  et  al.,  1978;  P f i e s t e r , 1984)  deprivation.  The  dinoflagellate species)  or phosphorus (Anderson and  available  species  in  evidence  which  sexual  uptake  variations,  kinetics,  been  cultured  observed  (<30  changes  rate,  i n growth r a t e , n u t r i e n t  excretion,  production  etc.,  may  occur i n response t o r e l a t i v e l y s h o r t term e n v i r o n m e n t a l changes,  within  a  of  expressed  morphologically,  botanists  as  tney  may  not as  or  to  which  would  genotypically  filter  to  consistency  allow  for  the  perturbation, must  through  several  (Brand,  1981).  variation  be  designated  not  distinct  within  the  elimination  of  Since  differences  are  s t a b l e environment s e r v e s v a r i a t i o n due  r a t h e r than g e n o t y p i c d i f f e r e n c e s .  followed  by  ecological,  species.  a r i s e from g e n e t i c  h e r i t a b l e , long term growth i n a c o n s t a n t and a  be  When  I f v a r i a n t s become g e n e t i c a l l y f i x e d ,  varieties do  synthesis,  to s e v e r a l c e l l d i v i s i o n s .  variants  define  morphological  modifications  environmental  of  "formae".  considered  physiological,  hours  phenotypic  alternative  be  phenotypic  several  nucleic acid  of  metabolites,  scale  the r a t e of p r o t e i n and  the  secondary  time  and  has  on  1985)  r a r e ( P f i e s t e r , 1984).  including  photosynthetic  studies  fusion  suggests t h a t s e x u a l i t y i s r a t h e r  Physiological  from  Lindquist,  Phenotypic  and  preferably  c u l t u r e t r a n s f e r c y c l e s , to s t a b i l i z e g e n o t y p i c  expression  In t h i s way, within  f o r several c e l l generations,  s o l e l y to  i t i s p o s s i b l e t o e s t i m a t e the t y p i c a l  a morphospecies, and  to r e c o g n i z e  of  fixed  t r u l y aberrant  range cells  (Bursa, 1963). The  stability  dinoflagellates characters polyedra 1975),  has  been  genotypically noted  i n c l u d e the p e r s i s t e n c e in  and  in  previous  long-term  studies.  characters  in  Examples of  such  of rhythmic b i o l u m i n e s c e n c e i n Gonyaulax  c u l t u r e , even a f t e r s h i f t s the  variant  stability  i n e n v i r o n m e n t a l phasing in  acclimated  growth  (Hastings,  rates  among  Protogonyaulax  tamarensis  Crypthecodinum content well  per  as  Rae,  cell  base  and  enzymatic  Beam  et  Beam  and  differences  r e s t r i c t i o n endonuclease  p o i n t and buoyant d e n s i t y have been observed b).  1976;  al.,  stable  DNA  cleavage  ( S t e e l e and  R e c e n t l y , g e l e l e c t r o p h o r e t i c s t u d i e s have r e v e a l e d within  Daggett  and  among  1984;  dinoflagellate  morphospecies  and Nerad, 1980; Schoenberg and Trench,  1982; Hayhome and P f i e s t e r ,  Himes,  in  For  1980; Beam and Himes, 1982; 1984), as  p a i r composition,  variation  (Schoenberg,  1981; Brand e t a l . , 1981a).  dinoflagellates,  (Himes and O'Brien,  melting  1980a  Whitten  cohnii-like  i n DNA  patterns,  i s o l a t e s (Brand,  Cembella  and  1980a;  1983; Watson and L o e b l i c h , 1983;  T a y l o r , 1985a and b; Hayhome, 1985;  and Hayhome, 1985).  The  use  nucleic  of  acid  circumscribe  biochemical and  techniques,  protein  including  sequences,  and  analysis of  secondary  isozymes,  metabolites,  to  s p e c i e s and uncover c r y p t i c p h e n e t i c and g e n e t i c r e l a t i o n s h i p s  has  merit  the  r e s u l t s o f a number o f b i o c h e m i c a l approaches a r e compared f o r t h e same  group are  when  o f organisms. more  qualified, If also  the  the  to stable t r a i t s .  I n a sense,  "objective" and  since  T h i s i s p a r t i c u l a r l y t r u e when  b i o c h e m i c a l l y - b a s e d c l a s s i f i c a t i o n schemes  variation  can  be  be  applied.  species.  the It  biochemical  possibility considered  of in  readily  quantified  data a r e amenable t o a n a l y s i s by n u m e r i c a l  i n t e r m e d i a r y pathways a r e known, meaningful  regarding  all  applied  techniques.  character weighting  U n f o r t u n a t e l y , an a r b i t r a r y d e c i s i o n must s t i l l  threshold  level  i s n o t obvious characters biochemical  of  difference  required  to  and  may  be made  d e f i n e a new  t h a t t h i s l e v e l s h o u l d be f i x e d t h e same f o r  or  even  f o r a l l groups  parallelism  and  o f organisms.  coincidence  should  e f f o r t s made t o r e c o n c i l e contemporary m o r p h o l o g i c a l  The  a l s o be species  12  with biochemical  variants.  Biochemical affinities  approaches  within  of  toxicity  Schmidt  et  al., 1979;  1985;  Oshima  L o e b l i c h , 1975; Other studied than  and  1978;  Shimizu,  al.,  toxin  and  et  Schmidt and  1985)  and  Loeblich,  L o e b l i c h , 1979a  Loeblich,  opposed  to  observed  term  et  a l . , 1981a  1971;  Nelson  et  Belastock,  1979a).  Brand  rates  of  et  (Guillard  (morphological, homology organisms.  and  Genetic  has  also  interbreeding)  morphological  vitamin  B-^  and  been  to  unite  biochemical)  species  to  and  1980;  and G u i l l a r d , 1968),  Murphy  Brand, 1979)  among  and and  Murphy,  reproductive i s o l a t e s from  Brand e t a l . , 1981a). the  concepts  species  circumscribe  the f a c t t h a t c l a s s i c a l taxonomy has similarities  Brand,  G u i l l a r d , 1976;  examined  1982;  been  (Guillard,  i n the  as  have  F i s h e r , 1977;  differentiation  1981;  essential  physiological  Given  responses,  Ryther, 1962;  enzymes (Murphy and  1981b.  is  to  complex -  differences,  p e r i o d i c i t y (Nelson and  d i f f e r e n t water masses (Brand, 1980; it  and  ( F i s h e r e t a l . , 1973;  division  coccolithophores  Ultimately,  genetic  acclimation  response  detectable  al.,  nana),  characters  - or s p e c i e s  b ) , n u t r i e n t uptake r a t e s (Carpenter 1976),  cell  electrophoretically 1978;  and  rates  tolerance  1980),  diatom s p e c i e s  physiological  al.,  stress  the  and  diatoms, have been more i n t e n s i v e l y  (=Cyclotella  reproduction  Brand  pollution  pseudonana short  in  Within  and  Cembella e t  f o r i n t r a s p e c i f i c g e n e t i c v a r i a n t s over a wider range o f  dinoflagellates.  1975;  bioluminescence ( L o e b l i c h  Schmidt and  particularly  taxonomic comparative  a l . , 1982a; Boyer e t a l . , 1985;  Yasumoto,  phytoplankton,  investigate  group have i n v o l v e d  Alam e t a l . , 1979;  Oshima  to  c o m p o s i t i o n ( L o e b l i c h and  Schmidt et a l . , 1978;  Thalassiosira  upon  applied  the t a m a r e n s i s / c a t e n e l l a  studies  b;  previously  d i f f e r e n c e s , and  and the  of  phenetic  genetic same  set  (DNA of  been l a r g e l y based since morphological  13  features  are  at  generalization,  least  a  indirectly  correlation  (1963)  sibling  criticized  species,  similarity  general  genetic  and  First, and  Gottlieb,  1977).  1977). (Avise  genetic  al.,  with  Kornfield genetic  generic,  specific,  and  subspecific  p l a n t s ( G o t t l i e b , 1977; Since  not  identifiable and  b;  Robinson,  1983;  inadequate  to  varieties,  or  dinoflagellates, sibling  all  and Beam  et  of  more  morphological  the  result  of  Nevertheless,  the  populations  two  of  most  between g e n e t i c  ( A y a l a e t a l . , 1974;  Avise,  similarities  particularly  similarity  has  levels  in  among  been  1976;  (Gottlieb,  animal  both  confirmed animals  Lewontin, 1974;  differentiation  characteristics  Guillard, and  1976;  Himes,  groups  at  the  (Hubby and  A v i s e , 1975)  defined species  in protists  expressed  and  in  readily  (Borden e t a l . , 1973a  morphological  Soudek and  features  may  be  p a i r s of s i b l i n g species, geographical  Morphologically by  is  C o r l i s s and Daggett, 1983;  1984),  between  ecotypes.  "biological"  among  a l . , 1974;  genetic  distinguish  as  was  the a n a l y s i s of  1984).  phenotypic  Murphy  and  and Koehn, 1975), the c l o s e r e l a t i o n s h i p  and  Ayala  degree  morphological  exceptions,  morphology  1968;  high  to  similarity.  rank  between  Throckmorton,  a  the g e n e t i c s i m i l a r i t i e s between organisms a r e  correlated  1975;  as  be made r e g a r d i n g the c o r r e l a t i o n between  taxonomic  numerous  coding,  i s o f t e n a c l o s e correspondence  Second,  Despite et  there  applied  species  relationships  assigned  positively  when the  sibling  o b s e r v a t i o n s may  similarly  usually  view that  than  phenetic  organisms.  this  between  homeostasis  genomic  apparent.  suggesting  evident  developmental related  by  to  between g e n e t i c s i m i l a r i t y , morphology,  a s s i g n e d taxonomic rank s h o u l d be Mayr  subject  sexual  exhibiting  similar  breeding  c o m p a t i b i l i t y , may  groups  of  represent true  reproductive i s o l a t i o n  (Beam and  14  Himes, 1977;  1980a and  Corliss  and  separation their  of  and  1974)  1971;  Allen  1983a  and  as  and  designated  analysis  in  eventually  between  place led  the  of  to  gross  segregate  electrophoretic Tetrahymena  1975;  profiles  ( A l l e n and  Adams and A l l e n , 1975;  body  and For  (Allen  Weremiuk,  A l l e n et a l . ,  aurelia  delineate  L a t i n binomials  level,  it  may  protists  breeding  complex,  to  could  (Corliss  the  Powelson e t a l . , 1975;  variants  t o the syngens of  as s i b l i n g s p e c i e s (Sonneborn, 1975).  of  and  of  of e l e c t r o p h o r e t i c isozyme  indeed  in  congruence  affinities.  detailed  be  impossible  to  with  the  Among members o f  a n a l y s i s of s t r u c t u r a l  s i z e , the p o s i t i o n o f the c o n t r a c t i l e v a c u o l e  revealed  species  (Sonneborn, 1939), were l a t e r  strains  regarded  morphometric  binary  Paramecium  reference  assignation  evidence  species  with  the  Use  species  kinetosomes  multivariate  of  mating t y p e s .  morphological  pyriformis  of  agreement  of c i l i a t e s ,  to d i n o f l a g e l l a t e s as w e l l .  as " v a r i e t i e s "  living  the  sibling  eventually  species  i n the  as "syngens" sensu Sonneborn (1957), on the b a s i s of  Paramecium, which a r e now  sibling  inherent  Tetrahymena, n u c l e o t i d e sequences  Members  o r i g i n a l l y considered  including  pertain  1971;  problems  sibling  electrophoretic  strains.  affinities  The  may  enzyme  Gibson,  breeding  number  similar  the  b) have been used t o e s t a b l i s h l e v e l s of g e n e t i c d i v e r s i t y among  numerically  At  discussed  Paramecium and  and  related  complex,  (1983)  observations  such  Li,  closely  Daggett  1984).  morphologically  general  ciliates,  b; 1982;  the  features  pore, and  the  not r e l i a b l y be used t o d i s c r i m i n a t e between and  Daggett,  analysis  of  1983).  members  stable  morphological  previously  designated  However,  of the P. a u r e l i a  differences species  (Gates  detailed complex  essentially  in  e t a l . , 1974;  Gates and Berger, 1976).  mating type Paramecium a u r e l i a s p e c i e s complex may  not be  a  15  strictly  analogous  including  members  exhibit  multiple  common  within  the  basis  addition cohnii  1982;  to  the  half  t y p i c a l DNA  affinities  (Beam and  evidence  et  Cembella Rae, (Beam high that  al.,  1980a and  Himes  less  major  cohnii  O'Brien, divided  and  of  the  sexual  marine (Beam and  1980;  Himes,  1977;  Beam e t a l . ,  1982)  into three variant number  and  c e l l morphotype, yet  genetically but  heterotrophic  groups  on  c e l l size.  In  variants  C.  of  fewer chromosomes were d i s c o v e r e d .  have now  species?)  The  contained  on  been d i v i d e d the  only  typical larger-celled  d i f f e r e n t i a t e d groups, were  electrophoretically  Schoenberg  and  Hayhome and  Taylor,  1985a and  b ) , and  basis  i n t o 31  of  not  sexually  t h e i r breeding  1977;  genetic  P f i e s t e r , 1983;  and  isozyme  Watson and  b; Hayhome, 1985), DNA  1980a and  diversity  determined  Trench, 1980a; Daggett and  breeding a f f i n i t y  genetic d i v e r s i t y within this  exception  be  Himes, 1984).  from  Himes,  Stylonychia,  mating types may  the  of  chromosome  larger  1982;  and  t o C.  and  DNA  the  (sibling  1976;  and  isolates  referable  distinguishable,  strains  Beam  clonal  c o n t e n t were a l s o observed.  representing  (Schoenberg,  with  genus  ciliates,  unknown i n d i n o f l a g e l l a t e s .  content,  exhibited  compatible  The  DNA  much  which  morphologically  the  Multiple  yet,  Other  typical larger size strains, smaller-celled  Variants  variants,  complex and  species,  on  1984;  mean  possessing  the  bursaria  t h e s e s t r a i n s c o u l d be  of  dinoflagellates.  s p e c i e s group, the number of mating types and  tentatively  that  for  mating types (Nanney, 1980).  studies  b;  indicated  P.  groups i s c u r r e n t l y  dinoflagellates and  the  cohnii  Intensive  1980a  of  system  dinoflagellate  Crypthecodinium compatibility  model  b;  and  1982;  Nerad,  Loeblich,  analysis  1984), r e v e a l s  effectively  masked  by  1980; 1983;  (Steele  segregational genetic  and  studies  a conspicuously  among d i n o f l a g e l l a t e s p e c i e s . is  variation  In the  way  morphological  16  conservatism, (Allen  and  dinoflagellates Weremiuk,  Paramecium  may  1971;  Allen  ( A l l e n and Gibson,  a l s o f a i l t o be r e f l e c t e d  resemble and  ciliates,  Li,  1974;  to  i n the expressed  sufficient  exploitation  of  offered  maintaining  by  colonization genotypes (e.g.,  genetic  favourable  of  ecological  marginal  in  which c o u l d s u r v i v e adverse seasonal)  or  tolerance  limits  often  organisms  and  reversible  response  to  populations  must  retain  deleterious  effects  influence  been  actively  Bryant,  Ayala, evidence  1976),  1976;  in  but  at  present  t h a t would support  also  enable  phenotypic operate  phenotype,  genetic  load  -  can  the  responses within  the  shifts.  stability  " f i n e - t u n e " the  At the same time, to  tolerate  the  the s u r v i v a l c o s t o f m a i n t a i n i n g inviability.  h e t e r o g e n e i t y on g e n o t y p i c  (Levins,  Hedrick  s e l e c t i v e advantages  A d i v e r s e genome, accommodating r a p i d  sufficient  debated  must  environmental  of environmental  effective  shifts, either periodic  Acclimated  accumulated mutations - which can l e a d t o The  The  the  ability  ensure t h a t the p o p u l a t i o n i n c l u d e s  changes  genetic  permit  population  physiologically  transient  of  to  environmental  s e t by the genome.  freely  the  catastrophic.  morphologically  1975;  diversity  Stability  niches.  h a b i t a t s and  and  morphotype.  heterogeneity  variation  expressed  has  1984)  s u r v i v a l of d i n o f l a g e l l a t e s p e c i e s i s c o n t i n g e n t upon t h e i r  retain  and  Williams,  1975), f o r which s i m i l a r l e v e l s of  2. Mechanisms f o r M a i n t a i n i n g V a r i a b i l i t y and  The  such as Tetrahymena  1968;  Lewontin, 1974;  e t a l . , 1976;  V a l e n t i n e , 1976;  polymorphism Ayala et a l . , Valentine  and  t h e r e i s no preponderant body of e m p i r i c a l  a strict  causal relationship.  Nevertheless, i t  17  is  reasonable  to  variability  within  represents recent  conspecific  electrophoretic and  Gillespie  and  Valentine,  1976;  evidence  molecular Langley,  selective  Nevo,  effects  in  frequencies,  allele  adaptations  supports  1974;  of  in  Hedrick 1983).  chemical  ultimately  Attempts indicated  to  suggesting  produce  1969)  variation  supported  suggested  pressures and  electrophoretic  heterogeneity  niche-width  by  that  1976;  Nevo (1983) and thermal  found  populations  The  bulk of the  t h a t s t r u c t u r a l gene  adaptive  al.,  (Powell,  1972;  Johnson,  1976;  co-workers examined  pollutants  on  allozyme  statistically different  genetic  adaptation.  shifts  When  such  e c o l o g i c a l , or g e o g r a p h i c a l  ( L e v i n , 1970).  evidence,  have  with  temporal  and  followed v a r i o u s environmental  hypotheses studies  (Nevo,  primarily  populations  a r e reduced,  isolated  are  reproductive, occur  view  allelic  c o r r e l a t e the degree of g e n o t y p i c polymorphism, u s u a l l y as  from  environmental  the  et  and  marine organisms, and  i s o l a t i o n , s p e c i e s d i v e r g e n c e may  evidence,  dinoflagellate  polymorphisms  1978;  polymorphism  or  haploid  a s u r v i v a l strategy with adaptive s i g n i f i c a n c e .  modifications  the  s p e c u l a t e t h a t the maintenance of h i g h l e v e l s of  1978).  The  spatial amplitude  first  l i n e of  on D r o s o p h i l a (Prakash e t a l . ,  o f the c e n t r a l h a b i t a t , where s e l e c t i v e  m a i n t a i n h i g h e r l e v e l s of polymorphism than  marginal  p o p u l a t i o n s c a p a b l e of s u r v i v i n g o n l y w i t h i n narrow t o l e r a n c e  limits. The  second  populations, to  paradigm,  relates  increased higher  protein  variation  theoretical  the  genotypic  through  levels  also  based l a r g e l y on experiments on D r o s o p h i l a  s u r v i v a l of p o p u l a t i o n s i n u n s t a b l e environments - and hence p h e n o t y p i c  of  (Powell,  considerations,  genetic 1972; Levins  diversity,  - flexibility, with  Gillespie  and  (1968)  reasoned  as  expressed  consequently  Langley, that  1974).  greater From  organisms from  18  unstable niche it  utilization  is  that  environments  should e x h i b i t higher genetic v a r i a t i o n , to  and  t o maximize e n v i r o n m e n t a l t o l e r a n c e .  the r e p r o d u c i b i l i t y and is  crucial  polymorphism. offered  populations  the  aquatic  Euphausiids;  other  with  to  and  stability. that  the  in  stable  idea  that  are  phenotypic  four  protein of  marine environments i s variation  in  Pacific  electrophoretic diversity  homogeneous environments does not  was  and lend  g e n e t i c polymorphism n e c e s s a r i l y i n c r e a s e s  V a l e n t i n e and A y a l a  species,  a  species was  resource  possible  mutation,  genetic on  1976;  (1976) suggested t h a t  of  krill,  associated  hypothesis  Valentine,  with  as  compared  greater  ( V a l e n t i n e , 1976)  the to  resource proposes  m u l t i p l i c i t y of s p e c i a l i s t a l l e l e s may  be  mechanisms  for  the  observed  gene f l o w through m i g r a t i o n , E v i d e n c e has  equilibrium  now  recombination,  s h i f t e d away from the  and  classical  which proposes t h a t most a l l e l e s a r e h i g h l y  the b a s i s of t h e i r e n v i r o n m e n t a l goodness of f i t , and  variation in  intrapopulation genetic d i v e r s i t y .  resulting in It is likely  l a r g e number of a l l e l i c v a r i a n t s coded f o r by the genome are e i t h e r  selectively balance  level  tropical  trophic  intraspecific a  in  acclimation.  of  little  on  in  ( K o r n f i e l d and Koehn, 1975;  environments  dinoflagellates:  selected  model  i n the p o p u l a t i o n to cope w i t h d i f f e r e n c e s i n m i c r o h a b i t a t s .  There  model  study  apparently  antarctic This  maintained  that  from  heterozygosity  temperate  fluctuation  i n c r e a s i n g l y u n s t a b l e water masses.  1978a; Wright, 1978).  higher  this  the  environmental i n s t a b i l i t y  White,  case,  hand, a n a l y s i s of a l l e l e f r e q u e n c i e s of o t h e r marine  organisms  credibility  for  (1972)  p o s i t i v e l y c o r r e l a t e d with On  p e r i o d i c i t y i n the e n v i r o n m e n t a l  support  Bromley's of  In t h i s  to any p o t e n t i a l s e l e c t i v e advantage a r i s i n g from g e n e t i c Some  by  optimize  neutral  equilibrium.  (Kimura The  and  Ohta,  balance  1971)  hypothesis  or  subject  to a  shifting  (Dobzhansky e t a l . , 1977;  19  Wright,  1978;  alleles  subject  gene  loci,  complete of  Nevo, to  with  no  highly  the  by  either  polymorphism,  accumulation  c o n t r i b u t e an o v e r b e a r i n g  haploids of  haploid  favourable  environmental effectively diploid  evaluation. masked  -  a  as  less  mutation t o s u p p l y  be  which  a g a i n s t the  substitutions  homeostatic  structural  gene  et  may  be  not  detected  by  mutations  would not  load.  Noctiluca,  d i n o f l a g e l l a t e s are  (Pfiester,  1984).  The  functional  p r i n c i p l e advantage  i n the a b i l i t y of mutants to r a p i d l y u t i l i z e are  immediately  Since  expressed  unfavourable  and  subject  d i n o f l a g e l l a t e s may  to  a l l e l i c v a r i a n t s cannot  express  a  be  with  multiplicity  of  c o n s e r v a t i v e s t r a t e g y , based p r i m a r i l y upon r e c u r r e n t  genetic v a r i e t y .  to  more  stringent  mechanisms  may  constraints.  permit  According  Population the  exogenous  variation  deme gene  1970).  (local flow  arising  from recombination  interbreeding  from  V a r i a n t s and  other  to  Mayr (1970)  the morphotype t o remain t r u e to form as  l o n g as g e n e t i c v a r i a t i o n does not exceed p r o h i b i t i v e !  (Mayr,  Hedrick  c o n t r a s t t o the genotype, the morphotype i n d i n o f l a g e l l a t e s seems t o  subject  within  fitness  hypothesis  s e l e c t i v e l y n e u t r a l r e c e s s i v e s , as i s the case  heterozygotes,  genotypes  In  of  strategy l i e s  mutations,  ("wild t y p e " ) g a i n i n g  evolutionary  minor  of  i n the v e g e t a t i v e stage  the  of  i n t e r p r e t a t i o n of the p e r s i s t e n c e of g e n e t i c  genetic  exception  that  the  In  the  allele  Arguments f o r and  claim  electrophoresis.  With  adaptive  have been w e l l debated (Johnson, 1973;  influenced  the  the e x i s t e n c e of a m u l t i p l i c i t y  s e l e c t i v e advantage, f o r a l a r g e number of  dominance.  including  substantially  in  single  neutrality  1976),  postulates  shifts  monomorphic  selective  al.,  1983)  population),  non-reproductively  aberrants  boundaries. can occur e x c l u s i v e l y or  as  a  result  of  isolated  populations  in natural dinoflagellate  populations  20  could  also  cells  differing  water  masses.  of  genetic  in  situ  conceivably  of  sexual  cohnii 1984),  has  been determined. significant  but  to  cytoplasmic  material  fluctuations  in  and  also  caused  be  particularly, A genetic  number  or  mechanism  partitioning 1971;  1977).  large  There  differences  may  be  unequal  (Beam  and  populations  similar  be and  random  in  i n a population  could  c e l l c y c l e phasing,  a species Wiley,  may  be  and  invoked to account f o r  polyploidy. may  1981),  V a r i a t i o n i n chromosome  s e r v e as a s t a s i p a t r i c s p e c i a t i o n whereby  such  g i v e r i s e t o post-mating r e p r o d u c t i v e have  species  e v e n t s , i n c l u d i n g intrachromosomal gene  p o l y t e n y and  1978a;  a  notoriously  and  Fritz,  inefficient  1984),  commonly  major chromosomal isolation.  mitosis  (Shyam  resulting  and  in  numbers of chromosomes i n daughter n u c l e i  the  (Silva,  O f t e n t h e r e i s an apparent random v a r i a t i o n i n the number of  chromosomes d i s t r i b u t e d per The  homothallic  d i s t r i b u t i o n of f u n c t i o n a l m e t a b o l i t e s  mechanisms  speciation  Triemer of  The  stages.  structure within  1978;  known.  V a r i a t i o n among c e l l s  asynchronous  aneuploidy,  Dinoflagellates  maintenance  i n the p a r t i t i o n i n g of n u c l e a r  mitosis.  p r o d u c t i o n and  and  (White,  r e c o m b i n a t i o n t o the  i n t r a s p e c i f i c m o r p h o l o g i c a l v a r i a t i o n may  chromosomal  rearrangements c o u l d  Sarma,  of  in l i f e cycle of  from d i f f e r e n t  frequency of s e x u a l i t y i n n a t u r a l  during  the  by  genotype d e r i v e d  a g g r e g a t i o n of  sexually active in culture  non-genetic v a r i a b i l i t y  variation  duplication,  the  Koshland, 1976).  variety  and  are not  unusually  amount  attributed  (Spudich  fusion  is  1977;  A  p h y s i c a l m i x i n g and  i n d i n o f l a g e l l a t e s cannot be a s s e s s e d , s i n c e t y p i c a l  Himes, not  to  c o n t r i b u t i o n of g e n e t i c  diversity  Crypthecodinium  due  i n p h y s i o l o g i c a l status The  rates  arise  cell  (Dodge, 1963).  number of chromosomes i n some d i n o f l a g e l l a t e s ( r a n g i n g  up  to  21  approximately  220;  Shyam  and  1976a)  as  evidence  copies  of  homologous chromosomes b e a r i n g  structural volume, Soyer  features  and  the  (1973)  dinoflagellates,  evidence as  but  the  populations  among  related  e t a l . , 1981;  Trench, 1985) On  variant a l l e l e s .  On  the b a s i s  s t a t e of chromosomes, Haapala  polyploidy  existence  belonging  species H o l t and  allows  as  a s p e c i a t i o n mechanism i n  chromosomal  in  to the same m o r p h o l o g i c a l s p e c i e s ,  and  Sarma, 1978;  P f i e s t e r , 1982;  Loper e t a l . ,  1980;  Himes, 1984;  Blank  Beam and  this intriguing possibility. e v i d e n c e from Symbiodinium  1985)  does  support an argument f o r p o l y p l o i d y i n t h i s s p e c i e s  since  the  number of 25,  detailed  atypical)  information sequences of  on in  polyteny  Hinnebusch indicated repeated sequences,  chromosomes  varied  an  r a t h e r than i n an e x p o n e n t i a l  Although (possibly  i t s a l l i e d possible s i b l i n g species  (=Gymnodinium)  and  of  the or  et that DNA, as  genomic  evidence  dinoflagellate  the  frequency,  polyploidy  is  complexity  1980).  The  a p p r o x i m a t e l y 60%  et  data  Trench, complex,  progression. available  species,  (Roberts  (Blank and  i n t e g r a l m u l t i p l e of a base  o n l y from a s i n g l e  Crypthecodinium and  location  d i n o f l a g e l l a t e chromosome does not  al.,  spp.,  multiples  microadriaticum  number  and  model f o r the d i n o f l a g e l l a t e  of  (Shyam and  the other hand, the r e c e n t  not  of  l a r g e chromosomal  suggested L o e b l i c h e t a l . (1981) f o r Heterocapsa  dinoflagellate  and  condensed  for  (Taylor,  c o u l d be polygenomic, w i t h m u l t i p l e  proposed a h i g h l y p o l y t e n e  inconclusive,  Loeblich  were p r e v i o u s l y c i t e d  by e l e c t r o n microscopy, the  permanently  The  closely  such s p e c i e s  visible  have  chromosome.  is  that  Sarma, 1978)  al., from  of repeated  suggest a h i g h  1974; DNA  c o h n i i , the  degree  A l l e n et a l . ,  renaturation  of the genome c o n s i s t e d of low  DNA  1975;  kinetics complexity  w i t h mismatched r e p e a t e d sequences i n t e r s p e r s e d among unique in  eukaryotes.  The  remaining  40-45%  of  the  DNA  was  22  considered highly al.,  to  represent  complex sequences. 1974;  hypothesis nucleus  Tuttle that  many  that  dinoflagellate preceded a  and  DNA (number o f c o p i e s p e r c e l l = 1.17) o f  The f r e q u e n c i e s o f induced mutations (Roberts e t Loeblich,  genes  1974;  occur  as  only  were  c o n s i s t e n t with the  single  by  genotypic  variation  populations,  is  the  c o p i e s , and t h a t t h e  basis  differences  between  1968;  precede  follow  or  between  individuals  Avise  and  genetic  Ayala,  p u t a t i v e cases o f sympatric  shifts  ecology,  parapatric potential  not  1976)?  diversification?  discounted  are  (Mayr, 1963)?; o r can  serve  as  an  and among m o r p h o l o g i c a l l y s i m i l a r p o p u l a t i o n s  Throckmorton,  in  speciation i n  massive genomic r e o r g a n i z a t i o n i n v o l v i n g a l l e l i c s u b s t i t u t i o n s  allelic  mechanism  of  a fundamental q u e s t i o n i s posed: i s s p e c i a t i o n  l a r g e number, i f n o t t h e m a j o r i t y , o f gene l o c i  minor'  which  1977)  i s functionally haploid.  Given  at  unique  isolation (Hubby and  I n s h o r t , does s p e c i a t i o n Mayr  (1963,  1970) has  speciation - speciation arising  p h y s i o l o g y , r e p r o d u c t i v e mode, e t c . , w i t h i n  from  populations  g e o g r a p h i c a l l y i s o l a t e d - as c r y p t i c cases o f a l l o p a t r i c or  speciation. importance  Yet  Mayr  has  generally  not  acknowledged  the  o f chromosomal rearrangements as i n i t i a t i n g events i n  s p e c i a t i o n (White, 1978a).  3. P h e n e t i c  An methods  Linkage  i n v e s t i g a t i o n of the l i t e r a t u r e r e v e a l s a large array of a l t e r n a t i v e for  relationships phenetic  and P h y l o g e n e t i c Taxonomic  representing among  taxa.  and  interpreting  taxonomic  and  systematic  These methods may g e n e r a l l y be d i v i d e d i n t o t h e  c l u s t e r i n g and o r d i n a t i o n t e c h n i q u e s  of numerical  taxonomy  (Sneath  23  and  Sokal,  1962;  1973)  phylogeneticists as  the  (Farris,  algorithms  (1978)  are  and  of  hybrids,  the  1970;  cladograms o r p h y l o g e n e t i c t r e e s o f  1972;  Fitch  and  Wiley,  1981).  Margoliash  Other t e c h n i q u e s ,  (1967) and  claims  evaluate,  due  critical  the  to  used  the  to  in  data  computation  analysis,  and  and  data  set.  Nevertheless,  the r e s u l t a n t t r e e s t o the i n p u t data  Avise,  1983).  technique,  by  parallelism trees  for  phenetic 1978;  been the  and  shown  to  yield  addition  of  new  same  clustering  methods  taxa methods  1981).  have  some use,  wishes t o draw from the Cluster indicating 1975) ,  a  1976) .  analysis  data  as  f i t " and  superior  alternative  (Mickevitch  and  1981;  the  Wagner  in stability  homoplasy  -  character Johnson,  (less  convergence,  congruence ( t h e a b i l i t y to generate  from  1976;  Swofford,  using  of  similar  data  sets),  than  1976;  Mickevitch,  i t seems t h a t most of the commonly used l i n k a g e  depending upon the i n f e r e n c e s t h a t the i n v e s t i g a t o r data.  of c h a r a c t e r data i s c l e a r l y an a p p r o p r i a t e method o f  acknowledged a  1981;  particularly  and  of  ( A v i s e and A y a l a ,  Farris,  trees  algorithms  "goodness  p h e n e t i c s i m i l a r i t y between t a x a (Sneath fact  techniques  Yet,  for  1978;  analysis,  r e g r e s s i o n ) and  the  Wiley,  Wilson,  Phylogenetic  has  affected  and  compared  different  parsimony  Prager  been  different  f o r the phenogram o r cladogram  analysis  1978;  have  to  i n t e r p r e t a t i o n , the l a c k of  phylogenetic  Mickevitch,  distance.  the i n c o m p a t i b i l i t y o f  judge "goodness o f f i t "  original  of  Wilson  proponents of a l t e r n a t i v e methods a r e d i f f i c u l t  errors  comparative  statistics to  of  and  such  t h a t b e g i n by p h e n e t i c c l u s t e r i n g from d i s t a n c e d a t a ,  then modify the r e s u l t i n g t r e e t o r e f l e c t p h y l o g e n e t i c The  Prager  the  even  representation  by of  those phylogeny  who  and S o k a l , 1973; oppose  (Mickevitch  Avise,  the use of such and  Johnson,  T h i s i s p r o v i d e d t h a t the c h a r a c t e r s chosen a r e r e l a t i v e l y numerous  24  and  due c o n s i d e r a t i o n i s g i v e n t o t h e q u e s t i o n o f " w e i g h t i n g " . Although  principal  i t  i s not, s t r i c t l y  components  cluster  analysis  analysis  (PCA)  among  characters.  This  taxa is  because  i n multidimensional  artificial  hierarchical  use  divergence on  the  of  allows  similarity very  similar  relationships, or  identical  f o r the co-occurrence  cluster  analysis  may  o f taxa  impose  s t r u c t u r e on t h e d a t a , s i n c e c l u s t e r i n g  phenetic  relationships  an  algorithms  similarity  to  reveal  recent  ancestral  from c h a r a c t e r and taxonomic d i s t a n c e data  and  relies  t h a t e v o l u t i o n a r y r a t e s a r e u n i f o r m among t h e d i v e r g i n g  According  strict  technique,  i n some r e s p e c t s be s u p e r i o r t o  many  space;  clustering  even when c h a r a c t e r s e t s a r e i d e n t i c a l .  assumption  groups.  PCA  a  phenetic  exhibiting  ordinated  The  may  for illustrating  particularly  f o r c e dichotomies  speaking,  to  constancy  proponents o f t h i s method (Sneath and S o k a l , 1973)  is  not  necessarily  required  for  an  accurate  representation. On  the  other  phylogenetic  relationships  phylogeneticists 1979;  rather to  that  strict  imply  Nei's  genetic  particularly lengths  in  have  been  phenetic  analysis  rests  relationships I  f o r example, subject  phylogenetic  substitutions,  a  and in  to  repeatedly  such  by  et a l . ,  1981), who c l a i m , among o t h e r upon  e v o l u t i o n a r y r a t e constancy.  identity  attacked  1972; 1981; M i c k e v i t c h , 1978; Baverstock  and M i t t e r , 1981; Swofford,  phylogenetic  reflected,  gene  (Farris,  Mickevitch  objections,  hand, those who would use p h e n e t i c c l u s t e r i n g t o a n a l y z e  a  p r i o r i assumptions o f  The use o f p h e n e t i c  clustering  based upon gene frequency  data u s i n g  distance  D  values  ( N e i , 1972; 1975), as  e l e c t r o p h o r e t i c d a t a , f o r branch valid criticism (Farris,  fitting, is  1981).  The branch  t r e e s s h o u l d r e p r e s e n t t h e number o f e v o l u t i o n a r y metric  property  obeying  the  Euclidean  triangle  25  inequality, taxa  where  the  A, B and C.  taxonomic  distance  D(A,C)  N e i ' s d i s t a n c e i s nonmetric  >  D(A,B) + D(B,C) f o r  and thus can y i e l d  misleading  phylogenetic reconstructions. The  construction  phylogeny aims  i s based  to  evolved 1983;  find  maximum  not  always  phylogeny  1983).  changes  the  maximum  t h e minimum  Sober,  character  on  the  with  o f Wagner t r e e s ( F a r r i s ,  parsimony c r i t e r i a .  The parsimony method  f o r which t h e observed  c h a r a c t e r s c o u l d have  evolutionary  Accordingly,  that  likelihood explicitly  1970) as a r e p r e s e n t a t i o n o f  change (Wiley, 1981; F e l s e n s t e i n ,  the  tree  w i t h t h e minimum number o f  adequately  r e p r e s e n t s t h e data i s c o n s i d e r e d t o be  tree  Haen and Neurath, 1976), a l t h o u g h  (De  assumed  it is  t h a t e v o l u t i o n proceeds by t h e most d i r e c t  path. Farris required cannot  (1972; of  character  be  simply  Nevertheless, (Farris,  stability One related an  1981;  matrices and  the  t h e minimal l e n g t h t r e e ,  to  rigid  Wagner  phylogenetic  high  assumptions level  of  the  greatest  technique. analysis  of f i t ,  regarding  the  congruence and  problems i n t h e p h y l o g e n e t i c a n a l y s i s o f c l o s e l y  e x t a n t t a x a i s t h e d e t e r m i n a t i o n o f t r e e p o l a r i t y and t h e c h o i c e o f taxon t o r o o t t h e t r e e .  Conventional phylogenetic a n a l y s i s  requires  t h e assemblage o f t a x a t o be monophyletic  for  root  character  criterion  ( M i c k e v i t c h , 1978).  appropriate  the  parsimony  i s capable o f y i e l d i n g t r e e s from  without a  the  distance  approach  1981)  with  that  yielding  to  Wagner  Swofford,  evolution,  out  analysis,  distance  distance of  data  pointed  extrapolated  the  1972;  Manhattan constancy  1981) has  to  represent  state(s).  plesiomorphic  state,  This a  the  plesiomorphic  necessarily  sensu Hennig (1966), and  ( p r i m i t i v e or generalized)  i m p l i e s a p r i o r i knowledge o f t h e  s i t u a t i o n n o t always r e a l i z a b l e f o r c h a r a c t e r data  26  (Baverstock presence  et  al.,  a  given  of  1979).  For  isozyme its  u s u a l l y c o n s i d e r e d to be the apomorphic  state  knowledge  of the g e n e t i c and e v o l u t i o n a r y i m p l i c a t i o n s of the isozyme bands  convergence,  appropriate  root  izozyme  for  bands.  plesiomorphic  more  etc.,  By  taxon,  under  study  range  of  is -  coded  to an  this  reductio  Without  prior  leads  to  the  assumption  as  ad  absurdum  arguments,  the  method  select  f o r e s t a b l i s h i n g p o l a r i t y and  a taxon  "out-group"  other c r i t e r i a ,  character  (operational possessed  c l o s e l y r e l a t e d t o those  i n the group  a p a r t from the s p e c i f i c c h a r a c t e r s e t a n a l y z e d  state  trait.  phylogenetic  - p r e f e r a b l y chosen on the b a s i s of a wide  in  both  the  taxonomic u n i t s ) i s taken  the  ultimate  (000...n) where n = number of c h a r a c t e r s  t r e e b u i l d i n g a l g o r i t h m , to serve as the r o o t of the t r e e . given  t h a t the  f u n c t i o n a l isozymes!  appropriate  relationships  a  state.  the t r e e i s the taxon e x p r e s s i n g the l e a s t number of  d e s c r i b e d , would have no  the  the p l e s i o m o r p h i c  d u p l i c a t i o n s , number of f u n c t i o n a l a l l e l e s per l o c u s , p a r a l l e l i s m s ,  regression,  A  absence  the  (derived)  gene  and  is  b i n a r y coded e l e c t r o p h o r e t i c d a t a ,  However,  n e c e s s a r i l y t r u e i f convergence has  it  out-group and  i n one  by  Presence of or more OTUs  as evidence  t h a t the common a n c e s t o r  should  noted  be  that  t h i s i s not  occurred.  C. G e n e r a l D e s c r i p t i o n and Taxonomic P o s i t i o n of the genus PROTOGONYAULAX  Members thecate and  many  of  the Protogonyaulax t a m a r e n s i s / c a t e n e l l a s p e c i e s complex a r e  gonyaulacoid forms  dinoflagellates  are known  dinoflagellates. bioluminescent. to  produce  the  A l l gonyaulacoids The  gonyaulacoids  are  photosynthetic  encompass a l l the  s a x i t o x i n analogues  (gonyautoxins)  27  a s s o c i a t e d w i t h p a r a l y t i c s h e l l f i s h p o i s o n i n g (PSP). According  t o a h y p o t h e t i c a l e v o l u t i o n a r y scheme based upon l i v i n g  forms  (Taylor,  1980a; 1985), p r o r o c e n t r o i d s and d i n o p h y s o i d s a r e l o c a t e d near  base  the  of  peridinoids through  tree, by  while  loss.  However,  primitive  dinoflagellate  (Loeblich,  1984).  upon a  taxonomy  the  form  a  stem  group  of  group,  thecate  others and  consider  would  gymnodinoids  essentially  number, shape, p o s i t i o n and o r i e n t a t i o n o f the t h e c a l p l a t e s . g o n y a u l a c o i d s may  including  the  p e r i d i n o i d s , which they s u p e r f i c i a l l y resemble,  morphological  relationships,  features.  such  gonyaulacoids  (1979;  divergences  as  rather  well  which  g r e a t e r t o r s i o n a l asymmetry i n  a p l a t e homology model from which major  the  the  they  of  than  g e n e r a l i z e d p e r i d i n o i d and g o n y a u l a c o i d forms can 1985  also d i f f e r  new  f o r examples o f i t s a p p l i c a t i o n ) .  morphological  the removal a  by t h e s e major In  for  symmetrical  1980a) has developed  as  (1980a),  creation  by a v a r i e t y  Most of t h e s e d i f f e r e n c e s i n v o l v e tendency  forms,  the d o r s a l s u r f a c e , as i n p e r i d i n o i d s .  between  peridinoids, view  be d i s t i n g u i s h e d from o t h e r t h e c a t e  As  on  be r e c o g n i z e d (see E v i t t , As  the  tree  and the t y p i c a l o c c u r r e n c e o f i n t e r c a l a r y p l a t e s on the r i g h t  epitheca,  Taylor  most  d i n o f l a g e l l a t e s has t r a d i t i o n a l l y been based  the  ventral  the  invert this  group,  of  leading to  a d a p t i v e r a d i a t i o n , and u l t i m a t e l y t o a t h e c a t e gymnodinoids  plate  The  gonyaulacoids  the  distinction  between g o n y a u l a c o i d s  i n r e p r o d u c t i o n and c y s t t y p e s .  In T a y l o r ' s  of the g o n y a u l a c o i d s from the P e r i d i n a l e s and  o r d e r Gonyaulacales  and  t o embrace t h i s group was  the  warranted  distinctions.  g o n y a u l a c o i d s , the t h e c a i s d i v i d e d i n t o an a n t e r i o r group o f p l a t e s form  the  e p i t h e c a , and a p o s t e r i o r s e r i e s c o m p r i s i n g the  hypotheca.  28  In  the  conventional  gonyaulacoids posterior apical  pore  in  pore  plate.  1  epitheca  (lp)  The  be  although  that  typical  the  surface  the  typical  flagellum  lies  along  in  is  antapical  usually  one  1  rather  (l  1  1  1  1  )  plate.  The  hook-shaped; a v e n t r a l  near the r i g h t margin of the f i r s t  is  displaced  Gonyaulax.  the  on  the  The  sulcus,  point of f l a g e l l a r  dinokont the  flagellation;  cingular  a l l dinoflagellates,  oblique.  ventral  a  apical  surface  longitudinal  groove  insertion. a  in than  groove  and  a trailing  on  Gonyaulacoids  ribbon-like  certain  cell  transverse  whiplash-type  plane of f i s s i o n  monilatum  e.g. Protogonyaulax  (=Gonyaulax  Protogonyaulax,  monilata),  f a i l u r e of daughter  (=Gonyaulax) c a t e n e l l a and  to  form  c e l l s may  flattened composed  c e l l s to separate a f t e r  transdiameter amphiesmal vesicles. of  chains of c e l l s .  occur.  v e g e t a t i v e c e l l s o f Protogonyaulax  an  i s shared w i t h the  Gessnerium  In the genus  u n i c e l l s a r e f r e q u e n t l y observed, but d o u b l e t s , q u a d r u p l e t s  even l o n g c h a i n s o f >30  in  i n gonyaulacoids i s  d u r i n g m i t o s i s ( d e s m o s c h i s i s ) , which e x p l a i n s the a b i l i t y o f  species,  The  the  In g o n y a u l a c o i d s , the w a l l of the parent c e l l  daughter  lies  single  s i x postcingulars ( 6 ' ' ) ,  longitudinally.  As  range  ),  1911),  t h i s displacement i s c h a r a c t e r i s t i c a l l y l e s s  at  possesses  and  1 1  plate notation (Kofoid,  from the p o s t e r i o r end t o the equator, i n t e r s e c t s the cingulum  ventral  extends  of  cingulum, a t r a n s v e r s e e q u a t o r i a l groove s e p a r a t i n g the  hypotheca,  in  a  present  Protogonyaulax,  extending  and  gonyaulacoids may  and  found  system  possess s i x p r e c i n g u l a r s ( 6  intercalary  epithecal (l )  Kofoidean  from  layer  ~20-45  Beneath  sporopollenin,  the  which  cytokinesis. a r e o v o i d i n form, and  um.  containing  These c h a i n s a r i s e due t o the  Under  thin  cellulosic  amphiesma forms  the  part  lies  a  of  the  typically  e x t e r n a l membrane thecal  plates in  resistant outer  pellicle  cyst  wall  29 following ecdysal  ecdysis. (pellicular)  Pellicular  cysts  regenerated cells  in  are  motile shape  The t h e c a l p l a t e s a r e r e a d i l y shed i n t h e f o r m a t i o n  can a  again  usually  mucilageanous  (~15  are  and  fusion  length)  Hypnocysts, formed from  of  gametes, a r e s i m i l a r i n  to p e l l i c u l a r cysts.  their  thicker  species  wall,  pigmentation.  Y e t , they can  often  covered  Hypnocysts  by  typically  c e l l s a r i s e from  1979).  complex,  ( F i g . 2).  of  i f the  two dominant morphotypes e x p r e s s e d w i t h i n t h e Protogonyaulax  of  catenella  members  in  darker  features P.  conditions.  thecal plates are often  o b l i g a t e dormancy p e r i o d b e f o r e m o t i l e t h e c a t e  catenelloid  and  urn  material,  c y s t s (Dale,  the  conditions.  sexual  by  ecdysing  since  environmental  o f hours and m o t i l i t y i s r a p i d l y r e s t o r e d  distinguished  an  tamarensis  unfavourable  transitory,  following  require  There  under  exposed t o f a v o u r a b l e  size  be  be  matter  planozygotes and  plate  cysts  of  the  designated  h e r e i n as tamarensoid ( F i g . 1) and  They a r e based s o l e l y on t h e morphology and t h e c a l  the  species described  (Whedon genus  and  Kofoid)  Gonyaulax  as P. t a m a r e n s i s (Lebour) T a y l o r Taylor,  Diesing.  both o r i g i n a l l y named as  In t h e o r i g i n a l d e s c r i p t i o n o f  tamarensis (Lebour, 1925), t h e K o f o i d e a n p l a t e formula was g i v e n a s : 4',  Oa,  6",  acatenella differed by  only  diagnosis  Kofoid,  The f i r s t 1936):  t a b u l a t i o n f o r c a t e n e l l a and  4', Oa, 6 " ,  6c, 6'",  lp, 1 " " ,  i n the i n c l u s i o n of s i x small c i n g u l a r p l a t e s , not described  by  adding  using  plates  additional  complement  and  f o r tamarensis.  observations  an  lp, 1 " " .  1  (Whedon  Lebour  small  6 ",  was  Subsequently,  several  electron  associated apical  plates  (1971) emended t h e  (7-8s).  microscopy l a t e r r e v e a l e d  Ultrastructural t h e presence o f two  w i t h t h e f l a g e l l a r pore r e g i o n o f t h e s u l c u s , and  platelet  therefore  sulcal  Steidinger  (Postek  expanded  to  and  Cox, 1976).  The t o t a l  plate  y i e l d t h e formula: l a p (= P o ) ,  lcp  10  Ji  TAMARENSOID Fig.  1  G e n e r a l morphology o f the tamarensoid morphotype o f Protogonyaulax. C e l l s may o r may not have a v e n t r a l pore a s s o c i a t e d w i t h the margin o f the f i r s t a p i c a l p l a t e .  CATENELLOID Fig.  2  G e n e r a l morphology o f the c a t e n e l l o i d morphotype o f Protogonyaulax, showing a n t e r o - p o s t e r i o r l y compressed in a c h a r a c t e r i s t i c chain configuration. Catenelloid l a c k a v e n t r a l pore a s s o c i a t e d w i t h the margin o f the apical plate.  cells cells first  31  (=  Pc),  4',  scanning  15  other  sulcal  Kofoid's  conventional  intercalary  as  antapical  be  Some  1985;  Fukuyo  for  identical  for  " t a m a r e n s i s " and (Taylor,  1979;  other  g o n y a u l a c o i d s , shown by T a y l o r noted  aritapically Taylor, In  seen  presence  profile  1975;  B a l e c h and  some  type  (1936),  a  apparent. membrane consistent  This  diagnostic  the  number of  p l a t e s , t o 9-10s.  Fukuyo  11 s u l c a l p l a t e s from  1985)  1985; have  (1936) i s now  Balech  and  departed from  The  The  (l  1  1  the )  1  is  posterior  considered  as  the  basic thecal plate  " c a t e n e l l a " are g e n e r a l l y acknowledged 1983;  Fukuyo, 1985;  Fukuyo e t  (1979).  of two  Tangen, 1985; of  "curtain most  a n t a p i c a l spines being  likely  feature  for  but  L o e b l i c h and  Loeblich,  1975;  Fukuyo, 1985).  catenella  fin"  i n tamarensis,  the s u l c a l l i s t s which extend  examined  covering a  remnant  s t r e t c h e d between the c i n g u l a r r i d g e s .  c h a i n of c e l l s Balech  was  and  f i r s t postcingular  (Braarud, 1945;  specimens  delicate  Tangen, 1985),  assigning only f i v e p l a t e s to  Kofoid  i l l u s o r y , apparently in  of  i n d i c a t e d by the p l a t e homologies, i n comparison w i t h  the  " s p i n e s " are  and  area  (Balech,  al.,  Steidinger,  1985).  these  T h i s was  sulcal  increased  recognize  authors et  and  a t o t a l of 2'"'.  al.,  Lebour  the  a n t e r i o r s u l c a l p l a t e ( F i g . 3).  -  1  has  p r e v i o u s l y assigned  left  (I" ')  now  by now  ( l p ) p l a t e of Whedon and  configurations to  spp.  The  the  1985),  a l . , 1985)  designations  series.  reinterpreted  on  i n c l u d i n g accessory  et  Fukuyo,  postcingular  first  (Fukuyo  1985;  D e t a i l e d l i g h t microscopy  observations  (Balech,  plates,  Protogonyaulax  Tangen,  lp, 1 " " .  from O s l o f j o r d , Norway ( B a l e c h  countries  co-workers  Japanese  7s, 6"',  microscopic  material  identifiable and  6c,  electron  tamarensoid from  6",  catenella  the  by Whedon and  girdle  depression  of the e x t e r n a l The and  Kofoid was  amphiesmal  " c u r t a i n f i n " i s not  a  may  a  v a r y even w i t h i n  ( T a y l o r , 1975).  noted  the  presence of a v e n t r a l pore a s s o c i a t e d w i t h the  right  32  Fig. 3  T h e c a l p l a t e c o n f i g u r a t i o n o f Protogonyaulax i n the K o f o i d n o t a t i o n system, as a p p l i e d by Fukuyo (1985). A. V e n t r a l view; B. D o r s a l view; C. E p i t h e c a ; D. Hypotheca. Po, a p i c a l pore p l a t e ; l ' - V , a p i c a l p l a t e s ; l - 6 , p r e c i n g u l a r p l a t e s ; 1' ' ' -5 » ' *, postcingular plates; l -2' , antapical plates; lc-6c, c i n g u l a r p l a t e s ; s.p., p o s t e r i o r s u l c a l p l a t e ; v.p., v e n t r a l pore. l l  1  1 , 1  , ,  1 , 1  33  34  margin  of  the  morphotypes, fratercula  Balech character  1979a;  Fukuyo,  1979;  Fukuyo  et  Braarud,  Kofoid,  caused  be  cells  it  unlikely and  round  ventral  (1971),  G.  treatments o f the " t a m a r e n s i s " s p e c i e s  1975;  1985;  G.  pore has been used as a major  T a y l o r , 1975;  B a l e c h , 1985;  Schmidt  and  Loeblich,  1925;  Braarud, 1945), a c a t e n e l l a and c a t e n e l l a (Whedon no  reference  the  to  absence  be  of  overlooked  (1985)  have  to  a v e n t r a l pore.  I t i s unclear  the f e a t u r e , or merely t h a t i t s or  disregarded  as t a x o n o m i c a l l y  reasoned c o n v i n c i n g l y t h a t a l t h o u g h Lebour  missed the presence of a pore i n t a m a r e n s i s , i t  that • Kofoid  would have f a i l e d t o note t h i s f e a t u r e i n  ( i f p r e s e n t ) , as he had d e s c r i b e d such pores i n  species.  or e l l i p s o i d a l a n t e r i o r attachment tamarensoid  division  and  catenelloid  proceeds r a p i d l y (Fukuyo,  pore  pore i s f r e q u e n t l y observed  morphotypes, p a r t i c u l a r l y when  1985).  A corresponding p o s t e r i o r  on the p o s t e r i o r s u l c a l p l a t e of c e l l s  i n c h a i n s , noted i n  o r i g i n a l d e s c r i p t i o n o f c a t e n e l l a (Whedon and K o f o i d , 1936), appears t o  m a i n t a i n c y t o p l a s m i c c o n t i n u i t y between newly d i v i d e d Although reference essentially was  Balech  The e a r l y d e s c r i p t i o n s o f t a m a r e n s i s (Lebour,  acatenella  the  attachment the  (Braarud)  tamarensoid  1985;  make  easily  o t h e r Gonyaulax  both  ( F i g . 3) of r e l a t e d  B a l e c h and Tangen,  Fukuyo  might  catenella  in  1980;  represents  insignificant.  A  plate  excavata  This  Loeblich,  1935;  1936)  this  obscurity  would  G.  subsequent  a l . , 1985).  and  (1925)  as  in  and  whether  1  (1967).  (Loeblich  and  (l )  B a l e c h (1967), G. p e r u v i a n a B a l e c h and De M e n d i o l a (1977) and  taxonomic  Gran  apical  described  cohorticula  group  first  Lebour to  (1925)  transdiameter,  isodiametrical  described  gave  as  and  prominently  only  her  mean  iconotypes  non-chain  cells.  c e l l l e n g t h (36 um), showed  cells  without  which  were  f o r m i n g . In c o n t r a s t ,  catenella  a p i c a l l y / t r a n s a p i c a l l y compressed  ( r a t i o of  35  l e n g t h : t r a n s d i a m e t e r = 0.71-0.88: 1 ) , w i t h c e l l s m o s t l y i n c h a i n s . Gonyaulacoid complex,  some  acatenella  species o f which may  Whedon  concava  (Gaarder)  Taylor,  1975;  Lebour  Balech  G.  (1985).  P.  Conrad  affinis  There  (1939),  cohorticula  be  a  B a l e c h (1967),  Fukuyo  is  1984,  1985;  G. dimorpha  1985),  but  (Paulsen)  B i e c h e l e r (1952), G.  kutnerae  complex  conform  (1911)  group  (3-6',  0-3a,  is  shape  and  the  apical of  the  pore  Fukuyo e t a l .  i b e r i c u m , A.  lusitanicum  list.  (Steidinger,  T a y l o r , 1975,  1971,  1983;  B a l e c h , 1985;  appropriate.  6c, 7-8s, 0-4a,  6'",  6",  species,  1979,  B a l e c h and Tangen,  t h a t the t r a n s f e r from the genus  clearly  from, Gonyaulax position  (1954),  t o c i r c u m s c r i b e the  the b a s i c Gonyaulax 6",  Wood  d i v e r s i t y o f o p i n i o n on how  disagreement  T a y l o r (1976) (pp, 3-4", differ  conjuncta  1985), A. l e e i , A.  and L o e b l i c h , 1979;  stricto to  G.  r e c e n t l y d e s c r i b e d and a s s i g n e d t o the genus  - c o u l d a l s o be added t o t h i s  little  sensu  (1967),  Parke  (1985) - A. t r o p i c a l e , A. insuetum, A. f u k u y o i  considerable  Diesing  Hsu  et a l . ,  Loeblich  G.  m o t i l e stage of P y r o c y s t i s ,  Fukuyo e t a l . (1985) and P. compressa  Balech  tamarensis/catenella  shape  (may  G.  Gonyaulax  ( P a u l s e n ) P a u l s e n (1949), Pyrodinium phoneus  morphospecies  (Halim)  and A. fundyense  they  the t a m a r e n s i s / c a t e n e l l a  G. f r a t e r c u l a B a l e c h (1964), G. o r i e n t a l i s  affinis  Other  Alexandrium  of  (1967)  washingtonensis  Protogonyaulax  by  (1936),  G. o s t e n f e l d i i  and  with  be synonymous or c o n s p e c i f i c , i n c l u d e  Kofoid  Balech  allied  (1979), G. p e r u v i a n a B a l e c h and De M e n d i o l a (1977), G. t r y g v e i  (1976),  (=  and  1979),  (1925),  Woloszynska  closely  Gonyaulax  Members of t h i s s p e c i e s  p l a t e f o r m u l a as emended by K o f o i d lp, 1 " " ) ,  and l a t e r as m o d i f i e d  6c, 5-10s, 5 - 6 ' " , l p , 1 " " ) . as t y p i f i e d by G. s p i n i f e r a ,  Yet, i n the  o f both e p i t h e c a l and h y p o t h e c a l p l a t e s , the s t r u c t u r e complex,  nucleus.  the degree of c i n g u l a r d i s p l a c e m e n t and the  The v e g e t a t i v e c e l l s o f the t a m a r e n s i s / c a t e n e l l a  36  group  also  epithecal  differ  markedly  from  i n t e r c a l a r y p l a t e and  those  of  conspicuous  G.  spinifera  t h e c a l ornamentation.  their  r e s p e c t i v e c y s t types were unknown when c a t e n e l l a and  first  described,  cysts,  with  1945;  excysting  Wall,  Fukuyo, types  both s p e c i e s a r e now  1975;  1985).  cells  Turpin  In sharp  i n l a c k i n g an  tamarensis  known to produce smooth w a l l e d  e x i t i n g through an i r r e g u l a r s p l i t  et  Although  al.,  1978;  c o n t r a s t , G.  Dale,  1979;  Yoshimatsu,  1981;  s p i n i f e r a produces a v a r i e t y of c y s t dorsal  archeopyle.  Unfortunately, Gonyaulax  has  incompatible strictest  ovoid  (Braarud,  w i t h s p i n y p r o j e c t i o n s , w h i l e excystment o c c u r s v i a an a n g u l a r  precingular  were  the  transfer  been  ways.  the t a m a r e n s i s / c a t e n e l l a complex from  accomplished The  taxonomic  of  in  proposed  sense,  in  three  solutions  different are  and  equally  presently  valid  i n the  t h a t they a l l adhere t o the b a s i c r u l e s of  nomenclature  (Taylor,  description,  t h e r e i s no a b s o l u t e l e v e l of d i f f e r e n t i a t i o n r e q u i r e d f o r the  appropriate genus,  or  generic its  shift to  1985).  As  long  of a s p e c i e s complex t o an e n l a r g e d p r e v i o u s l y d e s c r i b e d  a newly c r e a t e d genus.  assignation  usefulness,  "marketplace".  as the organisms f i t the g e n e r i c  is  determined  appropriateness The  arguments  U l t i m a t e l y , adoption  of a p a r t i c u l a r  by pragmatic c o n s i d e r a t i o n s , i n c l u d i n g and  for  common  the  usage  choice  in  of one  the  scientific  g e n e r i c name over  another  t o embrace t h i s s p e c i e s complex a r e based upon v a l i d d i f f e r e n c e s of  opinion  regarding  previous  historical  descriptions,  and  precedence,  the  accuracy  and  the s i g n i f i c a n c e of m o r p h o l o g i c a l  l e g i t i m a c y of variation in  certain thecal plates. Three Tangen,  generic 1985),  Protogonyaulax  names,  Alexandrium  Gessnerium Taylor  Halim  (Taylor,  Halim  (Loeblich  1979;  1984;  (Balech, and 1985)  1985;  Loeblich,  Balech  and  1979),  and  have been proposed t o  37  include  the  tamarensis/catenella  placed  this  Halim,  rejecting  the  group  grounds  (Halim,  1985;  the  1985;  which  rather  1979;  1984).  1985;  B a l e c h and  this  Tangen, 1985)  G.  type  and  the  absence  1967)  and  was  species, this  allegedly  ( T a y l o r , 1979;  as  i s the  botanical  the  Gonyaulax  of i n f o r m a t i o n more  not  (smooth-walled)  The  fact  that  and  automatically Loeblich  s i n c e the  to  type  is  now  known (Walker  and  the type s p e c i e s d e s c r i p t i o n (Halim,  is  invalidate  (1979),  From  L o e b l i c h (1979) i s abundantly  type  Howell,  or  questionable  on c y s t morphology.  r e a d i l y supportable,  (Howell) L o e b l i c h and  monilata  (Balech,  l a c k of o r i g i n a l h o l o t y p e m a t e r i a l  cyst  1979).  under  code of nomenclature ( T a y l o r ,  l o c a l i t y f o r r e - e x a m i n a t i o n of  is  1984;  l a c k of L a t i n  based upon o p t i c a l l y i n v e r t e d specimens of a p r e v i o u s l y  does  Loeblich  Gessnerium  monilatum  Steidinger,  1985),  The  on  described  i f the type s p e c i e s were c o n s i d e r e d  i s the  the  standpoint,  available,  not p r o v i d e d .  criticism  Tangen,  the  from  plates,  species,  and  inadequately  More troublesome f o r the proponents of Alexandrium  specimens  thecal  questionable  required than  (1979)  Halim, the name w i t h h i s t o r i c a l p r i o r i t y ,  a  Balech  i s not  zoological  new  is  Loeblich  d e s c r i p t i o n of the genus Gessnerium  t h a t a L a t i n d i a g n o s i s was  description  diagnosis,  L o e b l i c h and  the type s p e c i e s , A. minutum, was  and  Balech,  an e n l a r g e d  Alexandrium  that  1960)  inadequate  within  complex.  taxonomically using  circumscribe  described  uncomfortable,  but  Gessnerium, as emended by the  tamarensis/catenella  group. The newly  argument created  hinges can  largely be  Pyrodinium,  split  f o r the t r a n s f e r of the t a m a r e n s i s / c a t e n e l l a  genus,  as proposed by T a y l o r  upon the e f f i c a c y and from  closely  Alexandrium/Gessnerium,  (1979) (see a l s o E v i t t ,  reliability  related and  complex t o a 1985),  w i t h which Protogonyaulax  pre-existing Heteraulacus.  genera, The  including  brief  generic  38  diagnosis  f o r Protogonyaulax T a y l o r  of  notation,  plate  reproduced as  rather  than  (1979), which employs the T a y l o r the  more c o n v e n t i o n a l  K o f o i d system, i s  follows:  G o n y a u l a c o i d c e l l s w i t h a g i r d l e d i s p l a c e m e n t o f one w i d t h or l e s s , t h r e e a n t e r i o r p o l a r p l a t e s ( a p i c a l s ) , c o n t a c t i n g P, V/VI  Z  plate  between  to as  p o s t e r i o r s u l c a l homologue, w h i l e V/VI 5 ''  and  1  acknowledges  Is  archeopyle.  the  6' '  of the K o f o i d e a n system.  1  i s the  suture  T h i s emended  c o r r e c t i o n of a t y p o g r a p h i c a l e r r o r ( T a y l o r ,  i n the o r i g i n a l d i a g n o s i s , where the Z p l a t e was  incorrectly referred  Y.  In  the  isolating first  apical 1  complex these  known  cyst  differences,  the  critical  feature  from Alexandrium/Gessnerium i s whether or not 1  the  p l a t e - K o f o i d system = Is - T a y l o r system = l u  (1985) m o d i f i c a t i o n of the T a y l o r system) touches the a p i c a l pore The  plates,  as  1979;  Balech  appropriately generic  of  homologue ( l  (APC).  Loeblich, 1985;  absence  Protogonyaulax  Evitt s  be  the  postcingulars  description 1984)  is  girdle  c o n t a c t i n g Z. C y s t s are smooth w a l l e d  w i t h o u t an a n g u l a r  The  system  discussed Taylor,  and  in  1979;  Tangen,  of  the c o n t a c t  detail 1984;  1985)  is  d e l i n e a t i n g the genera.  criterion  effectively  consistency  by  1985; a  to  all  of  s e v e r a l authors ( L o e b l i c h and S t e i d i n g e r , 1983;  debatable,  Balech  as too v a r i a b l e , but T a y l o r  applied  ( o r l a c k of c o n t a c t )  but  (1979; 1985)  Balech,  1979;  c r u c i a l point i n objected  to  this  (1984) countered t h a t i t can  c e l l s which are not o b v i o u s l y abnormal or  megacytic. There  are a l s o pragmatic c o n s i d e r a t i o n s  f o r the use  of the g e n e r i c name  39  Protogonyaulax, prefix  as  opposed  and  non-taxonomist, arbitrariness Gessnerium  may who  of  Loeblich  such  and  and  still  to  become  North  be name  group,  Loeblich,  and  G.  by  these  of  by  confusing  as  a  reference  understandably  bewildered  changes.  contrast,  are the  In  rather  most  monilatum  obscure.  at  the  the  taxonomist  for  for  the  apparent  g e n e r i c names  Exclusive  common member o f Gessnerium  of  the  (Halim)  - an important i c t h y o t o x i c s p e c i e s -  non-taxonomists.  Alexandrium  its  Alexandrium i s  The use o f the g e n e r i c names  the t a m a r e n s i s / c a t e n e l l a complex has been  proponents.  On the o t h e r hand, Protogonyaulax  w i d e l y a c c e p t e d , p a r t i c u l a r l y i n Japan, on the P a c i f i c c o a s t o f  America,  adopted  less  Alexandrium  unheard  Gessnerium  For  A d d i t i o n o f the  f r e q u e n t l y i n c o r r e c t l y r e f e r r e d t o as "Gonyaulax";  virtually  limited  be  may  tamarensis/catenella  has  o r Alexandrium.  " P r o t o " t o the former genus name e s s e n t i a l l y conserves n o m e n c l a t u r a l  continuity,  is  t o Gessnerium  and  more  physiologists, reasons,  the  recently toxin present  in  A t l a n t i c Canada, where i t has been  chemists, studies  p a l y n o l o g i s t s and will  ecologists.  r e f e r the i s o l a t e s o f the  t a m a r e n s i s / c a t e n e l l a s p e c i e s complex t o the genus Protogonyaulax.  D. H i s t o r i c a l and Recent P e r s p e c t i v e s on Taxonomic V a r i a n t s W i t h i n the Protogonyaulax t a m a r e n s i s / c a t e n e l l a S p e c i e s Complex  Protogonyaulax  (=Gonyaulax)  "tamarensis/catenella" Lebour this  (1925) a r e a was  confirmed  that  from  complex  tamarensis,  the  member  t o be d e s c r i b e d ( F i g . 4 ) , was  the Tamar e s t u a r y near Plymouth,  not noted f o r PSP  first  of  identified  the by  England.  A t the time  o u t b r e a k s , a l t h o u g h subsequent  r e p o r t s have  t o x i c i t y does o c c u r i n the v i c i n i t y .  A f t e r considering net  40  Fig. 4  H i s t o r i c a l taxonomic scheme f o r some members o f t h e Protogonyaulax t a m a r e n s i s / c a t e n e l l a s p e c i e s complex.  41  Fig. 4  HISTORICAL TAXONOMIC SCHEME FOR SOME MEMBERS OF THE PROTOGONYAULAX TAMARENSIS / CATENELLA SPECIES COMPLEX Gonyaulax catenella Whedon and Kofoid, 1936; San Francisco Bay Bioluminescence (?) Cells compressed (+) Ventral pore (-?) Chain forming (•) Toxicity (•)  Gonyaulax tamarensis Lebour 1925; Tamar estuary, Plymouth, England Bioluminescence (?) Cells compressed (-) Ventral pore (?) Chain forming (-) Toxicity (?)*  Gonyaulax tamarensis var. globosa Braarud, 1945; Norway Bioluminescence (?) Cells compressed (-) Ventral pore (+?)*** Chain forming (-) Toxicity (?)  Gonyaulax tamarensis var. typica Braarud, 1945 Bioluminescence (?) Cells compressed (-) Ventral pore (?) Chain forming £-) Toxicity (?)*  Gonyaulax acatenella Prakash and Taylor, 1966; Loeblich and Loeblich, 1975; Malaspina Inlet, B.C. Bioluminescence (?) Cells compressed (-) Ventral pore (-) Chain forming (-) Toxicity ( ) Hypothecal flanges  Gonyaulax tamarensis var. excavata Braarud, 1945; Norway, Gulf of Maine, Bay of Fundy. Bioluminescence (?) Cells compressed (-) Ventral pore (+?)*** chain forming (-) Toxicity (?)**  Gonyaulax tamarensis (Braarud) Balech, 1971 Bioluminescence (?) Cells compressed (-) Ventral pore (?) Chain forming (-) Toxicity (?)  Gonyaulax acatenella Whedon and Kofoid, 1936; San Francisco Bay Bioluminescence (?) Cells compressed (-) Ventral pore (-?) Chain forming (-) Toxicity (?)* Weakly pigmented  +  Gonyaulax excavata (Braarud) Balech, 1971; East equatorial Atlantic Biolunlnescence (?) Cells compressed (-) Ventral pore (*) Chain forming (-) Toxicity (?)  I  REDEFINITION  Gonyaulax tamarensis (Lebour) Loeblich and Loeblich, 1975; England, East coast of North America Bioluminescence (-) Cells compressed (-) Ventral pore (+) Chain forming (-) Toxicity (-)  Gonyaulax excavata (Braarud) Loeblich and Loeblich, 1975; East coast of North America Biolunlnescence (+) Cells compressed (-) Ventral pore (-) Chain forming (-) Toxicity (*)  I  I r  "Catenella complex", Steidinger, 1971/ '*Tamarensis complex", Taylor, 1975 Bioluminescence (±) Ventral pore (±) Hypothecal flanges (±) Toxicity (±) Cells compressed (±) Chain forming (±)  NOT GONYAULAX Taylor, 1979; loeblich and Loeblich, 1979  Alexandrium (Halim); Egypt, 1960 anended by Balech and Tangen, 1965 Gonyaulacoid ln which 1' may or may not contact APC Cyst of type species unknown  Protogonyaulax Taylor, 1979, gen. nov. Gonyaulacoid in which 1' contacts APC Smooth cyst  Gessnerium (Halim); Venezuela, 1969 emended by Loeblich and Loeblich, 1979 Gonyaulacoid In which l may or may not contact APC Smooth cyst 1  REDEFINITION  Protogonyaulax tamarensis (Taylor) Fukuyo. 1980, 1985; Japan. Bioluminescence (?) Chain forming (-) Ventral pore (•) Toxicity ( ) Cells compressed (-) APC rectangular or narrow triangular Posterior pore (+) or (-) at plate margin +  not s p e c i f i c a l l y tested, but not associated with PSP t o x i c i t y i n the region.  Protogonyaulax catenella (Taylor) Fukuyo. 1980, 1985; Japan. Bioluminescence (?) Chain forming ( ) Ventral pore (-) Toxicity (*) Cells compressed (-) APC dorsally wide, triangular Poster i o r pore (•) or (-) away from plate margin  not s p e c i f i c a l l y tested, but associated with PSP t o x i c i t y i n the region.  +  ***  not noted i n o r i g i n a l description, but presence i s suggested by re-examination of micrographs.  42  haul and  specimens  collected  Braarud,'  1935)  Braarud  (1945)  general  morphology:  tamarensis G.  tamarensis was  specifically  from  prevalent  var.  excavata;  on  particularly associated  (=var.  with  into  (Gran  t h r e e v a r i e t i e s based upon  tamarensis),  toxicity,  "tamarensis"  synonymous w i t h and v a r .  G.  excavata.  but the organism was  morphotypes sensu Braarud  not  (1945)  the e a s t c o a s t of North America, namely v a r . t y p i c a and d i s c r i m i n a t e d the l a t t e r form by i t s deeply blooms  tamarensoid  of  the G u l f of Maine  a Norwegian form c h a r a c t e r i z e d by i t s rotund  Scandinavia,  with  description  typica  Two  Toxic  in  species  associated  Braarud  region.  this  globosa,  not  of Fundy and  the type l o c a l i t y , v a r . g l o b o s a  tested.  were  sulcal  var.  var.  the Bay  examining c u l t u r e d i s o l a t e s from O s l o f j o r d , Norway,  subdivided'  Lebour  morphology,  and  in  G.  causing eastern  PSP  sculpted  i n A t l a n t i c coastal regions,  Canada  and  New  England were l a t e r  d i n o f l a g e l l a t e s o f t e n conforming to B r a a r u d s 1  tamarensis  v a r . excavata  (Needier,  1949;  Prakash;  1963;  1967). The  impetus  described by  the  by  producing  a  chain-forming  Sommer  et  al.,  antero-posteriorly  catenella. as  taxonomic work on two  circumstantial  of  1937;  the  formation,  dinoflagellate  link  between  PSP  gonyaulacoid 1937).  toxic  c e l l s was  provided  California  dinoflagellate  The  compressed  in  was  species  and  (Sommer  chain-forming  the and  species  i d e n t i f i e d as Gonyaulax  A second s p e c i e s , G. a c a t e n e l l a , w i t h the same p l a t e t a b u l a t i o n  "catenella",  superficially  new  Whedon and K o f o i d (1936) from San F r a n c i s c o Bay,  strong  appearance Meyer,  for  was  differentiated  resembling and  the  descriptions  of  the  "acatenella"  were  not  G.  tamarensis  production  of  epithecal in  accord  as  a  Lebour  almost plate with  weakly in  pigmented  size,  lack  isodiametrical patterns  that  given  for  species of c h a i n  cells.  The  "catenella"  and  by Lebour (1925) f o r  43  G.  tamarensis.  also  A f t e r examining the type m a t e r i a l from C a l i f o r n i a ,  acknowledged  "tamarensis",  the  and  distinctness  further  of  " c a t e n e l l a " and " a c a t e n e l l a " from  strengthened  the  case f o r the c r e a t i o n o f  species  by  to  the  e s t u a r i n e waters of the Plymouth  of  " t a m a r e n s i s " ( o r "excavata") a l o n g the P a c i f i c c o a s t of B r i t i s h  (Taylor, b),  her o r i g i n a l a s s e r t i o n t h a t " t a m a r e n s i s " had a range  1975;  1984;  Alaska (Hall,  Oshima  and  Oshima  et  Turpin  1982;  et a l . ,  1979;  a l . , 1982a,  toxicity  in  Magnusson, associated "acatenella" to  be  the  California,  and l a t e r  in  toxic  of  1985;  Ogata e t a l . ,  1982;  i n other P a c i f i c was  until  neither  extracts  chains  in  l o c a t i o n s (Schantz and  observed  from  a  the  Braarud's globosa,  (1945) var.  description small  the  of  their  three  typica  zoological varieties  and  var.  samples,  Gonyaulax  excavata,  to  form from the e a s t e r n e q u a t o r i a l A t l a n t i c .  plates  of  G. of  and  commented on the "catenella".  B a l e c h (1971), r a i s e d  tamarensis  sulcal  region,  using  Lebour, v a r .  the l e v e l o f s p e c i e s .  His  based upon e x a m i n a t i o n o f a B a l e c h (1977) completed a  t a m a r e n s i s Lebour, i n c l u d i n g a d e t a i l e d the  as  i n 1965, were determined  nomenclature,  of  identified  nor  Prakash and T a y l o r (1966) noted  o f G. e x c a v a t a (Braarud) B a l e c h was  redescription of  system  elsewhere,  bloom  p o s s i b i l i t y o f c o n s p e c i f i c a f f i n i t y between " a c a t e n e l l a " and Using  dispelled  as the major s o u r c e o f s h e l l f i s h  and T a y l o r , 1966).  short  1980;  1985), has s i n c e  M a l a s p i n a I n l e t , B r i t i s h Columbia  (Prakash  presence  considered  "acatenella"  toxicity,  Columbia  g e o g r a p h i c a l l y c o n f i n e d t o the A t l a n t i c .  was  with  discovery  Cembella and T a y l o r , 1985a and  b and c; Fukuyo e t a l . ,  "catenella"  1964),  1978;  new  restricted  The more r e c e n t  T o r i u m i and Takano, 1979;  the b e l i e f t h a t " t a m a r e n s i s " was Although  region.  T a y l o r , 1984), and Japan (Fukuyo, 1979;  Yasumoto,  Lebour  investigation  c u l t u r e d m a t e r i a l from the  44  Plymouth  type  description  locality. on  Unfortunately,  Lebour's  holotype  he  was  specimens,  not as  able  these  to  base  are  his  no  longer  d e s c r i p t i o n o f Protogonyaulax  species  available. Much has  confusion  arisen  due  "tamarensis"  was  of  173a  was  dismissed  recognized  the  epithecal plates  independently  morphotypes  by  adding  new  (Plymouth  173)  and  original optically  corrected Loeblich  s u r r o u n d i n g the  criteria  to  (1925) an  a  that  They a r b i t r a r i l y  possessed  based  oversight  stable  these  England pore. to  unreliable. were  on  a  Plymouth  ventral  pore;  d i a g n o s i s makes no r e f e r e n c e t o a v e n t r a l (Loeblich  difference  isolates  (1975) r e j e c t e d B a l e c h ' s  critical  and  the  from the type l o c a l i t y o f G.  in  a  and major  Loeblich,  small  isolates, The  not  be  degree  were  toxic,  The.  conspecific.  Loeblich  and  (Braarud)  i n favour of a d e s c r i p t i o n of bioluminescent,  and w i t h o u t a  diagnostically  N e v e r t h e l e s s , t h e s e i s o l a t e s from the A t l a n t i c  c o a s t of N o r t h  to  represent  the  discarded  was  as  considered  was  pore  feature  o f e x c a v a t i o n i n the s u l c a l r e g i o n , which  description,  The  i n t e r p r e t e d as an  (1971) d i a g n o s i s o f G. e x c a v a t a  t r o p i c a l specimens,  which  Braarud's  may  1975).  biochemical  as w e l l as a m o r p h o l o g i c a l c h a r a c t e r i s t i c was  indication  America  the  of  t o r e s o l v e the d i f f i c u l t i e s  isolates  as  of  (toxicity),  ventral  iconotype  b i o l u m i n e s c e n t but d i d not possess a v e n t r a l pore.  Lebour's  persistence  New  with  non-bioluminescent,  was  that  Balech,  was  Lebour's  Lebour as t y p i c a l o f the o r i g i n a l l y d e s c r i b e d s p e c i e s .  fact  Loeblich  drawn  attempted  two  non-toxic,  Plymouth  that  " t a m a r e n s i s " , " e x c a v a t a " , and " a c a t e n e l l a " .  one  tamarensis  point  tamarensoid  of  selected  fact  erroneously  (1975)  of  diagnosis  the  by L o e b l i c h and L o e b l i c h (1975) and T a y l o r (1975).  Loeblich  separation  173  the  This  simultaneously and  to  was  reversed.  surrounding  " t r u e " e x c a v a t a sensu  Braarud  45  (1945).  This  was  bioluminescent ventral  "excavata"  morphological  highly  plates  domed  narrowed  the  presence  (1977)  o b s e r v a t i o n s on  was  problems  prominent  i n a d v e r t e n t o p t i c a l r o t a t i o n o f the  in  acatenella.  T h i s has c o n s i d e r a b l y  and  (Loeblich  tamarensis,  and  hypothecal  Loeblich,  flanges  To r e - e s t a b l i s h the the  description  1975)  along  the  to  these  used  unworkable.  redescriptions  with  criteria  pore  species  of  did  not  distinctions  toxicity,  serve  bioluminescence,  the and  tamarensoid the l a c k o f a  isolates  toxicity  were observed.  but  had  a  clearly  one  defined  (Schmidt  o f the v e n t r a l  T u r p i n e t a l . (1978)  (NEPCC 71) resembled ventral  pore  and  G. was  t o be weakly t o x i c by the mouse b i o a s s a y , w h i l e the o t h e r (NEPCC  resembled reported  Ofunato  and  i s o l a t e s from B r i t i s h Columbia;  Lebour,  determined  (Anderson  not  t o a l l e v i a t e the  within  In a d e t a i l e d examination o f tamarensoid  on two  tamarensis  strains  the  t o s e p a r a t e " t a m a r e n s i s " from " e x c a v a t a " have proven t o  bioluminescence,  254),  include  p l a t e margins,  L o e b l i c h , 1979a and b ) , almost a l l p o s s i b l e combinations  reported  of  i n the o r i g i n a l d e s c r i p t i o n (Whedon and K o f o i d , 1936).  The  ventral  epitheca  acatenella  associated  group.  once Lebour's  r e c o g n i z e d , r e s t s s o l e l y upon the presence of a more  emended  Unfortunately,  pore,  toxic  between G. a c a t e n e l l a Whedon and K o f o i d  m o r p h o l o g i c a l gap between t h e s e s p e c i e s .  of  identified  was  between  acatenella  and  Dale's  the Norwegian type l o c a l i t y which l a c k e d a  distinction  cone-shaped  distinction  pore  from  G. t a m a r e n s i s Lebour,  epithecal  be  with  pore.  The and  congruent  with  in and  and Bay  G.  e x c a v a t a B a l e c h but d i d not possess the c h a r a c t e r i s t i c Balech's without  Wall,  (Fukuyo,  1978; 1979;  (1971)  description.  In Massachusetts,  the v e n t r a l pore were i s o l a t e d from nearby Alam  et  a l . , 1979).  T o r i u m i and Takano, 1979)  toxic bays  In Japan,  strains  from  conformed  generally to  46  the  description  of  var.  excavata  Braarud, but d i f f e r e d from G.  excavata  sensu L o e b l i c h and L o e b l i c h (1975) i n t h a t they possessed v e n t r a l p o r e s . A var.  r e c e n t r e - e x a m i n a t i o n o f Braarud's excavata  specimens has  and  conforming  revealed  According from  globosa,  to  ventral  as  well  as  excavata  pores  are  Braarud  more  in  both  forms  (1945),  rotund  which  specimens  they  Tangen,  1985).  excavatum; Braarud's  s p e c i e s , A. o s t e n f e l d i i .  synonyms i l l u s t r a t e s the d i f f i c u l t i e s  a s t a b l e taxonomic Shellfish  The  Canada  was  i n the Bay o f Fundy and the S t . Lawrence e s t u a r y o f  reportedly  (Prakash,  strains  involved i n assigning i t  position.  toxicity  "tamarensis"  caused  1963).  (referred  by  a  non-bioluminescent  Both b i o l u m i n e s c e n t and  form o f  non-bioluminescent  t o as "excavata") were i s o l a t e d from the 1972  New  bloom and found t o be m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e (Schmidt e t  1978).  non-toxic between  locality  h i s t o r i c a l l y , A. o s t e n f e l d i i B a l e c h and Tangen has had a t l e a s t  proposed  al.,  and  of unknown t o x i c i t y , conforming t o  that,  England  observations of  have renamed Alexandrium  fact  toxic  (Balech  of  i n a c c o r d w i t h the d e s c r i p t i o n of G. tamarensis v a r .  g l o b o s a , were t r a n s f e r r e d t o another new  eastern  detailed  h i s d e s c r i p t i o n s from the Norwegian type  var.  ten  and micrographs  t o B a l e c h and Tangen (1985), some t o x i c , b i o l u m i n e s c e n t specimens  Oslofjorden  other  var.  (1945) drawings  Similarly,  forms  Yentsch  et  al.  (1978)  found  both  o f " t a m a r e n s i s " from the c o a s t o f Maine.  toxicity  and  bioluminescence  is  obscure  at  The  toxic  and  relationship  b e s t , and p r o b a b l y  nonexistent. Fukuyo  (1980;  specimens,  has  "catenella"  by  These  include  1985;  Fukuyo  attempted using the  to  features  shape  et  of  a l . , 1985),  discriminate not  the  working  between  (dorsally  wide  Japanese  "tamarensis"  p r e s e n t i n the o r i g i n a l APC  with  and  descriptions.  and t r i a n g u l a r i n  47  "catenella"; pore  narrow t r i a n g u l a r or r e c t a n g u l a r i n " t a m a r e n s i s " ) ,  (present  plate, the  and  plate  only  margin  Whedon  and  to  both  pore  are  (1936)  of the p o s t e r i o r s u l c a l  d i d not r e g a r d  classic  of the c a t e n e l l o i d morphotype  particularly and  the  significant,  " c a t e n e l l a " a r e of  chain  length  and  as  his  approximately the  posterior  not always d i a g n o s t i c a l l y u s e f u l , s i n c e these a r e Thus, t o date,  the f r e q u e n t  not  emendments  d i a g n o s i s of members of the " t a m a r e n s i s / c a t e n e l l a " complex have not  1. G e o g r a p h i c a l  s e p a r a t i o n between morphotypic v a r i a n t s .  Paralytic  shellfish  dinoflagellates deaths  1975).  It  channel is  i n t o x i c a t i o n and Blooms  poisoning  of  (PSP),  attributable  of the genus Protogonyaulax, has  fatalities the  E n v i r o n m e n t a l S i g n i f i c a n c e of  Blooms  throughout the w o r l d .  of in  Studies  D i s t r i b u t i o n and  Protogonyaulax spp.  1799,  as  Unfortunately,  E. R a t i o n a l e f o r E x p e r i m e n t a l  number  shape  characteristic  i n mature c e l l s .  y i e l d e d an u n e q u i v o c a l  300  the  Fukuyo  "tamarensis"  cells.  features  the  "tamarensis").  Kofoid  of  isodiametrical attachment  in  compression  re-descriptions  constant  "catenella"),  the p o s i t i o n of the p o s t e r i o r attachment pore ( l o c a t e d c l o s e r t o  antero-posterior sensu  in  the v e n t r a l  in  most cases  been the cause of a t  Based on h i s t o r i c a l r e c o r d s , the  a s s o c i a t e d w i t h a s i n g l e outbreak o f PSP  r e p o r t e d t h a t more than 100  least  highest  occurred  known as P e r i l S t r a i t s near S i t k a , A l a s k a  to  in  (Fortune,  A l e u t h u n t e r s succumbed to  PSP  d i e d a f t e r e a t i n g t o x i c mussels. Protogonyaulax  spp.  responsible  for  PSP  are  widely  48  distributed  in  (Fig.  There a r e o c c a s i o n a l r e c o r d s o f Protogonyaulax from t h e A r c t i c  and  5). the  coastal  tropics,  including  recently  from  tendency  f o r blooms  (Taylor,  in  Thailand  directly  the B r i t i s h  (Woloszynska al.,  1985;  Balech  and  et al.,  England  coast  1978; On  Alam  north  of  1985), but t h e r e i s a s t r o n g  a r e t h e prime s u s p e c t s as c a u s a t i v e agents f o r PSP (Gemmill  Atlantic,  Protogonyaulax  and Manderson,  1939),  Sanchez,  North  blooms  Portugal ( S i l v a ,  1963), S p a i n ( B l a n c o e t  1985), Norway ( B r a a r u d , 1945; D a l e , 1977;  A t l a n t i c Canada ( N e e d i e r , 1949; Prakash, 1963;  America (Anderson and W a l l , 1978; Y e n t s c h e t a l . ,  (Whedon and K o f o i d , 1936; Sommer e t a l . ,  1975),  through  the A l e u t i a n  1978; et  Chew,  1975),  and  1982a,  PSP  British  Columbia  from A l a s k a ( H a l l e t a l . ,  1937), Washington  (Prakash  and  1980; H a l l ,  c h a i n t o t h e c o a s t o f Japan (Oshima,  b  and  a l . , 1982).  c;  State  T a y l o r , 1966;  1982), e x t e n d i n g 1976; Oshima e t  Oshima and Yasumoto, 1979; Nishihama, 1980;  I n the s o u t h e r n hemisphere, Protogonyaulax blooms  i n temperate waters a r e known from s o u t h e r n C h i l e (Lembeye e t  al.,  1975), Uruguay  al.,  1983;  Carreto  ( D a v i s o n and Y e n t s c h , 1985) and A r g e n t i n a (Benavides e t et  a l . , 1985),  as w e l l as t h e c o a s t o f South A f r i c a  ( G r i n d l e y and N e l , 1968; 1970; G r i n d l e y and S a p e i k a , 1969; T a y l o r ,  of  1982).  P a c i f i c c o a s t s , t o x i c Protogonyaulax spp. have been r e c o r d e d from  Taylor,  The  are  1960; Wood, 1968), Belgium  1979; Anderson and M o r e l , 1979; Anderson e t a l . ,  and  causing  a l . , 1985), and  1971; White, 1982; White and White, 1985) and a l o n g t h e New  (Norris  Ogata  et  zones  or  1985),  et a l . ,  California  al.,  (Ferraz-Reyes  temperate  the north  and  Tangen,  Prakash  i n the  In  Conrad,  Fraga  Venezuela  (Tamiyavanich e t a l . ,  Isles  and  particularly  t o be more p r e v a l e n t between 30-60° N and S l a t i t u d e s  1984).  implicated  waters,  present  southwestern  study British  1984).  i s based l a r g e l y upon t o x i c i s o l a t e s from t h e c o a s t Columbia  and  Washington  State ( F i g . 6 ) . In the  49  Fig. 5  G l o b a l d i s t r i b u t i o n o f some members o f t h e genus Protogonyaulax. A- P. a c a t e n e l l a ; C- P. c a t e n e l l a ; F- P. f r a t e r c u l a ; H- P. c o h o r t i c u l a ; K- "Gonyaulax" kutnerae; P- P. p e r u v i a n a ; T- P. t a m a r e n s i s ; N- Protogonyaulax s p . : New Z e a l a n d tamarensoid i s o l a t e . R e v i s e d a f t e r T a y l o r , 1984.  51  Fig. 6  Map o f the n o r t h e a s t P a c i f i c c o a s t showing o r i g i n of Protogonyaulax i s o l a t e s . Morphotype a t time o f i s o l a t i o n : C = c a t e n e l l o i d ; I = i n t e r m e d i a t e ; T = tamarensoid.  130  125  53  southwestern  British  attributable  to  frequently,  1942, in  and  four  and  of  fatalities. poisonous  have Three  clams  sporadic  resulted deaths  from  red  tide  outbreaks  in  illness,  and,  were  reported  after  B a r k l e y Sound, Vancouver  an i n c i d e n t a t M a l a s p i n a I n l e t  i n Geogia S t r a i t  less the  Island, i n  i n 1965  resulted  i l l n e s s e s and one f a t a l i t y a f t e r t o x i c c o c k l e s were e a t e n (Prakash  Taylor,  Strait  region,  Protogonyaulax  in  consumption  Columbia  1966).  i n 1957 was  although  no  British  A  poorly  documented r e p o r t of r e d water  a s s o c i a t e d w i t h PSP  i n Georgia  i n t o x i c a t i o n i n more than 60 p e o p l e ,  deaths o c c u r r e d d u r i n g t h i s outbreak. The most r e c e n t death i n  Columbia  due t o PSP was  t h a t o f a n a t i v e I n d i a n a t H e a l t h Harbour,  G i l f o r d I s l a n d i n 1980. The  frequency,  caused  by  Protogonyaulax  research, program  for  toxicity.  of  (Prakash  1982;  White,  In  general,  of  British  et  PSP  Columbia  Local  specific  locations  toxicity  trends  Sound  need  f o r continuing  intensive  of a v i g i l a n t and e f f e c t i v e m o n i t o r i n g  i s p a r t i c u l a r l y worrisome  Anderson,  t h a t i n some a r e a s ,  1984), p a r t i c u l a r l y  i n s o u t h e r n New  i n s t e a d , a p e r i o d i c p a t t e r n of t o x i c i t y ,  seven  year  conditions on  England.  British to  tendencies 1984),  cycle,  fluctuating  has been noted (Gaines and T a y l o r ,  appeared t o have a marked e f f e c t on t o x i c i t y i n  the  difficult  recent  (Anderson,  Anderson and M o r e l , 1979; Anderson e t a l . ,  i n the r e c e n t y e a r s s i n c e r o u t i n e s h e l l f i s h m o n i t o r i n g  i n 1942;  approximate  shown  the  t o x i c i t y has not shown a c o n s i s t e n t i n c r e a s e on the c o a s t  1985).  have  It  a l . , 1971;  1982;  initiated an  underscores  PSP a r e i n c r e a s i n g i n i n t e n s i t y and e x t e n d i n g i n g e o g r a p h i c a l  range  on  d i s t r i b u t i o n , and s e v e r i t y o f PSP r e d t i d e s  as w e l l as the maintenance  outbreaks  was  widespread  but  Columbian  predict. to this  c o a s t , making the long-term  In Washington  S t a t e , t o x i c blooms  encroach i n t o the lower e x t e n t o f Puget has been somewhat o f f s e t by d e c r e a s i n g  5 4  toxicity  in  some  and N i s h i t a n i ,  the  Protogonyaulax  a  of  the  potent  neurotoxins  produced  o f contaminated s h e l l f i s h ,  presence,  inhibitory  effect  shellfish. not  public  health of  on  hazard.  these  both  poses  I n a d d i t i o n , t h e presence, o r even  dinoflagellate  the  by  f o r which  s c i e n c e has, as y e t , f a i l e d t o produce an e f f e c t i v e a n t i d o t e ,  suspected  commercial  toxins and  has  had a markedly  recreational  harvest of  The s t a t e o f knowledge r e g a r d i n g bloom i n i t i a t i o n and dynamics yet  provided  distribution. the  ingestion  through t h e consumption  substantial  has  (Erickson  1985).  Obviously,  medical  o f t h e t r a d i t i o n a l l y more t o x i c n o r t h e r n areas  In  reliable  many  p r e d i c t i v e models t o account f o r t o x i c i t y  c o u n t r i e s , t h e e x t e n t o f t h e problem has r e q u i r e d  implementation o f an expensive and o f t e n i n e f f i c i e n t m o n i t o r i n g system,  which  i s o n l y m a r g i n a l l y adequate  concerns, potential  much  less  to  search  t o d e a l w i t h t h e immediate f o r causal  relationships  public health and t o pursue  solutions.  2. The Problem o f Morphotypic G r a d i e n t s and S t a b i l i t y i n Protogonyaulax  In coast  certain of  areas of the northeast P a c i f i c  British  Columbia,  difficulties  in  particularly  acute.  have  reliably  found  in  Washington  State  there  morphotypes,  with  these  the  i n Washington  State  and  Alaska - the  d i s t i n g u i s h i n g " c a t e n e l l a " from " t a m a r e n s i s " a r e  Both  been  and  - i n Japan, on the southwest  t h e c a t e n e l l o i d and t h e tamarensoid morphotype waters.  is a  I n southwestern B r i t i s h Columbia and  tendency  tamarensoid  form  for spatial generally  separation  o f the  predominating i n more  55  estuarine  waters  (Taylor, these  and  1984).  alternate  isodiametrical Cembella  et  al.,  1982;  often can  species  intermediate  o f t e n predominating b).  coasts  occur  Oshima  (short  i n boundary areas  (Taylor,  to  be t e m p o r a l l y as w e l l as  e t a l . , 1982c; Fukuyo, 1985).  1980;  chains  of  1984; and  spatially  i n the same l o c a t i o n (Kodama e t a l . , 1982;  descriptors  (Fukuyo, 1979;  forms  In Japan, blooms of the tamarensoid  tend  have a l s o been noted  "tamarensis"  open  with  they  taxonomists  on  morphotypes,  but  the  dominating  t h e r e i s an apparent o v e r l a p i n the range of  cells)  intermediates  form  Nevertheless,  morphotype  separated,  that  catenelloid  and T a y l o r , 1985a and  catenelloid  Ogata  the  i n Japan, a l t h o u g h currently  1985;  used  Morphological  i t must be  by  recognized  prominent  Fukuyo e t a l . , 1985)  Japanese  to d i s c r i m i n a t e  from " c a t e n e l l a " have r e s u l t e d i n c o n s i d e r a b l e r e d e f i n i t i o n of  these s p e c i e s . In  addition  field  to  populations  the of  Protogonyaulax,  morphological  conversion  or  that  complete,  isolate except  for  also  the  with  and  is  the  which can occur i n problem of apparent  one morphotype to the o t h e r , e i t h e r  occur  partial  when i s o l a t e s a r e brought i n t o c u l t u r e .  (F.J.R.  field,  became rounder and approached  (Nishihama,  1980).  An  the  S i m i l a r transformations  T a y l o r and A. Cembella, p e r s . obs.)  for  from B r i t i s h Columbian and Washington S t a t e p o p u l a t i o n s . within  to  into  the genus Protogonyaulax , expressed  c e l l p r o p o r t i o n s , c h a i n l e n g t h and  including  level,  culture  observed  variation  calls  prosaic  in  been  respect  plates,  there  l a c k of c h a i n f o r m a t i o n ,  form  i s o l a t e s taken The  can  from  intergradation  from Hokkaido, Japan, which appeared as a " c a t e n e l l a " i n the  tamarensoid have  morphological  pores,  question the  particularly  f e a t u r e s of the t h e c a l  tends t o b l u r the d i s t i n c t i o n between morphotypes conventionally  reliability  of  these  used  species descriptors.  features  involves  At a  the narrow  56  taxonomic  question:  "To  nonetheless  important  toxicity.  There  biological defined?"  what  species  does  this  belong?"  -  which  is  f o r t h e i d e n t i f i c a t i o n o f blooms a s s o c i a t e d w i t h PSP  are  questions,  a l s o wider i m p l i c a t i o n s c o n c e r n i n g more fundamental such  a s : "What a r e s p e c i e s ? " , "How a r e good s p e c i e s  and "What i s the b a s i s f o r v a r i a t i o n among and w i t h i n s p e c i e s ? " -  as they may be a p p l i e d t o d i n o f l a g e l l a t e s .  3. Research O b j e c t i v e s  The aspects the  research of  presented  discriminate  between  among  objectives  polymorphism  among  approaches.  members  of  are  address  to  In  particular,  epifluorescence  microphotometry  (performance)  biochemical  soluble  liquid  and  are  criteria.  regime  used  morphotype  to  Answers  isolates or  Protogonyaulax  isolates,  and  s p e c i e s complex.  regarding by  biochemical isozymes,  c h a r a c t e r s used t o  are  techniques,  including g e l  analysis  used  from  one  assigned  compared  with  Physiological  to  estimate to  genotypic  DNA by  study  microscopic growth  v a r i a b i l i t y and chemotaxonomic  quantitative toxin  typical  by  high-pressure  The r e s u l t s o f t h e observations  studies  variation  analysis  chemotaxonomic  using  under a c o n s t a n t within  the f o l l o w i n g s p e c i f i c questions  location  The  applying  p o p u l a t i o n v a r i a t i o n w i t h i n t h i s group.  morphological  populations.  and b i o c h e m i c a l  questions  chromatography  analyses  are  the  Protogonyaulax  of  are  t h e s i s i s an attempt t o examine some  morphological  electrophoresis  affinities  this  the b i o l o g y o f the organisms c a u s i n g PSP r e d t i d e s , f o c u s i n g on  relationship  general  in  and  among  a r e sought: 1)  of the geographical  population,  species, e i t h e r morphologically or biochemically?;  57  2)  what  and  i s the degree of d i f f e r e n t i a t i o n  among  populations  morphological 4)  of  variation  the  same  reflected  ( p h e n o t y p i c and g e n o t y p i c ) w i t h i n  morphospecies?;  3)  i s the degree of  i n the amount of b i o c h e m i c a l v a r i a t i o n ? ;  do d i f f e r e n t b i o c h e m i c a l c h a r a c t e r s i n d i c a t e the same l e v e l o f v a r i a t i o n  and  d i f f e r e n t i a t i o n ? ; 5) would a taxonomic scheme f o r t h i s group based  phenetic accord  or  phylogenetic  with  different  one  constructed  biochemical  discriminated  as  relationships  between  genetic these  variation questions  underlying  the  dinoflagellates.  criteria  from  from  characters?;  "good"  species?;  the  natural  the  biochemical  morphological 6)  can  and  7)  a n a l y s e s be i n  characters,  contribute  processes  of  or  from  P. c a t e n e l l a / P . tamarensis  be  are t h e r e any c l e a r l y d e f i n a b l e  environment, morphotype, the degree of  and the b i o c h e m i c a l c h a r a c t e r s i n v e s t i g a t e d ? will  upon  to  the  understanding  differentiation  and  Answers t o  of the mechanisms speciation  in  58  CHAPTER I I  MATERIALS AND  A. C u l t u r e and  METHODS  Maintenance  1. I s o l a t e O r i g i n s and Growth C o n d i t i o n s  Protogonyaulax (Harrison North  et  East  Columbia, from  cultures  a l . , 1980) Pacific  Atlantic  Canada White  (Clone  A.  and  Oceans, Canada). from  Collection  northeast  the  Pacific  coast  University  an  isolate  from  S t a t i o n , S t . Andrews, N.B.,  Twenty-three  author  (NEPCC),  were o b t a i n e d from the of B r i t i s h  the  Bay of Fundy  y  = NEPCC 545) p r o v i d e d through the c o u r t e s y o f Dept. o f F i s h e r i e s  Protogonyaulax c l o n e s , i s o l a t e d i n June  were s u p p l i e d by G. Gaines (Oceanography  of B r i t i s h Columbia). by  s e v e r a l y e a r s on ESNW medium  A d d i t i o n a l i s o l a t e s were a v a i l a b l e by exchange  //7  (Biolog i c a l  for  - S i , +0.5uM Mo)  including  B a m f i e l d , B.C.,  University isolated  B.C.  collections,  Dr.  1984  (-Tris,  Culture  Vancouver,  other  maintained  from  Other c u l t u r e s were i n i t i a t e d from natural  (Fig.  phytoplankton  assemblages  Dept., cells on the  6 ) , p a r t i c u l a r l y those i n E n g l i s h Bay, a t  Vancouver. Inocula cultures  in  experimental maximum flasks.  cell  from 125  the NEPCC r e f e r e n c e c u l t u r e s were used t o i n i t i a t e s t o c k ml  purposes, density  Erlenmeyer  flasks.  To produce s u f f i c i e n t c e l l s f o r  the s t o c k c u l t u r e s were added a t a p p r o x i m a t e l y the t o two  litres  of c u l t u r e medium i n 2800 ml Fernbach  59  All at  s t o c k and e x p e r i m e n t a l c u l t u r e s were i n c u b a t e d i n a growth chamber  16  m~2  °C  on  a  14:10 h  s ~ l (measured  probe;  with  Biospherical  fluorescent  l i g h t / d a r k c y c l e , a t an i r r a d i a n c e o f 120 u E i n  a  submerged  Instruments,  lights.  Biospherical  San Diego,  QSL-100P  CA) p r o v i d e d by c o o l - w h i t e  The c u l t u r e s were n o t s t i r r e d , but were p e r i o d i c a l l y  shaken g e n t l y t o r e - e s t a b l i s h homogeneous c e l l Natural  Instruments  seawater  f o r a l l experiments  distribution.  was o b t a i n e d from B u r r a r d  Inlet  off  the p i e r a t the Westwater L a b o r a t o r y , Canadian Department o f F i s h e r i e s  and  Oceans,  through 20  a  litre  West 0.45  Vancouver, Mm  Millipore  polyethylene  culturing.  The  B.C., a t a depth o f 15 m.  After  filtration  f i l t e r , seawater was s t o r e d i n darkness i n  carboys f o r a t l e a s t a month b e f o r e b e i n g used f o r  ambient  s a l i n i t y was t y p i c a l l y  i n the narrow range from  26-28°/oo and was n o t a d j u s t e d .  2. Development and F o r m u l a t i o n o f NWSP-7 Growth Medium  A the  search culture  1968;  of of  1984).  totally  new  existing  and h i g h  enrichment  L o e b l i c h , 1975;  For this  medium  ( P r o v a s o l i e t a l . , 1957;  to  reason,  cell  cultures  were  f o r natural  the a r t i f i c i a l  G u i l l a r d and  i t was n o t n e c e s s a r y t o f o r m u l a t e a  c u l t u r e Protogonyaulax  yield  P r o v a s o l i , 1963;  H a r r i s o n e t a l . , 1980;  spp.,  but r a t h e r t o modify  r e c i p e s t o a c h i e v e the most e f f e c t i v e combination  experimental  from  dinoflagellates  M c L a c h l i n , 1973;  Keller,  rate  t h e l i t e r a t u r e r e v e a l e d no l a c k o f growth media used f o r  f o r experimental  o f r a p i d growth  requirements.  Stock and  grown on NWSP-7 ( T a b l e 1 ) , a newly f o r m u l a t e d  seawater.  seawater  medium  T h i s f o r m u l a t i o n was h i g h l y m o d i f i e d ASP-7 ( P r o v a s o l i , 1963), which has  60  T a b l e 1.  F o r m u l a t i o n o f NWSP-7 n a t u r a l seawater M.W.  enrichment medium. 8 1"  [ f i n a l ] uM  I. N u t r i e n t Stock (add 1 ml l " ) 1  NaN0 Na gIycerophosphate•5H2O NaH P0 -H 0  85.0 306.1 138.0  3  2  2  4  2  85.00 19.90 4.83  1000.0 65.0 35.0  I I . T r a c e M e t a l s Stock (add 1 ml l " ) C h e l a t o r : T r a c e M e t a l r a t i o 1  Na EDTA-2H 0 FeCl '6H 0 H3BO3 MnCl -4H 0 ZnS0 -7H 0 CoCl '6H 0 Na Mo0 •2H 0 2  2  2  3  2  2  2  4  2  2  2  4  2  372..2 270.,3 61.8 197.9 287.0 238.0 242.0  16.75 2.70 3.09 0.99 0.58 0.12 0.12  45.0 10.0 50.0 5.0 2.0 0.5 0.5  191.1  19.11  100.0  2.5:1  pH 5.0  I I I . N i t r i l o t r i a c e t i c a c i d (NTA) Stock (add 1 ml l " ) 1  IV. V i t a m i n s Stock (add 1 ml l " ) 1  Cyanocobalamin ( B ^ ) Thiamine HC1 Nicotinic acid Ca pantothenate p-aminobenzoic a c i d Folic acid Biotin Inositol Thymine pH 7.8 2  mg 1" 1.0 500.0 100.0 100.0 10.0 2 0 1.0 5000.0 3000.0  A d j u s t pH o f seawater t o 6.0 a f t e r t h e a d d i t i o n o f m a c r o n u t r i e n t s and t r a c e metals. A f t e r a u t o c l a v i n g , add s t e r i l i z e d NaHCC^ t o y i e l d a f i n a l C - c o n c e n t r a t i o n o f 2.0 mM.  61  been  extensively  Prakash  et  used  i n t h e c u l t u r e o f d i n o f l a g e l l a t e s (Prakash, 1967;  a l . , 1971; Tomas, 1974; N o r r i s and Chew, 1975), but which gave  unacceptably  low  growth  rates  and  a  high  percentage o f m o r p h o l o g i c a l  a b e r r a n t s i n t h e p r e s e n t s t u d y ( s e e Appendix I ) . In  a d d i t i o n t o t h e replacement o f t h e a r t i f i c i a l  natural  seawater  important buffer a  ways.  to  result  base, Tris  from  required trace  (hyroxymethyl  from  ASP-7  aminomethane),  i n several added  other  t o ASP-7 as a  o f p h o t o s y n t h e t i c a c t i v i t y , was o m i t t e d a f t e r i t was shown t o be  i s often  deleted  differs  minimize t h e i n c r e a s e i n pH o c c u r r i n g d u r i n g a u t o c l a v i n g and as  i n h i b i t o r y t o growth Si  NWSP-7  seawater s a l t s w i t h a  added  NWSP-7,  nutrient  to  broad-spectrum a l g a l growth media, but i t was  since  this  element  f o r the culture  requirement  contributions  ( s e e Appendix I ) .  from  f o r S i would the natural  i s not u s u a l l y considered a  of dinoflagellates. be  readily  seawater  satisfied  base  or  I n any event, a by  background  by l e a c h i n g from t h e  c u l t u r e glassware. The reflect (Cembella increased yield. only  ratio  of  typical et to  N:P  i n t h e medium was a d j u s t e d t o 10:1 t o a c c u r a t e l y  intracellular  a l . , 1984b). 1000  uM  P-source  ratios  100  uM,  i s often  (Provasoli,  found i n marine p h y t o p l a n k t o n  The c o n c e n t r a t i o n s o f N (as NaNOj) and P were  and  Glycerophosphate  N:P  respectively,  added  to  maximize  t o a l g a l c u l t u r e media as t h e  1963; H a r r i s o n e t a l . ,  1980), due t o i t s l e s s e r  tendency  t o form c o p r e c i p i t a t e s w i t h metals d u r i n g a u t o c l a v i n g  1968).  However,  as  the sole  enzymatic activity  (Provasoli,  t h e u t i l i z a t i o n o f g l y c e r o p h o s p h a t e , a phosphate  P-nutrient  could  potentially  cleavage  of  P^  (Cembella  et  a l . , 1984a).  cell  be  limited  from t h e o r g a n i c moiety by  by  ester,  the rate of  phosphomonoesterase  Phosphomonoesterase  a c t i v i t y was  62  undetectable comparison and  i n non-P-limited  be  (A. Cembella,  released  through  autoclaving, (as  (2:1 molar  unpublished thermal  obs.).  96)  Presumably,  degradation  and  costatum  (NEPCC  of  inorganic  in  P-limited  linkage during  I n NWSP-7, both o r g a n i c P  ( P ^ ) were  supplied  seawater  of  that  (McLachlin,  typically  1973),  added  to  i t can s a f e l y be reduced t o a synthetic  seawater  base.  The  o f B added t o NWSP-7 was decreased by more than 90% r e l a t i v e  ASP-7. The  P  I I metals  given  i n Table  2H2O),  which  Compared  solution  1. was  o f ASP-7 was r e p l a c e d by the f o r m u l a t i o n  The m o d i f i e d not present  t o ASP-7,  NWSP-7  metals  mix i n c l u d e d Mo (as Na2MoO^*  i n Provasoli's  contained  a  (1963)  two-fold  In  the  siderophores  progress (with  Fe-limitation diatoms This  and  suggests  requirement. tide  presence  G.  can be other  minimal  experiments  that Indeed,  on  i n Fe ( a s  substantially.  the a c t i v i t y  of Fe-binding  more  readily  this  species  induced (Trick may  i n Protogonyaulax et a l . ,  have  Iwasaki (1979) c l a s s i f i e d such  species  display  a  1983;  Fe quota ( Q i m  n  Fe^»  than i n  M u e l l e r , 1985).  comparatively  high  Fe  " t a m a r e n s i s " as a "Type 2" accelerated  h i g h c o n c e n t r a t i o n s o f d i s s o l v e d Fe and/or Mn. cell  I I mixture.  Boyer, u n p u b l i s h e d e x p e r i m e n t s ) , i t was noted t h a t  dinoflagellates  flagellate; of  of  P  increase  F e C l 3 ) , w h i l e t h e e x c e s s i v e Mn c o n c e n t r a t i o n was reduced  of  (NEPCC 18c)  B i s n o r m a l l y p r e s e n t i n excess o f p o t e n t i a l growth requirements  concentration  red  low i n  some f r e e P^ may  the ester  but t h i s has never been e s t a b l i s h e d .  natural  fraction  minimum  255, and  ratio).  Since  to  Prorocentrum  Na2~glycerophosphate*5H20)  in  isolate  t o t h a t d e t e c t e d i n t h e diatom Skeletonema  the d i n o f l a g e l l a t e  culture  Protogonyaulax  growth  i n the  Measurements  c a l c u l a t e d p e r u n i t c e l l volume f o r  6 3  Fe-depleted value  batch  cultures  approximately  pseudonana,  in a  of  twice  Protogonyaulax  as h i g h as f o r a n e r i t i c diatom, T h a l a s s i o s i r a  similarly  Fe-depleted  Cembella and G. Boyer, u n p u b l i s h e d The metal  chelator:  trace  availability to  McLachlin,  1973; H a r r i s o n  NWSP-7  was  be  toxicity.  considered  i n t h e range  formulated  et  to  condition  (Mueller,  1985; A.  obs.).  metal r a t i o  and  i s o l a t e NEPCC 255, gave a  i s a c r i t i c a l f a c t o r governing Since  of  ratios  are usually  1.5-3.0: 1 ( P r o v a s o l i e t a l . ,  a l . , 1980),  yield  optimum  trace  1957;  t h e t r a c e metals s o l u t i o n o f  an EDTA ( e t h y l e n e d i a m i n e t e t r a a c e t a t e ) :  metals r a t i o o f 2.5: 1. Although  i t i s n o t as  triacetic  acid)  Reducing  the f i n a l  present NTA  effective  possesses  considerable  concentration  i n ASP-7,  to  i n stimulating  100  a c h e l a t o r as EDTA, NTA ( n i t r i l o -  of  NTA  metal  rate  capacity.  i n NWSP-7 from 366 LIM, as i s  MM, s i m u l t a n e o u s l y  t h e growth  complexing  acknowledges t h e e f f e c t o f  o f Protogonyaulax (Yentsch e t a l . ,  1975),  and t h e p o t e n t i a l f o r i n h i b i t i o n a t e x c e s s i v e c o n c e n t r a t i o n s .  growth  inhibition  chelation the  of  nominal  assuming  may  be  required total  due  either  metals.  ratio  of  m e t a l complexation  to  direct  toxicity  This  o r t o over-  Even w i t h t h e lowered NTA c o n c e n t r a t i o n , c h e l a t o r binding s i t e s t o t r a c e metal ions,  o n l y w i t h t h e added s y n t h e t i c c h e l a t o r s , was a  r e l a t i v e l y h i g h 27: 1. The final  NTA  s t o c k s o l u t i o n was prepared  concentration  NaOH t o enhance The (Na ) 2  at  trace  distilled  t h e f r e e a c i d a t 1,000X  water and t i t r a t i n g t o n e u t r a l i t y  with  solubility. metals  i n warm  1.000X  to  by adding  final  stock  distilled  s o l u t i o n was prepared  water  concentration.  (~60  by d i s s o l v i n g t h e EDTA  ° C ) , then adding  t h e t r a c e metals  The pH was a d j u s t e d t o ~2, a f t e r which  64  the  chelator-trace  minutes  to  to  mildly  a  metal  solution  ensure complexation. acidic  pH  was  heated  to  boiling  f o r several  A f t e r c o o l i n g , t h e s o l u t i o n was a d j u s t e d  5.0  and  stored  refrigerated  i n one  litre  polycarbonate b o t t l e s . Nutrients trace  metal  precipitation the  pH  was  were  added  and NTA  (1.0 ml  stock  1"^) from t h e m a c r o n u t r i e n t ,  solutions p r i o r to autoclaving.  i n t h e medium, due t o an i n c r e a s e adjusted  to  6.0  using  1.0  To minimize  i n pH w h i l e  N  HC1  n o t respond  well  after  chelated  autoclaving,  the addition of  nutrients. Protogonyaulax (White,  cultures  1976) which  C-limitation  may  particularly  those  volume To  unstirred  restore  pH  C-limitation added  might  n o t be with  balance  i n large  vials  serve a  (8  requirement  to  stimulate  growth  volume  metabolites.  1~1  ^>12>  filtered filter  a  S3 s  solution  a  and  to  NaHCC^  eliminate inorganic  was  the p o t e n t i a l f o r C  autoclaved  ( a s Na2HC03) was f o r 15  min i n  autoclaving. t h a t Protogonyaulax has an o b l i g a t o r y t  n  e  providing  specified  through  Although  ml) and then a s e p t i c a l l y added t o t h e medium (2.0 mM  The v i t a m i n  Provasoli's  C-limitation.  areas f o r gas exchange, i n l a r g e  cultures,  Anhydrous  f o r ^>\2> by  surface  8.2,  i t i s likely only  alleviate  reduced growth r a t e and c e l l y i e l d may r e s u l t .  f i n a l C-concentration) a f t e r Although  to  t o constant a g i t a t i o n  s i g n i f i c a n t f a c t o r i n s m a l l volume c u l t u r e s ,  large  cultures  t o NWSP-7.  screw-cap  do  vitamin  precursors f o r NWSP-7  (Provasoli, f o r ASP-7  syringe  and s t o r e d f r o z e n  mix  complex  1963),  medium.  mixture  i n NWSP-7 may  f o r the biosynthesis was  adopted  supplemented  vitamin  o f other  directly with  The v i t a m i n s were  from  1.0 M g sterile-  equipped w i t h a d i s p o s i b l e 0.22 Mm M i l l i p o r e  i n 50 ml p o l y c a r b o n a t e b o t t l e s .  The s o l u t i o n was  65  added a s e p t i c a l l y a f t e r a u t o c l a v i n g As  a  "semi-defined"  enrichment  of  associated  with  which  soil  1975),  of  duplicated for  mass  other  NWSP-7  avoids  some  a  of  n a t u r a l seawater media.  i s added, such as E r d - S c h r e i b e r suffer  c u l t u r e , based upon  severe  between  N a t u r a l media t o  (Fjflyn, 1934)  d i s a d v a n t a g e i n t h a t the  nutritional  NWSP-7 may be p r e p a r e d i n l a r g e volumes  culture, while avoiding  t h e time-consuming and expensive p r o c e s s  totally artificial  seawater base.  I s o l a t i o n and C l o n i n g  cultures  were  initiated  from  natural  samples  using a glass micropipette  chamber  under low power (160X) o f t h e i n v e r t e d microscope.  phytoplankton  t o i s o l a t e c e l l s from a two ml c o u n t i n g Isolated  isolation  nutrients)  i n g l a s s spot p l a t e s u n t i l v i s i b l y f r e e o f a s s o c i a t e d  Individual  cells trays,  isolation  medium.  lid  the  of  approximately sterile allowed  medium  (filtered  seawater  enriched  w i t h 10% ESNW  were then t r a n s f e r r e d t o the w e l l s o f s t e r i l i z e d  isolation  were  cells  passed s e q u e n t i a l l y through a s e r i e s o f f o u r t o s i x s t e r i l e washes i n  autoclaved  of  and GPM  preparations.  Protogonyaulax  were  t h e problems  t h e enrichment i s n o t o n l y unknown, b u t cannot be c o n s i s t e n t l y  of p r e p a r i n g  3.  1  f o r dinoflagellate  seawater,  certain  extract  (Loeblich, quality  natural  medium  (1 ml l " ) .  with  each  well  containing  approximately  Growth was monitored d a i l y by o b s e r v a t i o n  plastic  trays  using  a  dissecting  species. plastic  one ml o f through t h e  microscope.  After  20 c e l l d i v i s i o n s , t h e c u l t u r e s were t r a n s f e r r e d t o t h r e e ml isolation to  medium i n 15 ml screw-cap t e s t tubes.  reach l a t e exponential  The c u l t u r e s  phase, as determined by f o l l o w i n g  66  the  i n vivo  Then  they  fluorescence  were  Erlenmeyer  curve ( s e e Chapter I I C . l ) t o a maximum v a l u e .  transferred  flasks  to  75  and m a i n t a i n e d  ml  of  f u l l - s t r e n g t h ESNW i n 125 ml  as r e f e r e n c e i s o l a t e s w i t h i n the NEPCC  through r o u t i n e t r a n s f e r s a t t h e end o f each c u l t u r e  4.  P r e p a r a t i o n and T e s t i n g o f A x e n i c C u l t u r e s  Two  Protogonyaulax  antibiotics  and  medium  culture  concentration (mixed  1:1) o f  Thus,  medium  (Table  added  to 1.0  tubes drop  and  255, were t r e a t e d w i t h  t h e method (Table  2)  o f Droop (1967). was  p r e p a r e d i n NWSP-7  in  screw-cap  15  ml  progressively  reduced  so  solution  tube  on  test by  contained  half  the cultures,  3 ) , m o d i f i e d from STP medium  each tube.  a  culture  through a sequence  with  tubes.  The  dilution  i n untreated cultures. the o r i g i n a l  o f s i x tubes.  antibiotic  A f t e r mixing  one drop o f s t e r i l i t y  (Provasoli et a l . ,  each  of  bacterial  growth.  A n t i b i o t i c - t r e a t e d c u l t u r e s were i n c u b a t e d f o r 24 h,  these  subcultures  and kept  test  A f t e r two weeks, one  was added t o t h r e e ml o f a u t o c l a v e d  i n t h e dark a t room temperature t o t e s t f o r  Any i n c r e a s e i n t u r b i d i t y over a p e r i o d o f t h r e e weeks  noted by v i s u a l i n s p e c t i o n o f t h e tubes, and by f o l l o w i n g density  test  1957), was  ml from each was t r a n s f e r r e d i n t o d u p l i c a t e 15 ml screw-cap  medium  optical  antibiotic  two-fold s e r i a l  c o n t a i n i n g e i g h t ml o f a n t i b i o t i c - f r e e medium. of  The  i n a 1:1 r a t i o w i t h 6 ml o f e x p o n e n t i a l l y  culture  sterility  was  solution  253  aseptically  and  antibiotic  by  the a n t i b i o t i c - t r e a t e d  the f i r s t  concentration,  then  was  NEPCC  axenic  antibiotic  and mixed  growing  isolates,  rendered  sterile-filtered  the  cycle.  (Spectronic  20  spectrophotometer;  Bausch  increases i n and Lomb,  T a b l e 2.  A n t i b i o t i c mix f o r Protogonyaulax  purification.  C o n c e n t r a t i o n (mg ml Benzyl p e n i c i l l i n sulfate Streptomycin s u l f a t e Chloramphenicol  T a b l e 3.  8.0 1.6 0.2  M o d i f i e d STP s t e r i l i t y t e s t medium f o r t h e m o n i t o r i n g o f p o s s i b l e b a c t e r i a l growth i n a n t i b i o t i c - t r e a t e d Protogonyaulax cultures.  For 100 ml A u t o c l a v e d NWSP-7* , V i t a m i n mix f o r NWSP-7"' Autoclaved s o i l extract Yeast a u t o l y s a t e Sucrose Na-glutamate DL-alanine Trypticase Glycine pH 7.5 "see T a b l e 1.  95.0 ml 0.1 ml 5.0 ml 20 mg 100 mg 50 mg 10 mg 20 mg 20 mg  Rochester, results  NY)  of  at  the  turbidity),  620  nm.  sterility  Cultures tests  were  were  judged t o be a x e n i c  negative  ( i . e . no  increase  and i f no b a c t e r i a were d e t e c t e d by p h a s e - c o n t r a s t  examination  under  high  power  (1,000X)  using  a  Zeiss  i f the in  microscopic oil-immersion  objective. In  the  counts al., ml  two l i t r e u n i a l g a l Protogonyaulax c u l t u r e s , p e r i o d i c b a c t e r i a l  were  carried  out  using  the e p i f l u o r e s c e n c e t e c h n i q u e  (Hobbie e t  1977), t o ensure t h a t the c u l t u r e s were not h i g h l y contaminated. of  unialgal  pre-treated  with  Greensboro,  least  The  St.  f i l t e r e d onto a Nuclepore f i l t e r  Black  acridine  Sigma,  ten  were  Irgalan  NC).  concentration) chloride;  culture  (0.22 urn)  ( C . I . , a c i d b l a c k 107; Ciba-Geigy  cultures orange  were  incubated  (3,6-bis  with  0.01%  [dimethylamino]  Two  Corp., (final  acridinium  L o u i s , MO) f o r two minutes p r i o r t o f i l t r a t i o n .  At  h i g h power (1,000X) microscope f i e l d s and a t l e a s t 200 b a c t e r i a  for  each  sample  were  The  concentration  of  counted  u s i n g a Z e i s s e p i f l u o r e s c e n c e microscope.  b a c t e r i a p e r ml and t h e t o t a l b a c t e r i a l c e l l volume  r e l a t i v e t o t h a t o f the d i n o f l a g e l l a t e s were  B. M o r p h o l o g i c a l  estimated.  Examination o f Protogonyaulax I s o l a t e s  1. I s o l a t e I d e n t i f i c a t i o n and M o r p h o l o g i c a l C h a r a c t e r i s t i c s  Cultures conditions medium. phase,  f o r morphological (Chapter  Only by  A.l)  cultures  following  morphological  II  studies.  the  that  examination in  natural  were  growth  were seawater  grown  under  standard  e n r i c h e d w i t h NWSP-7  v e r i f i e d t o be i n e x p o n e n t i a l growth  curve  fluorometrically,  were used f o r  69  Protogonyaulax however occurs for  they  do  toward  cells  of  the  mitotic  interphase.  case  of  the  catenelloid  of  measured  maximum  was  between  The  as  when  was  cell  using  the  and  (2b) axes  apical  dimension  was  measured as t h e  t h e a p i c a l and a n t a p i c a l p o l e s ; t h e t r a n s a p i c a l  determined  calculated minor  near  The c e l l s were i m m o b i l i z e d i n 0.5% f o r m a l i n  immediately.  cingulum,  In the  i s o l a t e s which formed c h a i n s , a s i n g l e c e l l  measurements.  diameter  dimension the  s e l e c t e d , a l t h o u g h obvious a b e r r a n t s  each c h a i n was taken t o be a r e p r e s e n t a t i v e i n d i v i d u a l f o r  dimension  and  when t h e m a j o r i t y o f t h e c e l l s were i n  which appeared t o be undergoing d i v i s i o n were r e j e c t e d .  middle  cell  cycle,  examined were randomly  the  Cells  examination were c o l l e c t e d near t h e middle o f t h e l i g h t  light/dark  cells  those  usually  t h e end o f t h e dark c y c l e (Tomas, 1974; p e r s . o b s . ) .  period  and  c u l t u r e do n o t d i v i d e i n a b s o l u t e synchrony,  d i s p l a y a tendency f o r phased d i v i s i o n ; d i v i s i o n  morphological  The  in  t h e maximum e q u a t o r i a l diameter p a r a l l e l t o oriented  i n v e n t r a l view.  The volume was  a p i c a l and t r a n s a p i c a l dimensions as t h e major (2a) in  the  formula  for a  prolate  spheroid,  where  V = A/3irab . 2  Epifluorescence thecae made the  treated  m i c r o s c o p i c o b s e r v a t i o n s o f i n t a c t c e l l s and d i s c a r d e d  w i t h 0.1% c a l c o f l u o r white M2R (Sigma, S t . L o u i s , MO) were  t o c o n f i r m t h e presence or absence o f a v e n t r a l pore a t t h e margin o f first  complex position  apical  (APC) of  ( l  1  ) plate.  The  size  and shape o f t h e a p i c a l pore  and t h e p o s t e r i o r s u l c a l p l a t e , as w e l l as t h e p r e s e n c e and  the  anterior  and  p o s t e r i o r attachment pores (when p r e s e n t )  were a l s o n o t e d . Isolates taxonomic  designated  status  by  the  NEPCC number were a s s i g n e d a p r o v i s i o n a l  w i t h i n t h e Protogonyaulax s p e c i e s complex (P. c a t e n e l l a ,  Table 4.  Isolate  Location of O r i g i n and Morphological C h a r a c t e r i s t i c s of Isolates of the Protogonyaulax tamarensis/catenella species complex.  O r i g i n of Isolate  Ventral pore  Apical Diameter (um) 3? ± s.d.  Transapical Ratio Mean Diameter (im) A p i c a l : Volume X" ± s.d. Transapical (urn-*) Diameter (X10 )  Shape o f epicone and hypocone  Morphotype***  # c e l l s per chain****  3  71*  P a t r i c i a Bay, B.C. Canada, Aug., 1973  Present  35.84 1 3.40  32.34 1 1.96  1.11  19.63  high domed epicone; rounded hypocone  Tamarensoid  SI  180  Brentwood Bay, B.C. Canada, Aug., 1973  Absent  37.96 t 3.94  37.25 i 3.05  1.02  27.60  Rounded  Tamarensoid  S2  183*  Tamar estuary, Plymouth, England, June, 1957  Present  36.21 t 2.17  33.84 ± 1.89  1.07  21.72  Rounded  Tamarensoid  52  253*  Laguna Obldos Portugal, 1962  Absent  24.88 ± 1.75  22.38 ± 2.09  1.11  6.53  Rounded  Tamarensoid  $2  255  Lummi Island, HA, USA. Aug., 1976  Present  37.09 i 1.41  33.34 t 3.08  1.11  21.60  Rounded  Tamarensoid  52  Isolate  O r i g i n of Isolate  Ventral pore  Apical Transapical Ratio Mean Diameter (|ia) Diameter (urn) Apicalt Volume Jf t a.d. X t s.d. Transapical (MB') Diameter (X10 )  Shape of epicone and hypocone  Morphotype*  I c e l l s per chain****  3  400  E n g l i s h Bay, B.C., Canada, June, 1981  Absent  33.25 t 2.34  31.66 ± 2.58  1.05  17.46  Usually rounded; few flattened  Tamarensoid  S2  401  E n g l i s h Bay, B.C., Canada, June, 1981  Absent  36.04 i 2.80  32.00 t 1.88  1.13  19.33  Rounded  Tamarensoid  S2  403*  English Bay, B.C., Canada June, 1981  Absent  38.09 t 3.53  35.13 i 2.89  1.08  24.64  Rounded  Tamarensoid  52  404*  E n g l i s h Bay, B.C., Canada June, 1981  Present  33.54 ± 3.13  33.41 1 2.88  1.00  19.62  Usually rounded; few flattened  Tamarensoid  32  405*  E n g l i s h Bay. B.C., Canada June, 1981  Absent  28.42 ± 2.80  25.91 t 2.48  1.10  10.00  Rounded  Tamarensoid  406*  E n g l i s h Bay. B.C.. Canada June, 1981  Absent  27.50 t 3.38  26.38 t 2.34  1.04  10.02  Rounded  Tamarensoid  22  407*  English Bay, B.C., Canada, June, 1981  Absent  28.33 ± 2.35  26.25 ± 2.28  1.08  10.24  Rounded  Tamarensoid  52  Isolate  O r i g i n of Isolate  Ventral pore  Apical Transapical Ratio Mean Diameter (tan) Diameter (MB) A p i c a l : Volume X 1 s.d. JT t s.d. Transapical (ml Diameter (X10 )  Shape of eplcone and hypocone  Morphotype*  # c e l l s per chain****  3  409  English Bay, B.C., Canada, June, 1981  Absent  30.09 t 2.36  27.21 ± 2.36  1.11  11.68  Rounded  Tamarensoid  52  412  English Bay, B.C., Canada, June, 1981  Present  28.66 i 2.75  25.96 t 2.06  1.10  10.12  Rounded  Tamarensoid  52  508  Whangarei, North Island, New Zealand, Feb.. 1983  Absent  29.38 ± 2.68  26.71 t 3.02  1.10  10.99  Rounded  Tamarensoid  52  545*  Bay of Fundy, N.B., Canada J u l y , 1976  Absent  35.95 t 3.09  32.00 ± 3.35  1.12  19.06  Rounded  Tamarensoid  402  E n g l i s h Bay, B.C., Canada, June, 1981  Absent  32.91 t 2.58  33.46 t 3.34  0.98  18.99  Epicones/ hypocones rounded or flattened  Intermediate  52  516  E n g l i s h Bay, B.C., Canada, June, 1982  Present  30.84 ± 6.73  29.66 i 3.19  1.04  14.21  Epicone egg- or bellshaped; hypocone tapered**  Intermediate  52  52  Isolate  O r i g i n of Isolate  Ventral pore  Apical Diameter (um) JT ± s.d.  Mean Transapical Ratio Diameter (um) A p i c a l i Volume x"± s.d. Transapical (um ) Diameter (X10 ) 3  Shape of epicone and hypocone  Morphotype***  # c e l l s per chain****  3  254  Hidden Basin. Nelson Island B.C., Canada Oct., 1976  Absent  28.33 t 3.05  30.21 t 3.20  0.94  12.70  Rounded  Catenelloid  S2  355*  Penn Cove, Whldbey Island, WA. USA, 1980  Absent  29.34 ± 2.45  32.50 t 2.13  0.90  14.65  Flattened  Catenelloid  i4  356*  Quartermaster Harbor, Vashon Island. WA, USA 1980  Absent  29.17 t 4.60  30.92 t 2.02  0.94  13.78  Flattened  Catenelloid  £4  435  Friday Harbor, San Juan Island, WA, USA, Aug., 1982  Absent  33.91 t 3.33  36.46 i 3.43  0.93  21.97  Flattened  Catenelloid  24  529  Friday Harbor, San Juan Island, WA, USA, Aug., 1983  Absent  32.13 t 2.66  32.50 t 2.23  0.99  17.58  Flattened  Catenelloid  £4  543  Friday Harbor, San Juan Island, WA, USA Aug., 1983  Absent  30.29 t 2.88  33.50 t 2.04  0.90  16.10  Flattened  Catenelloid  £4  • c l o n a l I s o l a t e | **some specimens have f l a t t e n e d epicones and hypocones; ***aorphotype as presently i n culture; * * * * t y p i c a l number of c e l l s per chain a t time of o r i g i n a l i s o l a t i o n .  74  P.  tamarensis,  morphological  or  Protogonyaulax  characteristics  intermediate)  (Table  4).  on the b a s i s of g e n e r a l  C e l l s of the " c a t e n e l l a "  type  were  those  originally  i s o l a t e d i n c h a i n s t y p i c a l l y c o n s i s t i n g of f o u r or  more  cells  and  remained somewhat a n t e r o - p o s t e r i o r l y compressed i n  culture  (ratio  embraced than  those  a  few  usually  which  apical:transapical isolates  c e l l s , and  >1:1.  which  cells  <1:1).  The  "tamarensis"  type  d i d not o r i g i n a l l y form c h a i n s of g r e a t e r  i n which the a p i c a l : t r a n s a p i c a l diameter  Isolates  isodiametrical  diameter  that  characteristically  made  ratio  was  s h o r t c h a i n s of  w h i l e i n e x p o n e n t i a l growth phase were c o n s i d e r e d t o  r e p r e s e n t the " i n t e r m e d i a t e " morphotype.  2. Chain Length Experiments  The  effects  low  l e v e l s of p a r t i c u l a r n u t r i e n t s (N, P, and Fe)  the  length  355  (Penn Cove, Whidbey I s l a n d , WA)  cycle.  The  (Harrison  (5.5  (as  1980)  the  form  by  had  i s o l a t e NEPCC  were i n v e s t i g a t e d throughout  an  (-Tris, of  reducing  medium,  to  1%  the  N:P  FeCl3).  proportionately, Fe and  al.,  nutrient  uM)  medium Fe  in  prepared  limiting  c h a i n s formed i n c u l t u r e s of the c a t e n e l l o i d  on  a culture  c o n t r o l c u l t u r e s c o n t a i n e d n u t r i t i o n a l l y complete ESNW medium  et  exclusively were  of  of  the of  the ratio  +0.5  pM  Mo),  (6.6  uM).  Low  N, P, and Fe c u l t u r e s  added  was  with  Fe  concentration.  0.25:1, The  For the low  w h i l e the low P (0.2  low Fe medium c o n t a i n e d 0.07  c o n c e n t r a t i o n of EDTA i n the metals mix was  keep  the c h e l a t o r : metal  c o n t r o l c u l t u r e s (1.6:1).  added  c o n c e n t r a t i o n s of the p r o s p e c t i v e  control  r a t i o of 2500:1. The  to  N:P  FeCl^  -Si,  N  pM) pM  reduced  r a t i o e q u i v a l e n t i n the  low  75  Ten  ml  o f NEPCC 355 m a i n t a i n e d on ESNW were t r a n s f e r r e d a t t h e end o f  exponential  growth  containing  50  maintained  under  intervals advanced  o f medium. standard  t o minimize  length of  exponential,  early  stationary  and  The f l a s k s were g e n t l y Four ml o f  u s i n g a 7 mm diameter l e n g t h o f s t e r i l e s e l e c t i o n against longer chains,  a narrow bore p i p e t t e .  random  I I A . l ) , and sampled a t  b e f o r e t h e samples were removed.  non-random  was examined  25  late  (Chapter  of the culture cycle.  were withdrawn  t h e use o f  made  conditions  phases  flasks  T r i p l i c a t e c u l t u r e s f o r each treatment were  t o ensure homogeneity  tubing,  chain  i n t o 125 ml b o r o s i l i c a t e g l a s s Erlenmeyer  i n mid-exponential,  culture  from  ml  senescence  swirled each  phase  After fixation  glass  arising  (0.5% f o r m a l i n ) ,  under p h a s e - c o n t r a s t m i c r o s c o p y and counts were  fields  (125X)  i n d u p l i c a t e 2 ml i n v e r t e d microscope  c o u n t i n g chambers.  C. V a r i a t i o n i n Growth Rates  1. D e t e r m i n a t i o n o f A c c l i m a t e d Growth Rates  For  the determination  cultures NWSP-7 the  natural a  t h e NEPCC  were  acclimated  growth  rates,  unialgal  effects  of  f o r a l l isolates seawater depth  exponential  of  phase,  m)  To minimize  water q u a l i t y d i f f e r e n c e s on growth, t h e growth were  (collected 15  stock  m a i n t a i n e d i n 125 ml Erlenmeyer f l a s k s on  medium under s t a n d a r d c o n d i t i o n s (see Chapter I I A . l ) .  possible  rates  at  from  of  determined  s i m u l t a n e o u s l y , u s i n g t h e same  from B u r r a r d I n l e t , Vancouver, J u l y 15, 1983  f o r the preparation  of  t h e medium.  1.0 ml o f each c u l t u r e was t r a n s f e r r e d i n t o  In l a t e triplicate  25  X  150 mm  screw cap b o r o s i l i c a t e g l a s s c u l t u r e tubes each c o n t a i n i n g 25  ml of medium. to  allow  The tubes were p l a c e d a t a 45° angle w i t h caps for  gas  exchange.  of  the  standard  modification proceed  through  maximal  cell  early  containing  second  transfer  Growth  was  fresh  achieved.  medium.  experimental essentially  (1981a)  determination  room  the  through  incubation  the  chamber  light  and  before  held the  was  corresponded  approximately  (White,  and were a l l o w e d t o  u n t i l approximately  that  the  control  can  to  cycle,  the  f o r 15 min  fluorometric determined to  cultures the  was  method  10% of the  the  cause  were  At t h r e e  day  removed from  the  i n a 16 °C water bath a t ambient measurements  during middle  the of  were  late the  made.  The  afternoon,  which  light  p e r i o d o f the  i s unusually s u s c e p t i b l e to turbulence  the  Rather  tubes  monitored  of Brand e t a l .  of a c c l i m a t e d growth r a t e s .  Protogonyaulax  i n growth r a t e .  homogeneity, to  always  cycle.  1976)  reduction  regime  without  Growth r a t e s were compared d u r i n g t h i s  according  growth  fluorescence  light/dark  incubated  Then they were r e - i n o c u l a t e d i n t o  and  fluorometrically,  intervals  phase  were  series.  in  for  cultures  environmental  exponential  density  tubes  The  loosened  formation  of p e l l i c u l a r c y s t s and  than v o r t e x mixing  the samples t o  a  ensure  tubes were g e n t l y i n v e r t e d t h r e e times immediately  prior  i n s e r t i o n i n t o the f l u o r o m e t e r . In  (Turner  v i v o f l u o r e s c e n c e was Designs,  photomultiplier, Corning 2-64) maximal  5-60;  Mountain and  the  reference  determined View,  CA)  appropriate filter:  u s i n g a Turner equipped filter  Corning  10-000R f l u o r o m e t e r  w i t h an i n f r a r e d  system ( e x c i t a t i o n  3-66;  emission f i l t e r :  f o r the d e t e c t i o n of i n v i v o c h l o r o p h y l l a f l u o r e s c e n c e . growth  rates  were  determined  from  sensitive  the  semi-log  filter: Corning  Acclimated plots  of  77  fluorescence  versus  time  over  the  time  interval  ( t j - t ) indicated by 0  the linear portion of the growth curve. As  a  between  calibration for cell  number  self-absorption  departure  and  in  vivo  from  linearity in the relationship  fluorescence,  which can arise due to  by cells, particularly in dense cultures, and at the lower  detection  limit  plots  c e l l number versus in vivo fluorescence were constructed using a  of  series  of  Since  of  the fluorometer when the c e l l density is low, log-log  two-fold  cell  dilutions of  densities  ml"^-, self-absorption  in  a  dense culture in exponential phase.  Protogonyaulax  effects were  cultures  rarely  exceeded ~10^  less than would be expected for many  other phytoplankton species. For  comparison,  previously k  =  cell  described,  and  lnCN-i/NQXl.AAS/t),  and  N  the  time  others  are  Q  the  interval  (Brand  et  cell  counts  of  in  vivo  increases  in  numbers  (div at  Control  a l . , 1981a;  increases  k  densities Q  d" ) 1  tj  and  is  cultures,  as  can  et  the  growth  rate, Nj_  t , respectively, and t is Q  experiments  Watras  fluorescence of  several  the growth rates calculated using the formula, where  t^-t .  were made on  performed  a l . , 1982)  have  here and by shown that  be used to accurately represent  Protogonyaulax cells through division, provided  sufficient time is allowed for acclimation in a constant environment.  78  D.  DNA A n a l y s i s by E p i f l u o r e s c e n c e  Microphotometry  1. N u c l e a r DNA D e t e r m i n a t i o n  The  DNA  buffered  of  Protogonyaulax c e l l s was s t a i n e d by i n c u b a t i n g c e l l s i n a  solution  2-phenyl-indole) by  dissolving  mg  ml  of  DAPI  - 1  )  culture  of  the  (Sigma, DAPI  distilled  stock  were  (=  S t . L o u i s , MO).  (0.1  in  1:50  n u c l e i c a c i d fluorochrome DAPI ( 4 , 6 - d i a m i d i n o -  mg  ml" ), 1  The s t o c k s o l u t i o n was p r e p a r e d  Tris  d e i o n i z e d water.  added  directly  dilution)  and  - 1  ) , and EDTA (0.37  F o r t h e working s o l u t i o n , 0.2 ml  to  the  (0.60 mg m l  10  ml o f e x p o n e n t i a l l y  mixture  was  incubated  growing at  room  temperature.  The curve o f r e s u l t a n t e p i f l u o r e s c e n c e i n t e n s i t y v e r s u s time  was  over  followed  binding. as  no  this  A  s t a n d a r d i n c u b a t i o n p e r i o d o f not l e s s than 2 h was  further elapsed  time.  (200X)  programmed  in  yield  effects the  a  and  to  examined u s i n g a Z e i s s a  computerized  was  photomultiplier  system  Zoology D e p t . ) ,  fluorescence  from background and n o n - n u c l e a r  DNA,  stopped down t o c i r c u m s c r i b e o n l y t h e n u c l e a r a r e a .  filter  the  epifluorescence  readout o f r e l a t i v e e p i f l u o r e s c e n c e . To minimize t h e  extraneous  barrier  stage  connected  were  BASIC (J.D. Berger, Univ. B r i t i s h Columbia,  aperature  blue  Cells  digital  of  adopted,  i n c r e a s e i n n u c l e a r DNA e p i f l u o r e s c e n c e was d e t e c t e d a f t e r  microscope  to  48 h t o determine s a t u r a t i o n k i n e t i c s f o r fluorochrome  was i n t e r p o s e d between the e p i f l u o r e s c e n t  d e t e c t o r t o suppress the b r i l l i a n t  A  microscope  red autofluorescence of  the c h l o r o p l a s t s . The  detector  erythrocytes determined  system was q u a n t i t a t i v e l y c a l i b r a t e d u s i n g the n u c l e a t e d  o f the domestic c h i c k e n , G a l l u s g a l l u s domesticus, which were to  be  uniform  in  size  and  nuclear fluorescence after  DAPI  79  staining. 0.43  The  s.e.m.)  techniques  erythrocytes  (2.55 ±  was c a l c u l a t e d from l i t e r a t u r e v a l u e s o b t a i n e d u s i n g  various  (Fasman,  Protogonyaulax (NEPCC  from  cells,  202a;  (n=30  mean  DNA c o n t e n t o f c h i c k e n  1976).  Nuclear  epifluorescence  for individual  and those o f an i s o l a t e o f Gonyaulax p o l y e d r a  Stein  ex. B. Sweeney, La J o l l a , CA), was measured i n q u i n t u p l i c a t e  cells the  nuclear  per  isolate).  ventral  Epifluorescence  equatorial  view  of  o f i n t a c t n u c l e i was measured  the  c e l l s , a f t e r applying  gentle  p r e s s u r e t o t h e cover s l i p t o l i n e a r l y extend t h e n u c l e u s . In  addition  cells 183,  identified relative  of  vegetative  r e s u l t i n g from s e x u a l 255,  from  to  403, by  to  dark  their  and  435 was determined.  large  diameter cells.  planomeiocytes  reddish  the nuclear  DNA c o n t e n t o f z y g o t i c  f u s i o n i n senescent c u l t u r e s o f NEPCC i s o l a t e s  normal v e g e t a t i v e  excysted a  405  cells,  (50-70  Zygotes were o p e r a t i o n a l l y nm)  and  Planozygotes could  lumpy morphology, be d i s t i n g u i s h e d  by t h e i r d a r k e r brown p i g m e n t a t i o n and l a c k  accumulation  body  (Anderson e t a l . , 1983; Anderson,  1984).  E.  Method f o r P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s  1. E l e c t r o p h o r e s i s  of Soluble  a. C e l l c u l t u r e , h a r v e s t  For  Enzymes  and s t o r a g e  t h e e l e c t r o p h o r e t i c s t u d i e s , Protogonyaulax i s o l a t e s were c u l t u r e d  under  the  standard  yield  cultures  conditions  f o r isozyme  already  analysis  specified that  were  (Chapter I I A . l ) . subject  To  t o t h e minimal  80  accumulation isolates  of  from  English  progressively  larger  soon  as  were  followed  these  possible  Cell cycle day,  Bay,  NEPCC  the  were c u l t u r e d  i n enzyme p a t t e r n s  the  when  i n long-term c u l t u r e , two c l o n a l  403  and  407,  were t r a n s f e r r e d  was  original isolation.  Their  cells  qualitative  confined  By h a r v e s t i n g  were  in  following optical  enzyme  cell  counts  and  approximately  applied equal  X  10^ for  GSA  Dupont  rotor;  loose  pellets  activity  within  rinsed  and  buffer  [dithiothreitol];  was  cycle,  growth, determined by By comparing  a conversion  f a c t o r was  the c u l t u r e volume h a r v e s t e d , such t h a t cells  (~2 X 10^) were o b t a i n e d f o r each  cell  d e n s i t i e s were t y p i c a l l y between  upon  the  isolate.  (3,000  -  Sorvall,  Cultures  were  Wilmington, DE) a t 4 °C; the  resuspended  in  (100 mM T r i s , Sigma,  St.  a  50-fold  1 mM EDTA, 10 mM  removed  volume  of  Cleland's  L o u i s , MO) a t pH 7.5 (4 ° C ) ,  X g, S o r v a l l SS-34 r o t o r ; Dupont Instruments -  Wilmington, DE) i n p r e - c h i l l e d 8 ml g l a s s v i a l s .  buffer  cell  was minimized.  curve t o a maximum v a l u e .  depending  Instruments  washing  recentrifuged  Sorvall, wash  DTT  the  min a t 5,000 X g i n a r e f r i g e r a t e d c e n t r i f u g e ( S o r v a l l  were  sterile-filtered reagent,  5  of  harvest,  ml,  clonal  the p o t e n t i a l f o r q u a n t i t a t i v e  vivo fluorescence,  to adjust  At per  centrifuged  in  numbers  extraction.  1.0-5.0  then  with  of  a t a p p r o x i m a t e l y t h e same time o f  a t t h e end o f e x p o n e n t i a l  the i n v i v o f l u o r e s c e n c e  calculated  enzyme  harvested  subcultures  to the middle o f the l i g h t / d a r k  were i n i n t e r p h a s e ,  variation  patterns  produced.  p a r t i c u l a r l y f o r those enzymes which may be p h o t o i n d u c i b l e , Cells  isozyme  i n d e p e n d e n t l y t o compare any p o s s i b l e  always  incubator.  most  into  c u l t u r e volumes- 1 ml, 8 ml, 75 ml, then 2000 ml- as  after  collection  of  modifications  over a p e r i o d o f more than one year; m u l t i p l e  isolates  instability  and  genotypic  The s u p e r n a t a n t  by a s p i r a t i o n , then the c e l l p e l l e t s were q u i c k  81  frozen  i n d r y i c e and l y o p h i l i z e d o v e r n i g h t .  desiccator  a t -40 °C u n t i l  V i a l s were s t o r e d  i n a vacuum  extraction.  , b. B a c t e r i a l c o n t a m i n a t i o n  Although subsequent (1,000X) the  every  effort  growth,  low  typically  <  indicated  that  volume.  1  x  made t o reduce b a c t e r i a l c o n t a m i n a t i o n and  numbers  phase-contrast  epifluorescence  was  of  microscopy  bacteria  were noted under high-power  i n a l l cultures.  P e r i o d i c counts u s i n g  t e c h n i q u e (Hobbie e t a l . , 1977) (Chapter I I A . 3 ) , were 10^  bacteria  bacteria  were  per  ml.  always  <  T o t a l c e l l volume c a l c u l a t i o n s 1%  of  the t o t a l d i n o f l a g e l l a t e  As a c o n t r o l , 1.0 l i t r e o f u n i a l g a l c u l t u r e was f i l t e r e d through a  Whatman  filter  allowing  the  refiltered  under  gentle  bacteria  through  a  to  vacuum  pass  into  nm GSWP  0.22  t o remove t h e d i n o f l a g e l l a t e s , w h i l e the  filtrate.  Millipore  The  membrane  f i l t r a t e was  to  bacteria.  The f i l t e r e d b a c t e r i a were then c o l l e c t e d i n s t e r i l e  buffer  electrophoresed  and  further extracts, were  test  of  possible  with  bacterial  dinoflagellate contamination  extraction  extracts. of  the  As a  dinoflagellate  two i s o l a t e s p u r i f i e d by a n t i b i o t i c treatment, NEPCC 253 and 255,  electrophoresed  supernumerary p u t a t i v e  in  p a r a l l e l with extracts  b a c t e r i a l bands were ever  c. D e t e r m i n a t i o n o f t o t a l  In  along  retain  order  to  ensure  consistently  applied  samples  periodically  was  to  from u n i a l g a l c u l t u r e .  No  observed.  protein  that the  sufficient  gels,  determined  total  quantities  of  protein  s o l u b l e p r o t e i n from  were  extracted  by t h e B r a d f o r d t e c h n i q u e ( B r a d f o r d ,  82  1976). in  The  method i n v o l v e s s p e c t r o p h o t o m e t r i c a l l y  absorbance  of  the g e n e r a l  (Sigma,  St.  Protein  samples were s o n i c a t e d  the  L o u i s , MO),  protocol  were  given  determined  albumin prepared  in  absorbance  was  in  Spectronic  595  a  enzyme e x t r a c t i o n .  in  the  range  curve  0.1-1.0  Sample p r o t e i n  plotted  mg  ml .  and  subtracted. 21  Brilliant  Blue  G-250,  Due  t o the h i g h  sample  blank  absorbance the  measured i n 1 cm p a t h l e n g t h  spectrophotometer (Bausch and  values  u s i n g bovine serum  a f t e r the a d d i t i o n of the s t a i n , and  Absorbance was  to  Reagent b l a n k s were  - 1  b u f f e r used f o r enzyme e x t r a c t i o n .  before  blank  for  t o sample p r o t e i n s .  c l a r i f i e d by c e n t r i f u g a t i o n a c c o r d i n g  calibration  Coomassie  measured . both  and  the  the ,of  caused by n o n - s p e c i f i c b i n d i n g  herein  increase  p r o t e i n s t a i n Coomassie B r i l l i a n t B l u e G-250  from  standards  measuring the  was sample  cuvettes  Lomb, R o c h e s t e r , NY)  at  nm.  d. Enzyme e x t r a c t i o n  Cell 'pellets 0.35 50  ml •uM  of  [nicotinamide  +  [nicotinamide  an  bursts. examination fragments, Sonicated  adenine  (Bronwill  ice-ETOH  a p p r o x i m a t e l y 2 X 10^  i c e - c o l d e x t r a c t i o n b u f f e r (100  NAD  sonicated  containing  bath This  dinucleotide Biosonik  at  adenine  60%  procedure  of  minimized extracts  and  virtually  no  and  1 mM  dinucleotide]  phosphate],  maximum  sonicated  4 °C f o r 15 min  Tris,  pH  EDTA, 10 mM  and  7.5  50  at  4  nM  DTT, NADP  °C),  output enzyme  indicated  intact  cells  (= 210  W)  f o r 90 s i n 10  denaturation. that  only  +  and  Model I I I , W i l l S c i e n t i f i c , R o c h e s t e r , NY)  of  extracts  mM  c e l l s were suspended i n  in s  Microscopic minute membrane  or major o r g a n e l l e s  remained.  were c e n t r i f u g e d a t 27,000 X g ( S o r v a l l SS-34 r o t o r ) a t the s u p e r n a t a n t was  d i r e c t l y a p p l i e d to the  gels.  83  e. Electrophoretic  Electrophoretic polyacrylamide vertical  separation  separation o f Isozymes was performed i n 1.5 mm thick 7%  g e l (PAG) (T=7.2$;  g e l apparatus  C=2.6$)  (Table  5), using a dual-slab  (Model VS-14, Proteus Technology, Richmond, B.C.),  with plate dimensions o f 180 X 140 nun.  Table 5.  Polyacrylamide Gel Formulation  Separating gel (7$) 7.0 ml acrylamide: Bis-acrylamide (30$: 0.8?) 15.4 ml d i s t i l l e d H 0 7.4 ml 1.67 M T r i s , pH 8.8 at 4°C 0.15 ml ammonium persulfate (10$) 15 f l TEMED 2  All  electrophoresis g e l buffer  reagents  were  was  M  0.4  obtained  from  Sigma (St. Louis,  M0).  The  T r i s , pH 8.8 at 4 °C; the electrode  buffer  f o r a l l enzyme systems was 0.19 M glycine - 0.05 M T r i s , pH 8.3 at  4 °C. Electrophoresis 4  °C, with  ice-water pump. phoresed  was  carried  out i n a  additional  cooling  provided  refrigerator by  a  compartment at  internally  circulating  bath driven from a two l i t r e external reservoir by a p e r i s t a l t i c  P r i o r to the application of enzyme extracts, gels were pre-electrof o r 50  Vh  at 2 W per gel constant power (voltage max.: 100 V;  current max.: 40 mA per g e l ) . Glycerol was added to equal 10$ of the t o t a l sample volume, a f t e r which  84  80  nl  loaded  of  extract  into  p a r a l l e l wells  added  to  10%  wells  to  serve  between  (=  glycerol  runs,  as a  characteristics  Vh  to  increased gel)  until  was  required  8 a  per  mm  of  were  standard  (see  6):  dehydrogenase (G6PDH,  E.C.  1.1.1.30),  malic  discrepancies  of known e l e c t r o p h o r e t i c Electrophoretic  strain.  m a i n t a i n e d a t 2 W per g e l f o r  w i t h i n the g e l . 400  elapsed.  V;  The  power, was  c u r r e n t max.:  50  then  75 mA  E l e c t r o p h o r e t i c runs  per  normally  origin.  (GDH,  All  E.C. gels  1970;  dehydrogenase  E.C.  1.4.1.2),  1.1.1.49),  malate  Sing,  (AlaDH,  isocitrate  1.1.1.40), and were  E.C.  gels  (MDH,  dehydrogenase  dehydrogenase  E.C.  (HBDH,  E.C.  E.C.  1.1.1.42),  1.1.1.37), NADP-dependent  s u c c i n a t e dehydrogenase (SucDH, i n the dark a t 37 °C f o r one  extracts  1976)  1.4.1.1), glutamate  dehydrogenase (IDH,  incubated  containing  Hopkinson,  glucose-6-phosphate  B-hydroxybutyrate  dehydrogenase  H a r r i s and  then a t room temperature u n t i l maximum r e s o l u t i o n was Duplicate  end  a t which time the t r a c k i n g dye m i g r a t e d a  (Brewer and  Alanine  enzyme (ME,  1.3.99.1).  a p p l i e d t o the  band s c o r i n g  NADP-dependent  NAD-dependent  Vh had h,  was  s t a i n e d f o r the f o l l o w i n g enzymes, w i t h minor m o d i f i c a t i o n s  techniques  Table  255,  were  (0.01%)  i n the same g e l p o s i t i o n .  g e l ( v o l t a g e max.:  from the  f . G e l s t a i n i n g and  Gels  NEPCC  alignment  4.5  isolate,  Bromophenol b l u e  t o a p r i m a r y band from t h i s  protein  approximately  each  To s t a n d a r d i z e minor  l o a d i n g , the power was  W  from  e x t r a c t i o n b u f f e r and  strain,  always run  t o t a l of 750  d i s t a n c e of 105  the  reference  for  to  protein)  the t r a c k i n g dye.  extract  allow  Mg  i n random o r d e r .  in  m o b i l i t i e s were n o r m a l i z e d After  ~120  of  E.C. hour,  achieved. each  isolate  were  TABLE 6.  Stain protocols f o r dehydrogenase Isozymes  Components added to 60 ml buffer Enzyme  Stain buffer  Alanine dehydrogenase AlaDH (B.C. .1.4.1.1)  0.1 M phosphate  pH at 37°C Substrate 8.0  Chelator  Metal cofactor Nucleotide cofactor  DL-alanine 500 mg  NAD 10 mg  Stain MTT (10 mg m l " ) 1.5 ml PMS (5 mg m l " ) 0.5 ml 1  1  Glucose-6-phosphate dehydrogenase G6PDH (E.C. 1.1.1.49)  0.1 M T r i s  Glutamate dehydrogenase GDH (E.C. 1.4.1.2)  0.1 M T r i s  8.0  8.0  D-glucose6-phosphate 400 mg  MgCl (0.5 M) 1.0 ml 2  NADP 10 mg  MTT (10 mg m l " ) 1.5 ml PMS (5 mg ml" ) 0.5 ml 1  1  NAD 10 mg  L-glutamate 1000 mg  MTT (10 mg m l " ) 1.0 ml PMS (5 mg m l " ) 0.5 ml 1  1  B-hydroxybutyrate dehydrogenase HBDH (E.C. 1.1.1.30)  0.1 M phosphate  Isocitrate dehydrogenase IDH (E.C. 1.1.1.42)  0.1 M T r i s  Malate dehydrogenase MDH (E.C. 1.1.1.37)  0.1 M T r i s  Malic enzyme ME (E.C. 1.1.1.40)  0.1 M T r i c i n e  Succinate dehydrogenase SucDH (E.C. 1.3.99.1)  0.1 M phosphate  7.5  8.0  8.0  8.0  7.0  NAD 10 mg  3-DL-hydroxybutyrate(Na) 500 mg  MTT (10 mg ml* ) 1.5 ml PMS (5 mg m l " ) 1.0 ml 1  1  MgCl (0.5M) 1.0 ml  DL-isocitric acid(Na) 500 mg  2  NADP 10 mg  MTT (10 mg m l " ) 1.0 ml PMS (5 mg m l " ) 0.5 ml 1  1  NAD 10 mg  L-malate(Na) 500 mg  MTT (10 mg ml" ) 1.0 ml PMS (5 mg m l " ) 0.5 ml 1  1  MgCl,(0.5 M) 1.0 ml  L-malate 500 mg  NADP 10 mg  MTT (10 mg m l " ) 1.0 ml PMS (5 mg m l " ) 0.5 ml 1  1  succinic acid (Na ) 400 mg 2  EDTA(Na ) 200 mg 2  NAD 20 mg ATP 30 mg  MTT (10 mg m l " ) 2.0 ml . PMS (5 mg m l " ) 1.0 ml 1  1  00  86  electrophoresed least  two  band  intensity.  involving faint  or  migration and  obvious  between  were  was  staining  enzyme,  and  measured  intensity  film,  illuminated fixed  migration  runs,  the r e s u l t s o f a t  from was  A  ASA by  anomalies  samples  obtained.  Professional table  each  the  origin  recorded.  with  In cases  the absence o f s t a i n i n g a c t i v i t y , the q u e s t i o n a b l e appearance o f  or  results  Band  dividers,  bands  within  for  s e p a r a t e runs were used t o c a l c u l a t e mean m i g r a t i o n d i s t a n c e and  architect's  were  simultaneously  of  o f an  were r e - e l e c t o p h o r e s e d u n t i l  photographic  50)  f o r isozymes  record  (Kodak  M staining  F. T o x i n A n a l y s i s by H i g h - P r e s s u r e  EPY  made on a  light  (3,200°K).  Gels  and p r e s e r v e d i n i s o p r o p a n o l : g l y c e r o l : a c e t i c a c i d  mixed w i t h an e q u a l volume o f 0.2  congruent  Ektachrome  c e r t a i n s t a i n e d g e l s was  an i n c a n d e s c e n t t u n g s t e n floodlamp  isolate  (50%:20%:7%)  buffer.  (Performance)  Liquid  Chromatography  1. HPLC A n a l y s i s o f T o x i n s  a. C e l l c u l t u r e and h a r v e s t  Unialgal of  two  Protogonyaulax  species,  (=Protoceratium San  Juan  Gonyaulax  Island,  WA,  for  the  profiles  and  toxicity  medium  in  from  presence o f PSP  2800  ml  (NEPCC  NEPCC 535,  1983),  by  from the NEPCC, as w e l l as  polyedra  reticulatum;  examined  NWSP-7  isolates  HPLC,  a  202a)  G. Gaines related  toxins.  and  G.  grindleyi  i s o l a t e , F r i d a y Harbor, g o n y a u l a c o i d genus, were  For the d e t e r m i n a t i o n o f t o x i n  i s o l a t e s were c u l t u r e d i n two  Fernbach  cultures  flasks  under  standard  litres  of  conditions  87  (Chapter which To  II  was  A.l).  determined by m o n i t o r i n g i n v i v o  determine  samples were  of  the  seven  and  length  of  maximal  cell were  used  extraction,  g)  a  in  and  the  varied  and  end  lag period  respectively. confirm  culture  that  cycle  volume  to  was  and  The  508,  and  516,  the e x p o n e n t i a l phase of  markedly  one-half,  409,  S i n c e the  late-exponential  of the  402,  composition,  growth r a t e s  and  among i s o l a t e s , samples phase c u l t u r e s were taken  t h r e e - q u a r t e r s of the  and  time  the time c o r r e s p o n d i n g  samples c o l l e c t e d a t  cells  in duplicate  by  centrifugation  the t o x i n p r o f i l e s were s t a b l e enough be  used  f o r chemotaxonomic a n a l y s i s .  removed t o y i e l d 1.0  x 10^  cells for  the  pellet  transferred clinical  pellet  Palmer-Maloney c o u n t i n g chambers. (5  was  min  at  suspended  resuspended  in  The 3  5,000  i n 5 ml  to an 8 ml v i a l and  centrifuge.  to  various  toxin  as determined by p h a s e - c o n t r a s t microscope counts (160X) of  and was  phase  mid-  to  culture  pellet  401,  d i f f e r e n t times d u r i n g  lag  density,  Sufficient  rotor),  three  the  the  collected  253,  NEPCC 183,  one-quarter,  throughout  400  isolates,  early-,  between  least  on t o x i n c o n t e n t and  the  elapsed  (Chapter I I C . l ) .  e f f e c t of c u l t u r e age  at  approximately  times  fluorescence  a f t e r 10 days i n s t a t i o n a r y phase.  representing at  the  collected  growth,  C e l l s were h a r v e s t e d i n l a t e e x p o n e n t i a l growth phase,  X g, 5 °C, of dR^O.  recentrifuged  s u p e r n a t a n t was ml  0.03  N  at  C e l l s were Sorvall  GSA  Each suspended (5 min  a t 2,000 X  removed by  aspiration  acetic  acid  for  toxin  extraction.  b. T o x i n  Toxins Model  extraction  were  III),  at  extracted 60%  of  from the  c e l l s by s o n i c a t i o n  maximum output (= 210  W),  (Bronwill  using  Biosonik  f i v e 10 s  bursts  88  with  the  vial  confirmed  by  Sonicated  examination  samples  particulate a  immersed i n an  were  debris,  syringe-mounted  (20  ice-ETOH bath. under  phase-contrast  recentrifuged  and  Complete c e l l d i s r u p t i o n  (10  min  microscopy  at  \Jia M i l l i p o r e  filter  (400X).  2,000 X g) t o remove  the s u p e r n a t a n t a c i d e x t r a c t was  0.45  was  prior  filtered  through  to d i r e c t i n j e c t i o n  u l ) i n t o the HPLC.  c. A n a l y t i c a l method  The was  HPLC  method  developed  experiments Research  by  for  Dr.  J.J.  by  performing  Centre,  USFDA,  described  in detail  the d e t e r m i n a t i o n Sullivan, the  toxin  Seattle,  ( S u l l i v a n and  of t o x i n s from Protogonyaulax  who  c o l l a b o r a t e d a c t i v e l y i n the  analysis  WA.  The  Iwaoka, 1983;  at  the Seafood P r o d u c t s  method has  been p r e v i o u s l y  S u l l i v a n and W e k e l l ,  1984;  S u l l i v a n e t a l . , 1985). In  the  separation  most  recent  was  achieved  divinylbenzene (hexane  sulfonate,  Milford,  MA)  fluorescence, the  basic  follows: PO4)  as  with  column  Na)  and  ion-pair  they  Phase  A:  ammonium  (Kratos  by  (Sullivan  reverse  (Hamilton PIC  B7  phase PRP-1,  5.0  HPLC  on  Since  the PSP  1985), a  NV),  (heptane s u l f o n a t e , Na)  reagents.  H 0, 2  w i t h 1.5 (pH  substituted post-column mM  al.,  toxin  polystyrene with  PIC  alkaline  mM  7.00);  for  H2O.  reaction periodate  PIC  B6  and  B6  (Waters Assoc.,  toxins lack  native  were o x i d i z e d t o f l u o r e s c e n t d e r i v a t i v e s , a c c o r d i n g  phosphate  URS-051  et  Reno,  method of Bates and Rapoport (1975).  acetonitrile  NJ)  resin  modification  to  The  mobile phases were as  1.5  mM  Phase B:  PIC  B7,  as Phase A,  Following  1.0  mM  (as  but w i t h  post-column  20%  oxidation  system; K r a t o s A n a l y t i c a l , Ramsey, i n 50 mM  Na PO 3  A  (pH  7.80), to  yield  89  fluorescent were  derivatives,  detected  Norwalk, CT) The  by  concentration  hydroxy  HPLC  nm;  method  e m i s s i o n : 400  employed  a  (Sullivan for  CA)  and  Iwaoka,  fluorescent  toxins  (Table  Spectra  a  to  shoulder  7),  Physics  miss on  was  evident  the  PSP  Rf  toxins  values  standards. deemed  NEO,  Model  more  the  GTX-^  having  and  conditions  were  by  Baselines  a  which  GTX4,  a  The  1984). Jose,  integrator occasionally displayed a to  GTX4,  B 2 peak.  as i t o f t e n appeared  For t h e s e  and  STX,  reasons,  i n t e g r a t o r were checked manually u s i n g  response  factors  from  as  Substantial baseline d r i f t  l o n g e s t r e t e n t i o n times.  the  N-1  produce weakly  4270 i n t e g r a t o r ( S p e c t r a P h y s i c s , San  prominent  the  toxin  appropriate.  the  most  recently  the run  were redrawn, and peaks areas were r e c a l c u l a t e d as  The HPLC was standard  r e c a l i b r a t e d a f t e r every f i v e samples by  mixture  of  toxins diluted  i n 0.03  M  P u r i f i e d t o x i n standards were o b t a i n e d from Dr. Sherwood H a l l  Toxin  total  These  and  Food and Drug A d m i n i s t r a t i o n , Washington,  the  periodate  r e l a t i v e to t h a t p r e v i o u s l y  r e g i o n of the column c h a r a c t e r i s t i c f o r NEO  plotted  chromatographing acid.  of lower  by f a v o u r i n g o x i d a t i o n of the  the peak c o r r e s p o n d i n g  the  in  chromatograms  Perkin-Elmer,  nm).  1983).  derivatives  used to p l o t peak a r e a s .  tendency  LS-4;  acid), toxins  the d e t e c t i o n of t o x i n s , y i e l d i n g adequate s e n s i t i v i t y f o r  all  was  M nitric  combination  f l u o r e s c i n g d e r i v a t i v e s ( S u l l i v a n and Wekell, A  (0.75  and h i g h e r r e a c t i o n temperature,  recommended compromise  acidification  spectrofluorometer (Perkin-Elmer  ( e x c i t a t i o n : 340  current  detecting  a  and  acetic (U.S.  D.C).  p r o f i l e s were run i n d u p l i c a t e , u s i n g samples from  approximately  same phase of the growth curve o f c o n s e c u t i v e c u l t u r e t r a n s f e r s , f o r a of  compounds  four  determinations.  (C^-C^)  (Table  7)  The were  C - l l s u l f a t e d C-13 not  adequately  carbamyl-N-sulfo-  s e p a r a t e d by HPLC, as  T a b l e 7. A b b r e v i a t i o n s and names o f PSP t o x i n s from Protogonyaulax spp.; s a x i t o x i n and i t s n a t u r a l l y - o c c u r r i n g d e r i v a t i v e s .  Abbreviation  Common Name  Semi-systematic Name  saxitoxin  STX  saxitoxin  neosaxitoxin  NEO  N-1-hydroxysaxitoxin 1  lla-hydroxyneosaxitoxin  sulfate  GTX  :  gonyautoxin  GTX  2  gonyautoxin 2  lla-hydroxysaxitoxin  sulfate  GTX  3  gonyautoxin 3  HB-hydroxysaxitoxin  sulfate  GTX  4  gonyautoxin 4  HB-hydroxyneosaxitoxin  »1  gonyautoxin 5  21-sulfosaxitoxin  B  gonyautoxin 6  21-sulfoneosaxitoxin  C  2  l  c  2  sulfate  epigonyautoxin 8  21 -sulfo-1 l c t - h y d r o x y s a x i t o x i n s u l f a t e  gonyautoxin 8  21-sulfo-113-hydroxysaxitoxin s u l f a t e  C  3  21-sulf o-1 loc-hydroxyneosaxitoxin s u l f a t e  c  4  21-sulf0-113-hydroxyneosaxitoxin s u l f a t e  91  they  c o - e l u t e d near the s o l v e n t f r o n t ( S u l l i v a n and W e k e l l ,  al.,  1985).  for  data  Consequently,  analysis.  their  respective  avoid  the  problem Hall,  epimers,  facile  GTX^  GTX4  in  f o r each i s o l a t e was  STX  and  the  lower  hot  NEO,  toxicity by  hot  (Proctor  et  al.,  values. [MU]  (Boyer  GTX /GTX 1  4  Previous  HC1  et  work  t o x i n complex  x  the  epimers  equilibrium 1980;  from  were p o o l e d t o ratio,  and  H a l l et a l . ,  the 1980;  Boyer e t a l . , 1985).  c a l c u l a t e d from unhydrolyzed  samples not  treatment, which would c o n v e r t B^ and B and  C  to  x  GTX^GTX,^.  compounds  to  The  higher  2  to  c o n v e r s i o n of  toxicity  carbamate  h y d r o l y s i s i s the b a s i s of the P r o c t o r enhancement  1975;  (1700),  profiles  values made  acid  for  -  follows  N  sulfamate  Conversion umol -'-)  HPLC  0.1  respectively,  products  the  e p i m e r i z a t i o n a t C - l l (Boyer,  Toxicity to  GTX3,  establishing  H a l l and R e i c h a r d t , 1984;  Boyer e t  and G T X 2 were c l e a r l y s e p a r a t e d  and  1982;  subjected  the  Although  difficulty  of  they were amalgamated as the C  1984;  Hall,  factors the  1985):  GTX /GTX 2  used t o r e p o r t PSP  calculations C /C 1  NEO  B  :  (2100)  toxicity  ( i n Mouse u n i t s  (MMU c e l l  (250),  2  (1400),  3  mouse  of NEPCC 255  often  for specific toxin a c t i v i t y  toxicity  al.,  comparing  1982)  - 1  (150), and  )  were B  STX  (180),  2  (2050).  bioassay data with values c a l c u l a t e d  (Boyer e t a l . , 1985)  as  from  indicated that calculated  agree q u i t e w e l l w i t h b i o a s s a y r e s u l t s , c o n s i d e r i n g the assumptions regarding  lower  Sullivan  the  level and  of  specific  replicability  Iwaoka, 1983).  tend t o u n d e r e s t i m a t e  t o x i c i t y of the v a r i o u s t o x i n analogues of  mouse  assays  (±20%; H a l l ,  and 1982;  I f a n y t h i n g , the c a l c u l a t e d v a l u e s from HPLC  t o x i c i t y as determined  by the b i o a s s a y .  92  CHAPTER I I I  EXPERIMENTAL RESULTS AND DISCUSSION  A.  Morphological  1.  Variation  Characteristics  in Cell  Protogonyaulax size  among  given  c e l l s cultured  when  exponential  growth,  variation).  Within  under u n i f o r m c o n d i t i o n s v a r i e d  ( T a b l e A), y e t v a r i a t i o n  isolates  isolate,  Size  measurements was a  were  limited  chain consisting  considerably  less  size  variation.  isolate  been  well  conserved  years  has in  culture.  Protogonyaulax  The  isolates  approximately  normal  distribution  of  apical  followed  transapical  on  interphase c e l l s  ±10%  ~  of c e l l s  C.V.  a  during  (coefficient  of  i n i n t e r p h a s e , t h e r e was  The c h a r a c t e r i s t i c c e l l over  ( F i g . 7).  i n l i n e a r dimensions w i t h i n a  made  to  widely i n  s i z e w i t h i n an  time, even a f t e r a p e r i o d o f many and  transapical  frequency  The  only  diameters  distribution  notable  of  diameters  NEPCC  which  was  was  the  exception 529,  within  that  showed  a  pronounced skewness t o t h e r i g h t . Morphometric A00-A12)  (NEPCC cell The  size 1981  measurements clearly  among  smaller  forms  A05-A12,  and  revealed  isolates  isolates  from  with those,  of  from  such  mean as  the p o t e n t i a l  English  Bay  Bay volume  NEPCC  were d i v i d e d ~1  X  isolates  for clonal variability i n  t h e same g e o g r a p h i c a l l o c a t i o n  English a  contemporaneous  10^  ( T a b l e A).  i n t o two s i z e unP,  including  classes: NEPCC  A00-A0A, w i t h a volume a p p r o x i m a t e l y  93  Fig.  7  Frequency d i s t r i b u t i o n o f a p i c a l and t r a n s a p i c a l (um) of Protogonyaulax i s o l a t e s (n=30).  diameters  20NEPCC  NEPCC 2 5 3  183  u ID-  S'  O 301 34|36|42 1  32 36 40  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  30 34 38 32 36  TRANSAPICAL  20 24|28 22 26 30  APICAL  TRANSAPICAL  24 26 30 34  APICAL  TRANSAPICAL  APICAL TRANSAPICAL  APICAL  26 30  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  00'  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  APICAL  TRANSAPICAL  100  double t h i s v a l u e . The size  New Zealand i s o l a t e , NEPCC 508, corresponded i n s i z e t o t h e s m a l l e r  class  Obidos, size  from  Portugal)  was  Bay.  The  smallest  approximately  half  i s o l a t e , NEPCC 253 (Laguna  t h e volume o f i s o l a t e s i n t h i s  class. A  one-way  (Sokal  and  volume  between  showed  that  together.  analysis  Rohlf,  groups  less  from  clones  was  (ANOVA)  performed  among  Fisher's  among  isolates  than  among  catenelloid  exhibit  less  either  size  within  a  F-statistic  t o compare t h e v a r i a t i o n i n c e l l  from  English  a l l  isolates  isolates  among tamarensoid forms.  isolates,  using  o f Protogonyaulax i s o l a t e s ( T a b l e 8 ) .  (a=0.05)  that  d i d not  multiclonal  variance  variations  Variance  different  of  1981)  size  significantly  as  English  was  The a n a l y s i s Bay were n o t considered  not s i g n i f i c a n t l y  Finally, isolates  variation  than  those  initiated begun  as  c u l t u r e o r among a l l t h e i s o l a t e s  compared from each group.  2. V a r i a t i o n and S t a b i l i t y  i n C e l l Shape and Other  Characteristic  Features  Among  Protogonyaulax  transapical from  diameter  isodiametrical  particularly  ratio  transfer  cycles,  usually  ratios  maintained  in  culture,  the  ranged from 0.90-1.13: 1, a maximum  p r o p o r t i o n s o f about 10% ( T a b l e 4 ) .  Certain  apical: deviation  isolates,  among t h e c h a i n - f o r m i n g c a t e n e l l o i d s , v a r i e d more from t h e 1:1  diameter  cells  isolates  when the  ceased,  originally  isolated.  After  the f i r s t  p r o d u c t i o n o f markedly a n t e r o - p o s t e r i o r l y in  favour  of  few c u l t u r e compressed  forms which more c l o s e l y approached  Table 8.  ANOVA comparing v a r i a n c e i n c e l l volume between Protogonyaulax i s o l a t e s grouped by l o c a t i o n o f o r i g i n , p r e s e n t morphotype i n c u l t u r e and c l o n a l i t y the time o f o r i g i n a l c u l t u r e i n i t i a t i o n , n = number o f i s o l a t e s i n each group.  Mean Volume ± s.e.m. (um X10 )  Isolates  3  d.f. (n-1)  F  g  3  E n g l i s h Bay/ a l l isolates  15..12 + 1..54 16..26 + 1..07  10 23  1,.05  Catenelloid/ tamarensoid  16.,13 + 1,.36 16.,26 + 1,.57  5 15  3,.55  Clonal/ non-clonal  15.,44 + 1..70 16.,95 + 1..40  10 12  1..33  "not s i g n i f i c a n t  S i g n i f i c a n c e of variance difference oc=0.05  F  a(2)  > F  s5  n s  '  F  a(2)  > F  s5  n s >  F  a(2)  > F  s5  n s  '  102  isodiametric 435,  529  proportions.  and  T h i s t r e n d was observed f o r c a t e n e l l o i d  543 from F r i d a y Harbor  isolates  (San Juan I s l a n d , WA), as w e l l as f o r  the group o f c l o n e s r e c e n t l y i s o l a t e d from B a m f i e l d , B.C. Nevertheless, catenelloid flattened the  isolates  derived  from  compressed  morphotypes d i d r e t a i n a v e s t i g i a l tendency t o produce cells,  modified  observed  Protogonyaulax  even  i n long-term c u l t u r e .  catenelloid  to  slightly  Once e s t a b l i s h e d i n c u l t u r e ,  morphotypes appeared t o be s t a b l e and were never  r e v e r t t o t h e h i g h l y compressed  forms o r i g i n a l l y o b t a i n e d from  natural populations. The binds  use  of  the ' fluorescent  selectively  to  hydrate-iodine-hydriodic Stosch, plate from or  1969  cellulose, acid  technique),  (Y.  made  t h e c a l s t a i n c a l c o f l u o r w h i t e M2R, rather  than  Fukuyo,  possible  pers.  cells.  t h e c a l squashes  chloral  comm., based on t h e Von  1  v e n t r a l pore ( F i g s . 1 and 3 ) ,  When c a l c o f l u o r M2R was used on empty thecae ( F i g . 8) ( F i g . 9 ) , d e t a i l s o f t h e APC, attachment pores and p l a t e  margins were r e a d i l y Significantly,  of  the r e s o l u t i o n of d e t a i l s a t the  margins, such as t h e presence o f t h e l intact  formulations  which  visible. the  v e n t r a l 1' pore was absent from a l l t h e c a t e n e l l o i d  i s o l a t e s examined under e p i f l u o r e s c e n c e and p h a s e - c o n t r a s t microscopy. Among  the  ventral  1"  English  Bay,  station  in  Thus,  tamarensoid and i n t e r m e d i a t e i s o l a t e s , t h e o c c u r r e n c e o f t h e  pore  was  404  and  not 412  geographically consistent. from  the  1981  Three i s o l a t e s  from  bloom, and 516, from t h e same  1982, d i s p l a y e d t h i s f e a t u r e , w h i l e t h e r e s t d i d n o t ( T a b l e 4 ) .  isolates  geographically  possessing  the  isolated variants.  pore  may  not  simply  be  considered  as  Yet, t h i s feature ( i f present) i s stable  within  a g i v e n i s o l a t e i n c u l t u r e over time, and appears n o t t o be a c q u i r e d  by  isolate  an  i f not o r i g i n a l l y present.  F o r example, t h e e x i s t e n c e o f a  103  Fig. 8  A p i c a l view o f empty t h e c a o f NEPCC 403 s t a i n e d w i t h 0.1% c a l c o f l u o r showing e p i t h e c a l p l a t e s under e p i f l u o r e s c e n c e m i c r o s c o p y (400X). Exposure: 1 min; Ektachrome ASA 160.  Fig. 9  A p i c a l view o f e p i t h e c a l p l a t e s o f NEPCC 516 s t a i n e d w i t h 0.1% c a l c o f l u o r showing v e n t r a l pore on t h e f i r s t a p i c a l ( l ) p l a t e under e p i f l u o r e s c e n c e microscopy (800X). Exposure: 1 min; Ektachrome ASA 160. 1  105  ventral  pore  in  NEPCC  micrograph,  was  1979a)  examination  by  made  to  presence  the from  NEPCC  basis  culture,  of  (1975),  and  by  the  B.C.  under  epifluorescence microscopic  ventral  pore.  Nevertheless, t h i s feature to a v e s t i g i a l  notch  the  prominent  hypothecal  f l a n g e and h i g h l y domed  o f Gonyaulax a c a t e n e l l a sensu L o e b l i c h and L o e b l i c h described  by  Prakash  and T a y l o r (1966) from  I t s a c a t e n e l l a - l i k e c h a r a c t e r i s t i c s were r e c o g n i z e d  list.  The g e n e r a l form has been conserved i n c u l t u r e  and c o n t i n u e s t o be i n a c c o r d w i t h t h e d e s c r i p t i o n l a t e r g i v e n  et  a l . (1978).  of dark  acatenella  The major d i s c r e p a n c i e s between t h e o r i g i n a l (Whedon  and  K o f o i d , 1936) and NEPCC 71 i s t h e  brown p i g m e n t a t i o n and t h e v e n t r a l l  isolate.  The  acatenella  type  specimens  1  Isolate  180  forms  large  ( F i g . 10).  The  egg-shaped cells  of  cells this  pore i n t h e c u l t u r e d  were  p i g m e n t a t i o n , a l t h o u g h t h i s was n o t thought t o be h i g h l y  hypocones  thecae  diameter r a t i o s and i t s i n a b i l i t y t o form c h a i n s i n  previously  species  description consistent  stained  upon i s o l a t i o n by R. Waters and were noted i n a b r i e f r e c o r d i n  1973,  Turpin  NEPCC  as  Inlet,  immediately  since  present  acid  even among c e l l s from t h e same c u l t u r e .  the  characteristic  NEPCC  The  (Schmidt and L o e b l i c h ,  71 was p r o v i s i o n a l l y c l a s s i f i e d as a " t a m a r e n s i s " t y p e ,  epicone  the  of  i t possesses  Malaspina  iodine-hydriodic  1979  a c l e a r l y d e f i n e d pore i n some specimens,  Although the  in  almost a decade a f t e r t h e o r i g i n a l i s o l a t i o n , c o n t i n u e d  a t t h e 1' p l a t e margin,  on  of  shown by T u r p i n e t a l . (1978) i n a SEM  confirmed  microscopy.  observations,  varied  first  independently  phase-contrast  show  71,  almost  significant.  w i t h rounded  isolate  without  e p i c o n e s and  are usually heavily  pigmented.  by  In  NEPCC  R.  Waters  181(b) ( F i g . 11), i s o l a t e d as P. c a t e n e l l a from Pat Bay, B.C. in  1977,  the  f o r m a t i o n o f c h a i n s has ceased c o m p l e t e l y i n  106  Fig.  10  Photomicrograph o f NEPCC 180, a tamarensoid morphotype from Brentwood Bay, B.C., e x h i b i t i n g a rounded apex and antapex i n e q u a t o r i a l view (250X). Exposure: f l a s h 1/125 s; Ektachrome EPY ASA 50.  Fig.  11  Photomicrograph o f NEPCC 181(b), i s o l a t e d as a c h a i n - f o r m i n g c a t e n e l l o i d morphotype from P a t r i c i a Bay, B.C., i n e q u a t o r i a l view (400X). Exposure: f l a s h 1/125 s; Ektachrome EPY ASA 50.  108  culture.  The  hypocone  remains  maintained ESNW  or  has  rather  i n culture NWSP-7.  from f u r t h e r NEPCC locality  become  high-domed and b e l l - s h a p e d , w h i l e t h e  flattened. on  This  isolate  c o u l d be o n l y p o o r l y  seawater from B u r r a r d I n l e t , B.C. e n r i c h e d w i t h  S i n c e t h e d i v i s i o n r a t e was <0.1 d i v d  - 1  , i t was o m i t t e d  experimentation.  183  (=  of  tamarensis,  morphological with  epicone  Plymouth  change  isolate has  173) from t h e Tamar e s t u a r y , t h e type  been  maintained  i n c u l t u r e s i n c e 1957.  without  substantial  I t c o n t i n u e s t o conform w e l l  t h e o r i g i n a l d e s c r i p t i o n by Lebour (1925) i n s i z e , c e l l dimensions and  the l a c k o f c h a i n f o r m a t i o n . The  Portuguese  morphology  and  isolate  plate  patterns,  "tamarensis".  This  densities  culture  in  greenish-hued  of  pigments:  Cembella,  unpublished 254  individuals, et  al.,  form  G.  pore.  tamarensoid  i s capable o f a c h i e v i n g u n u s u a l l y h i g h X  10^ m l  - 1  ) and  uncharacteristic  i t always  appropriately flattened 254  apex  maintains  o f P. t a m a r e n s i s .  a  Thin-layer  Island,  B.C.),  o r i g i n a l l y d e r i v e d from  excysted  i n a previous p u b l i c a t i o n (Turpin resembling the t r o p i c a l  sensu B a l e c h (1971), except f o r t h e absence o f a v e n t r a l  long-term  and  cell  results).  was p r o v i s i o n a l l y c l a s s i f i e d  In  overall  c h l o r o p h y l l , w i t h u n d e t e c t a b l e l e v e l s o f p e r i d i n i n (A.  (Nelson  excavata  in  i s s i g n i f i c a n t l y s m a l l e r than t h e t y p i c a l  1978) as a s t r a i n o f Gonyaulax tamarensis  morphology  NEPCC  (>1  253, w h i l e  i t s pigments on c e l l u l o s e p l a t e s r e v e a l e d a low r a t i o o f  accessory  Isolate  isolate  pigmentation  chromatography  NEPCC  culture,  this  isolate  has  t h e a b i l i t y t o form c h a i n s o f c e l l s .  designated and  as  antapex,  a  "catenella",  with  cells  by  wider  assumed  a  flattened  I t would now be more  virtue  of i t s angularly  than l o n g .  Nevertheless,  has n o t v a r i e d a p p r e c i a b l y i n o v e r a l l s i z e from t h a t r e p o r t e d by  109  Turpin  et  present  time.  The  isolate  Portugal, forms  a l . (1978), two y e a r s a f t e r i t was i s o l a t e d i n t o c u l t u r e , t o t h e  is a  chains  the  narrow  Zealand,  tamarensoid  culture,  rather  isolate.  Isolate  precingular  516  than  plate,  occur.  other  253  from  form l a c k i n g a v e n t r a l pore t h a t  never  New  which  NEPCC  B.C.)  and  are  For  t o appear g r e e n i s h  may  serve  to  separate  toward  than  i t from  1984).  i s more m o r p h o l o g i c a l l y v a r i a b l e i n isolates.  intermediate  most  specimens,  Many  cells  are of  i n morphology  between  t h e e p i c o n e i s egg- o r  a l t h o u g h c e l l s w i t h f l a t t e n e d e p i c o n e s a r e a l s o observed. tapers  cell  Zealand i s o l a t e p o s s e s s e s a v e r y  (Taylor,  Protogonyaulax  tamarensis.  usually  t h e antapex,  The  yet flattened antapices also  T h i s i s o l a t e appears t o l a c k t h e a b i l i t y t o form c h a i n s l o n g e r than  four c e l l s NEPCC the  Bay,  proportions  and  bell-shaped,  (English  the  isodiametric  hypocone  like  but t h e d i f f e r e n c e i s l e s s s t r i k i n g  The  " t a m a r e n s i s " as a new morphospecies  catenella  508),  y e t i s s i m i l a r l y capable of a t t a i n i n g high  than brownish,  Portuguese  sixth  culture  (NEPCC  The c e l l s o f NEPCC 508 a l s o have a tendency  colour,  for  New  small  in  densities. in  from  i n culture. 545,  a  tamarensis morphotype from t h e Bay o f Fundy, conforms t o  d e s c r i p t i o n o f Gonyaulax excavata sensu L o e b l i c h and L o e b l i c h (1975) i n  t h a t i t i s t o x i c , b i o l u m i n e s c e n t and l a c k s a v e n t r a l pore. The or  shape  wide  of  subrectangular,  lamb-chop  shaped,  shape  the  of  t h e APC among Protogonyaulax  were  examined  some  notable  APC  t o r e g u l a r o v o i d , t e a r - d r o p shaped,  depending was  i s o l a t e s v a r i e d from narrow  upon  t h e i s o l a t e ( F i g . 12).  In general, the  conserved w i t h i n an i s o l a t e , p a r t i c u l a r l y i f c e l l s  a t t h e same p o i n t o f t h e c u l t u r e c y c l e . exceptions.  triangular or  However, t h e r e were  In NEPCC 508 (New Z e a l a n d ) , t h e shape o f t h e APC  110  Fig.  12  C h a r a c t e r i s t i c morphology of the a p i c a l pore complex and the p o s t e r i o r s u l c a l p l a t e o f Protogonyaulax i s o l a t e s i n c u l t u r e .  NEPCC  71  NEPCC  180  o a NEPCC  255  o  NEPCC  355  D  NEPCC  183  o NEPCC  400  &  NEPCC  253  a NEPCC  ®  401  o D  NEPCC  402  NEPCC  403  406  o  404  NEPCC  405  ®  0 NEPCC  NEPCC  NEPCC  407  NEPCC  409  NEPCC  412  O M K3  NEPCC  435  NEPCC  508  516  a  o NEPCC  NEPCC  543  NEPCC  545  (3)  a o  NEPCC  529  114  varied  from  toward  the  516  was  base  showed  (most common), to an o v o i d  c o n t a c t e d the f i r s t  an  (San  apical plate.  varied  tapering  APC  i n NEPCC  i n t e r g r a d a t i o n between a round-contoured and  from  lamb-chop  to  forms were  a chain-forming " c a t e n e l l a " , an  angular  i n the shape of the a s s o c i a t e d  In another c a t e n e l l o i d morphotype from the APC  The  form  o c c a s i o n a l l y regular ovoid  Juan I s l a n d , WA),  with c h a r a c t e r i s t i c differences  pore. the  that  In NEPCC 529  APC  form,  rectangular  u s u a l l y lamb-chop shaped, but  observed. the  clearly  apical  same l o c a t i o n , NEPCC  t e a r - d r o p shaped i n e x p o n e n t i a l  543, phase  cultures. Occasionally, apical  pore  sulcal  of  England  not  consistent  they  position  be  Japan  pore  is  (Fig.  13).  evident  in  and  isolate.  The  posterior  the  pores were i n d i c a t e d i n the  p o s t e r i o r s u l c a l p l a t e of the  present.  the  These c o r r e s p o n d i n g attachment pores were  There was pore  no  relative  i s o l a t e s ( F i g . 12) i f  c l e a r r e l a t i o n s h i p between to  the  the p o s t e r i o r s u l c a l p l a t e  o f the p o s t e r i o r s u l c a l p l a t e i n c u l t u r e d  linked  natural  posterior  posterior  posterior  morphotype.  shape  (Y.  A  f e a t u r e s w i t h i n an  the  closely  from  a p o s t e r i o r attachment pore on the  the h y p o t h e c a l p l a t e s of a tamarensoid i s o l a t e from  region  of  and  observed.  commonly  margin and The  of  of the APC  were  APC,  were  photomicrograph New  a n t e r i o r attachment pore, a d j a c e n t to the hook-shaped  the  plate,  drawings  an  populations  Fukuyo, sulcal  compressed and  w i t h the a s s i g n e d morphotype.  pers.  from B a m f i e l d , B.C. comm.  regarding  p l a t e appeared u n u s u a l l y  i n chains,  i s o l a t e s could  not  In c a t e n e l l o i d specimens  (A. Cembella, p e r s . Japanese  obs.)  Protogonyaulax),  and the  wide i n c e l l s t h a t were markedly  compared to t h a t of  tamarensoids.  115  Fig.  13  A n t a p i c a l view o f h y p o t h e c a l p l a t e s from tamarensoid specimens s t a i n e d w i t h c h l o r a l h y d r a t e - i o d i n e - h y d r i o d i c a c i d , examined under p h a s e - c o n t r a s t microscopy (250X). The p o s t e r i o r s u l c a l p l a t e i s shown w i t h a pore near the p l a t e margin. Massachusetts specimens from D.M. Anderson; c o u r t e s y o f F.J.R. T a y l o r .  Fig.  IA  Photomicrograph o f c h a i n - f o r m i n g P. c a t e n e l l a i n a p h y t o p l a n k t o n sample from Puget Sound, WA (250X). C o u r t e s y o f F.J.R. T a y l o r .  116  117  3. Chain  The  Length  tendency  for  antero-posteriorly populations  catenella-like  flattened  (Fig.  14),  cells,  was  isolates a  lost  to  form  characteristic  following  long trait  initiation  chains in  of  of  natural  laboratory  cultures, usually after several transfers. There  was  progression NEPCC  an apparent  i n v e r s e c o r r e l a t i o n between c h a i n l e n g t h and  from e x p o n e n t i a l through  355  (Fig.  15  and  experimental  medium  reached  boundary  the  0),  inoculation,  >90%  were  present  duplets;  mid-exponential ~60%, the  growth  concomitant control  chains  of  singlets through  up  to  and  of  cells  of  no  the  longer  the  of the t r a n s f e r t o the  stock  c u l t u r e inoculum  s t a t i o n a r y phase.  chains  were  observed.  16a).  Statistically  Fe  the  i n t h i s phase.  remained a p p r o x i m a t e l y  phase, y e t t h e r e was  growth curves  conditions  resulted  lower  initial  cells in  of  mid-exponential  s l i g h t decrease  i n the  13.  e v i d e n t t h a t growth under  low  growth r a t e , r e l a t i v e t o the  control,  probably  Day  the growth r a t e i n c r e a s e d i n the low Fe c u l t u r e s t o g i v e a maximal  cell Fe the  13,  through  a  During  The percentage  c o n s t a n t from  an apparent  ( F i g . 15), i t was  in  hours  i n s i g n i f i c a n t numbers of  r e l a t i v e number of c h a i n s of f o u r c e l l s or more a f t e r Day From  Two  had  - the remainder  appearance o f c h a i n s l o n g e r than two  (Fig.  duplets  time  13), the p r o p o r t i o n of s i n g l e t s d e c l i n e d t o  12 c e l l s were observed  stationary  the  the c e l l s e x i s t e d as s i n g l e t s  (by Day  with  culture  At  between e x p o n e n t i a l and  after  as  s t a t i o n a r y growth phase i n c u l t u r e s of  16a-d).  (Day  the  yield  a t Day  cultures  were  medium,  or  p r o l o n g a t i o n of the l a g phase.  28 a p p r o x i m a t e l y apparently  to  draw  Nevertheless,  e q u a l t o the c o n t r o l c u l t u r e s .  The  by  low  a b l e t o scavenge s u f f i c i e n t r e s i d u a l Fe from  on i n t e r n a l r e s e r v e s , t o e v e n t u a l l y a c h i e v e  high  118  Fig.  15  Growth curves o f NEPCC 355 on c o n t r o l ESNW medium ( • ) , low N ( A ) , low P ( • ) and low Fe ( x ) . E r r o r b a r s : ±1.0 s.d.  6TT  120  Fig.  16  Histograms o f t h e percentage o f c e l l s p r e s e n t i n c h a i n s o f v a r i o u s l e n g t h s throughout t h e growth c y c l e o f NEPCC 355. 16a, s t a n d a r d ESNW medium; 16b, low Fe; 16c, low P; 16d, low N. Number o f c e l l s p e r c h a i n : l - singlets; 2 - duplets; 3 - t r i p l e t s ; A - quadruplets, e t c . c  C  C  c  121  122  O  >< I  5 D  I  I  o  O)  !  o 00  o  N  SIT30  I  •  O CD  O IO  1  o  T V 1 0 1 JO  1  i  o  o 10  CM  %  t o  o  124  S  1  1  3  0  1  V  1  0  1  JO  %  125  growth  rates.  Between  approximately cells  in  i n multiples  remaining decline in  equal  a  the  increased  the  those  maximal were  in  clearly  this  the  low  the  end  values  comprised  >75%  in  numbers  low in  the  rates  f o r the  cells  decreased constant In  entered  stationary  The  mirrored  exponential  phase,  and then  rose.  Fe and c o n t r o l c u l t u r e s , and reached a much lower  f o r t h e low N and low P c u l t u r e s .  of exponential  limitation  In t h e P - l i m i t e d  growth was approached around Day 13. i n m u l t i p l e s had i n c r e a s e d  P-limited  cultures  rose  marginally.  low  N  was  slightly  By  from  low  36,  and  S i n g l e t s always  medium,  The growth  lower than t h a t o f low P c u l t u r e s , but c e l l continued  t o i n c r e a s e s l o w l y u n t i l Day 28  t o g i v e a p p r o x i m a t e l y t h e same c e l l y i e l d .  and 16c  c o n t r o l s ( F i g . 16a and b ) .  o f more than two c e l l s d u r i n g  i n chains  cultures  the  15),  (Fig.  the  o f t h e t o t a l c e l l s throughout t h e growth c u r v e .  N  (Fig.  13  were  ( F i g . 1 6 c ) . I n s t a t i o n a r y phase, t h e r a t i o o f s i n g l e t s t o  in  rate  rates  ( F i g . 15). Both growth r a t e and c e l l y i e l d  evident  multiples  Day  in  time, t h e p e r c e n t a g e o f c e l l s  initial  growth  t h e low N and low P c u l t u r e s d i v i d e d a t a markedly lower r a t e  c e l l density  cultures,  the  senescence ( a p p r o x i m a t e l y Day 24-36), t h e percentage o f  singlets relative to c e l l s  than  when  s i g n i f i c a n t l y i n t h e low Fe c u l t u r e s , w h i l e  i n chains  As  through  in  17,  p e r c e n t a g e o f s i n g l e t s i n t h e low Fe c u l t u r e s was  ( F i g . 16b).  Cells  and  constant  r e l a t i v e increase  progressed  13  t h e low Fe and c o n t r o l c u l t u r e s , t h e p e r c e n t a g e o f  approximately in  phase  Day  A l t h o u g h t h e growth  N and low P c u l t u r e s were s i m i l a r ( c l o s e t o 0) between  the  d).  dramatically  c h a i n l e n g t h h i s t o g r a m s were s u b s t a n t i a l l y d i f f e r e n t D u r i n g t h i s p e r i o d , t h e percentage o f c e l l s in  the  low  i n chains  N c u l t u r e s , w h i l e i t remained  roughly  i n t h e low P c u l t u r e s . general,  t h e s e r i e s o f percentage h i s t o g r a m s showing t r e n d s  i n chain  126  length 16b  f o r t h e low N c u l t u r e s resembles  and  d).  exponential a  in  I n both c a s e s , t h e g r e a t e s t p r o p o r t i o n o f c h a i n s o c c u r r e d i n  phase, w h i l e c h a i n f o r m a t i o n decreased i n senescence.  greater  found  t h a t f o r t h e low Fe c u l t u r e s ( F i g .  percentage low  Fe  of  than  cells  However,  i n c h a i n s l o n g e r than two i n d i v i d u a l s was  i n low N c u l t u r e s .  No c h a i n s o f g r e a t e r than  five  c e l l s were ever observed i n low N c u l t u r e s .  4. D i s c u s s i o n  In  Protogonyaulax,  daughter  throughout  (Tomas,  constant marked altered, isolates  a  the  chain during  the  formation  immediately  of  following  in until  post-mitotic the  next  growth. mitotic  This s i z e i s constant division  is  initiated.  observed  uniformity  in  the s i z e of interphase c e l l s  i s n o t unexpected.  The f a c t t h a t c e l l  exponential  within  phase  a  size i s essentially  g i v e n i s o l a t e , and t h a t t h e  d i f f e r e n c e s among i s o l a t e s a r e m a i n t a i n e d when t h e c u l t u r e regime i s strongly is  suggests  fundamentally  t h a t t h e c h a r a c t e r i s t i c s i z e o f Protogonyaulax determined  by  genetic,  rather  than  by  factors.  accumulated  studies  in  genetic  basis.  and  in  r e p r e s e n t c l o n e s o f c e l l s d i v i d i n g a p p r o x i m a t e l y i n synchrony  1974),  environmental The  isolate  interphase,  chains  within  results  However, t h e c e l l s r a p i d l y a c h i e v e t h e s i z e c h a r a c t e r i s t i c o f  particular  Since  division  c e l l s which a r e s m a l l e r than t h e p a r e n t c e l l  cytokinesis. the  mitotic  evidence  dinoflagellates  Symbiodinium  Within  from also  DNA  analysis  and  breeding  affinity  i n d i c a t e s t h a t such s i z e v a r i a t i o n has a  t h e Crypthecodinium  m i c r o a d r i a t i c u m (Schoenberg,  c o h n i i (Beam and Himes, 1982) 1976; Schoenberg  and Trench,  127 1980b)  species  complexes,  morphologically be  segregated  such  as  from  English  reported  NEPCC  distinct  253,  cell  that  abundant  in  complete  and the  do  cysts  cell  rate. (Prakash, dividing  F o r example, White (1978b)  the  growth  rate  varied  a  marked  effect  on  mean c e l l s i z e .  The  and  of  those  of  which  reduction  obs.).  large  It is difficult vegetative  a  i n vegetative  cells  that  to morphologically  appear  size  On t h e o t h e r hand, P - l i m i t a t i o n  lumpy  cells  cell  formed  in  appear unable t o  d i s t i n g u i s h between  clonal  i n multiclonal  c u l t u r e s , under  cultures  following  gametes p a r t i c u l a r l y when t h e q u a d r i f l a g e l l a t e stage has obs.; Anderson and L i n d q u i s t ,  1985).  d i f f e r e n t i a l e f f e c t o f N- and P - l i m i t a t i o n suggests t h a t changes i n  size  specific  although  Boyer, p e r s .  passed (A. Cembella, p e r s . The  have  formation  and  fusion  size.  a P. t a m a r e n s i s i s o l a t e was c o n s t a n t w i t h i n t h e  6-43°/oo,  G.  immotile  P-depletion,  e n v i r o n m e n t a l f a c t o r s which can a f f e c t  o f Protogonyaulax c e l l s t o N - l i m i t a t i o n i s the p r o d u c t i o n  mitosis.  large  of  pellicular  Cembella  some  forms" o f d i n o f l a g e l l a t e s  Nevertheless, c e r t a i n other e x t r i n s i c f a c t o r s , p a r t i c u l a r l y  response  results  Some Protogonyaulax i s o l a t e s ,  (1971; 1977).  i n f l u e n c e on c e l l  from  limitation,  typical  sexual  size classes.  isolates  and perhaps i s o l a t e s 508 and t h e s m a l l e r - c e l l e d forms  size  range  nutrient  different  Bay, may be analogous t o t h e " s m a l l  substantially.  the  could  i s evidence  that  salinity  (A.  d i s t i n g u i s h a b l e o n l y on t h e b a s i s o f s i z e d i f f e r e n c e s  r a t e have l i t t l e  found  of  were  from European waters by S i l v a  There growth  that  into  genetically  a r e more nutrient,  l i k e l y a d i r e c t m e t a b o l i c response t o l i m i t a t i o n by a  than  a  According  to  volume  1967;  Watras  et  rapidly  during  non-specific  f u n c t i o n o f t h e a t t e n u a t e d growth  measurements  made  with the Coulter  a l . , 1982), i t was p r e v i o u s l y  early  exponential  counter  found t h a t  phase were s m a l l e r  cells  than those  128  formed  in  cells  mature  as  the  observed  cultures.  cultures  (White  and  Yet,  a d e c r e a s e i n the  a  Maranda, 1978).  cautionary  cultures  age,  (~12-20  nm),  gametes those the  the  thecal  isolates Coulter  cultures  counter  of  size".  spherical  with  the  In  the  volume  of  to  diagnostic  is  cells,  al.,  1982),  may  and  length  culture cycle.  distributions  cysts  bacteria  Furthermore, c h a i n  Use  in of  i n Protogonyaulax lead  to  severe  c o n s i d e r e d as s t r i c t l y i n d i c a t i v e of  counter y i e l d s o n l y  indiscriminant  u n d e f i n e d p a r t i c l e s , which may  integrate  not  measurements  correlate  well  c e l l s , p a r t i c u l a r l y f o r mature  genetically unequivocally  over in  of  particular  and  apparently  c h a r a c t e r i s t i c s f o r use  plate features  linkages  and  general  to d i s t i n g u i s h between i s o l a t e s , i n t h a t  years,  within  regardless  leaves  However,  little it  "tamarensis" lacking  thecal  in  presence or absence of a v e n t r a l pore i s  maintained  culture,  between  The  character  many  fixed.  morphological  i t i s tempting t o s p e c u l a t e upon the p o s s i b l e  consistently  stage  is  Coulter  occurrence  persistence  pore  et  Protogonyaulax  morphotypes, p e l l i c u l a r ecdysed  size  the r e s u l t s are  i n Protogonyaulax .  useful  growth  c l e a r l y deduced.  increases.  Watras  The  analysis,  morphology  trait  from  determine  1967; if  effort  taxonomic  a  aberrant  fragments  to  specific factor(s)  cultures.  an  between  of  s i z e of d i s t r i b u t i o n v e g e t a t i v e  senescent  previously  U n f o r t u n a t e l y , the  forming c h a i n s v a r i e s throughout the  (Prakash,  a l s o been  i t s h o u l d be r e c o g n i z e d t h a t as  anisogamous),  misinterpretations "cell  note,  percentage  (possibly  tamarensis  approached s t a t i o n a r y phase has  l i m i t i n g growth i n t h e s e experiments cannot be As  s i z e o f P.  is and  a  given  of  the  doubt of  strain.  Its  stable  e n v i r o n m e n t a l regime or that  little  use  this to  character  is  discriminate  " c a t e n e l l a " , since although  i n " c a t e n e l l a " , i t may  the  the  be p r e s e n t or absent i n  129  "tamarensis". the  SEM  State  micrographs  (Postek  specimens  microscope also  general Cox  and  that  and  conceivable  longer  sealed  of  are  descriptors Taylor,  filled  the species  was  the c e l l s depicted  in  in  l e a v e s open t h e  and c r y p t i c under (Taylor,  light  1984).  It  o r i g i n a l l y m i s i d e n t i f i e d - the i n t h e micrographs o f Postek and  sulcal plate  i s c h a r a c t e r i s t i c a l l y associated  p a i r s or chains.  The p o s t e r i o r pore i s t y p i c a l l y  c e l l s and t h e t e r m i n a l  i n d i v i d u a l i n duplets  Both t h e a n t e r i o r and p o s t e r i o r attachment pores a r e become most  pores  invisible  of  the  i s not  function  such  indirectly,  be  This  with  as  NEPCC  cells  mature  Protogonyaulax  and  cease  isolates  chain existed  as s i n g l e t s i n c u l t u r e , t h e r e l a t i v e l y uncommon appearance o f  is a  factors,  pores  may  individual  Since  attachment  culture  Washington  "tamarensis"-like.  and  predominantly  as " c a t e n e l l a " from  i n some Protogonyaulax specimens  chains.  formation.  the  pores  that  existing  i n mature  often  to  such  observation  cells  identified  t h e c a t e n e l l o i d morphotype.  pore on t h e p o s t e r i o r  absent  are  to  (1976) i s r a t h e r  with  cells  Cox, 1976), v e n t r a l pores have n o t been a s s o c i a t e d  morphology  The  in  of  conforming  possibility  is  With t h e n o t a b l e e x c e p t i o n o f t h e v e n t r a l 1' pore v i s i b l e i n  as  linked  of  unexpected.  limitation,  to the d i v i s i o n rate. features  (Loeblich  and  occurrence of chains i n  t h e growth s t a g e , i s a f f e c t e d by e n v i r o n m e n t a l  nutrient  transient  The  and  i s perhaps,  As a consequence,  directly  or  t h e attachment  and a r e d i a g n o s t i c a l l y u n r e l i a b l e as s p e c i e s  Loeblich,  1975;  Schmidt  and L o e b l i c h ,  1979a;  1984).  The  posterior  the  compressed  not f l a t t e n e d .  sulcal  p l a t e and t h e d o r s a l end o f t h e APC may be wider  c e l l s o f the c a t e n e l l o i d morphotype than i n c e l l s The g r e a t e r  distance  from t h e p o s t e r i o r attachment  which pore  t h e margin o f t h e p o s t e r i o r s u l c a l p l a t e , as i n " c a t e n e l l a " sensu Fukuyo  130  (1985),  may  be  due  characteristic examined,  was  may  to  the  not  reflect  evident  decrease  populations 1980b).  mechanical  chains  cultured  itself.  That  "catenella"  isolates  wide  into  culture  is  true  f o r the l o s s of chain formation  Possible  explanations  genetic  observations  has of  to  selection  be c o n s i d e r e d  i n c u l t u r e remain include: physical/  i n c u l t u r e , environmentally  induced  shown  o f r a p i d l y s t i r r e d and a i r - b u b b l e d Protogonyaulax  that  mechanical  a g i t a t i o n r e s u l t s i n the breaking of  The  contribute  somewhat  to  cultures.  However, t h i s e x p l a n a t i o n f o r t h e f a i l u r e t o produce long  culture  natural as the  is  of  favourable are  brought  usually  somewhat  differences  genetic  in  i n c h a i n l e n g t h , even i n u n s t i r r e d  the  found  o b s e r v a t i o n s o f long c h a i n s i n  i n h i g h l y t u r b u l e n t waters, such  chain-forming  tendency  among  possessed  by  certain  conditions  culture, in  During  this  selective  pressures  isolates,  strains  c h a i n s , n e v e r t h e l e s s r e q u i r e s t h e proper  resumed.  chains  nor  i n the c u l t u r e c y c l e .  environmental  resulting  by  i n those  predisposition  long  into  reduction  Furthermore, m e c h a n i c a l breakage does n o t account f o r  between d i f f e r e n t stages  formation  the  even  i n t h e s u r f zone. stable  impact o f c h a i n s a g a i n s t t h e f l a s k w a l l s may a l s o  weakened  populations,  The  cells,  a l s o o f diatoms ( T a y l o r ,  cells.  in  this  changes and t h e e f f e c t o f growth r a t e per s e .  Cursory cultures  brought  factors,  phenotypic  the  plate  p o s t e r i o r s u l c a l p l a t e s , t o cease i n c u l t u r e .  reason(s)  speculative.  the  i n c h a i n l e n g t h when Protogonyaulax i s o l a t e s from n a t u r a l  are The  in  of  t h e tendency f o r f o r m a t i o n o f extremely  w i t h c o r r e s p o n d i n g l y broad The  widening  an  they  for  expression.  f o r the  combination  When i s o l a t e d  are subject to short-term  of  cells  shock e f f e c t s ,  extended l a g phase b e f o r e r a p i d c e l l d i v i s i o n i s initial  against  acclimation the  long-chain  period  t h e r e may be g e n e t i c  forming  phenotypes.  In  131 multiclonal  cultures,  short-chain  forming  cycles  after  production the If  clone  could with  isolation.  result  in  the  rapid  dominance  of a  a h i g h growth r a t e , w i t h i n a few t r a n s f e r  I n c l o n a l c u l t u r e s , t h e s w i t c h from t h e i n i t i a l  of long-chains  t o s h o r t - c h a i n s and s i n g l e t s may be t h e r e s u l t o f  f i x a t i o n o f a random f a v o u r a b l e o r n e u t r a l mutation f o r s h o r t e r c h a i n s . the formation  increasing it  this  may  buoyancy,  be  homogeneous  less  as has been h y p o t h e s i z e d  advantageous  cultures  interpretations culture  o f long c h a i n s i n n a t u r a l p o p u l a t i o n s  where  hypothesize  results  from  the  as  a  f o r diatoms ( T a y l o r , 1980b),  survival  sinking  is  that  the  loss  of  i s an a d a p t a t i o n f o r  strategy  less  in  significant.  lack of chain-forming capacity  f o r chain  s m a l l volume These g e n e t i c phenotypes i n  formation  and  represents the "genetics of s u r v i v o r s " . As  any  a l t e r n a t i v e h y p o t h e s i s , perhaps i t i s n o t t h e g e n e t i c  for  the  the  conditions  e x p r e s s i o n o f t h e l o n g - c h a i n phenotype t h a t i s l o s t , but o n l y t h a t  Protogonyaulax phytoplankton always  conformed  in  the  expression  forming  long  Inlet  of  chains  the  genotype  have  never  t o t h e tamarensoid from  rarely  formed  grown  on  from  Southern  California)  observed  in  of  enriched  seawater  from  long c h a i n s , even when they were i s o l a t e d as It  may  be  significant  as a " c a t e n e l l a " , resumed t h e f o r m a t i o n  seawater  been  unfavourable.  o r i n t e r m e d i a t e morphotypes ( T a b l e A ) .  elsewhere  catenelloids.  University  are  from B u r r a r d I n l e t , and i s o l a t e s from t h i s a r e a have  isolates  chain-forming isolated  f o r the  samples  Furthermore, Burrard  potential  t h a t NEPCC  181(b),  o f c h a i n s when s u b c u l t u r e d  Washington ( S e a t t l e ) c u l t u r e c o l l e c t i o n on e n r i c h e d  Puget Sound, and when grown by Dr. M a r i a Ross ( U n i v e r s i t y o f on  water  from  the  Los  Angeles  catenelloid  form i s dominant i n n a t u r a l p o p u l a t i o n s .  "catenella"  morphotype  may  be  r e g i o n , where t h e  The e x p r e s s i o n o f t h e  i n h i b i t e d by growth on "tamarensis  water".  132  However, water,  since it  isolate  is difficult  181(b)  survives  o n l y m a r g i n a l l y on B u r r a r d  t o f a c t o r out the e f f e c t of growth r a t e per se from  the d i r e c t e f f e c t of water q u a l i t y on c h a i n The  higher  during noted  (Tomas,  merely  due  focuses  to  on  imply  that  well  and  of  evidence  that  implication,  indicated  Since  the  for  P.  of  c a t e n e l l a occurring i n chains  stationary  cells rate  phase has  been p r e v i o u s l y  of i n c r e a s i n g c h a i n l e n g t h  natural  situ this  growth  per  se,  rather  is  the  the  case  tamarensoid than  " c a t e n e l l a " populations. rates  are d i f f i c u l t (Norris  morphotypes  catenelloids,  than  on  has  and have  specific T h i s would formed a r e  Although  reliable  t o o b t a i n , t h e r e i s no  Chew, a  1975).  lower  Another  growth r a t e i n  a l s o not been supported.  growth r a t e s t u d i e s i n c u l t u r e (Chapter  growth  as  t o s e p a r a t e when d i v i d i n g r a p i d l y ,  i n c u l t u r e s where l o n g c h a i n s a r e not  acclimated  that  significantly  r a t e s of " c a t e n e l l a " and  "tamarensis"  The  III B.l) were not  different. progressive  the low  Fe and  trends  in  c h a i n l e n g t h throughout the c u l t u r e  low N c u l t u r e s were s i m i l a r , y e t the growth r a t e s  c e l l y i e l d s were markedly d i f f e r e n t , c h a i n l e n g t h appeared to be more a  function On  in  that  from  of  interpretation  division  rates  populations  evidence  cycle  in  in  production.  g e n e t i c f a c t o r s t h a t can a f f e c t growth r a t e .  those  estimates  The  cell  cells  than  failure  growth  below  natural  the  the  of  growth  1974).  environmental  and  percentage  exponential  Inlet  of  the  the phase of the c u l t u r e c y c l e , than the a b s o l u t e growth r a t e .  o t h e r hand, the i n f l u e n c e of h i g h e r growth r a t e on c h a i n l e n g t h  expressed  in  the  longer  c h a i n s formed i n e x p o n e n t i a l phase i n the low  was Fe  than the low N c u l t u r e . Nutrient growth  rate  limitation  may  affect  c h a i n l e n g t h by d i r e c t a f f e c t s on  or by i n d i r e c t a f f e c t s on the morphology.  In the former  the  case,  133  for  example,  N-  undergo  rapid  nucleic  acid  limitation  P-limitation  mitosis, and  could  distortions  or  and  due  protein  lead  to  aberrant  to  may r e s u l t the  lack of these r e q u i r e d n u t r i e n t s f o r  synthesis. structural morphotypes,  i n the i n a b i l i t y of c e l l s t o  In  the  latter  deficiencies which  case,  causing  nutrient  thecal  plate  are incompatible with chain  formation. The 1'  use  o f m o r p h o l o g i c a l f e a t u r e s , such as t h e presence o f t h e v e n t r a l  pore, t h e presence and p o s i t i o n o f t h e a n t e r i o r and p o s t e r i o r  attachment  pores,  and t h e shape o f t h e APC and p o s t e r i o r s u l c a l p l a t e , as advocated by  Fukuyo  and co-workers (Fukuyo,  "catenella" specimens  from from  applicable of  the  other  to  Protogonyaulax  regions. as  " t a m a r e n s i s " , cannot a l l environments.  Japanese  Pacific,  1980; 1985; Fukuyo e t a l . , 1985) t o s e p a r a t e  coast, At  in  be viewed  as s t r i c t l y d i a g n o s t i c f o r  A l t h o u g h t h e s e c r i t e r i a may be n a r r o w l y  spp. w i t h i n t h e r e s t r i c t e d g e o g r a p h i c a l range  severe  problems  a r i s e when they a r e extended t o  l e a s t w i t h i n and among p o p u l a t i o n s from t h e n o r t h e a s t  culture,  i t i s not  clear  that  r e p r e s e n t a r a t h e r continuous m o r p h o l o g i c a l spectrum There  diagnosis, isolates  f o r "tamarenis" the  from  isolates  the  would  "tamarensis" which  while  would and  do  presence English  "tamarensis",  plate  f e a t u r e s do n o t  i n f i e l d populations.  are other d i f f i c u l t i e s i n v o l v e d i n the a p p l i c a t i o n of the r e v i s e d  descriptions  sample,  these  Bay,  "catenella".  F o r example, by Fukuyo's  t h e v e n t r a l pore i n o n l y some contemporaneous would  assign  the  pore-bearing  shift  to  "catenella".  lack  of  a v e n t r a l pore i n some Massachusetts  reassign  have  isolates to  those without t h e pore, p r e s e n t even i n t h e same water  isolates  not  of  and  them  to  The shape o f t h e p o s t e r i o r  " c a t e n e l l a " ( T a y l o r , 1984).  from t h e type l o c a l i t y a  sulcal  "tamarensis" Even those  i n t h e Tamar e s t u a r y , England  v e n t r a l pore ( e . g . , Plymouth 173a),  and some o f the  134  non-pore Fundy, would  b e a r i n g tamarensoid  and O s l o f j o r d , Norway ("excavata" sensu L o e b l i c h and L o e b l i c h , move  to  speciation  "catenella".  mechanism  morphologically, At  morphotypes from the G u l f of Maine, the Bay  This  whereby  would  revision  morphological  stable  species  differences,  arise within sympatrically distributed  and  distortion,  differences  Unfortunately, shape  and  the  chain  more  of  while  expressed  populations. l e v e l of  r e l y i n g too h e a v i l y upon minor  unknown e c o l o g i c a l and  conventionally  formation,  1975)  r e i n t e r p r e t a t i o n requires invoking a  p r e s e n t , the r e d e s c r i p t i o n appears to i n t r o d u c e an u n a c c e p t a b l e  taxonomic  of  genetic  used m o r p h o l o g i c a l  significance. criteria,  cell  a r e a p p a r e n t l y even more s u b j e c t to v a r i a t i o n  and i n t e r g r a d a t i o n .  B. Growth Rates  1. A c c l i m a t e d  Growth Rates of Protogonyaulax  isolates  a. R e s u l t s  Protogonyaulax (X  =  data  0.66 were  Rolhf, by time  ±  analyzed  location  varied  s.d.;  range:  by  one-way  a  widely  0.06  -  in 1.42  acclimated d i v d" ) 1  growth  ( F i g . 17).  ANOVA u s i n g F i s h e r ' s F - t e s t ( S o k a l  rate The and  t o compare v a r i a n c e i n growth r a t e s between i s o l a t e s grouped of  o r i g i n , p r e s e n t morphotype i n c u l t u r e and  isolation  considerably were  0.42  1981),  of  isolates  (Table 9).  among groups, and  sometimes  significantly  c l o n a l i t y a t the  S i n c e the number of i s o l a t e s examined v a r i e d the v a r i a n c e s i n growth r a t e s between groups different,  the  nonparametric  Mann-Whitney  135  Fig.  17  Histogram o f a c c l i m a t e d growth r a t e s o f Protogonyaulax i s o l a t e s from B a m f i e l d and E n g l i s h Bay i n B r i t i s h Columbia, and o t h e r regions.  Table 9.  ANOVA comparing v a r i a n c e i n a c c l i m a t e d growth r a t e s between Protogonyaulax i s o l a t e s grouped by l o c a t i o n o f o r i g i n , p r e s e n t morphotype i n c u l t u r e and c l o n a l i t y a t t h e time o f i s o l a t i o n , n = number o f i s o l a t e s i n each group.  Variance ( s ) in k (div d" ) 2  1  d.f. (n-1)  F  s  S i g n i f i c a n c e of variance difference a=0.05  Isolates A l l isolates/ Bamfield  0.18 0.02  43 22  7.29  F  s> a(2);  A l l isolates/ E n g l i s h Bay  0.18 0.17  43 10  1.05  F  s  E n g l i s h Bay/ Bamfield  0.17 0.02  10 22  8.50  F  s> a(2)5  s  **  Bamfield/ non-Bamfield  0.02 0.23  22 20  9.54  F  s> a(2);  s  **  E n g l i s h Bay/ n o n - E n g l i s h Bay  0.17 0.11  10 32  1.55  F  s< a(2)5  A l l tamarensoid/ a l l catenelloid  0.17 0.07  12 28  2.43  F  s  Non-EB/BF tamarensoid/, non-EB/BF c a t e n e l l o i d "  0.16 0.26  3 5  1.63  F  a(2)5  < F  F  F  F  < F  s  **  ns***  n s  ***  a ( 2 ) 5 ns^  s~a(2)  S i g n i f i c a n c e of variance difference <x=0.05  V a r i a n c e (s~") in k (div d ,  d.f. (n-1)  A l l clonal/ a l l non-clonal  0.18 0.19  32 10  1.06  F  E n g l i s h Bay c l o n a l / E n g l i s h Bay n o n - c l o n a l  0.12 0.18  4 5  1.50  F  Isolates s< cx(2);  ^***  s  "s***  F  < F  a(2);  ''only tamarensoid and c a t e n e l l o i d i s o l a t e s which were not i s o l a t e d from e i t h e r E n g l i s h Bay o r B a m f i e l d were compared. ""significant not s i g n i f i c a n t  139  U-test  ( Z a r , 1974)  was  used  t o t e s t f o r s i g n i f i c a n t d i f f e r e n c e s i n mean  a c c l i m a t e d growth r a t e between groups ( T a b l e 10). When recent  grouped by g e o g r a p h i c a l l o c a t i o n o f o r i g i n , t h e growth r a t e s among contemporaneous  significantly among  variable  a l l isolates  Bamfield growth of  less  group rate  isolates  from  B.C.  were  shown  t o be  than among p o p u l a t i o n s n o t from B a m f i e l d , and  considered  was  Bamfield,  together  (a=0.05).  In  p a r t i c u l a r , the  s u b s t a n t i a l l y less g e n e t i c a l l y diverse with respect to  than t h e i s o l a t e s from E n g l i s h Bay, B.C.  the Bamfield c a t e n e l l o i d  The mean growth r a t e  i s o l a t e s was a l s o s i g n i f i c a n t l y lower  than t h a t  of i s o l a t e s o r i g i n a t i n g elsewhere (a=0.05).  in  Within  t h e group o f i s o l a t e s o r i g i n a t i n g from E n g l i s h Bay, t h e v a r i a n c e  growth  r a t e s was n o t s i g n i f i c a n t l y d i f f e r e n t from t h a t o f i s o l a t e s  other  regions  nor  Bay  isolates  English  among  a l l isolates.  was  notably  from  However, t h e mean growth r a t e o f  higher  than f o r B a m f i e l d  i s o l a t e s and  among a l l i s o l a t e s . When growth  grouped rate  neither isolates  by  variation  when  d i d not  compared.  Among  was  than  apparent  the  were  come  there  were  no  catenelloid  apparent and  differencesi n  tamarensoid  c o n s i d e r e d t o g e t h e r , nor when o n l y  from  either  English  a l l i s o l a t e s , t h e mean growth r a t e o f tamarensoid that  of  derived  forms, those  Bay or B a m f i e l d were forms  c a t e n e l l o i d s , but t h i s d i f f e r e n c e was no l o n g e r  when t h e E n g l i s h Bay and B a m f i e l d p o p u l a t i o n s were  Isolates mean  between  a l l isolates  which  higher  morphotype,  excluded.  from a s i n g l e c e l l e x h i b i t e d no marked d i f f e r e n c e s i n  growth r a t e s o r g e n o t y p i c v a r i a t i o n compared w i t h those o r i g i n a t i n g as  multiclonal  isolates.  T h i s was t r u e among a l l t h e i s o l a t e s , and when o n l y  i s o l a t e s from E n g l i s h Bay were c o n s i d e r e d . The  maximal  growth r a t e s p r e s e n t e d  i n T a b l e 11 were c o l l e c t e d from t h e  Table 1 0 .  Nonparametric Mann-Whitney U - t e s t f o r the s i g n i f i c a n c e of t h e d i f f e r e n c e i n mean a c c l i m a t e d growth r a t e s f o r Protogonyaulax i s o l a t e s grouped by l o c a t i o n o r i g i n , p r e s e n t morphotype i n c u l t u r e and c l o n a l i t y a t t h e time of i s o l a t i o n , n = number of i s o l a t e s i n each group.  S i g n i f i c a n c e of d i f f e r e n c e i n means  Mean growth r a t e k (div d-1) X + s.d.  Isolates  E n g l i s h Bay/ Bamfield  1 .06 0 .44  + 0.41 + 0.15  11 23  Bamfield/ non-Bamfield  0 .44 + 0.15 0 .91 + 0.47  23 21  E n g l i s h Bay/ n o n - E n g l i s h Bay  1 .06 0 .54  + 0.41 + 0.33  11 33  A l l tamarensoid/ a l l catenelloid  1 .05 0 .46  + 0.41 + 0.26  13 29  Non-EB/BF tamarensoid/ non-EB/BF c a t e n e l l o i d *  1 .05 0 .56  + 0.40 + 0.51  4 6  U  0 > 0 5  (2)>U(and U );  ns***  A l l clonal/ a l l non-clonal  0 .58 0 .90  + 0.38 + 0.42  33 11  U  0 > 0 5  ( )>U(and U );  ns***  E n g l i s h Bay c l o n a l / E n g l i s h Bay n o n - c l o n a l  1 .24 + 0.34 0 .91 + 0.43  5 6  U  0 - 0 5  ( ) > U ( a n d U');  ns***  u  o.o5(2)< <  u  o.o5(2)  u  o.o5(2)< <  U  0.05(2)  u  significant;  '''"''not s i g n i f i c a n t  u  u  < U  <  o  u  r  o r  (°  u  u  r  u  ' ) ;  '>;  ' ) ;  '>;  1  1  2  'only tamarensoid and c a t e n e l l o i d i s o l a t e s which were not from e i t h e r E n g l i s h Bay or B a m f i e l d were compared. > % x  <  o r  2  isolated  s  **  s  **  s  **  s  **  T a b l e 11.  Maximum growth r a t e s of Protogonyaulax batch c u l t u r e experiments.  Species  P. tamarensis  O r i g i n of isolate  Cape Ann, USA  k (div  MA,  b  spp  d" )  Growth medium  1  f/2 f/2 f/2 f F o g e l and H a s t i n g s Aquil GPM  White, 1976 White, 1978b White and Maranda, 1978 S h i m i z u e t a l . , 1975a Y e n t s c h and Mague, 1980 Y e n t s c h and Mague, 1980 Schmidt and L o e b l i c h , 1979b  F o g e l and H a s t i n g s  C o l e e t a l . , 1975 Y e n t s c h e t a l . , 1975  P.  tamarensis  Ipswich Bay, G u l f of Maine, USA  0.45  P.  tamarensis  Bay of Fundy, N.B., Canada  0.41 0.36  P.  tamarensis  Bay of Fundy, N.B., Canada or Plymouth, England (?)  0.21 0.32 0.36 0.52  P.  tamarensis  Perch Pond, Falmouth, MA, USA  0.55 0.19-0.66  M i l l Pond, O r l e a n s , MA, USA  0.60 0.73 0.53  tamarensis  Reference 3  0.29 0.36 0.29 0.22 0.30-1.05 0.10-2.00° 0.48 c  P.  i n e x p o n e n t i a l phase from  Erd-Schreiber ASP-7 ASW ASW+humic a c i d ESWA ESWA+humic a c i d d  e  Prakash, Prakash, Prakash Prakash Prakash Prakash  1967 1967 and and and and  Rashid, Rashid, Rashid, Rashid,  1968 1968 1968 1968  f/2 f/2  Brand e t a l . , Brand, 1981  1981a  f/2 f/2 f/2  Watras e t a l . , 1982 Anderson and L i n d q u i s t , Anderson e t a l . , 1984  1985  Species  Origin of isolate  k (div d" ) 1  Growth medium  Reference 3  P. tamarensis  Ofunato Bay, Honshu, Japan  0.48  P. tamarensis  Lummi I s l a n d , WA, USA  0.68  ESNW  Boyer e t a l . , 1985  P. tamarensis  Tamar e s t u a r y , Plymouth, England  0.50  ESNW  Boyer e t a l . , 1985  P. c a t e n e l l a  Provasoli i s o l a t e  P. c a t e n e l l a  Singh e t a l . , 1982  0.28 0.26  Erd-Schreiber organic^  Sequim Bay, WA, USA  0.50 0.52  ASP-7 ( N 0 " ) ASP-7 ( N H )  P. c a t e n e l l a  Whidbey I s l a n d , WA, USA  0.49  Protogonyaulax sp.  Porpoise Island, A l a s k a , USA  0.41  1  3  +  4  ESNW  Prakash, 1967 P r o c t o r e t a l . , 1975 N o r r i s and Chew, 1975 N o r r i s and Chew, 1975 Boyer e t a l . , 1985  H a l l , 1982  R e f e r e n c e s f o r n u t r i e n t media: f , f / 2 ( G u i l l a r d and Ryther, 1962); GPM ( L o e b l i c h , 1975); E r d - S c h r e i b e r (Foyn, 1934); F o g e l and H a s t i n g s medium ( F o g e l and H a s t i n g s , 1971); A q u i l (Morel e t a l . , 1979); ASP-7 ( P r o v a s o l i , 1963); ASW (Prakash and R a s h i d , 1968); ESWA (Prakash and Rashid, 1968); ESNW ( H a r r i s o n e t a l . , 1980) ^ b e l i e v e d t o be i s o l a t e 429 ( L o e b l i c h and L o e b l i c h , 1975) i s o l a t e d by C. M a r t i n , G l o u c e s t e r Marine S t a t i o n , G l o u c e s t e r , MA. T h i s s t r a i n has been d e s i g n a t e d a l t e r n a t i v e l y as G. excavata ( L o e b l i c h and L o e b l i c h , 1975; Yentsch and Mague, 1980; White, 1976 and 1978b; Schmidt e t a l . , 1978; White and Maranda, 1978), G. t a m a r e n s i s (Shimizu e t a l . , 1975a) and G. tamarensis v a r . excavata (Schmidt and L o e b l i c h , 1979a and c  s e a s o n a l o s c i l l a t i o n s i n growth r a t e  ^mean growth r a t e among i s o l a t e s (n=83) e  r a n g e o f growth r a t e s among i s o l a t e s (n=75)  fgeographical  o r i g i n o f i s o l a t e ( s ) not given  Shigh c o n c e n t r a t i o n s  of organic  n u t r i e n t s , no i n o r g a n i c n u t r i e n t s  added.  144  literature,growth with  as  curves time.  the  tamarensis  indicating  changes  study,  since,  i n c e l l density or i n vivo fluorescence  be s t r i c t l y compared w i t h those measured i n  with  t h e e x c e p t i o n o f the growth r a t e s f o r P.  from  Falmouth,  MA  necessarily  represent  acclimated  varied  in  medium, the  by t h e o r i g i n a l a u t h o r s , o r were c a l c u l a t e d from t h e  These v a l u e s cannot  current  not  given  irradiance  and c u l t u r e volume.  range  of  isolates. basis  level,  growth  When  of  morphotype,  growth.  temperature,  1981a), they do  The e x p e r i m e n t a l  salinity,  nutrient  regimes  enrichment  N e v e r t h e l e s s , these d a t a g i v e an i m p r e s s i o n o f  rates  the  (Brand, 1981; Brand e t a l . ,  anticipated  values there  f o r " c a t e n e l l a " and " t a m a r e n s i s "  a r e s e g r e g a t e d i n t o two groups s o l e l y on t h e are  no c l e a r d i f f e r e n c e s i n t h e growth r a t e s  between c a t e n e l l o i d and tamarensoid  isolates.  b. D i s c u s s i o n  As  used  rates  in  within  t h e p r e s e n t study t o determine  satisfies  precision.  The  phytoplankton  dinoflagellates,  results that  the  (1980) P.  with of  the necessary c r i t e r i a of r e p l i c a b i l i t y ,  studies  (Brand,  that  constant  i n growth  and among p h y t o p l a n k t o n p o p u l a t i o n s , t h e i n v i v o f l u o r e s c e n c e  technique  indicated  genetic variation  of  1980;  acclimated such time,  Brand 1981;  growth  as although  and 1982; rates  co-workers on s e v e r a l s p e c i e s o f Brand of  Protogonyaulax, those  of  s t a b i l i t y and  et  a l . , 1981a  haploid and  diploid  species,  and b ) , including  coccolithophores, diatoms  were n o t .  were The  t h e c u r r e n t i n v e s t i g a t i o n s on Protogonyaulax  supported t h e view  a c c l i m a t e d growth r a t e s a r e g e n e t i c a l l y f i x e d .  Y e n t s c h and Mague  reported  tamarensis  an from  annual p e r i o d i c i t y i n t h e growth r a t e o f an i s o l a t e o f Cape  Ann, MA, but no evidence o f such c y c l i c a l  events  145  was observed among t h e NEPCC Protogonyaulax It  isolates.  might be argued t h a t t h e use o f u n i a l g a l r a t h e r than a x e n i c i s o l a t e s  to  determine a c c l i m a t e d growth r a t e s i s i n v a l i d , because o f t h e p o s s i b i l i t y  of  differential  conditioning  Protogonyaulax flora  which  1985). when  had  differ  found  isolates  distinctively Brand  that  were  (1980;  the  virtually  the  medium caused by b a c t e r i a .  from  each  bacterial  o t h e r ( D i m a n l i g and T a y l o r ,  1981) e f f e c t i v e l y c o u n t e r e d t h i s argument  a c c l i m a t e d growth r a t e s o f a x e n i c and non-axenic  i d e n t i c a l , and t h a t b a c t e r i a i n low c o n c e n t r a t i o n s  little  effect  on  exponential  growth  phase,  numbers  on  c u l t u r e s from t h e NEPCC a r e known t o have dominant  However, he  effects  growth.  S i n c e t h e growth r a t e s were determined i n  when  d i s s o l v e d o r g a n i c n u t r i e n t s and b a c t e r i a l  were lower than t h e l a t e r growth s t a g e s , t h e p o t e n t i a l i n f l u e n c e o f  b a c t e r i a on d i n o f l a g e l l a t e growth was minimized. In  any  associated which  case, with  could of  although  axenic  the  the  bias  benefits  had  the  use  significantly  of  genotypic  changes  and  axenic  extended  much  from  greater  1981),  growth  cultures.  rate,  l a g phase.  (Brand,  in English  than  among  indicating  that  genotypically 1981;  selection  the p o s s i b l e  Y e n t s c h e t a l . (1975) found t h a t  non-axenic P. t a m a r e n s i s c u l t u r e s o f t h e same i s o l a t e  maximal  variation  populations  clonal  of a n t i b i o t i c s to p u r i f y phytoplankton c u l t u r e s ,  the  axenic  acclimated Bay (C.V.:  cultures  a  growth  i n acclimation. rates  within  Protogonyaulax  38.7%) and B a m f i e l d (C.V.:  i s o l a t e s from Falmouth, MA (C.V.: the  displayed  T h i s i s e v i d e n c e t h a t such a n t i b i o t i c -  t r e a t e d c u l t u r e s were e x p e r i e n c i n g d i f f i c u l t i e s The  and  " n a t u r a l " growth r a t e s , more than outweighs  preparing  same  risk  British  Columbian  34.1%) was  10.4%) (Brand,  populations  were  more  heterogeneous. The Protogonyaulax i s o l a t e s from Massachusetts Brand  et  a l . , 1981a) were d e r i v e d from b e n t h i c h y p n o c y s t s ,  146  whereas  all  originally  isolates from  heterogeneity that  from  isolated  in  the  from  expected that  to  less  "filter"  environmental  conditions,  population of  hypnocysts.  represent  an  It  cells.  i n d i v i d u a l chains  the  may  have  natural a  not  clear  estimator  that  of  the  If this  i s not the case, and  of  highly  selected  alternative  genotypes  may  already  for  cultures  survivors" genetic  problem  cells  transfer  from  cells  form s e x u a l  hypnocysts r e p r e s e n t  survival,  only  may  also  have  a  the  loss  of i s o l a t e s due  culturing  process  itself  the  which can  be s u c c e s s f u l l y c u l t u r e d .  from 100%; obtain  the  genotypes  case of the B a m f i e l d  single of  of  be  limiting  In  range  may  to  chains  the  cultures  isolated  English by  Bay  the  culture  is  the  This  may  isolates.  "genetics  of  impact on e s t i m a t e s of to i n v i a b i l i t y  sufficiently  as a g e n e t i c  available  product  through  high,  the  sieve, necessarily  f o r e x p e r i m e n t a t i o n t o those  i s o l a t e s , the r a t e of s u c c e s s f u l t r a n s f e r s  from  the n a t u r a l environment to c u l t u r e  isolates,  isolating  cysts  a r e s t r i c t e d range of  cells,  significant  viewed  i n the  formation  be e x p r e s s e d i n c y s t - d e r i v e d c u l t u r e s .  samples  a  t o t a l range of genotypes i n the  vegetative  natural  be  Under u n f a v o u r a b l e  which  from  If  would  through  f u s i o n r e q u i r e d f o r the  initiated  variation.  Cultures  conceivable  passed  be r e f l e c t e d i n reduced v a r i a t i o n i n growth r a t e s among c u l t u r e d For  genotypic  It is  f r a c t i o n of the v e g e t a t i v e  population. genotypes  lower  are c l o n a l , and  environment.  undergo the s e x u a l  unbiased  The  mating types (= d i f f e r e n t genotypes).  only  is  B r i t i s h Columbia came  c u l t u r e s i s somewhat s u r p r i s i n g , g i v e n  isolates  in  s u r v i v e and  in  p o t e n t i a l f o r heterogeneity.  cyst-derived  pre-selective  locations  vegetative  s i n g l e c e l l s and  exhibit  such  two  cyst-derived  hypnocysts c o n t a i n two  initiated  the  single  o n l y two  was  i s o l a t e s of 12 attempts t o  c e l l s were l o s t  i n the  initial  sub-  147  culturing.  The  germinated 1981;  hypnocysts  Brand  10-50%  p e r c e n t success i n c u l t u r i n g Protogonyaulax  of  et the  was  not  given  in  t h e Massachusetts  a l . , 1981a), however Brand single  cell  isolates study  from  (Brand,  (1982) l a t e r r e p o r t e d t h a t o n l y  i s o l a t i o n s o f o t h e r p h y t o p l a n k t o n s p e c i e s were  s u c c e s s f u l - m o s t l y due t o c o n t a m i n a t i o n . Another growth  rates  observed situ of  interpretation  on  acclimated  species  huxleyi  differentiation  from  and  was  were  significant  differences  the  growth  rates  with  diverse  habitats.  Gephyrocapsa  oceanica,  found  populations  to  i n genotypic v a r i a t i o n i n  the f i n d i n g s f o r other  For the coccolithophores, no  intraspecific  among o c e a n i c i s o l a t e s , but n e r i t i c and o c e a n i c  clearly  distinct  (Brand e t a l . ,  1982).  There were a l s o  i n t h e growth r a t e o f E. h u x l e y i i s o l a t e s from t h e  and those from o t h e r n e r i t i c p o p u l a t i o n s from warmer waters  south.  I n c o n t r a s t t o t h e s e o b s e r v a t i o n s , i s o l a t e s o f t h e diatom pseudonana  varied  only  slightly,  i fat a l l ,  from g e o g r a p h i c a l l y s e p a r a t e d n e r i t i c h a b i t a t s (Brand e t a l . , Regardless  waters,  of  examined yet  the  geographical important such  genetic  Maine  Thalassiosira  isolates  of i n  I n t h i s c o n t e x t , i t i s i n s t r u c t i v e t o compare t h e r e s u l t s  study  Emiliana  of  differences  i n c u l t u r e as r e a l , and as u n b i a s e d e s t i m a t o r s o f t h e degree  phytoplankton  Gulf  the  among samples from n a t u r a l p o p u l a t i o n s t r e a t s t h e d i f f e r e n c e s  diversity. this  of  their in  the  variation  regions  as  geographical  was  origin,  among i s o l a t e s 1981b).  a l l o f t h e Protogonyaulax  p r e s e n t study were r e p r e s e n t a t i v e s from in  acclimated  substantial.  growth  neritic  r a t e s w i t h i n and among  A h i g h growth r a t e might be l e s s  a s u r v i v a l s t r a t e g y f o r Protogonyaulax  than o t h e r mechanisms,  as a l t e r n a t i v e l i f e c y c l e s t a g e s , i n c l u d i n g encystment, and may n o t be  selected  f o r as  strategies,  rigidly.  In  Protogonyaulax  spp.  the  terminology  may  be  less  of  ecological  "r-selected"  than  survival neritic  148  diatoms  ( G u i l l a r d and Kilham,  The  paired-group  analyze  factors,  1977).  comparisons  including  and  10)  geographical  location  in  and  of  origin,  the  data  coincident  to  show  4  that  mean  origin,  significantly  related  single as  location,  well  When  in  as  t o minimize d i s t o r t i o n s due t o  incautious  rather  mind  growth  this  conforming  look rate  at  the d a t a i n T a b l e 10  o f tamarensoid  than  morphotype diversity  per  isolates is  se,  may  be  more  i n growth r a t e components.  t h a t most o f the c a t e n e l l o i d  i s o l a t e s were from a  w h i l e the m a j o r i t y of the tamarensoid  to  that  the i n f l u e n c e o f f a c t o r s a s s o c i a t e d w i t h  genotypic  Bamfield,  linked,  example, a l t h o u g h T a b l e 9 i n d i c a t e s  However,  to  be g e n e t i c a l l y  isolates  the n a t u r a l water f o r the growth medium were from E n g l i s h  isolates  compared,  the  the time o f  s i g n i f i c a n t l y more v a r i a b l e i n growth r a t e s  an  geographical  bear  not  isolates,  higher.  must  cautiously  For  are  significantly  One  viewed  bias.  isolates  tamarensoid  appears  be  sampling  catenelloid than  must  some c a s e s , t h e s e f a c t o r s may  at  and  t o account f o r the d i f f e r e n c e s i n v a r i a n c e and mean growth r a t e s In  clonality  age  isolation,  groups.  culture,  make i t p o s s i b l e t o  of  but  isolates  9  morphotype  between  the  (Table  either  Bay.  morphotype from o t h e r l o c a t i o n s were  apparent c o n n e c t i o n between morphotype and mean growth r a t e  i s no l o n g e r s i g n i f i c a n t . Brand usually and that  (1980)  the  recent  clonal  isolates  for  a l o n g e r time.  I t was  suggested  of  P.  tamarensis  (Brand e t a l . ,  d e c l i n e i n growth r a t e w i t h time i n c u l t u r e was  depression  effects  that  had h i g h e r growth r a t e s than those i n i t i a t e d as n o n - c l o n a l i s o l a t e s  maintained  separate  noted  among a few genotypes. the  of  effects clonality  T h i s may  1981b)  due t o i n b r e e d i n g  be t r u e , but i t i s n e c e s s a r y t o  of time, as measured by a s e x u a l g e n e r a t i o n s , and on  the  p o t e n t i a l f o r sexual recombination.  the  In the  149  present  study,  versus been  there  non-clonal retained  important the  summer  culture  consider  of  no  s i g n i f i c a n t d i f f e r e n c e s between a l l c l o n a l  i s o l a t e s from d i f f e r e n t g e o g r a p h i c a l  in  to  were  1984  f o r varying  periods  t h a t t h e most r e c e n t  l o c a t i o n s which have  o f time.  However, i t i s  i s o l a t e s - a l l from B a m f i e l d i n  - a l s o comprised t h e m a j o r i t y  of the c l o n a l i s o l a t e s .  Comparison  of c l o n a l versus non-clonal  to  out t h e e f f e c t o f t h e l e n g t h o f time i n c u l t u r e and  factor  origin  on  growth  r a t e , since, with the exception  contemporaneous  isolates.  suggests  the  that  cultures result  mean  geographical  o f NEPCC 516, these were  A g a i n t h e r e was no s i g n i f i c a n t d i f f e r e n c e .  potential  f o r sexual  recombination  in  issue  growth  addition  of rate  to  growth r a t e s as a form o f " h y b r i d how  the  Bamfield  geographical  were  that  could  vigor".  t o account f o r t h e s i g n i f i c a n t l y lower v a r i a n c e and  in  their  This  multiclonal  does n o t have a l a r g e impact on g e n o t y p i c m o d i f i c a t i o n s  i n increased  The  i s o l a t e s from E n g l i s h Bay a l l o w s one  isolation  since, i n  from o t h e r p o p u l a t i o n s ,  Given  t h e above comments on t h e e f f e c t s o f c l o n a l i t y on t h e growth r a t e s o f  observed  were  enhancement isolates  through were  reasonable accumulation these before several lower  strictly  to  of  cultures the  variance  most  mitotic were  growth  months,  due  postulate  not rate  t o t h e c a t e n e l l o i d morphotype.  i t i s unlikely to  genetic  those  belonged  a l l of  isolates  isolates,  and  i s problematic,  these  contemporaneous  clonal  isolates  the  lack  that  the  the  lower  of opportunity  recombination. recently  DNA  that  brought  since  culture,  these  i t i s not  slower growth can be a t t r i b u t e d t o t h e  replication  completely experiments,  errors.  acclimated since  I t i s not l i k e l y to  existence  that  i n culture  they were n o t u t i l i z e d  and s e v e r a l t r a n s f e r s , a f t e r i s o l a t i o n . in  f o r growth r a t e  Furthermore, into  growth r a t e s  until  The s i g n i f i c a n t l y  growth r a t e s among i s o l a t e s from t h i s p o p u l a t i o n  may be  150  evidence  that  the  time e l a p s e d s i n c e i s o l a t i o n was  g e n e t i c d i v e r g e n c e t o have o c c u r r e d i n t h e s e c l o n a l It  is  is/are  tempting  genetically  but  without  for  comparison,  that  cultures.  the i n s i t u B a m f i e l d p o p u l a t i o n s )  l e s s heterogeneous than the p o p u l a t i o n s from  this  it  elsewhere,  cannot  be  i s reasonable  conclusively  to suggest  demonstrated or d i s p r o v e d .  that differences i n reproductive  between a l l o p a t r i c a l l y d i s t r i b u t e d p o p u l a t i o n s a r e based upon g e n e t i c  differences  arising  habitats. attained base the  conclude  for large  a l a r g e number of contemporaneous i s o l a t e s from o t h e r l o c a t i o n s  Nevertheless, rates  to  insufficient  It by  for  may  be  isolates  the  pattern  culture  from  culture of  evolution  significant  that  of the  populations highest  in  different  growth r a t e s were  from the same n a t u r a l m i l i e u from which the seawater medium was  growth  regime  the  rates  was  obtained.  among  selected  Crossovers  and  inversions in  i s o l a t e s c o u l d p o s s i b l y occur  to  favour  conditions  more  i f the closely  r e p r e s e n t a t i v e of o t h e r environments.  C. Q u a n t i t a t i v e N u c l e a r DNA  Content  1. R e s u l t s and D i s c u s s i o n  Epifluorescence tool and  in  the  the  estimation  clarification  Although cells,  microphotometry  the as  sacrificed,  ability  with  flow  of  individual  cells  i s an  of i n t r a s p e c i f i c v a r i a t i o n i n n u c l e a r DNA of  to  stages rapidly  cytometry  epifluorescence  in  the  important content  l i f e c y c l e of  dinoflagellates.  measure the n u c l e a r DNA  content of many  (Yentsch e t a l . , 1983;  Hayhome, 1985), i s  microphotometry does o f f e r s e v e r a l advantages.  151  Selection  of  appropriate pigments  the  correct  barrier  and  aperature  filter  pre-fixation  r e q u i r e d f o r f l o w cytometry  nuclei.  chlorophyll  autofluorescence  co n t e n t  is  necessary  caused  (Yentsch  et  by  values  procedure  is  microscope visual  with rather  The  exposure  al.,  more  degree involved  of  of  cysts  gametes  interpretation  from of DNA  from  of the s t a i n e d c e l l s  to  The measurement of  DNA  1983).  (Hayhome, 1985)  precision  and  undoubtedly  accuracy,  but  the  than t h a t employed f o r e p i f l u o r e s c e n t  D i r e c t m i c r o s c o p i c o b s e r v a t i o n s a l l o w f o r the  obvious  aberrants  and  l y s e d (presumably  e p i f l u o r e s c e n t microscope t e c h n i q u e  pellicular  fusing  high  determinations.  rejection  cells.  a  o f n u c l e a r DNA  to minimize the i n t e r f e r e n c e o f  from e x t r a c t e d n u c l e i by flow cytometry  yields  of  This  wavelengths  the  e l i m i n a t e s the need f o r the p h o t o - o x i d a t i o n o f  unextracted  excitation  s i z e f o r n u c l e a r measurement and  from  non-viable)  a l s o p e r m i t s the s e p a r a t i o n  s m a l l v e g e t a t i v e c e l l s , and of p l a n o z y g o t e s  abnormally  large vegetative c e l l s .  and  Furthermore, the  v a l u e s from i n d i v i d u a l c e l l s i n a c h a i n c o n f i g u r a t i o n  is possible. When  viewed  DAPI-stained and  were  nuclear of  morphologically  nuclei  Gavrila,  al o n g  view,  nucleus, plane in  in 1977;  the  under  the  n u c l e i o f a l l Protogonyaulax  shape  ventral  apically  agrees  w i t h p r e v i o u s l i g h t and  1982;  (Dodge, 1964; Balech  and  microscope,  This  o b s e r v a t i o n upon the  electron microscopic studies  Silva,  1971;  Tangen, 1985;  1977;  Tomas,  1974;  Fukuyo, 1985).  In  the n u c l e u s appeared as a t h i c k b l u e - f l u o r e s c i n g l i n e a r band  cell  equator  particularly  ( F i g . 19).  of  the  The  p o t e n t i a l f o r s e l f - s h a d i n g of the  n u c l e a r a p i c e s , which curved back below the  of o b s e r v a t i o n , or f o r f l u o r e s c e n c e a b s o r p t i o n by c e l l u l a r  intact  the  i s o l a t e s were C-shaped ( F i g . 18),  indistinguishable.  Protogonyaulax Hall,  epifluorescence  cells,  was  estimated  by  comparing  DNA  components  values obtained  from  152  Fig.  18  A p i c a l view o f NEPCC 516 s t a i n e d w i t h DAPI showing c r e s c e n t shaped n u c l e u s and r e d a u t o f l u o r e s c e n c e under e p i f l u o r e s c e n c e m i c r o s c o p y (800X). Exposure: 1 min; Ektachrome ASA 160.  Fig.  19  E q u a t o r i a l view o f NEPCC 516 s t a i n e d w i t h DAPI showing f l u o r e s c e n t n u c l e a r bar a t the cingulum under e p i f l u o r e s c e n c e m i c r o s c o p y (500X). Red a u t o f l u o r e s c e n c e has been s u p p r e s s e d with a blue b a r r i e r f i l t e r . Exposure: 1 min; Ektachrome ASA 160.  153  154  various  angles  and  factors  d i d not  nucleus  was  produced  increased  the  chromosomes,  with  from  from  ventrally  by  applying  nuclear  lay  close  the  DNA to  (21.42-63.29  due  the  observer  overlying  Nuclear  cover  pg  cell  - 1  G.  value  DNA  content  content  of  ).  as ever  Amphidinium  greater and be  than  polyedra  was  The  quantity  identical  ± 2.68 s.d. pg c e l l ! ) -  within of  isolates  the  nuclear  range DNA  (45.76 ± of  values  c o n t e n t i n G.  contrasted sharply with the o l d e r report i n  total  where DNA, measured by an e x t r a c t i v e  DNA,  reported  was  for a given  determined  error.  well  1969),  1978)  (36.13  f o r a l l Protogonyaulax  carterae,  that  Durrand, in  tended  t h e v e n t r a l s u r f a c e exposed, t h i s  and  (Holm-Hansen,  highest  those  spp., as was p r e v i o u s l y r e p o r t e d (Dodge, 1964).  cell"-'-),  technique  may  with  slip,  DNA measurements  Nevertheless, since c e l l s n a t u r a l l y  slip  from t h e s e experiments  (Galleron  at  t o uneven d i s r u p t i o n o f t h e  i n c r e a s e t h e mean DNA v a l u e s .  of  t h e mean  fluorometric  assuming  the  f o r o b t a i n i n g h i g h l y c o n s i s t e n t DNA measurements.  content  pg  literature  values  to  measurements  cover  Protogonyaulax  s.e.m.  times  toward  o f t h e n u c l e u s o f Gonyaulax p o l y e d r a was v i r t u a l l y  of  The  the  Only when t h e c r e s c e n t - s h a p e d  projecting  pressure  oriented c e l l s .  under  shape  polyedra  of  d i d not  p e r s p e c t i v e was adopted  2.35  tips  These  a p o l a r view were n o t u s u a l l y s u b s t a n t i a l l y d i f f e r e n t from  orient  that  the  non-compressed c e l l s .  from t h e c e n t r a l p o r t i o n o f t h e n u c l e u s .  variance  but  made  to  t o be s i g n i f i c a n t .  especially  squashes,  The  versus  t o t h e e q u a t o r i a l p l a n e was t h e r e a marked decrease i n e m i t t e d  fluorescence,  to  compressed  appear  viewed  right-angles  from  given  as 200 pg c e l l "  dinoflagellate.  1  That t h e DNA  i n t h e same p u b l i c a t i o n , was 1.6  subsequently  by o t h e r  investigators  lends credence t o t h e s u g g e s t i o n t h a t  This  - the  discrepancy  cannot  be accounted  these f o r by  t h a t i t r e p r e s e n t s t h e c o n t r i b u t i o n o f non-nuclear DNA, s i n c e t h i s  155  would  typically  conceivable was  of the t o t a l c e l l u l a r DNA.  mean  mean  values  value  of  DNA  pg  with  consistent probable  requires  further  DNA  isolations  the  range  through  Yentsch  explanation.  1979;  extraction  (1979),  determined  the DNA  No apparent  analysis  (r  c  =  i n Protogonyaulax  -0.15;  that  cultures  autodiploidy.  On  In the  c o n t e n t per c e l l  varied  decrease  I f t h i s phenomenon i s r e a l , i t changes i n mean DNA  content  per  i n the c u r r e n t study among  and time e l a p s e d s i n c e the  s l o p e = -0.25). (Loper  et  c o r r e l a t i o n between  T h i s may  a l . , 1980;  be s i g n i f i c a n t  may  be  indicative  of  aneuploid  i n view 1982)  increase  or  the o t h e r hand, m o r p h o l o g i c a l l y s i m i l a r i s o l a t e s of the  m i c r o a d r i a t i c u m s p e c i e s complex were s i m i l a r t o  in  relationship  no  original  H o l t and P f i e s t e r ,  Symbiodinium  1985).  i n the p r e s e n t  the appearance of chromosomal i n c r e a s e s i n long-term  dinoflagellate  that  an  are considered.  ( F i g . 20) r e v e a l e d no apparent  observations  suggested  that  by  Y e n t s c h e t a l . , 1983),  of v a l u e s found  e x p o n e n t i a l phase were observed  content  previous  which  and  tamarensis,  and Yentsch,  with  losses  P.  A p r e v i o u s mean  isolates.  Regression nuclear  for  age d u r i n g e x p o n e n t i a l phase.  throughout  multiple  found  and G. p o l y e d r a .  c l o s e t o the  w i t h the stage i n c u l t u r e , and even e x h i b i t e d a f o u r - f o l d  culture  cell  1  by S p e c t o r (1984) i s remarkably  (Mickelson  Mickelson  markedly  - 1  1969)  pg c e l l " ) of 11 i s o l a t e s of n i n e o t h e r  f o r Protogonyaulax  cell  technique  when by  course  isolate.  (= 41.20  given  obtained  reasonably  study  content  species  ~20  extractive  study,  I t i s of  t h a t the e a r l i e r measurement i n G. p o l y e d r a (Holm-Hansen,  dinoflagellate  of  <5%  based upon a h i g h l y p o l y p l o i d The  is  comprise  Protogonyaulax  between chromosome number and the l e n g t h of  an i s o l a t e had been m a i n t a i n e d  i n c u l t u r e was  found  (Blank and  time  Trench,  156  F i g . 20  L i n e a r r e g r e s s i o n a n a l y s i s o f t h e r e l a t i o n s h i p o f mean n u c l e a r DNA content t o time e l a p s e d s i n c e the o r i g i n o f i s o l a t e s i n culture. DNA v a l u e s f o r contemporaneous i s o l a t e s NEPCC 400-412 from E n g l i s h Bay (1981) were combined as a s i n g l e p o i n t . (X ± s.e.m.). R e g r e s s i o n c o e f f i c i e n t r = -0.15, s l o p e = -0.25. E r r o r bars = ±1.0 s.d.; n=30. c  70.0r  60.0\  400-412  ,5  i  <D O  x  50.0  355 255  516  Q. 40.01 529J  30.0 O  zo.o\  O 3  lOJOf  ,180 71  • 545  183,  435  j  i 253  $543 ^508  J  L.  J  10  '  '  I  I  I  15  Years in culture  I  L  '  20  L  _J  25  1  i_  158  Although least  functionally  (Dodge, is  t h e v e g e t a t i v e c e l l s o f Protogonyaulax  a r e c o n s i d e r e d t o be a t  h a p l o i d , t h e chromosome s i z e ( G a v r i l a , 1977) and number.  1963) a r e v a r i a b l e .  In P. t a m a r e n s i s , t h e s p l i t t i n g o f chromosomes  asynchronous ( G a v r i l a , 1977), which may e x p l a i n some o f t h e v a r i a t i o n i n  chromosome  number  population. lead  to  content  found  even among c e l l s from t h e same  Unequal d i s t r i b u t i o n o f chromatin  of  fragment  nuclei  of  unequal  during mitotic d i v i s i o n  The  Protogonyaulax  size (Silva,  1977).  The s m a l l e r n u c l e a r  for  tamarensis  (=  number  mean  the  NEPCC  haploid  183).  DNA  chromosome  contained  that  1  =  DNA  the  genome  of  the  Plymouth  isolate  Assuming t h a t no changes i n e i t h e r  2.01  approximately X  10^  1973), t h i s i s e q u i v a l e n t t o Without  reduced  chromosome count from a member o f t h e  evidence  pg DNA.  P.  isolate,  Using t h e e s t i m a t e  n u c l e o t i d e base p a i r s (Haapala and Soyer,  ~6 X 10^ base p a i r s p e r chromosome.  from  accurate  counts,  i t was not  to  isolates  r e f l e c t e d d i f f e r e n c e s i n t h e number o f chromosomes p e r n u c l e u s , o r in  chromosomes. chromosomal apparent  the In  quantity  vary  relationship 1985).  content  was  necessarily  In  usually with  cell  t h e v a r i a t i o n i n n u c l e a r DNA c o n t e n t among  of  Symbiodinium  volume  Trench,  whether  chromosome  possible  variations  determine  0.3  of  chromosome  c o n t e n t per chromosome have o c c u r r e d i n t h i s  each  pg  available  c e l l with  s p e c i e s complex i s t h e v a l u e o f 134-152 chromosomes g i v e n by  (1963)  or  only  may  n u c l e i (Dodge, 1963) and t h e  can g i v e r i s e t o a s m a l l e r , y e t v i a b l e , daughter  content.  Dodge  DNA  uneven chromosome numbers i n daughter  formation  DNA  and  DNA  microadriaticum,  irregularly  between the well  contained  among  chromosome  Crypthecodinium correlated  volume  (Beam  with and  in  e q u i v a l e n t numbers o f  not  only  isolates, number  and  but  d i d the t o t a l there  volume  was no  (Blank and  c o h n i i s p e c i e s complex, t h e DNA chromosome Himes, 1984).  number,  but  not  Among P e r i d i n i u m  159  isolates  examined  by  nuclear  DNA  content  groups,  but  these  flow  cytometry  indicated  two  (Hayhome, 1985), t h e d i s t r i b u t i o n o f statistically  significant  different  d i f f e r e n c e s were n o t c o r r e l a t e d w i t h c e l l volume, which  remained c o n s t a n t . The mean  n u c l e a r DNA c o n t e n t among Protogonyaulax  cell  prolate  volume  spheroid  tamarensis failed 508  =  c  (Table  flow  0.19, A).  slope  = 0.00) ( F i g . 2 1 ) , c a l c u l a t e d as a  I n a p r e v i o u s a n a l y s i s o f one i s o l a t e o f P.  cytometry  (Yentsch  to c o r r e l a t e c l o s e l y with c e l l  et  size.  a l . , 1983), DNA p e r c e l l  their  2.60  lower 12  mean  DNA  pg  cell  s.d.  content, - 1  ,  25.55  ±  respectively,  2.58  was  s.d.  judged  pg c e l l " to  than t h e mean v a l u e o b t a i n e d from a l l Protogonyaulax  and  half  13).  The  to  morphotype o r g e o g r a p h i c a l l o c a t i o n o f o r i g i n .  of  nuclear from  DNA  those  a=0.05)  from  DNA  in  Protogonyaulax  catenelloid  (Tables  isolates  12  from  isolates  was  isolates  (Student's  stationary  diameter phase,  IA).  This  in  was  and 13). The mean DNA c o n t e n t o f t h e t e n  the  cultures  1981  of  approximately  appears  Mean  t-test,  E n g l i s h Bay bloom d i d n o t d i f f e r combined.  amount o f n u c l e a r DNA c o n t a i n e d i n t h e l a r g e d a r k l y pigmented in  not  i s o l a t e s were n o t s i g n i f i c a n t l y  o b t a i n e d f o r tamarensoid  s i g n i f i c a n t l y from t h e mean o f a l l i s o l a t e s  zygotes.  (1971; 1977).  related  contemporaneous  (Table  (Tables  nuclear  two-tailed;  nm  isolates  of  different  >50  be s i g n i f i c a n t l y  quantity  obviously  The  and 21.A2  These " s m a l l form" d i n o f l a g e l l a t e s  may r e s u l t from r e d u c t i o n a n e u p l o i d y as r e p o r t e d by S i l v a  values  1  spectrum,  DNA c o n t e n t o f i s o l a t e s 253 and 508 was almost e x a c t l y  t h e mean v a l u e f o r Protogonyaulax.  The  also  N e v e r t h e l e s s , i s o l a t e s 253 and  were noteworthy i n t h a t they l a y a t t h e lower end o f t h e s i z e  while ±  by  ( r  i s o l a t e s d i d not vary with  cells  183,  255, A03, A05, and A35 d u r i n g  double  that of the vegetative c e l l s  t o c o n f i r m t h e i r p r o v i s i o n a l i d e n t i f i c a t i o n as  160  F i g . 21  N u c l e a r DNA c o n t e n t o f Protogonyaulax i s o l a t e s i n r e l a t i o n t o mean c e l l volume. The c o r r e l a t i o n c o e f f i c i e n t r = 0.19; s l o p e = 0.00. E r r o r bars = ±1.0 s.d.; n=30. c  Q) O  0)80.0 a  •412  §60.0  405  £40.0  I  J409  o  z  [406  {253  20.0  5.0  255  J180  4aa_  ^29  1508  ± 10.0 15.0 20.0 25.0 Mean cell volume (um x 10 ) 3  3  162 Table 12. Mean nuclear DNA content of Protogonyaulax isolates by morphotype and location f origin, n = number of isolates in each group.  Mean nuclear DNA content (pg c e l l ) - 1  Isolates Catenelloid  39. 49 + 4.97 s . e.m.> n=4  Tamarensoid  47. 16 + 2. 92 s .e.m. 5 n=16  English Bay (1981)  51. 81 + 2. 70 s .e.m.  NEPCC 253  25. 55 + 2.58 s .d.; n =30  NEPCC 508  21.42 + 2. 60 s .d.; n =30  A l l isolates  45. 76 + 2. 35 s .e.m. J n=22  n=10  9  ;  Table 13. Pairwise comparison of the difference in mean nuclear DNA content between grouped Protogonyaulax isolates. Test of significance is the Student's t-test; two-tailed at a = 0.05; H : Mi= number of isolates in each group. n  =  Q  d.f. (ni+^-2)  ter[.05(2)]  Hypothesis  Catenelloid/ tamarensoid  1.20  18  2.10  H accepted'  English Bay (1981)/ a l l isolates  1.54  30  2.05  H accepted*  NEPCC 253/ a l l isolates  8.39  21  2.08  H  NEPCC 508/ a l l isolates  10.10  21  2.08  HQ rejected'  ns": the population means are not significantly different s'°': the population means are significantly different  Q  n  Q  rejected-  T a b l e 14. N u c l e a r DNA content o f v e g e t a t i v e c e l l s o f Protogonyaulax i s o l a t e s r e l a t i v e t o t h a t o f z y g o t e s . Number o f i s o l a t e s , n = 30.  N u c l e a r DNA c o n t e n t (X ± s. d.; n=30) (pg c e l l " ) 1  Isolate  Vegetative c e l l s  Zygotes  DNA R a t i o  NEPCC 183  43.06 ± 7.19  81.04 ± 8. 11  1.88:1  NEPCC 255  48.35 ± 4.00  101.89 ± 1. 61  2.11:1  NEPCC 403  43.55 ± 6.43  85.14 ± 5. 37  1.95:1  NEPCC 405  55.36 ± 4.16  92.13 ± 6. 53  1.66:1  NEPCC 435  37.61 ± 3.57  70.64 ± 3. 15  1.88:1  164  D.  Electrophoresis  of Soluble  Enzymes  1. G e n e r a l E l e c t r o p h o r e t i c  In  electrophoretic  electrical  field  system. the  The  is  electrical  effects;  of  of  the  rate  system  the  are  so-called composition stacking  migration  rate,  proportional  gel  Coincidently,  pH  of  with  from  "discs"  originally  recommended  to  the  (1964)  the  for  upon  are  their  also  t o s i e v i n g and  gel  and  electrophoresis  Davis  electrode  The  (1964), e l e c t r o p h o r e t i c DISC-PAGE t e c h n i q u e i s  a g e l b u f f e r which d i f f e r s  buffer.  i s superposed above the s e p a r a t i n g  s e p a r a t e d p r o t e i n bands f r e q u e n t l y  DISC-PAGE  is  t h i s method.  the  gel. Acrylamide  addition  of  continuous  performed  in  the  gel.  resemble  narrow  tubes  DISC-PAGE i s c a p a b l e of y i e l d i n g  buffer  systems,  since  the  sample  "leading"  i o n , r e s u l t i n g i n a moving f r o n t p r o c e e d i n g through  (CH^CH'CONI^) the  in  U s u a l l y a sample and/or  remain b r a c k e t e d w i t h i n a narrow zone formed between the "trailing"  important  (DISC-PAGE)  proteins  the  net  retardation  resolution  and  buffer  the medium to  sharper  ion  than  ionic  an  e l e c t r i c a l charge:mass r a t i o  gel f i l t r a t i o n .  porosity  when  shape  due  i s achieved using  well-defined  flattened  an  in  resistance.  Ornstein  higher  the  proteins  primarily  and  polyacrylamide  separation  and  of  medium u s i n g  depends  net  combined  since  charge  size  discontinuous  techniques  porous  molecular  is  by  a  migration  The  frictional  pioneered  in  the  r a t e of the p r o t e i n molecules w i t h i n  opposite  the  m u l t i p l i e d by the In  effected  charge.  determinants  separation,  migration  electrode  Theory  bifunctional  is  polymerized  and  cross-linked  by  the  reagent N,N'-methylenebisacrylamide ("Bis")  165  [(CH =CH-C0NH)2 CH2]•  Persulfate  ,  2  and  often  N,N,N',N'-tetramethylethylenediamine  polymerization. that to  is  achieved  (TEMED)  i n s t a r c h g e l s due  concentration  of a c r y l a m i d e and c r o s s - l i n k e r .  regulating  several  are  genes,  features  and  be  may  relatedness  upon  based  diversity,  gels  since  the p o l y p e p t i d e s  varying  the in  enzyme  variants.  in  there  gel  for  by  highly  complex  greater  adjusting  the  and  is  be  an  characters  i n t e r p r e t e d as  of  the  i n t e r a c t i o n s between  susceptible  electrophoresis  to  attempt  environmental to  establish  - p r i m a r y gene p r o d u c t s The  phenotypes expressed  i n d i c a t i v e of r e l a t i v e  on  genetic  i s a d i r e c t correspondence between enzyme l o c i  t h a t many enzymes e x i s t i n m u l t i p l e isozymic charge  and  electrophoresis  histochemical a  to  and  f o r which they code.  electrical  isozyme  by  the  t a x a are p h e n o t y p i c e x p r e s s i o n s  biochemical  may  discovery  1959),  with  accomplished  more d i r e c t l y l i n k e d t o the genome.  electrophoretic  With  size,  of  coded  Isozyme  are  accelerate  s i z e , and  i n Chemosystematics, Taxonomy  presuma