Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A1 C45.pdf [ 22.18MB ]
Metadata
JSON: 831-1.0053282.json
JSON-LD: 831-1.0053282-ld.json
RDF/XML (Pretty): 831-1.0053282-rdf.xml
RDF/JSON: 831-1.0053282-rdf.json
Turtle: 831-1.0053282-turtle.txt
N-Triples: 831-1.0053282-rdf-ntriples.txt
Original Record: 831-1.0053282-source.json
Full Text
831-1.0053282-fulltext.txt
Citation
831-1.0053282.ris

Full Text

VARIABILITY AND SPECIES DISCRIMINATION WITHIN THE PROTOGQNYAULAX TAMARENSIS/CATENELLA SPECIES COMPLEX: TOXIC RED-TIDE DINOFLAGELLATES By ALLAN DOUGLAS CEMBELLA B.Sc, Simon Fraser University, 1.977,. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Departments of Botany and Oceanography We accept t h i s thesis 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 t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her rep r e s e n t a t i v e s - . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date Q | J / r y Hag 1Q\ i ABSTRACT VARIABILITY AND SPECIES DISCRIMINATION WITHIN THE PROTOGONYAULAX TAMARENSIS/CATENELLA SPECIES COMPLEX: TOXIC RED-TIDE DINOFLAGELLATES A l l a n Douglas Cembella Un i v e r s i t y of B r i t i s h Columbia 1986 Thecate gonyaulacoid d i n o f l a g e l l a t e s r e f e r a b l e to the genus Protogonyaulax Taylor cause p a r a l y t i c s h e l l f i s h poisoning (PSP) i n coastal regions throughout the world. Isolates of the Protogonyaulax tamarensis/ c a t e n e l l a species complex from diverse geographical regions, including ten contemporaneous i s o l a t e s from the same l o c a t i o n , were subjected to chemotaxonomic analysis of soluble isozymes by gel electrophoresis, an analysis of toxin components using high-pressure (performance) l i q u i d chromatography and quantitative nuclear DNA determinations by epifluorescence microphotometry. The r e s u l t s were compared with conventional morphological c r i t e r i a used to discriminate among species, to e s t a b l i s h taxonomic linkages and to estimate phenotypic and genotypic v a r i a t i o n within t h i s group. These biochemical methods, along with measurements of acclimated growth rate, offered a means of d i s t i n g u i s h i n g between i s o l a t e s of t h i s species complex, for which the thecal plate patterns were s u b s t a n t i a l l y the same. The isozyme patterns revealed a high degree of genetic polymorphism within and among morphotypes and geographical populations. Yet, within the tamarensoid morphotype, i s o l a t e s from the same lo c a t i o n were more s i m i l a r than to those from elsewhere. This general trend was supported by evidence from toxin p r o f i l e s and DNA a n a l y s i s , although t o x i n heterogeneity was a more conservative measure of v a r i a t i o n than isozyme d i v e r s i t y . Protogonyaulax i s o l a t e s varied markedly i n t o t a l t o x i n concentration and t o x i c i t y , even through the culture cycle of i n d i v i d u a l i s o l a t e s , but the toxin r a t i o s were d i s t i n c t i v e and r e l a t i v e l y constant. The c a t e n e l l o i d and tamarensoid forms, the dominant morphotypes within t h i s species complex, were not well correlated with the biochemical characters investigated. Given the occasional presence of morphological intermediates, the morphological features presently used to i d e n t i f y P. c a t e n e l l a and P. tamarensis cannot always be used to r e l i a b l y discriminate between these morphospecies, and appear to be inadequate as stable species d e s c r i p t o r s . At l e a s t two smaller i s o l a t e s from a l l o p a t r i c populations exhibited morphological and biochemical differences large enough to indicate possible species divergence. The high l e v e l of genetic d i v e r s i t y r e f l e c t e d i n the biochemical heterogeneity within populations from a given geographical area suggests that s i b l i n g species may also have arisen within sympatrically d i s t r i b u t e d Protogonyaulax populations. i i i E sara mia colpa se c o s i e? - M a c h i a v e l l i The phosphorescence, that bluey greeny. Very good f o r the brain. -"Ulysses", James Joyce But not on us! the oysters c r i e d , Turning a l i t t l e blue. A f t e r such kindness, that would be A dismal thing to do! - "Through the Looking Glass", Lewis C a r r o l l i v TABLE OF CONTENTS ABSTRACT i TABLE OF CONTENTS i v LIST OF FIGURES ix LIST OF TABLES x i i i ACKNOWLEDGEMENTS x v i CHAPTER I. INTRODUCTION 1 A. C l a s s i f i c a t i o n and the Species Problem i n D i n o f l a g e l l a t e s 1 B. V a r i a b i l i t y and Speciation 7 1. Phenotypic versus Genotypic Evidence 7 2. Mechanisms f o r Maintaining V a r i a b i l i t y and S t a b i l i t y 16 3. Phenetic and Phylogenetic Taxonomic Linkage 22 C. General Description and Taxonomic P o s i t i o n of the Genus Protogonyaulax 26 D. H i s t o r i c a l and Recent Perspectives on Taxonomic Variants Within the Protogonyaulax tamarensis/catenella Species Complex 39 E. Rationale f o r Experimental Studies 47 1. Geographical D i s t r i b u t i o n and Environmental S i g n i f i c a n c e of Protogonyaulax spp. blooms 47 2. The Problem of Morphotypic Gradients and S t a b i l i t y i n Protogonyaulax 54 3. Research Objectives 56 CHAPTER I I . MATERIALS AND METHODS 58 A. Culture and Maintenance 58 1. Is o l a t e Origins and Growth Conditions 58 2. Development and Formulation of NWSP-7 Growth Medium 59 3. I s o l a t i o n and Cloning 65 4. Preparation and Testing of Axenic Cultures 66 B. Morphological Examination of Protogonyaulax Isolates 68 1. Isolate I d e n t i f i c a t i o n and Morphological 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. Determination of Acclimated Growth Rates 75 D. DNA Analysis by Epifluorescence Microphotometry I 78 1. Nuclear DNA Determination 78 E. Method for Polyacrylamide Gel Electrophoresis 79 1. Electrophoresis of Soluble Enzymes 79 a. C e l l c u l t u r e , harvest and storage 79 b. B a c t e r i a l contamination 81 c. Determination of t o t a l protein 81 d. Enzyme extraction 82 e. Ele c t r o p h o r e t i c separation 83 f. Gel s t a i n i n g and band scoring 84 F. Toxin Analysis by High-Pressure (Performance) L i q u i d Chromatography 86 1. HPLC Analysis of Toxins 86 a. C e l l culture and harvest 86 b. Toxin extraction 87 c. A n a l y t i c a l method 88 CHAPTER I I I . EXPERIMENTAL RESULTS AND DISCUSSION 92 A. Morphological 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 Size 92 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 C h a r a c t e r i s t i c Features 100 3. Chain Length 117 4. Discussion. 126 B. Growth Rates 134 1. Acclimated Growth Rates of Protogonyaulax i s o l a t e s . . . . 134 a. Results 134 b. Discussion 144 C. Quantitative Nuclear DNA Content 150 1. Results and Discussion 150 D. Electrophoresis of Soluble Enzymes 164 1. General Electrophoretic Theory 164 2. The Use of Electrophoresis i n Chemosystematics, Taxonomy, and Population Genetics 165 3. Interpretation of Electrophoretic Data 169 4. Enzyme Reactions: Pyr i d i n e - l i n k e d Dehydrogenases 173 5. Electrophoretic 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 Relationships among Protogonyaulax Isolates 191 a. Phenetic c l u s t e r analysis 191 b. Phylogenetic linkage analysis 204 7. Discussion 214 i . The dehydrogenase isozymes and genotype 214 i i . Isozyme v a r i a t i o n and genetic i d e n t i t y 218 v i i i i i . Evolutionary divergence and phylogeny 222 i v . E l e ctrophoretic evidence and speciation 224 E. V a r i a t i o n i n Toxin Composition and T o x i c i t y 225 1. Introduction 225 2. Toxin Concentrations and P r o f i l e s 231 3. Toxin-Based Chemotaxonomic Relationships among Protogonyaulax Isolates 260 a. Phenetic c l u s t e r analysis 260 b. Analysis of p r i n c i p a l components 266 4. Discussion 278 CHAPTER IV. GENERAL DISCUSSION AND IMPLICATIONS 287 A. Genotypic Polymorphism and Environmental Heterogeneity 287 B. Evolutionary Divergence, Phylogeny and Speciation 290 C. Suggestions for Further Research 300 CHAPTER V. SUMMARY AND CONCLUSIONS 303 REFERENCES 306 APPENDICES 333 APPENDIX 1 333 A. A l t e r n a t i v e Growth Media 333 1. Culture Methods 333 2. A r t i f i c i a l Seawater Enrichment 334 3. Natural Seawater Enrichment 342 4. The. E f f e c t of T r i s on Growth 345 v i i i 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 i x LIST OF FIGURES Fi g . 1 General morphology of the tamarensoid morphotype of Protogonyaulax.. 30 F i g . 2 General morphology of the c a t e n e l l o i d morphotype of Protogonyaulax, showing antero-posteriorly compressed c e l l s i n a c h a r a c t e r i s t i c chain configuration 30 Fi g . 3 Thecal plate configuration of Protogonyaulax i n the Kofoid notation system, as applied by Fukuyo (1985) 33 F i g . 4 H i s t o r i c a l taxonomic scheme for some members of the Protogonyaulax tamarensis/catenella species complex 41 F i g . 5 Global d i s t r i b u t i o n of some members of the genus Protogonyaulax 50 F i g . 6 Map of the northeast P a c i f i c coast showing o r i g i n of Protogonyaulax i s o l a t e s 52 F i g . 7 Frequency d i s t r i b u t i o n of a p i c a l and t r a n s a p i c a l diameters (nm) of Protogonyaulax i s o l a t e s (n=30) 94-99 F i g . 8 A p i c a l view of empty theca of NEPCC 403 stained with 0.1% c a l c o f l u o r showing epithecal plates under epifluorescence microscopy (400X) 104 F i g . 9 A p i c a l view of epithecal plates of NEPCC 516 stained with 0.1% c a l c o f l u o r showing v e n t r a l pore on the f i r s t a p i c a l ( l 1 ) p l ate under epifluorescence microscopy (800X) 104 Fi g . 10 Photomicrograph of 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 equatorial view (250X) 107 F i g . 11 Photomicrograph of NEPCC 181(b), i s o l a t e d as a chain-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., in e q u a t o r i a l view (400X) 107 F i g . 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 plate of Protogonyaulax i s o l a t e s i n culture 111-113 F i g . 13 Antapical view of hypothecal plates from tamarensoid specimens stained with c h l o r a l hydrate-iodine-hydriodic a c i d , examined under phase-contrast microscopy (250X). ... 116 F i g . 14 Photomicrograph of chain-forming P. ca t e n e l l a i n a phytoplankton sample from Puget Sound, WA (250X) 116 F i g . 15 Growth curves of NEPCC 355 on con t r o l ESNW medium ( • ) , low N ( • ), low P ( • ) and low Fe ( X ) 119 X F i g . 16 Histograms of the percentage of c e l l s present i n chains of various lengths throughout the growth cycle of NEPCC 355 121-124 F i g . 17 Histogram of acclimated growth rates of Protogonyaulax i s o l a t e s from Bamfield and English Bay i n B r i t i s h Columbia, and other regions 136 .Fig. 18 A p i c a l view of NEPCC 516 stained with DAPI showing crescent-shaped nucleus and red autofluorescence under epifluorescence microscopy (800X) 153 F i g . 19 Equatorial view of NEPCC 516 stained with DAPI showing fluorescent nuclear bar at the cingulum under e p i f luorescence microscopy (500X) 153 F i g . 20 Linear regression analysis of the r e l a t i o n s h i p of mean nuclear DNA content to time elapsed since the o r i g i n of i s o l a t e s i n culture 157 F i g . 21 Nuclear DNA content of Protogonyaulax i s o l a t e s i n r e l a t i o n to mean c e l l volume 161 F i g . 22 Zymograms of dehydrogenases extracted from Protogonyaulax i s o l a t e s 179-182 F i g . 23 Zymogram of NAD-dependent glutamate dehydrogenase (GDH) isozymes of some Protogonyaulax i s o l a t e s from English Bay, B.C 187 F i g . 24 Zymogram of NAD-dependent malate dehydrogenase (MDH) isozymes of Protogonyaulax i s o l a t e s 187 F i g . 25 Zymogram of NAD-dependent malate dehydrogenase (MDH) isozymes of some Protogonyaulax i s o l a t e s from English Bay, B.C 190 F i g . 26 Zymogram of NADP-dependent malic enzyme (ME) isozymes of some Protogonyaulax i s o l a t e s from English Bay, B.C 190 F i g . 27 UPGMA linkage dendrogram i n d i c a t i n g e l ectrophoretic s i m i l a r i t i e s among Protogonyaulax i s o l a t e s , constructed from S j values 194 F i g . 28 Unsealed Prim network i n d i c a t i n g minimum spanning distances between Protogonyaulax i s o l a t e s , based upon elec t r o p h o r e t i c p r o f i l e s of dehydrogenase isozymes 209 F i g . 29 Optimized midpoint rooted Wagner tree showing phylogenetic 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 electrophoretic p r o f i l e s of dehydrogenase isozymes 212 x i F i g . 30 Structures and abbreviations f o r saxitoxin (STX) and the eleven other PSP toxins obtained from Protogonyaulax spp 229 F i g . 31 Toxin composition of Protogonyaulax i s o l a t e s (as % t o t a l toxin ±1.0 s.d.; n=4) during exponential growth phase. ... 238-249 F i g . 32 HPLC chromatogram of PSP toxins extracted from a tamarensoid i s o l a t e , NEPCC 255 (Lummi Island, WA) 251 F i g . 33 HPLC chromatogram of PSP toxins extracted from a c a t e n e l l o i d i s o l a t e , NEPCC 529 (Friday Harbor, San Juan Island, WA) 253 F i g . 34 HPLC chromatogram of PSP toxins extracted from an i s o l a t e of intermediate morphotype, NEPCC 402 (English Bay, B.C.) 255 Fi g . 35 HPLC chromatogram of PSP toxins extracted from a highly t o x i c tamarensoid i s o l a t e , NEPCC 545 (Bay of Fundy, N.B.) 257 F i g . 36 Centroid-linkage dendrogram i n d i c a t i n g Euclidean distances between Protogonyaulax i s o l a t e s based upon r a t i o s of equally weighted toxins 262 F i g . 37 Ordination of Protogonyaulax i s o l a t e s by p r i n c i p a l component analysis based upon the percent of t o t a l toxin composition represented by each toxin component 273-275 F i g . 38 Growth curve of NEPCC 409 represented by i n vivo fluorescence 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 with natural seawater (ESNW) ( A ) enrichments during the second trans f e r cycle 341 F i g . 39 Zymogram of enzyme extracts of NEPCC 355 stained f o r NAD-dependent glutamate dehydrogenase (GDH) i n 0.1 M T r i s (tracks 1-5) versus 0.1 M T r i c i n e (tracks 6-10) s t a i n i n g buffer, pH 8.0 351 F i g . 40 Zymogram of enzyme extracts of NEPCC 355 stained f o r NADP-dependent malic enzyme (ME) comparing the e f f e c t of T r i s s t a i n i n g buffer with T r i c i n e ' b u f f e r at pH 8.0 351 F i g . 41 Zymogram of NAD-dependent GDH isozymes of NEPCC 409 extracted from frozen l y o p h i l i z e d versus non-lyophilized frozen c e l l s '. 354 F i g . 42 Zymogram of enzyme extracts of NEPCC 409 stained f o r a- and 3~esterases i n 0.1 M c i t r a t e buffer, pH 6.0 354 x i i F i g . A3 Densitometric scan of gels stained for NAD-dependent malate dehydrogenase (MDH) extracted from NEPCC 529 comparing enzyme extraction by mechanical homogenization (A3a) with high 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 F i g . AA Densitometric scan of gels stained for NAD-dependent malate dehydrogenase (MDH) extracted from NEPCC 529 showing the ' e f f e c t on band i n t e n s i t y of extract concentration and volume applied to gels 36A-366 Fi g . A5 Densitometric scan of gels stained f o r NAD-dependent glutamate dehydrogenase (GDH) extracted from NEPCC A09 showing the e f f e c t of a l t e r n a t i v e tetrazolium stains on band i n t e n s i t y : A5a, MTT; A5b, NBT 370 F i g . A6 Densitometric scan of gels stained for alanine dehydrogenase (AlaDH) extracted from NEPCC 25A showing the e f f e c t of s t a i n i n g buffer pH on band i n t e n s i t y : A6a, pH 8.0; A6b, pH 7.0 373 F i g . A7 Densitometric scan of gels stained f o r NADP-dependent malic enzyme (ME) extracted 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 buffers 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 F i g . A8 Densitometric scan of gels stained for NAD-dependent glutamate dehydrogenase (GDH) extracted 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 buffers 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 Special s i m i l a r i t i e s unweighted pair-group average (SUPGMA) linkage dendrogram for Protogonyaulax i s o l a t e s 383 F i g . 50 Regression analysis for the cophenetic c o r r e l a t i o n c o e f f i c i e n t (R = rcoph a s ^? 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 g . 51 Five a l t e r n a t i v e midpoint-rooted distance Wagner trees produced by the PHYSIS subprogram SWAG (TREES=5; TRIES=500), based on the Manhattan distance matrix derived from ele c t r o p h o r e t i c p r o f i l e s 390-39A F i g . 52 Optimized Wagner tree showing phylogenetic 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 elec t r o p h o r e t i c p r o f i l e s of dehydrogenase isozymes 398 F i g . 53 Ordination of Protogonyaulax i s o l a t e s by p r i n c i p a l component analysis based upon the absolute concentrations of toxin components per c e l l A06-A07 F i g . 5A Ordination of Protogonyaulax i s o l a t e s by p r i n c i p a l component analysis based upon the r a t i o s of equally weighted toxins A11-A12 x i i i LIST OF TABLES Table 1 Formulation of NWSP-7 natural 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 Modified STP s t e r i l i t y t e s t medium f o r the monitoring of possible 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 67 Table 4 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 i s o l a t e s of the Protogonyaulax tamarensis/catenella species complex 70-73 Table 5 Polyacrylamide gel formulation 83 Table 6 Stain protocols f or dehydrogenase isozymes 85 Table 7 Abbreviations and names of PSP toxins from Protogonyaulax spp.; sa x i t o x i n and i t s na 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 90 Table 8 ANOVA comparing variance i n c e l l volume between Protogonyaulax i s o l a t e s grouped by lo c a t i o n of o r i g i n , present morphotype i n culture and c l o n a l i t y the time of o r i g i n a l culture i n i t i a t i o n 101 Table 9 ANOVA comparing variance i n acclimated growth rates between Protogonyaulax i s o l a t e s grouped by lo c a t i o n of o r i g i n , present morphotype i n culture and c l o n a l i t y at the time of i s o l a t i o n '. 137-138 Table 10 Nonparametric Mann-Whitney U-test for the s i g n i f i c a n c e of the dif f e r e n c e i n mean acclimated growth rates f o r Protogonyaulax i s o l a t e s grouped by lo c a t i o n of o r i g i n , present morphotype i n culture and c l o n a l i t y at the time of i s o l a t i o n 140 Table 11 Maximum growth rates of Protogonyaulax spp. i n exponential phase from batch culture experiments 141-143 Table 12 Mean nuclear DNA content of Protogonyaulax i s o l a t e s by morphotype and lo c a t i o n of o r i g i n 162 Table 13 Pairwise comparison of the dif f e r e n c e i n mean nuclear DNA content 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 the Student's t - t e s t ; two-tailed at a=0.05; H Q: m= "2 1 6 2 Table 14 Nuclear DNA content of vegetative c e l l s of Protogonyaulax i s o l a t e s r e l a t i v e to that of zygotes 163 Table 15 S i m i l a r i t y c o e f f i c i e n t s (Sj) of Protogonyaulax i s o l a t e s , based upon dehydrogenase band patterns 183 xiv Table 16 Mean s i m i l a r i t y c o e f f i c i e n t s (Sj) of Protogonyaulax i s o l a t e s by geographical o r i g i n and morphotype f o r i n d i v i d u a l dehydrogenases 184 Table 17 Cophenetic c o r r e l a t i o n matrix contructed from UPGMA dendrogram ( F i g . 27) based on dehydrogenase banding patterns 195 Table 18 Binary character state matrix f o r dehydrogenase isozymes coded as presence(l)-absence(0) data 198-203 Table 19 Manhattan distance matrix computed from the character matrix (Table 18) showing distance 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) 207 Table 20 PSP toxin composition of Protogonyaulax i s o l a t e s expressed as the concentration of each toxin (fmol c e l l " ^ - ) , percent of t o t a l toxin composition represented 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 la t e exponential growth phase by HPLC of toxin extracts i n 0.03 N ac e t i c ac i d 232-236 Table 21 Amalgamation sequence f o r centroid-linkage c l u s t e r formation from unstandardized % toxin data 264 Table 22 Variable r a t i o s of toxin components of Protogonyaulax i s o l a t e s . The maximum r a t i o of the t o t a l toxin content among a l l i s o l a t e s f or each toxin i s a r b i t r a r i l y given as 1.0; r e l a t i v e values for other i s o l a t e s are expressed as a r a t i o of the maximum 265 Table 23 Univariate summary s t a t i s t i c s f o r % toxin data used i n the p r i n c i p a l components analysis 268 Table 24 Co r r e l a t i o n matrix constructed from % toxin data 269 Table 25 Eigenvalues giving variance explained by each factor i n the analysis of p r i n c i p a l components 270 Table 26 Orthogonal rotated factor 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 are the eigenvectors of the c o r r e l a t i o n matrix based on % toxin data m u l t i p l i e d by the square roots of the corresponding eigenvalues 271 Table 27 Estimated f a c t o r scores and Mahalanobis distances (chi-square s) from each case to the centroid of a l l cases f o r the o r i g i n a l data based on % toxin data composition (Table 20) 276 Table 28 C e l l density and y i e l d r a t i o s a f t e r 12 days following inoculation from reference cultures maintained 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 XV Table 29 Table 30 Table 31 Acclimated maximum growth rate (k) determined i n exponential growth phase during the second tran s f e r seri e s 336 Acclimated maximum growth rate (k) f o r NEPCC 180 grown on various 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 during exponential phase 337-338 Euclidean distance matrix computed from the character matrix (Table 18) showing distance 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 Table 32 Phenetic goodness of f i t s t a t i s t i c s f o r distance Wagner trees produced by the random addition sequence subprogram SWAG (TREES=5; TRIES=500) to the Manhattan distance matrix 396 Table 33 Phenetic goodness of f i t s t a t i s t i c s f o r distance Wagner trees produced by the random addition sequence subprogram SWAG (TREES=5; TRIES=500) to the Manhattan distance matrix a f t e r f i t t i n g trees to the character matrix using command DIAG to maximize parsimony 396 Table 34 Phenetic goodness of f i t s t a t i s t i c s f o r the two optimum distance Wagner trees i n Fig'. 51 comparing f i t to the Manhattan distance matrix a f t e r re-optimization to minimize percent standard deviation (PSDOPT) or the S - s t a t i s t i c (SOPT) of the PHYSIS command FIT 396 Table 35 Orthogonal rotated 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 are the eigenvectors of the c o r r e l a t i o n matrix based on absolute concentration of toxin components (Table 20) mu l t i p l i e d by the square roots of the corresponding eigenvalues 403 Table 36 Estimated f a c t o r scores and Mahalanobis distances (chi-square s) from each case to the centroid of a l l cases f o r the o r i g i n a l data based on absolute concentration of toxin components (Table 20) 404 Table 37 Orthogonal rotated factor loading for the 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 are the eigenvectors of the c o r r e l a t i o n matrix based on normalized v a r i a b l e r a t i o s of toxin components (Table 22) mu l t i p l i e d by the square roots of the corresponding eigenvalues 408 Table 38 Estimated f a c t o r scores and Mahalanobis distances (chi-square s) from each case to the centroid of a l l cases for the o r i g i n a l data from normalized v a r i a b l e r a t i o s of toxin components (Table 22) 409 ACKNOWLEDGEMENTS The ultimate r e s p o n s i b i l i t y f o r the research undertaken f o r t h i s d i s s e r t a t i o n rests with the author, but many others have contributed s i g n i f i c a n t l y to i t s successful completion. I wish to acknowlege the support and advice of my thesis committee, composed of Drs. F.J.R. Taylor and P.J. Harrison of the Un i v e r s i t y of B r i t i s h Columbia Depts. of Oceanography and Botany, and Dr. J.D. Berger of the Dept. of Zoology. In p a r t i c u l a r , my senior t h e s i s advisor, Dr. F.J.R. ("Max") Taylor has provided the i n t e l l e c t u a l framework within which these i n q u i r i e s were undertaken. His e c l e c t i c i n t e r e s t s i n the b i o l o g i c a l realm, which range from red, t i d e ecology and physiology to phytoplankton taxonomy, symbiosis, and the evolution of eukaryotes, with a p a r t i c u l a r focus (obsession?) on d i n o f l a g e l l a t e s , coupled with an unerring a b i l i t y to pose the b i o l o g i c a l l y relevant questions, have served to stimulate my own i n t e r e s t i n these to p i c s . Dr. Paul J. Harrison has been a prominent source of encouragement since I entered graduate school, and has been e s p e c i a l l y h e l p f u l i n giving advice r e l a t i n g to phytoplankton physiology. From a p r o t o z o o l o g i s t 1 s perspective, Dr. Jim Berger has helped me to appreciate the l i n k s and differences between d i n o f l a g e l l a t e s and other p r o t i s t s . In addition, he has generously permitted the use of his epifluorescent microphotometer f o r the determination of nuclear DNA. As a prospective s c i e n t i s t , I owe an enormous debt to Dr. Naval J. Antia - S c i e n t i s t , mentor, and f r i e n d - whose devotion to the pursuit of s c i e n t i f i c inquiry has never f a l t e r e d i n h i s long career and has ins p i r e d me to attempt to do likewise. x v i i Drs. Greg Boyer and John S u l l i v a n have lent t h e i r expertise i n the chemical analysis of toxins, enabling me to e s t a b l i s h the chemotaxomic linkages described i n t h i s t h e s i s . I am indebted to Drs. Dan Brooks, Richard 0'Grady and Paul Gabrielson f o r introducing me to the phylogenetic progam PHYSIS and for assistance i n i t s a p p l i c a t i o n . Dr. Gregory Gaines helped s i g n i f i c a n t l y with the i n t e r p r e t a t i o n of data by p r i n c i p a l components an a l y s i s . Judy Acreman, curator of the NEP Culture C o l l e c t i o n , has been a f a i t h f u l shepherd for my f a l t e r i n g cultures during my frequent absences from the laboratory. I am g r a t e f u l to Dr. J.-C. T h e r r i a u l t of the Centre Champlain des Sciences de l a Mer, Ministere des Peches et des Oceans (Quebec), Canada, for arranging t e c h n i c a l and f i n a n c i a l support i n the f i n a l stages of the thesis program. L i o n e l Corriveau was p a r t i c u l a r l y h e l p f u l i n a s s i s t i n g i n the d r a f t i n g of several f i g u r e s . Lucy Maurice has devoted s u f f i c i e n t time and e f f o r t i n the preparation of the f i n a l manuscript to be j u s t i f i a b l y considered as a co-author. She has t o l e r a t e d my frequent lapses of good humour with remarkable grace and tolerance. With gratitude, i t i s to her that I r e s p e c t f u l l y dedicate t h i s t h e s i s . F i n a l l y , to my long-suffering parents, who must have wondered many times i f I would ever " f i n i s h school", I hope that I can assure them that the time i s nigh. 1 CHAPTER I INTRODUCTION A. C l a s s i f i c a t i o n and the Species Problem i n D i n o f l a g e l l a t e s The obvious reproductive, morphological, and e c o l o g i c a l gaps that appear to separate most higher organisms led almost i n t u i t i v e l y to the Linnean view of species as f i x e d and e s s e n t i a l l y immutable, while imbued with "objective" s i g n i f i c a n c e . With the advent of evolutionary ideas, species came to be regarded as rather subjective aggregations of i n d i v i d u a l s possessing s i m i l a r derived characters, which change i n response to s e l e c t i v e pressure. Indeed, Darwin (1859) offered the following pragmatic, but nonetheless i n t e l l e c t u a l l y u n s a t i s f y i n g advice: "In determining whether a form should be ranked as a species or a v a r i e t y , the opinion of n a t u r a l i s t s having sound judgement and wide experience seems the only guide to follow." Later, the contributions of g e n e t i c i s t s and population b i o l o g i s t s (Haldane, 1929; Dobzhansky, 1950; Carson, 1957; Lewontin, 1974; Wright, 1978) introduced a species concept within which populations serve as the fundamental gene pool u n i t s . Species were defined as dynamic populations that interbreed, e i t h e r i n a c t u a l i t y or p o t e n t i a l l y , and are i s o l a t e d by reproductive b a r r i e r s . This perspective has been characterized as the " B i o l o g i c a l Species Concept" (BSC) (Mayr, 1940; 1957; 1963). Species concepts are i n e x t r i c a b l y linked to the assumptions underlying the c l a s s i f i c a t i o n schemes adopted to discriminate between species. In recent years, a phenetic species concept, e s s e n t i a l l y an extension of the 2 t y p o l o g i c a l species approach, u t i l i z i n g numerical analysis to define d i s c r e t e groupings of organisms on the basis of character s i m i l a r i t i e s , without implying evolutionary r e l a t i o n s h i p s , has been developed (Sneath and Sokal, 1962; 1973). The 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 "Phylogenetic Systematics", which advocated a c l a s s i f i c a t i o n system based on the possession of shared derived characters (synapomorphies), served to spawn a school of systematists known as c l a d i s t s or phylogenetic systematists with views a n t i t h e t i c a l to those of the p h e n e t i c i s t s . Advocates of c l a s s i c a l evolutionary c l a s s i f i c a t i o n (Mayr, 1963; 1970; Bock, 1974) have adopted methods of both phenetics and phylogenetics, but are severely 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 i n the development of a modern u n i v e r s a l l y applicable d e f i n i t i o n of "species", unifying phenotypic, genotypic, and phylogenetic aspects (Mayr, 1957; Doyen and Slobodchikoff, 1974; Sokal, 1974), remain i n t r a c t a b l e . In f a c t , s u r p r i s i n g l y l i t t l e consensus has emerged, as evidenced by a r t i c l e s appearing i n issues of Systematic Zoology during the past decade. Debates between the pheneticists (numerical taxonomists) (Sokal and Crovello, 1970; Sneath and Sokal, 1962; 1973), the phylogeneticists ( c l a d i s t s ) (Hennig, 1966; Funk and Brooks, 1981; Wiley, 1981) and c l a s s i c a l evolutionary taxonomists (Mayr, 1963; 1970; Bock, 1974) have been intense and often personal. Yet progress i n the d e f i n i t i o n of species concepts has often been l i m i t e d to the r e p r i n t i n g of " c l a s s i c " publications (as i n Mayr, 1976; Slobodchickoff, 1976 and Jameson, 1977). As an operational d e f i n i t i o n , species are often recognized as populations e x h i b i t i n g d i s t i n c t i v e morphological characters inhabiting a d i s c r e t e geographical region. The lack of a u n i f i e d species concept has led some taxonomists to c l a s s i f y populations which are geographically 3 d i s j u n c t ( a l l o p a t r i c ) or reproductively i s o l a t e d , but morphologically i n d i s t i n g u i s h a b l e , as separate species. A l t e r n a t i v e l y , d i f f e r e n t species have been recognized among sympatrically d i s t r i b u t e d populations that are morphologically d i s t i n g u i s h a b l e . I t often becomes impossible to separate such "species" from e c o l o g i c a l or geographical v a r i e t i e s of polymorphic species. Mayr (1942) coined the term " s i b l i n g species" f o r i n c i p i e n t species within which reproductive b a r r i e r s have arisen without concomitant morphological divergence. The s i b l i n g species concept was o r i g i n a l l y applied extensively to speciation among Drosophila populations, but has since been adopted f o r use within a wide v a r i e t y of other taxonomic groups, including p r o t i s t s (Sonneborn, 1975; Beam and Himes, 1977; 1980b; 1982; 1984). The problems of species d e f i n i t i o n , p a r t i c u l a r l y the de l i n e a t i o n of stable s i b l i n g species, and the s i g n i f i c a n c e to be a t t r i b u t e d to v a r i a b i l i t y , although acute f o r taxonomists of higher organisms, are even more profound f o r those dealing with lower eukaryotes, such as d i n o f l a g e l l a t e s . For d i n o f l a g e l l a t e s , taxonomists who would apply the term "species" as a unit of i d e n t i f i c a t i o n ( t y p o l o g i c a l species or morphospecies) are often confused by the simultaneous morphological p l a s t i c i t y of many species i n response to environmental changes, and the conservatism of general form within and among geographical populations. This conservation of morphotype may mask variants ( c r y p t i c species?) that are c l e a r l y genotypically or biochemically d i s t i n c t . Frequently a morphological continuum e x i s t s , rather than a d i s c r e t e gap between populations. I f morphological variants are merely considered as imperfect renditions of the master archetype which defines the species, then the s e l e c t i o n of characters and the l e v e l of deviation from the "normal" 4 required to segregate species i s an a r b i t r a r y decision of the i n d i v i d u a l taxonomist and i s subject to dispute. Decisions made regarding the s i g n i f i c a n c e of p a r t i c u l a r morphological characters are almost always made without knowledge of, or reference to, the genomic code required to produce them. Furthermore, the p l e i o t r o p i c e f f e c t of many genes complicates the in t e r p r e t a t i o n of genotypic and phylogenetic r e l a t i o n s h i p s based upon the simultaneous numerical analysis of several morphological characters. C l e a r l y , morphological variants among d i n o f l a g e l l a t e populations are more d i s t a n t l y r e l a t e d , i n a phylogenetic sense, i f , f o r example, the thecal plate features used to separate them are the product of a coordinated multi-gene complex expressed through several metabolic reactions, than i f the 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 sing l e point-mutation. This i s also true i f the observed polyphenism i s simply the expression of an alternate morphotype due to environmental influence. The case f o r the establishment of the BSC i s based on the b e l i e f that the species i s a fundamental evolutionary u n i t comprising a common gene pool, a r i s i n g from sexual i n t e r f e r t i l i t y , rather than the subjective aggregation of i n d i v i d u a l s possessing shared s i m i l a r i t i e s . These d e f i n i t i o n s are not mutually exclusive, but they are not ne c e s s a r i l y synonymous, p a r t i c u l a r l y f o r the lower eukaryotes. The BSC, as conventionally conceived (Mayr, 1957), i s inappropriate for e x c l u s i v e l y asexual and o b l i g a t e l y s e l f - f e r t i l i z i n g organisms, since the gene pool i s r e s t r i c t e d to the l e v e l of the i n d i v i d u a l . This r e s t r i c t i o n e f f e c t i v e l y eliminates i t s a p p l i c a t i o n to a huge proportion of the p r o t i s t s . The usefulness of the BSC depends upon the a b i l i t y of the investigator to adequately describe the common gene pool. In f a c t , Mayr (1969), one of the leading proponents of the BSC, deleted references to p o t e n t i a l l y 5 interbreeding populations used i n h i s e a r l i e r d e f i n i t i o n (Mayr, 1940), which re f e r r e d mainly to geographically i s o l a t e d populations. The word " p o t e n t i a l l y " has a degree of imprecision which has l e f t i t open to various i n t e r p r e t a t i o n s . I f the p o t e n t i a l f o r interbreeding i s accepted as a v a l i d c r i t e r i o n f o r membership i n the same species, but interbreeding i s l i m i t e d to optimal laboratory s e t t i n g s , the BSC i s poorly applicable to natural populations which may be, i n r e a l i t y , reproductively i s o l a t e d by geographical or 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 . On the other hand, i f breeding " p o t e n t i a l " i s not accepted as part of the BSC, co-occurring members of the same morphospecies from the same geographical population cannot be accepted as belonging to the same b i o l o g i c a l species sensu s t r i c t o unless interbreeding i s a c t u a l l y observed or can be d e f i n i t i v e l y i n f e r r e d . The recognition of d i s c r e t e " b i o l o g i c a l species" among sexually a c t i v e p r o t i s t s i s hampered by the exceptional occurrence of i n t e r s p e c i f i c mating. Although t h i s often r e s u l t s i n progeny that are i n f e r t i l e or of low v i a b i l i t y (Sonneborn, 1957), i t demonstrates the p o s s i b i l i t y of gene flow between "species", a l b e i t i n a r e s t r i c t e d sense. To minimize ambiguity i n the use of the word "species" to apply to both i n t e r f e r t i l e groups and t y p o l o g i c a l aggregations of s i m i l a r i n d i v i d u a l s , Sonneborn (1957) favoured the use of the term "syngen" f o r organisms known to share a common gene pool, whether or not such groups could be d i f f e r e n t i a t e d as separate species on the basis of conventional taxonomic c r i t e r i a . There i s an a d d i t i o n a l problem i n the recognition of species among d i n o f l a g e l l a t e s which 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 from the f a c t that d i f f e r e n t taxonomists have adopted these organisms into the botanical or z o o l o g i c a l realms, or have assigned them an intermediate 6 p o s i t i o n as p r o t i s t s (Taylor, 1976b; 1985). Many d i n o f l a g e l l a t e species (approximately h a l f ) are c l e a r l y p l a n t - l i k e - as pigmented autotrophic primary producers, they are prominent members of the phytoplankton community. Yet the remaining species are non-photosynthetic symbionts, parasites, or f r e e - l i v i n g f a c u l t a t i v e or obligate heterotrophs. D i n o f l a g e l l a t e s have possible evolutionary a f f i n i t i e s with the c i l i a t e s (Taylor, 1976b; 1978; 1980a), and t h e i r f l a g e l l a r m o t i l i t y i s an animal-like c h a r a c t e r i s t i c . As "plants", d i n o f l a g e l l a t e s are us u a l l y accorded phylum status (Dodge, 1984) (DIVISION: PYRRHOPHYTA or DINOPHYTA), whereas t h e i r s i g n i f i c a n c e i s diminished to the order l e v e l [ORDER: D i n o f l a g e l l i d a (Barnes, 1968) or D i n o f l a g e l l a t a (Hyman, 1940; G r e l l , 1973)] within the phylum PROTOZOA, when they are considered as "animals". At the i n f r a s p e c i f i c l e v e l , anomalies can a r i s e due to the a p p l i c a t i o n of e i t h e r the International Code of Botanical Nomenclature (ICBN; Voss et a l . , 1983) or the International Code of Zoological Nomenclature (ICZN; S t o l l et a l . , 1964) to the same group of d i n o f l a g e l l a t e s (Taylor, 1976a; 1985). For example, the ICZN does not regulate or formally recognize the use of the i n f r a s p e c i f i c designations "forma" or "v a r i e t a s " , which botanists commonly use to designate minor morphological variants within a species, e.g., Pyrodinium bahamense var. compressum - for a t r o p i c a l d i n o f l a g e l l a t e e x h i b i t i n g a compressed morphotype. At the i n f r a s p e c i f i c l e v e l , the zoologist can only use "subspecies", which implies p a r t i a l reproductive i s o l a t i o n , and probably r e f l e c t s a greater degree of genetic d i f f e r e n t i a t i o n . A further discrepancy between the botanical and z o o l o g i c a l view of species i s r e l a t e d to the s i g n i f i c a n c e attached to the p o t e n t i a l f o r h y b r i d i z a t i o n . Among zo o l o g i s t s , evidence of h y b r i d i z a t i o n would generally 7 be considered to c o n s t i t u t e a de facto case for incomplete speciation (Mayr, 1970). Yet botanists, who tend to be more aware of v a r i a t i o n s i n reproductive s t r a t e g i e s , p a r t i c u l a r l y involving h y b r i d i z a t i o n among inbreeding versus outbreeding species, often de-emphasize the p o t e n t i a l for i n t e r f e r t i l i t y as the major c r i t e r i o n of speciation, i f e c o l o g i c a l and evolutionary d i s c o n t i n u i t y r e l a t i o n s h i p s remain i n t a c t (Grant, 1971). Recently, more emphasis has been placed on non-interbreeding with other populations, than on interbreeding within and among populations, to e s t a b l i s h species boundaries. This i n t e r p r e t a t i o n of the BSC r e l i e s on exclusion, and i s not t o t a l l y s a t i s f a c t o r y . As long as the breeding a f f i n i t i e s i n d i n o f l a g e l l a t e s remain l i t t l e studied, the divergent botanical and z o o l o g i c a l attitudes toward the s i g n i f i c a n c e of h y b r i d i z a t i o n present l i t t l e threat to our understanding of speciation i n t h i s group of organisms. However, as these r e l a t i o n s h i p s become better characterized, such unresolved issues w i l l prove inc r e a s i n g l y problematic. B. V a r i a b i l i t y and Speciation 1. Phenotypic versus Genotypic Evidence The f o s s i l record of d i n o f l a g e l l a t e s i s long - dating from the Mesozoic, and possibly the Paleozoic or Precambrian periods (Taylor, 1978; 1980a and b). In some species, i t i s also distinguished by a rather remarkable conservatism of form, p a r t i c u l a r l y of cyst morphotypes, (Taylor, 1980b; E v i t t , 1985). A few extant species can be recognized as v i r t u a l l y i d e n t i c a l to t h e i r f o s s i l i z e d progenitors (e.g., S p i n i f e r i t e s 8 spp.) (Taylor, 1976a). Nevertheless, the occurrence of morphotypic and ph y s i o l o g i c a l variants within and among d i n o f l a g e l l a t e populations apparently belonging to the same species i s also well known (Braarud, 1945; 1951; Bursa, 1963; Taylor, 1976a; 1980b), both from natural assemblages and in culture. Morphological variants may be generally recognized on the basis of c e l l s i z e and shape. For thecate forms, v a r i a t i o n may also be expressed i n differences i n plate tabulation and structure, p a r t i c u l a r l y those involving plate fusions or subdivisions, or the presence of supernumerary plates. D i n o f l a g e l l a t e taxonomists working with f i e l d material are often forced to make species decisions based upon a few specimens of unknown "prehistory", and with l i t t l e knowledge of the range of v a r i a t i o n i n morphological characters within the population. The dimensional changes and d i s t o r t i o n of thecal plate patterns that can occur following nutrient depletion or sexual events represent a su b s t a n t i a l deviation from the "normal" vegetative c e l l . An unwary observer of such specimens i n natural populations may be misled by t h e i r unusual appearance. Braarud's (1945; 1951) observations on cultures of Protogonyaulax (=Gonyaulax) tamarensis and S c r i p p s i e l l a trochoidea (=Peridinium  trochoideum) suggested that stable differences i n morphological characters (presumably g e n e t i c a l l y f i x e d ) , including mean c e l l s i z e and the shape of c e r t a i n t h e c a l p l a t e s , could be recognized among clones of the same morphospecies. Although some v a r i a t i o n i n these morphological characters was expressed within a given clone, primary features, e s p e c i a l l y thecal plate patterns, remained e s s e n t i a l l y unchanged i n long term cultures. This was p a r t i c u l a r l y true within the clone which he designated as G. tamarensis var. excavata. 9 Among d i n o f l a g e l l a t e s , the genus Ceratium provides the most extreme examples of morphological v a r i a t i o n i n s i z e , o v e r a l l shape, and external features (Taylor, 1976a; 1980b; E v i t t , 1985). Not only are the lengths and number of the a p i c a l horns subject to environmentally induced morphological changes, varying with changes of season, water temperature and n u t r i t i o n a l status, but s u b s t a n t i a l l y d i f f e r e n t morphotypes may coexist even within a contemporaneous natural population. Intergradations, forming a morphological continuum, are also observed between variants previously considered to be v a l i d l y separable species (Taylor, 1976a). Extreme v a r i a t i o n can occur to such an extent that, based upon morphological c r i t e r i a , the terminal c e l l of a c l o n a l chain, i f considered i n i s o l a t i o n , would be transferable to another subgenus ( E v i t t , 1985). Such int r a - c h a i n v a r i a t i o n can usu a l l y be a t t r i b u t e d to differences between parent and daughter c e l l s a r i s i n g through m i t o t i c d i v i s i o n . In some cases, v a r i a t i o n within a species may be due to l i f e h i s t o r y stage differences between vegetative c e l l s and gametes or sexual cysts. A d d i t i o n a l v a r i a t i o n may be accounted f o r by sexual dimorphism and anisogamy expressed i n opposite mating types (Taylor, 1980b). The above example serves to i l l u s t r a t e that the basis f o r v a r i a t i o n i n d i n o f l a g e l l a t e s , including alternate stages i n the l i f e h i s t o r y , cannot usually be d i r e c t l y determined from observations of f i e l d specimens, but i t may be e m p i r i c a l l y demonstrable through environmental manipulation of c e l l s i n c ulture. Many d i n o f l a g e l l a t e s (perhaps a l l ) have a sexual stage or are at l e a s t p o t e n t i a l l y sexual, but for the vast majority of species (>98%), sexua l i t y i s cur r e n t l y unknown ( P f i e s t e r , 1984). In the few cases where sex u a l i t y i s characterized i n d i n o f l a g e l l a t e s , i t can usually only be induced under conditions of environmental s t r e s s , such as nitrogen (Turpin 10 et a l . , 1978; P f i e s t e r , 1984) or phosphorus (Anderson and Lindquist, 1985) deprivation. The a v a i l a b l e evidence from studies on cultured d i n o f l a g e l l a t e species i n which sexual fusion has been observed (<30 species) suggests that s e x u a l i t y i s rather rare ( P f i e s t e r , 1984). P h y s i o l o g i c a l v a r i a t i o n s , including changes i n growth rate, nutrient uptake k i n e t i c s , photosynthetic rate, excretion, the production of secondary metabolites, and the rate of protein and n u c l e i c a c i d synthesis, etc., may occur i n response to r e l a t i v e l y short term environmental changes, within a time scale of several hours to several c e l l d i v i s i o n s . When expressed morphologically, phenotypic variants would be designated by botanists as a l t e r n a t i v e "formae". If variants become g e n e t i c a l l y f i x e d , tney may be considered to define genotypically d i s t i n c t e c o l o g i c a l , p h y s i o l o g i c a l , or morphological v a r i e t i e s within the species. Since phenotypic modifications which do not a r i s e from genetic differences are not h e r i t a b l e , long term growth i n a constant and stable environment serves as a f i l t e r to allow f o r the elimination of v a r i a t i o n due s o l e l y to environmental perturbation, rather than genotypic d i f f e r e n c e s . Phenotypic consistency must be followed f o r several c e l l generations, and preferably through several culture t r a n s f e r cycles, to s t a b i l i z e genotypic expression (Brand, 1981). In t h i s way, i t i s possible to estimate the t y p i c a l range of v a r i a t i o n within a morphospecies, and to recognize t r u l y aberrant c e l l s (Bursa, 1963). The s t a b i l i t y of genotypically f i x e d variant characters i n d i n o f l a g e l l a t e s has been noted i n previous studies. Examples of such characters include the persistence of rhythmic bioluminescence i n Gonyaulax  polyedra i n culture, even a f t e r s h i f t s i n environmental phasing (Hastings, 1975), and the long-term s t a b i l i t y i n acclimated growth rates among Protogonyaulax tamarensis i s o l a t e s (Brand, 1981; Brand et a l . , 1981a). For Crypthecodinum c o h n i i - l i k e d i n o f l a g e l l a t e s , stable differences i n DNA content per c e l l (Himes and O'Brien, 1980; Beam and Himes, 1982; 1984), as well as i n DNA base p a i r composition, r e s t r i c t i o n endonuclease cleavage patterns, melting point and buoyant density have been observed (Steele and Rae, 1980a and b). Recently, gel electrophoretic studies have revealed enzymatic v a r i a t i o n within and among d i n o f l a g e l l a t e morphospecies (Schoenberg, 1976; Daggett and Nerad, 1980; Schoenberg and Trench, 1980a; Beam et a l . , 1982; Hayhome and P f i e s t e r , 1983; Watson and Loeblich, 1983; Beam and Himes, 1984; Cembella and Taylor, 1985a and b; Hayhome, 1985; Whitten and Hayhome, 1985). The use of biochemical techniques, including analysis of isozymes, n u c l e i c acid and protein sequences, and secondary metabolites, to circumscribe species and uncover c r y p t i c phenetic and genetic r e l a t i o n s h i p s has merit when applied to stable t r a i t s . This i s p a r t i c u l a r l y true when the r e s u l t s of a number of biochemical approaches are compared f o r the same group of organisms. In a sense, biochemically-based c l a s s i f i c a t i o n schemes are more "objective" since v a r i a t i o n can be r e a d i l y q u a n t i f i e d and q u a l i f i e d , and the data are amenable to analysis by numerical techniques. I f the intermediary pathways are known, meaningful character weighting may also be applied. Unfortunately, an a r b i t r a r y decision must s t i l l be made regarding the threshold l e v e l of di f f e r e n c e required to define a new species. I t i s not obvious that t h i s l e v e l should be f i x e d the same for a l l biochemical characters or even f o r a l l groups of organisms. The p o s s i b i l i t y of biochemical p a r a l l e l i s m and coincidence should also be considered i n e f f o r t s made to r e c o n c i l e contemporary morphological species 12 with biochemical v a r i a n t s . Biochemical approaches previously applied to investigate taxonomic a f f i n i t i e s within the tamarensis/catenella group have involved comparative studies of t o x i c i t y and toxin composition (Loeblich and Loeblich, 1975; Schmidt et a l . , 1978; Alam et a l . , 1979; Schmidt and Loeblich, 1979a and b; Shimizu, 1979; Oshima et a l . , 1982a; Boyer et a l . , 1985; Cembella et a l . , 1985; Oshima and Yasumoto, 1985) and bioluminescence (Loeblich and Loeblich, 1975; Schmidt et a l . , 1978; Schmidt and Loeblich, 1979a). Other phytoplankton, p a r t i c u l a r l y diatoms, have been more i n t e n s i v e l y studied f o r i n t r a s p e c i f i c genetic variants over a wider range of characters than d i n o f l a g e l l a t e s . Within the diatom species - or species complex -T h a l a s s i o s i r a pseudonana (=Cyclotella nana), genetic d i f f e r e n c e s , as opposed to short term p h y s i o l o g i c a l acclimation responses, have been observed i n reproduction rates ( G u i l l a r d and Ryther, 1962; Brand, 1980; Brand et a l . , 1981a and b), nutrient uptake rates (Carpenter and G u i l l a r d , 1971; Nelson et a l . , 1976), response to vitamin B-^ ( G u i l l a r d , 1968), p o l l u t i o n stress tolerance (Fisher et a l . , 1973; Fisher, 1977; Murphy and Belastock, 1980), c e l l d i v i s i o n p e r i o d i c i t y (Nelson and Brand, 1979) and e l e c t r o p h o r e t i c a l l y detectable enzymes (Murphy and G u i l l a r d , 1976; Murphy, 1978; Brand et a l . , 1981b. Genetic d i f f e r e n t i a t i o n i n the reproductive rates of coccolithophores has also been examined among i s o l a t e s from d i f f e r e n t water masses (Brand, 1980; 1981; 1982; Brand et a l . , 1981a). Ultimately, i t i s e s s e n t i a l to unite the concepts of phenetic (morphological, p h y s i o l o g i c a l and biochemical) species and genetic (DNA homology and interbreeding) species to circumscribe the same set of organisms. Given the f a c t that c l a s s i c a l taxonomy has been l a r g e l y based upon morphological s i m i l a r i t i e s and d i f f e r e n c e s , and since morphological 13 features are at l e a s t i n d i r e c t l y subject to genomic coding, as a gen e r a l i z a t i o n , a c o r r e l a t i o n between genetic s i m i l a r i t y , morphology, and assigned taxonomic rank should be apparent. Mayr (1963) c r i t i c i z e d t h i s view when applied to the analysis of s i b l i n g species, by suggesting that the high degree of morphological s i m i l a r i t y evident between s i b l i n g species was more the r e s u l t of developmental homeostasis than genetic s i m i l a r i t y . Nevertheless, two r e l a t e d general observations may be made regarding the c o r r e l a t i o n between genetic and phenetic r e l a t i o n s h i p s among the populations of most organisms. F i r s t , there i s often a close correspondence between genetic s i m i l a r l y and assigned taxonomic rank (Ayala et a l . , 1974; Avise, 1976; G o t t l i e b , 1977). Second, the genetic s i m i l a r i t i e s between organisms are u s u a l l y p o s i t i v e l y correlated with morphological s i m i l a r i t i e s ( G o t t l i e b , 1977). Despite numerous exceptions, p a r t i c u l a r l y among animal groups (Avise et a l . , 1975; K o r n f i e l d and Koehn, 1975), the close r e l a t i o n s h i p between morphology and genetic s i m i l a r i t y has been confirmed at the generic, s p e c i f i c , and sub s p e c i f i c l e v e l s i n both animals (Hubby and Throckmorton, 1968; Ayala et a l . , 1974; Lewontin, 1974; Avise, 1975) and plants ( G o t t l i e b , 1977; 1984). Since not a l l genetic d i f f e r e n t i a t i o n i s expressed i n r e a d i l y i d e n t i f i a b l e phenotypic c h a r a c t e r i s t i c s i n p r o t i s t s (Borden et a l . , 1973a and b; Murphy and G u i l l a r d , 1976; C o r l i s s and Daggett, 1983; Soudek and Robinson, 1983; Beam and Himes, 1984), morphological features may be inadequate to d i s t i n g u i s h between p a i r s of s i b l i n g species, geographical v a r i e t i e s , or ecotypes. Morphologically s i m i l a r breeding groups of d i n o f l a g e l l a t e s , as defined by sexual compatibility, may represent true s i b l i n g " b i o l o g i c a l " species e x h i b i t i n g reproductive i s o l a t i o n (Beam and 14 Himes, 1977; 1980a and b; 1982; 1984). C o r l i s s and Daggett (1983) discussed the problems inherent i n the separation of morphologically s i m i l a r s i b l i n g species of c i l i a t e s , and t h e i r general observations may pertain to d i n o f l a g e l l a t e s as w e l l . For c i l i a t e s , such as Paramecium and Tetrahymena, nucleotide sequences ( A l l e n and L i , 1974) and enzyme electrophoretic p r o f i l e s ( A l l e n and Weremiuk, 1971; A l l e n and Gibson, 1971; 1975; Adams and A l l e n , 1975; A l l e n et a l . , 1983a and b) have been used to e s t a b l i s h l e v e l s of genetic d i v e r s i t y among c l o s e l y r e l a t e d s t r a i n s . Members of the Paramecium a u r e l i a species complex, o r i g i n a l l y considered as " v a r i e t i e s " (Sonneborn, 1939), were l a t e r numerically designated as "syngens" sensu Sonneborn (1957), on the basis of breeding a f f i n i t i e s between mating types. Use of electrophoretic isozyme analysis i n place of l i v i n g reference s t r a i n s to delineate variants eventually led to the assignation of L a t i n binomials to the syngens of Paramecium, which are now regarded as s i b l i n g species (Sonneborn, 1975). At the gross morphological l e v e l , i t may indeed be impossible to segregate s i b l i n g species of p r o t i s t s i n congruence with the e l e c t r o p h o r e t i c evidence and breeding a f f i n i t i e s . Among members of the Tetrahymena pyriformis complex, d e t a i l e d analysis of s t r u c t u r a l features including body s i z e , the p o s i t i o n of the c o n t r a c t i l e vacuole pore, and the number of kinetosomes could not r e l i a b l y be used to discriminate between s i b l i n g species ( C o r l i s s and Daggett, 1983). However, d e t a i l e d m u l t i v a r i a t e morphometric analysis of members of the P. a u r e l i a complex eventually revealed stable morphological differences e s s e n t i a l l y i n agreement with the previously designated species (Gates et a l . , 1974; Powelson et a l . , 1975; Gates and Berger, 1976). The binary mating type Paramecium a u r e l i a species complex may not be a 15 s t r i c t l y analogous model system for d i n o f l a g e l l a t e s . Other c i l i a t e s , including members of the P. bursaria complex and the genus Stylonychia, e x h i b i t multiple mating types (Nanney, 1980). Mult i p l e mating types may be common within d i n o f l a g e l l a t e species, yet, with the exception of the Crypthecodinium c o h n i i species group, the number of mating types and sexual c o m p a t i b i l i t y groups i s currently unknown i n d i n o f l a g e l l a t e s . Intensive studies on c l o n a l i s o l a t e s of marine heterotrophic d i n o f l a g e l l a t e s t e n t a t i v e l y r e f e r a b l e to C. c o h n i i (Beam and Himes, 1977; 1980a and b; 1982; 1984; Himes and O'Brien, 1980; Beam et a l . , 1982) indicated that these s t r a i n s could be divided into three variant groups on the basis of mean DNA content, chromosome number and c e l l s i z e . In addition to the t y p i c a l larger s i z e s t r a i n s , s m a l l e r - c e l l e d variants of C. cohnii possessing much less DNA and fewer chromosomes were discovered. Variants which exhibited the larger c e l l morphotype, yet contained only h a l f the t y p i c a l DNA content were also observed. The t y p i c a l l a r g e r - c e l l e d v a r i a n t s , representing major g e n e t i c a l l y d i f f e r e n t i a t e d groups, were not morphologically di s t i n g u i s h a b l e , but have now been divided into 31 sexually compatible s t r a i n s ( s i b l i n g species?) on the basis of t h e i r breeding a f f i n i t i e s (Beam and Himes, 1984). The evidence from e l e c t r o p h o r e t i c a l l y determined isozyme v a r i a t i o n (Schoenberg, 1976; Schoenberg and Trench, 1980a; Daggett and Nerad, 1980; Beam et a l . , 1982; Hayhome and P f i e s t e r , 1983; Watson and Loeblich, 1983; Cembella and Taylor, 1985a and b; Hayhome, 1985), DNA analysis (Steele and Rae, 1980a and b), and breeding a f f i n i t y and segregational genetic studies (Beam and Himes, 1977; 1980a and b; 1982; 1984), reveals a conspicuously high genetic d i v e r s i t y within and among d i n o f l a g e l l a t e species. In the way that t h i s genetic d i v e r s i t y i s e f f e c t i v e l y masked by morphological 16 conservatism, d i n o f l a g e l l a t e s may resemble c i l i a t e s , such as Tetrahymena (A l l e n and Weremiuk, 1971; A l l e n and L i , 1974; Williams, 1984) and Paramecium ( A l l e n and Gibson, 1975), for which s i m i l a r l e v e l s of d i v e r s i t y also f a i l to be r e f l e c t e d i n the expressed morphotype. 2. Mechanisms f o r Maintaining V a r i a b i l i t y and S t a b i l i t y The s u r v i v a l of d i n o f l a g e l l a t e species i s contingent upon t h e i r a b i l i t y to r e t a i n s u f f i c i e n t genetic heterogeneity to permit the e f f e c t i v e e x p l o i t a t i o n of favourable e c o l o g i c a l niches. The s e l e c t i v e advantages offered by maintaining v a r i a t i o n i n the population also enable the c o l o n i z a t i o n of marginal habitats and ensure that the population includes genotypes which could survive adverse environmental s h i f t s , e i t h e r p e r i o d i c (e.g., seasonal) or catastrophic. Acclimated phenotypic responses expressed morphologically and p h y s i o l o g i c a l l y must operate within the tolerance l i m i t s set by the genome. A diverse genome, accommodating rapid and often f r e e l y r e v e r s i b l e changes i n phenotype, can "fine-tune" the organisms response to transient environmental s h i f t s . At the same time, populations must r e t a i n s u f f i c i e n t genetic s t a b i l i t y to t o l e r a t e the deleterious e f f e c t s of genetic load - the s u r v i v a l cost of maintaining accumulated mutations - which can lead to i n v i a b i l i t y . The influence of environmental heterogeneity on genotypic polymorphism has been a c t i v e l y debated (Levins, 1968; Lewontin, 1974; Ayala et a l . , 1975; Bryant, 1976; Hedrick et a l . , 1976; Valentine, 1976; Valentine and Ayala, 1976), but at present there i s no preponderant body of empirical evidence that would support a s t r i c t causal r e l a t i o n s h i p . Nevertheless, i t 17 i s reasonable to speculate that the maintenance of high l e v e l s of a l l e l i c v a r i a b i l i t y within conspecific haploid d i n o f l a g e l l a t e populations represents a s u r v i v a l strategy with adaptive s i g n i f i c a n c e . The bulk of the recent e l e c t r o p h o r e t i c evidence supports the view that s t r u c t u r a l gene modifications and molecular polymorphisms are adaptive (Powell, 1972; G i l l e s p i e and Langley, 1974; Hedrick et a l . , 1976; Johnson, 1976; Valentine, 1976; Nevo, 1978; 1983). Nevo (1983) and co-workers examined the s e l e c t i v e e f f e c t s of chemical and thermal p o l l u t a n t s on allozyme polymorphism i n marine organisms, and found s t a t i s t i c a l l y d i f f e r e n t s h i f t s i n a l l e l e frequencies, suggesting genetic adaptation. When such adaptations u l t i m a t e l y produce reproductive, e c o l o g i c a l , or geographical i s o l a t i o n , species divergence may occur (Levin, 1970). Attempts to c o r r e l a t e the degree of genotypic polymorphism, usually as indicated from e l e c t r o p h o r e t i c evidence, with temporal and s p a t i a l environmental heterogeneity have followed various environmental amplitude or niche-width v a r i a t i o n hypotheses (Nevo, 1978). The f i r s t l i n e of evidence, supported by studies p r i m a r i l y on Drosophila (Prakash et a l . , 1969) suggested that populations of the c e n t r a l habitat, where s e l e c t i v e pressures are reduced, maintain higher l e v e l s of polymorphism than marginal and i s o l a t e d populations capable of surviving only within narrow tolerance l i m i t s . The second paradigm, also based l a r g e l y on experiments on Drosophila populations, r e l a t e s the s u r v i v a l of populations i n unstable environments to increased genotypic - and hence phenotypic - f l e x i b i l i t y , as expressed through higher l e v e l s of genetic d i v e r s i t y , with consequently greater protein v a r i a t i o n (Powell, 1972; G i l l e s p i e and Langley, 1974). From t h e o r e t i c a l considerations, Levins (1968) reasoned that organisms from 18 unstable environments should e x h i b i t higher genetic v a r i a t i o n , to optimize niche u t i l i z a t i o n and to maximize environmental tolerance. In t h i s case, i t i s the r e p r o d u c i b i l i t y and p e r i o d i c i t y i n the environmental f l u c t u a t i o n that i s c r u c i a l 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 genetic polymorphism. Some support f o r t h i s model i n marine environments i s offered by Bromley's (1972) study on protein v a r i a t i o n i n P a c i f i c populations of Euphausiids; the l e v e l of e lectrophoretic d i v e r s i t y was p o s i t i v e l y c o r r e l a t e d with i n c r e a s i n g l y unstable water masses. On the other hand, analysis of a l l e l e frequencies of other marine and aquatic organisms from apparently homogeneous environments does not lend c r e d i b i l i t y to the idea that genetic polymorphism n e c e s s a r i l y increases with environmental i n s t a b i l i t y ( K o r n f i e l d and Koehn, 1975; Valentine, 1976; White, 1978a; Wright, 1978). Valentine and Ayala (1976) suggested that the higher heterozygosity i n t r o p i c a l species of k r i l l , as compared to temperate and a n t a r c t i c species, was associated with greater resource s t a b i l i t y . This trophic resource hypothesis (Valentine, 1976) proposes that i n stable environments a 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 maintained i n the population to cope with differences i n microhabitats. There are four possible mechanisms for the observed v a r i a t i o n i n d i n o f l a g e l l a t e s : mutation, gene flow through migration, recombination, and phenotypic acclimation. Evidence has now s h i f t e d away from the c l a s s i c a l model of genetic equilibrium which proposes that most a l l e l e s are highly selected on the basis of t h e i r environmental goodness of f i t , r e s u l t i n g i n l i t t l e i n t r a s p e c i f i c and intrapopulation genetic d i v e r s i t y . I t i s l i k e l y that a large number of a l l e l i c variants coded for by the genome are e i t h e r s e l e c t i v e l y neutral (Kimura and Ohta, 1971) or subject to a s h i f t i n g balance equilibrium. The balance hypothesis (Dobzhansky et a l . , 1977; 19 Wright, 1978; Nevo, 1983) postulates the existence of a m u l t i p l i c i t y of a l l e l e s subject to s h i f t s i n s e l e c t i v e advantage, for a large number of gene l o c i , with no s i n g l e highly adaptive a l l e l e ("wild type") gaining complete monomorphic dominance. Arguments for and against the hypothesis of s e l e c t i v e n e u t r a l i t y have been well debated (Johnson, 1973; Hedrick et a l . , 1976), including the claim that evolutionary f i t n e s s may not be s u b s t a n t i a l l y influenced by the minor substitutions detected by electrophoresis. In e i t h e r i n t e r p r e t a t i o n of the persistence of genetic polymorphism, the accumulation of s t r u c t u r a l gene mutations would not contribute an overbearing genetic load. With the exception of Noctiluca, d i n o f l a g e l l a t e s are f u n c t i o n a l haploids i n the vegetative stage ( P f i e s t e r , 1984). The p r i n c i p l e advantage of the haploid strategy l i e s 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 favourable mutations, which are immediately expressed and subject to environmental evaluation. Since unfavourable a l l e l i c variants cannot be e f f e c t i v e l y masked as s e l e c t i v e l y neutral recessives, as i s the case with d i p l o i d heterozygotes, d i n o f l a g e l l a t e s may express a m u l t i p l i c i t y of genotypes - a less conservative strategy, based p r i m a r i l y upon recurrent mutation to supply genetic v a r i e t y . In contrast to the genotype, the morphotype i n d i n o f l a g e l l a t e s seems to be subject to more stringent constraints. According to Mayr (1970) homeostatic mechanisms may permit the morphotype to remain true to form as long as genetic v a r i a t i o n does not exceed prohibitive! boundaries. Population v a r i a t i o n a r i s i n g from recombination can occur e x c l u s i v e l y within the deme ( l o c a l interbreeding population), or as a r e s u l t of exogenous gene flow from other non-reproductively i s o l a t e d populations (Mayr, 1970). Variants and aberrants i n natural d i n o f l a g e l l a t e populations 20 could also conceivably a r i s e due to physical mixing and aggregation of c e l l s d i f f e r i n g i n p h y s i o l o g i c a l status and genotype derived from d i f f e r e n t water masses. The contribution of genetic recombination to the maintenance of genetic d i v e r s i t y i n d i n o f l a g e l l a t e s cannot be assessed, since t y p i c a l i n s i t u rates of sexual fusion are not known. The homothallic species Crypthecodinium c o h n i i i s unusually sexually a c t i v e i n culture (Beam and Himes, 1977; 1984), but the frequency of s e x u a l i t y i n natural populations has not been determined. A s i g n i f i c a n t amount of i n t r a s p e c i f i c morphological v a r i a t i o n may be a t t r i b u t e d to non-genetic v a r i a b i l i t y i n the p a r t i t i o n i n g of nuclear and cytoplasmic material during mitosis. There may be s i m i l a r random fl u c t u a t i o n s i n the production and d i s t r i b u t i o n of f u n c t i o n a l metabolites (Spudich and Koshland, 1976). V a r i a t i o n among c e l l s i n a population could also be caused by asynchronous differences i n c e l l c ycle phasing, and p a r t i c u l a r l y , i n l i f e c ycle stages. A v a r i e t y of chromosomal mechanisms may be invoked to account f o r genetic v a r i a t i o n and speciation events, including intrachromosomal gene du p l i c a t i o n , aneuploidy, polyteny and polyploidy. V a r i a t i o n in chromosome number or structure within a species may serve as a s t a s i p a t r i c speciation mechanism (White, 1978a; Wiley, 1981), whereby such major chromosomal rearrangements could give r i s e to post-mating reproductive i s o l a t i o n . D i n o f l a g e l l a t e s have a notoriously i n e f f i c i e n t mitosis (Shyam and Sarma, 1978; Triemer and F r i t z , 1984), commonly r e s u l t i n g i n the p a r t i t i o n i n g of unequal numbers of chromosomes i n daughter n u c l e i ( S i l v a , 1971; 1977). Often there 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 c e l l (Dodge, 1963). The large number of chromosomes i n some d i n o f l a g e l l a t e s (ranging up to 21 approximately 220; Shyam and Sarma, 1978) were previously c i t e d (Taylor, 1976a) as evidence that such species could be polygenomic, with multiple copies of homologous chromosomes bearing variant a l l e l e s . On the basis of s t r u c t u r a l features v i s i b l e by electron microscopy, the large chromosomal volume, and the permanently condensed state of chromosomes, Haapala and Soyer (1973) have proposed a highly polytene model for the d i n o f l a g e l l a t e chromosome. The evidence for polyploidy as a speciation mechanism i n d i n o f l a g e l l a t e s , as suggested Loeblich et a l . (1981) f o r Heterocapsa spp., i s inconclusive, but the existence of chromosomal multiples i n d i n o f l a g e l l a t e populations belonging to the same morphological species, and among c l o s e l y r e l a t e d species (Shyam and Sarma, 1978; Loper et a l . , 1980; Loeblich et a l . , 1981; Holt and P f i e s t e r , 1982; Beam and Himes, 1984; Blank and Trench, 1985) allows t h i s i n t r i g u i n g p o s s i b i l i t y . On the other hand, the recent evidence from Symbiodinium (=Gymnodinium) microadriaticum and i t s a l l i e d possible s i b l i n g species (Blank and Trench, 1985) does not support an argument for polyploidy i n t h i s species complex, since the number of chromosomes varied an i n t e g r a l multiple of a base number of 25, rather than i n an exponential progression. Although d e t a i l e d genomic evidence i s a v a i l a b l e only from a s i n g l e (possibly a t y p i c a l ) d i n o f l a g e l l a t e species, Crypthecodinium c o h n i i , the information on the frequency, complexity and l o c a t i o n of repeated DNA sequences i n the d i n o f l a g e l l a t e chromosome does not suggest a high degree of polyteny or polyploidy (Roberts et a l . , 1974; A l l e n et a l . , 1975; Hinnebusch et a l . , 1980). The data from DNA renaturation k i n e t i c s indicated that approximately 60% of the genome consisted of low complexity repeated DNA, with mismatched repeated sequences interspersed among unique sequences, as i n eukaryotes. The remaining 40-45% of the DNA was 22 considered to represent unique DNA (number of copies per c e l l = 1.17) of highly complex sequences. The frequencies of induced mutations (Roberts et a l . , 1974; T u t t l e and Loeblich, 1974; 1977) were consistent with the hypothesis that many genes occur as only s i n g l e copies, and that the nucleus i s f u n c t i o n a l l y haploid. Given that genotypic v a r i a t i o n i s the basis of speciation i n d i n o f l a g e l l a t e populations, a fundamental question i s posed: i s speciation preceded by massive genomic reorganization involving a l l e l i c s u b s t i t u t i o n s at a large number, i f not the majority, of gene l o c i (Mayr, 1963)?; or can minor' a l l e l i c d ifferences between i n d i v i d u a l s serve as an i s o l a t i o n mechanism between and among morphologically s i m i l a r populations (Hubby and Throckmorton, 1968; Avise and Ayala, 1976)? In short, does speciation precede or follow genetic d i v e r s i f i c a t i o n ? Mayr (1963, 1970) has discounted putative cases of sympatric speciation - speci a t i o n a r i s i n g from s h i f t s i n ecology, physiology, reproductive mode, etc., within populations which are not geographically i s o l a t e d - as c r y p t i c cases of a l l o p a t r i c or parapatric speciation. Yet Mayr has generally not acknowledged the p o t e n t i a l importance of chromosomal rearrangements as i n i t i a t i n g events i n speciation (White, 1978a). 3. Phenetic and Phylogenetic Taxonomic Linkage An 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 reveals a large array of a l t e r n a t i v e methods f or representing and i n t e r p r e t i n g taxonomic and systematic r e l a t i o n s h i p s among taxa. These methods may generally be divided into the phenetic c l u s t e r i n g and ordination techniques of numerical taxonomy (Sneath 23 and Sokal, 1962; 1973) and the cladograms or phylogenetic trees of the phylogeneticists ( F a r r i s , 1970; 1972; Wiley, 1981). Other techniques, such as the algorithms of F i t c h and Margoliash (1967) and Prager and Wilson (1978) are hybrids, that begin by phenetic c l u s t e r i n g from distance data, then modify the r e s u l t i n g tree to r e f l e c t phylogenetic distance. The claims of the proponents of a l t e r n a t i v e methods are d i f f i c u l t to evaluate, due to errors i n computation and i n t e r p r e t a t i o n , the lack of c r i t i c a l comparative data a n a l y s i s , and the i n c o m p a t i b i l i t y of d i f f e r e n t s t a t i s t i c s used to judge "goodness of f i t " f o r the phenogram or cladogram to the o r i g i n a l data set. Nevertheless, d i f f e r e n t algorithms of phylogenetic analysis have been compared for "goodness of f i t " and parsimony of the resultant trees to the input data (Avise and Ayala, 1976; Mickevitch, 1978; Prager and Wilson, 1978; F a r r i s , 1981; Swofford, 1981; Avise, 1983). Phylogenetic a n a l y s i s , p a r t i c u l a r l y using the Wagner technique, has been shown to y i e l d trees superior i n s t a b i l i t y ( l e s s a f f e c t e d by the addition of new data and homoplasy - convergence, p a r a l l e l i s m and regression) and congruence (the a b i l i t y to generate s i m i l a r trees for the same taxa from a l t e r n a t i v e character data s e t s ) , than phenetic c l u s t e r i n g methods (Mickevitch and Johnson, 1976; Mickevitch, 1978; Wiley, 1981). Yet, i t seems that most of the commonly used linkage methods have some use, depending upon the inferences that the investigator wishes to draw from the data. Cluster analysis of character data i s c l e a r l y an appropriate method of i n d i c a t i n g phenetic s i m i l a r i t y between taxa (Sneath and Sokal, 1973; Avise, 1975) , a f a c t acknowledged even by those who oppose the use of such techniques as a representation of phylogeny (Mickevitch and Johnson, 1976) . This i s provided that the characters chosen are r e l a t i v e l y numerous 24 and due consideration i s given to the question of "weighting". Although i t i s not, s t r i c t l y speaking, a c l u s t e r i n g technique, p r i n c i p a l components analysis (PCA) may i n some respects be superior to c l u s t e r a n alysis f o r i l l u s t r a t i n g phenetic s i m i l a r i t y r e l a t i o n s h i p s , p a r t i c u l a r l y among taxa e x h i b i t i n g many very s i m i l a r or i d e n t i c a l characters. This i s because PCA allows f o r the co-occurrence of taxa ordinated i n multidimensional space; c l u s t e r analysis may impose an a r t i f i c i a l h i e r a r c h i c a l structure on the data, since c l u s t e r i n g algorithms force dichotomies even when character sets are i d e n t i c a l . The use of phenetic s i m i l a r i t y to reveal recent ancestral and divergence r e l a t i o n s h i p s from character and taxonomic distance data r e l i e s on the assumption that evolutionary rates are uniform among the diverging groups. According to proponents of t h i s method (Sneath and Sokal, 1973) s t r i c t constancy i s not n e c e s s a r i l y required f o r an accurate representation. On the other hand, those who would use phenetic c l u s t e r i n g to analyze phylogenetic r e l a t i o n s h i p s have been attacked repeatedly by phylogeneticists ( F a r r i s , 1972; 1981; Mickevitch, 1978; Baverstock et a l . , 1979; Mickevitch and M i t t e r , 1981; Swofford, 1981), who claim, among other objections, that phenetic analysis rests upon a p r i o r i assumptions of rather s t r i c t evolutionary rate constancy. The use of phenetic c l u s t e r i n g to imply phylogenetic r e l a t i o n s h i p s based upon gene frequency data using Nei's genetic i d e n t i t y I and distance D values (Nei, 1972; 1975), as r e f l e c t e d , f o r example, i n ele c t r o p h o r e t i c data, f o r branch f i t t i n g , i s p a r t i c u l a r l y subject to such v a l i d c r i t i c i s m ( F a r r i s , 1981). The branch lengths i n phylogenetic trees should represent the number of evolutionary gene s u b s t i t u t i o n s , a metric property obeying the Euclidean t r i a n g l e 25 i n e q u a l i t y , where the taxonomic distance D(A,C) > D(A,B) + D(B,C) f or taxa A, B and C. Nei's distance i s nonmetric and thus can y i e l d misleading phylogenetic reconstructions. The construction of Wagner trees ( F a r r i s , 1970) as a representation of phylogeny i s based on maximum parsimony c r i t e r i a . The parsimony method aims to f i n d the phylogeny for which the observed characters could have evolved with the minimum evolutionary change (Wiley, 1981; Felsenstein, 1983; Sober, 1983). Accordingly, the tree with the minimum number of character changes that adequately represents the data i s considered to be the maximum l i k e l i h o o d tree (De Haen and Neurath, 1976), although i t i s not always e x p l i c i t l y assumed that evolution proceeds by the most d i r e c t path. F a r r i s (1972; 1981) has pointed out that the parsimony c r i t e r i o n required of character data a n a l y s i s , y i e l d i n g the minimal length tree, cannot be simply extrapolated to the distance Wagner technique. Nevertheless, the distance Wagner approach to phylogenetic analysis ( F a r r i s , 1972; 1981; Swofford, 1981) i s capable of y i e l d i n g trees from Manhattan distance matrices without r i g i d assumptions regarding the constancy of evolution, and with a high l e v e l of f i t , congruence and s t a b i l i t y (Mickevitch, 1978). One of the greatest problems i n the phylogenetic analysis of c l o s e l y r e l a t e d extant taxa i s the determination of tree p o l a r i t y and the choice of an appropriate taxon to root the tree. Conventional phylogenetic analysis requires the assemblage of taxa to be monophyletic sensu Hennig (1966), and for the root to represent the plesiomorphic ( p r i m i t i v e or generalized) character s t a t e ( s ) . This n e c e s s a r i l y implies a p r i o r i knowledge of the plesiomorphic state, a s i t u a t i o n not always r e a l i z a b l e f or character data 26 (Baverstock et a l . , 1979). For binary coded electrophoretic data, the presence of a given isozyme i s u s u a l l y considered to be the apomorphic (derived) state and i t s absence the plesiomorphic state. Without p r i o r knowledge of the genetic and evolutionary implications of the isozyme bands gene dup 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 locus, p a r a l l e l i s m s , regression, convergence, etc., t h i s leads to the assumption that the appropriate root f o r the tree i s the taxon expressing the l e a s t number of izozyme bands. By reductio ad absurdum arguments, the ultimate plesiomorphic taxon, coded as (000...n) where n = number of characters described, would have no f u n c t i o n a l isozymes! A more appropriate method for e s t a b l i s h i n g p o l a r i t y and phylogenetic r e l a t i o n s h i p s i s to s e l e c t a taxon c l o s e l y r e l a t e d to those i n the group under study - an "out-group" - preferably chosen on the basis of a wide range of other c r i t e r i a , apart from the s p e c i f i c character set analyzed by the tree b u i l d i n g algorithm, to serve as the root of the tree. Presence of a given character state i n both the out-group and i n one or more OTUs (operational taxonomic units) i s taken as evidence that the common ancestor possessed the t r a i t . However, i t should be noted that t h i s i s not n e c e s s a r i l y true i f convergence has occurred. C. General Description and Taxonomic P o s i t i o n of the genus PROTOGONYAULAX Members of the Protogonyaulax tamarensis/catenella species complex are thecate gonyaulacoid d i n o f l a g e l l a t e s . A l l gonyaulacoids are photosynthetic and many forms are bioluminescent. The gonyaulacoids encompass a l l the d i n o f l a g e l l a t e s known to produce the saxitoxin analogues (gonyautoxins) 27 associated with p a r a l y t i c s h e l l f i s h poisoning (PSP). According to a hypothetical evolutionary scheme based upon l i v i n g forms (Taylor, 1980a; 1985), prorocentroids and dinophysoids are located near the base of the tree, while gonyaulacoids form a stem group leading to peridinoids by adaptive r a d i a t i o n , and u l t i m a t e l y to athecate gymnodinoids through plate l o s s . However, others consider gymnodinoids the most p r i m i t i v e d i n o f l a g e l l a t e group, and would e s s e n t i a l l y invert t h i s tree (Loeblich, 1984). The taxonomy of thecate 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 upon the number, shape, p o s i t i o n and o r i e n t a t i o n of the thecal p l a t e s . As a group, the gonyaulacoids may be distinguished from other thecate forms, 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, by a v a r i e t y of morphological features. Most of these differences involve symmetrical r e l a t i o n s h i p s , such as the tendency f o r greater t o r s i o n a l asymmetry i n gonyaulacoids and the t y p i c a l occurrence of i n t e r c a l a r y plates on the r i g h t v e n t r a l epitheca, rather than on the dorsal surface, as i n p e r i d i n o i d s . Taylor (1979; 1980a) has developed a plate homology model from which major divergences between the generalized p e r i d i n o i d and gonyaulacoid forms can be recognized (see E v i t t , 1985 for examples of i t s a p p l i c a t i o n ) . As well as the morphological d i s t i n c t i o n between gonyaulacoids and pe r i d i n o i d s , they also d i f f e r i n reproduction and cyst types. In Taylor's view (1980a), the removal of the gonyaulacoids from the Peridinales and the creation of a new order Gonyaulacales to embrace t h i s group was warranted by these major d i s t i n c t i o n s . In gonyaulacoids, the theca i s divided into an anterior group of plates which form the epitheca, and a posterior s e r i e s comprising the hypotheca. 28 In the conventional Kofoidean system of plate notation (Kofoid, 1911), gonyaulacoids possess s i x precingulars ( 6 1 1 ) , s i x postcingulars ( 6 1 ' ' ) , one posterior i n t e r c a l a r y ( l p ) and a s i n g l e antapical ( l 1 1 1 1 ) p late. The a p i c a l pore i n gonyaulacoids i s u s u a l l y rather hook-shaped; a v e n t r a l e p i t h e c a l pore may be present near the r i g h t margin of the f i r s t a p i c a l ( l 1 ) p late. The cingulum, a transverse equatorial groove separating the epitheca and hypotheca, i s displaced on the v e n t r a l surface i n Protogonyaulax, although 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 less than that found i n t y p i c a l Gonyaulax. The sulcus, a l o n g i t u d i n a l groove extending from the posterior end to the equator, i n t e r s e c t s the cingulum on the v e n t r a l surface at the point of f l a g e l l a r i n s e r t i o n . Gonyaulacoids possesses the t y p i c a l dinokont f l a g e l l a t i o n ; a r i b b o n - l i k e transverse flagellum l i e s along the cingular groove and a t r a i l i n g whiplash-type extends l o n g i t u d i n a l l y . As i n a l l d i n o f l a g e l l a t e s , the plane of f i s s i o n i n gonyaulacoids i s oblique. In gonyaulacoids, the wall of the parent c e l l i s shared with the daughter c e l l during mitosis (desmoschisis), which explains the a b i l i t y of c e r t a i n species, e.g. Protogonyaulax (=Gonyaulax) ca t e n e l l a and Gessnerium  monilatum (=Gonyaulax monilata), to form chains of c e l l s . In the genus Protogonyaulax, u n i c e l l s are frequently observed, but doublets, quadruplets and even long chains of >30 c e l l s may occur. These chains a r i s e due to the f a i l u r e of daughter c e l l s to separate a f t e r cytokinesis. The vegetative c e l l s of Protogonyaulax are ovoid i n form, and t y p i c a l l y range i n transdiameter from ~20-45 um. Under the external membrane l i e s an amphiesmal layer containing t h i n c e l l u l o s i c thecal plates i n fl a t t e n e d v e s i c l e s . Beneath the amphiesma l i e s a r e s i s t a n t p e l l i c l e composed of sporopollenin, which forms part of the outer cyst wall 29 following ecdysis. The thecal plates are r e a d i l y shed i n the formation of ecdysal ( p e l l i c u l a r ) cysts under unfavourable environmental conditions. P e l l i c u l a r cysts can be t r a n s i t o r y , since the thecal plates are often regenerated i n a matter of hours and m o t i l i t y i s r a p i d l y restored i f the c e l l s are again exposed to favourable conditions. Hypnocysts, formed from motile planozygotes following sexual fusion of gametes, are s i m i l a r i n shape and s i z e (~15 urn i n length) to p e l l i c u l a r cysts. Yet, they can usu a l l y be distinguished by t h e i r thicker w a l l , often covered by mucilageanous material, and darker pigmentation. Hypnocysts t y p i c a l l y require an obligate dormancy period before motile thecate c e l l s a r i s e from ecdysing cysts (Dale, 1979). There are two dominant morphotypes expressed within the Protogonyaulax  tamarensis species complex, designated herein as tamarensoid ( F i g . 1) and c a t e n e l l o i d ( F i g . 2). They are based s o l e l y on the morphology and thecal plate features of the species described as P. tamarensis (Lebour) Taylor and P. c a t e n e l l a (Whedon and Kofoid) Taylor, both o r i g i n a l l y named as members of the genus Gonyaulax Diesing. In the o r i g i n a l d e s c r i p t i o n of tamarensis (Lebour, 1925), the Kofoidean plate formula was given as: 4', Oa, 6", 6 1 " , l p , 1 " " . The f i r s t t abulation f o r c a t e n e l l a and acatenella (Whedon and Kofoid, 1936): 4', Oa, 6", 6c, 6'", l p , 1 " " , d i f f e r e d only i n the i n c l u s i o n of s i x small cingular p l a t e s , not described by Lebour f or tamarensis. Subsequently, Steidinger (1971) emended the diagnosis by adding several s u l c a l plates (7-8s). U l t r a s t r u c t u r a l observations using electron microscopy l a t e r revealed the presence of two small plates associated with the f l a g e l l a r pore region of the sulcus, and an a d d i t i o n a l a p i c a l p l a t e l e t (Postek and Cox, 1976). The t o t a l plate complement was therefore expanded to y i e l d the formula: lap (= Po), lcp 10 Ji T A M A R E N S O I D F i g . 1 General morphology of the tamarensoid morphotype of Protogonyaulax. C e l l s may or may not have a v e n t r a l pore associated with the margin of the f i r s t a p i c a l p l a t e . C A T E N E L L O I D F i g . 2 General morphology of the c a t e n e l l o i d morphotype of Protogonyaulax, showing an 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 configuration. C a t e n e l l o i d c e l l s lack a v e n t r a l pore associated with the margin of the f i r s t a p i c a l p l a t e . 31 (= Pc), 4', 6", 6c, 7s, 6"', l p , 1 " " . Detailed l i g h t microscopy and scanning electron microscopic observations on the s u l c a l area of tamarensoid material from O s l o f j o r d , Norway (Balech and Tangen, 1985), and from 15 other countries (Balech, 1985), has increased the number of i d e n t i f i a b l e s u l c a l p l a t e s , including accessory plates, to 9-10s. Fukuyo and co-workers (Fukuyo et a l . , 1985) now recognize 11 s u l c a l plates from Japanese Protogonyaulax spp. Some authors (Balech, 1985; Balech and Tangen, 1985; Fukuyo, 1985; Fukuyo et a l . , 1985) have departed from Kofoid's conventional designations by now assigning only f i v e plates to the postcingular s e r i e s . The previously assigned f i r s t postcingular ( l 1 1 1 ) i s reinterpreted as the l e f t a n t e r i or s u l c a l plate ( F i g . 3). The p o s t e r i o r i n t e r c a l a r y ( l p ) plate of Whedon and Kofoid (1936) i s now considered as the f i r s t a n t apical ( I " 1 ' ) - for a t o t a l of 2'"'. The basic thecal plate configurations f o r "tamarensis" and " c a t e n e l l a " are generally acknowledged to be i d e n t i c a l (Taylor, 1979; Steidinger, 1983; Fukuyo, 1985; Fukuyo et a l . , 1985). This was indicated by the plate homologies, i n comparison with other gonyaulacoids, shown by Taylor (1979). Lebour noted the presence of two antapical spines i n tamarensis, but these "spines" are i l l u s o r y , apparently being the s u l c a l l i s t s which extend ari t a p i c a l l y seen i n p r o f i l e (Braarud, 1945; Loeblich and Loeblich, 1975; Taylor, 1975; Balech and Tangen, 1985; Fukuyo, 1985). In some type specimens of c a t e n e l l a examined by Whedon and Kofoid (1936), a d e l i c a t e " c u r t a i n f i n " covering the g i r d l e depression was apparent. This was most l i k e l y a remnant of the external amphiesmal membrane stretched between the cingular ridges. The " c u r t a i n f i n " i s not a consistent diagnostic feature for c a t e n e l l a and may vary even within a chain of c e l l s (Taylor, 1975). Balech noted the presence of a v e n t r a l pore associated with the r i g h t 32 F i g . 3 Thecal plate configuration of Protogonyaulax i n the Kofoid notation system, as applied by Fukuyo (1985). A. Ventral view; B. Dorsal view; C. Epitheca; D. Hypotheca. Po, a p i c a l pore plate; l ' - V , a p i c a l plates; l l l - 6 , , , precingular plates; 1' ' ' -5 » ' *, postcingular plates; l 1 1 , 1 - 2 ' 1 , 1 , antapical plates; lc-6c, cingular plates; s.p., posterior s u l c a l plate; v.p., ventral-pore. 33 34 margin of the f i r s t a p i c a l ( l 1 ) plate ( F i g . 3) of r e l a t e d tamarensoid morphotypes, described as G. excavata (Braarud) Balech (1971), G. f r a t e r c u l a Balech (1967), G. peruviana Balech and De Mendiola (1977) and G. coh o r t i c u l a Balech (1967). This v e n t r a l pore has been used as a major taxonomic character i n subsequent treatments of the "tamarensis" species group (Loeblich and Loeblich, 1975; Taylor, 1975; Schmidt and Loeblich, 1979a; Fukuyo, 1979; 1980; 1985; Balech, 1985; Balech and Tangen, 1985; Fukuyo et a l . , 1985). The e a r l y descriptions of tamarensis (Lebour, 1925; Gran and Braarud, 1935; Braarud, 1945), acatenella and c a t e n e l l a (Whedon and Kofoid, 1936) make no reference to a v e n t r a l pore. I t i s unclear whether t h i s represents the absence of the feature, or merely that i t s obscurity caused i t to be overlooked or disregarded as taxonomically i n s i g n i f i c a n t . Fukuyo (1985) reasoned convincingly that although Lebour (1925) might e a s i l y have missed the presence of a pore i n tamarensis, i t would be u n l i k e l y that • Kofoid would have f a i l e d to note t h i s feature i n c a t e n e l l a and acatenella ( i f present), as he had described such pores i n other Gonyaulax species. A round or e l l i p s o i d a l a n t e r ior attachment pore i s frequently observed i n both the tamarensoid and c a t e n e l l o i d morphotypes, p a r t i c u l a r l y when c e l l s d i v i s i o n proceeds r a p i d l y (Fukuyo, 1985). A corresponding posterior attachment pore on the posterior s u l c a l p late of c e l l s i n chains, noted i n the o r i g i n a l d e s c r i p t i o n of c a t e n e l l a (Whedon and Kofoid, 1936), appears to maintain cytoplasmic co n t i n u i t y between newly divided c e l l s . Although Lebour (1925) gave only mean c e l l length (36 um), without reference to transdiameter, her iconotypes showed c e l l s which were e s s e n t i a l l y i s o d i a m e t r i c a l and non-chain forming. In contrast, c a t e n e l l a was described as prominently 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 length: transdiameter = 0.71-0.88: 1), with c e l l s mostly i n chains. Gonyaulacoid species c l o s e l y a l l i e d with the tamarensis/catenella complex, some of which may be synonymous or c o n s p e c i f i c , include Gonyaulax  acatenella Whedon and Kofoid (1936), G. co h o r t i c u l a Balech (1967), G. concava (Gaarder) Balech (1967) (may be a motile stage of Pyrocystis, Taylor, 1975; 1979), G. f r a t e r c u l a Balech (1964), G. o r i e n t a l i s (Paulsen) Lebour (1925), G. o s t e n f e l d i i (Paulsen) Paulsen (1949), Pyrodinium phoneus Woloszynska and Conrad (1939), G. dimorpha Biecheler (1952), G. kutnerae Balech (1979), G. peruviana Balech and De Mendiola (1977), G. try g v e i Parke (1976), G. washingtonensis Hsu (1967), G. conjuncta Wood (1954), Protogonyaulax a f f i n i s Fukuyo et a l . (1985) and P. compressa Fukuyo et a l . (1985). Other morphospecies recently described and assigned to the genus Alexandrium (Halim) Balech (1985) - A. t r o p i c a l e , A. insuetum, A. fukuyoi (= P. a f f i n i s Fukuyo et a l . , 1985), A. l e e i , A. ibericum, A. lusitanicum and A. fundyense - could also be added to t h i s l i s t . There i s considerable d i v e r s i t y of opinion on how to circumscribe the tamarensis/catenella group (Steidinger, 1971, 1983; Taylor, 1975, 1979, 1984, 1985; Loeblich and Loeblich, 1979; Balech, 1985; Balech and Tangen, 1985), but l i t t l e disagreement that the trans f e r from the genus Gonyaulax Diesing sensu s t r i c t o i s c l e a r l y appropriate. Members of t h i s species complex conform to the basic Gonyaulax plate formula as emended by Kofoid (1911) (3-6', 0-3a, 6", 6c, 7-8s, 6'", l p , 1 " " ) , and l a t e r as modified by Taylor (1976) (pp, 3-4", 0-4a, 6", 6c, 5-10s, 5-6'", l p , 1 " " ) . Yet, they d i f f e r from, Gonyaulax species, as t y p i f i e d by G. s p i n i f e r a , i n the shape and p o s i t i o n of both epithecal and hypothecal pla t e s , the structure of the a p i c a l pore complex, the degree of cingular displacement and the shape of the nucleus. The vegetative c e l l s of the tamarensis/catenella 36 group also d i f f e r markedly from those of G. s p i n i f e r a i n lacking an e p i t h e c a l i n t e r c a l a r y p late and conspicuous thecal ornamentation. Although t h e i r respective cyst types were unknown when cat e n e l l a and tamarensis were f i r s t described, both species are now known to produce smooth walled ovoid cysts, with excysting c e l l s e x i t i n g through an i r r e g u l a r s p l i t (Braarud, 1945; Wall, 1975; Turpin et a l . , 1978; Dale, 1979; Yoshimatsu, 1981; Fukuyo, 1985). In sharp contrast, G. s p i n i f e r a produces a v a r i e t y of cyst types with spiny projections, while excystment occurs v i a an angular dorsal precingular archeopyle. Unfortunately, the t r a n s f e r of the tamarensis/catenella complex from Gonyaulax has been accomplished i n three d i f f e r e n t and presently incompatible ways. The proposed solutions are equally v a l i d i n the s t r i c t e s t taxonomic sense, i n that they a l l adhere to the basic rules of nomenclature (Taylor, 1985). As long as the organisms f i t the generic d e s c r i p t i o n , there i s no absolute l e v e l of d i f f e r e n t i a t i o n required f o r the appropriate s h i f t of a species complex to an enlarged previously described genus, or to a newly created genus. Ultimately, adoption of a p a r t i c u l a r generic assignation i s determined by pragmatic considerations, including i t s usefulness, appropriateness and common usage i n the s c i e n t i f i c "marketplace". The arguments f o r the choice of one generic name over another to embrace t h i s species complex are based upon v a l i d differences of opinion regarding h i s t o r i c a l precedence, the accuracy and legitimacy of previous d e s c r i p t i o n s , and the s i g n i f i c a n c e of morphological v a r i a t i o n i n c e r t a i n thecal p l a t e s . Three generic names, Alexandrium Halim (Balech, 1985; Balech and Tangen, 1985), Gessnerium Halim (Loeblich and Loeblich, 1979), and Protogonyaulax Taylor (Taylor, 1979; 1984; 1985) have been proposed to 37 include the tamarensis/catenella complex. Loeblich and Loeblich (1979) placed t h i s group within an enlarged d e s c r i p t i o n of the genus Gessnerium Halim, r e j e c t i n g Alexandrium Halim, the name with h i s t o r i c a l p r i o r i t y , on the grounds that the type species, A. minutum, was inadequately described (Halim, 1960) and that a L a t i n diagnosis was not provided. The a l l e g e d l y inadequate d e s c r i p t i o n i s a questionable c r i t i c i s m (Taylor, 1979; 1984; 1985; Balech, 1985; Balech and Tangen, 1985), as i s the lack of L a t i n diagnosis, which i s not required i f the type species were considered under the z o o l o g i c a l rather than the botanical code of nomenclature (Taylor, 1979; 1984). More troublesome f o r the proponents of Alexandrium (Balech, 1985; Balech and Tangen, 1985) i s the lack of o r i g i n a l holotype material or new specimens from the type l o c a l i t y f o r re-examination of questionable thecal p l a t e s , and the absence of information on cyst morphology. From t h i s standpoint, Gessnerium i s more r e a d i l y supportable, since the type species, G. monilatum (Howell) Loeblich and Loeblich (1979) i s abundantly a v a i l a b l e , and the cyst type (smooth-walled) i s now known (Walker and Steidinger, 1979). The f a c t that the type species d e s c r i p t i o n (Halim, 1967) was based upon o p t i c a l l y inverted specimens of a previously described species, Gonyaulax monilata Howell, i s taxonomically uncomfortable, but t h i s does not automatically i n v a l i d a t e using Gessnerium, as emended by Loeblich and Loeblich (1979), to circumscribe the tamarensis/catenella group. The argument for the t r a n s f e r of the tamarensis/catenella complex to a newly created genus, as proposed by Taylor (1979) (see also E v i t t , 1985), hinges l a r g e l y upon the e f f i c a c y and r e l i a b i l i t y with which Protogonyaulax can be s p l i t from c l o s e l y r e l a t e d pre-existing genera, including Pyrodinium, Alexandrium/Gessnerium, and Heteraulacus. The b r i e f generic 38 diagnosis f o r Protogonyaulax Taylor (1979), which employs the Taylor system of plate notation, rather than the more conventional Kofoid system, i s reproduced as follows: Gonyaulacoid c e l l s with a g i r d l e displacement of one g i r d l e width or l e s s , three anterior polar plates ( a p i c a l s ) , Is contacting P, V/VI contacting Z. Cysts are smooth walled without an angular archeopyle. The Z plate i s the p o s t e r i o r s u l c a l homologue, while V/VI i s the suture between postcingulars 5 1'' and 6'1' of the Kofoidean system. This emended de s c r i p t i o n acknowledges the c o r r e c t i o n of a typographical error (Taylor, 1984) i n the o r i g i n a l diagnosis, where the Z plate was i n c o r r e c t l y r e f e r r e d to as Y. In the absence of known cyst d i f f e r e n c e s , the c r i t i c a l feature i s o l a t i n g Protogonyaulax from Alexandrium/Gessnerium i s whether or not the f i r s t a p i c a l homologue ( l 1 plate - Kofoid system = Is - Taylor system = l u E v i t t 1 s (1985) modification of the Taylor system) touches the a p i c a l pore complex (APC). The consistency of the contact (or lack of contact) of these pla t e s , as discussed i n d e t a i l by several authors (Loeblich and Loeblich, 1979; Taylor, 1979; 1984; 1985; Steidinger, 1983; Balech, 1979; 1985; Balech and Tangen, 1985) i s a debatable, but c r u c i a l point i n appropriately d e l i n e a t i n g the genera. Balech (1979; 1985) objected to t h i s generic c r i t e r i o n as too v a r i a b l e , but Taylor (1984) countered that i t can be e f f e c t i v e l y applied to a l l c e l l s which are not obviously abnormal or megacytic. There are also pragmatic considerations f o r the use of the generic name 39 Protogonyaulax, as opposed to Gessnerium or Alexandrium. Addition of the p r e f i x "Proto" to the former genus name e s s e n t i a l l y conserves nomenclatural continuity, and may be less confusing as a reference for the non-taxonomist, who may be understandably bewildered at the apparent a r b i t r a r i n e s s of such name changes. In contrast, the generic names Gessnerium and Alexandrium are rather obscure. Exclusive of the tamarensis/catenella group, the most common member of Gessnerium (Halim) Loeblich and Loeblich, G. monilatum - an important icthyotoxic species -i s s t i l l frequently i n c o r r e c t l y r e f e r r e d to as "Gonyaulax"; Alexandrium i s v i r t u a l l y unheard of by non-taxonomists. The use of the generic names Gessnerium and Alexandrium for the tamarensis/catenella complex has been l i m i t e d to i t s taxonomist proponents. On the other hand, Protogonyaulax has become widely accepted, p a r t i c u l a r l y i n Japan, on the P a c i f i c coast of North America, and more recently i n A t l a n t i c Canada, where i t has been adopted by p h y s i o l o g i s t s , toxin chemists, palynologists and e c o l o g i s t s . For these reasons, the present studies w i l l r e f e r the i s o l a t e s of the tamarensis/catenella species complex to the genus Protogonyaulax. D. H i s t o r i c a l and Recent Perspectives on Taxonomic Variants Within the Protogonyaulax tamarensis/catenella Species Complex Protogonyaulax (=Gonyaulax) tamarensis, the f i r s t member of the "tamarensis/catenella" complex to be described ( F i g . 4), was i d e n t i f i e d by Lebour (1925) from the Tamar estuary near Plymouth, England. At the time t h i s area was not noted for PSP outbreaks, although subsequent reports have confirmed that t o x i c i t y does occur i n the v i c i n i t y . A f t e r considering net 40 F i g . 4 H i s t o r i c a l taxonomic scheme f o r some members of the Protogonyaulax tamarensis/catenella species complex. 41 F i g . 4 HISTORICAL TAXONOMIC SCHEME FOR SOME MEMBERS OF THE PROTOGONYAULAX TAMARENSIS / CATENELLA SPECIES COMPLEX Gonyaulax tamarensis Lebour 1925; Tamar estuary, Plymouth, England Bioluminescence (?) Cells compressed (-) Ventral pore (?) Chain forming (-) Toxicity (?)* Gonyaulax catenella Whedon and Kofoid, 1936; San Francisco Bay 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 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 tamarensis var. excavata Braarud, 1945; Norway, Gulf of Maine, Bay of Fundy. Bioluminescence (?) Cells com-pressed (-) 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 (Braarud) Balech, 1971 Bioluminescence (?) Cells compressed (-) Ventral pore (?) Chain forming (-) Toxicity (?) 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 1 may or may not contact APC Smooth cyst REDEFINITION Protogonyaulax tamarensis (Taylor) Protogonyaulax catenella (Taylor) Fukuyo. 1980, 1985; Japan. Biolumi-nescence (?) Chain forming (-) Ventral pore (•) Toxicity ( +) Cells compressed (-) APC rectangular or narrow triangular Posterior pore (+) or (-) at plate margin Fukuyo. 1980, 1985; Japan. Biolumi-nescence (?) Chain forming ( +) Ventral pore (-) Toxicity (*) Cells compressed (-) APC dorsally wide, triangular Poste-rior pore (•) or (-) away from plate margin not specifically tested, but not associated with PSP toxicity in the region. not specifically tested, but associated with PSP toxicity in the region. *** not noted in original description, but presence is suggested by re-examination of micrographs. 42 haul specimens c o l l e c t e d i n the Bay of Fundy and the Gulf of Maine (Gran and Braarud,' 1935) and examining cultured i s o l a t e s from Oslofjord, Norway, Braarud (1945) subdivided' t h i s species into three v a r i e t i e s based upon general morphology: var. t y p i c a (=var. tamarensis), synonymous with G. tamarensis Lebour from the type l o c a l i t y , var. globosa and var. excavata. G. tamarensis var. globosa, a Norwegian form characterized by i t s rotund morphology, was not associated with t o x i c i t y , but the organism was not s p e c i f i c a l l y tested. Two "tamarensis" morphotypes sensu Braarud (1945) were prevalent on the east coast of North America, namely var. t y p i c a and var. excavata; Braarud discriminated the l a t t e r form by i t s deeply sculpted s u l c a l region. Toxic blooms causing PSP i n A t l a n t i c coastal regions, p a r t i c u l a r l y i n Scandinavia, eastern Canada and New England were l a t e r associated with tamarensoid d i n o f l a g e l l a t e s often conforming to Braarud 1s de s c r i p t i o n of G. tamarensis var. excavata (Needier, 1949; Prakash; 1963; 1967). The impetus for the taxonomic work on two new d i n o f l a g e l l a t e species described by Whedon and Kofoid (1936) from San Francisco Bay, was provided by the strong circumstantial l i n k between PSP i n C a l i f o r n i a and the appearance of a chain-forming gonyaulacoid d i n o f l a g e l l a t e (Sommer and Meyer, 1937; Sommer et a l . , 1937). The to x i c chain-forming species producing antero-posteriorly compressed c e l l s was i d e n t i f i e d as Gonyaulax  ca t e n e l l a . A second species, G. acatenella, with the same plate tabulation as " c a t e n e l l a " , was d i f f e r e n t i a t e d as a weakly pigmented species s u p e r f i c i a l l y resembling G. tamarensis Lebour i n s i z e , lack of chain formation, and the production of almost i s o d i a m e t r i c a l c e l l s . The descriptions of the epithecal plate patterns for " c a t e n e l l a " and "acatenella" were not i n accord with that given by Lebour (1925) for 43 G. tamarensis. A f t e r examining the type material from C a l i f o r n i a , Lebour also acknowledged the d i s t i n c t n e s s of " c a t e n e l l a " and "acatenella" from "tamarensis", and further strengthened the case for the creation of new species by her o r i g i n a l a s s e rtion that "tamarensis" had a range r e s t r i c t e d to the estuarine waters of the Plymouth region. The more recent discovery of "tamarensis" (or "excavata") along the P a c i f i c coast of B r i t i s h Columbia (Taylor, 1975; 1984; Turpin et a l . , 1978; Cembella and Taylor, 1985a and b), Alaska ( H a l l , 1982; Taylor, 1984), and Japan (Fukuyo, 1979; 1980; 1985; Oshima and Yasumoto, 1979; Toriumi and Takano, 1979; Ogata et a l . , 1982; Oshima et a l . , 1982a, b and c; Fukuyo et a l . , 1985), has since d i s p e l l e d the b e l i e f that "tamarensis" was geographically confined to the A t l a n t i c . Although " c a t e n e l l a " was considered as the major source of s h e l l f i s h t o x i c i t y i n C a l i f o r n i a , and l a t e r i n other P a c i f i c locations (Schantz and Magnusson, 1964), "acatenella" was neither observed elsewhere, nor associated with t o x i c i t y , u n t i l extracts from a bloom i d e n t i f i e d as "acatenella" i n Malaspina I n l e t , B r i t i s h Columbia i n 1965, were determined to be t o x i c (Prakash and Taylor, 1966). Prakash and Taylor (1966) noted the presence of short chains i n t h e i r samples, and commented on the p o s s i b i l i t y of con s p e c i f i c a f f i n i t y between "acatenella" and " c a t e n e l l a " . Using the system of z o o l o g i c a l nomenclature, Balech (1971), raised Braarud's (1945) three v a r i e t i e s of Gonyaulax tamarensis Lebour, var. globosa, var. t y p i c a and var. excavata, to the l e v e l of species. His d e s c r i p t i o n of G. excavata (Braarud) Balech was based upon examination of a small form from the eastern equatorial A t l a n t i c . Balech (1977) completed a r e d e s c r i p t i o n of G. tamarensis Lebour, including a d e t a i l e d i n v e s t i g a t i o n of the plates of the s u l c a l region, using cultured material from the 44 Plymouth type l o c a l i t y . Unfortunately, he was not able to base hi s d e s c r i p t i o n on Lebour's holotype specimens, as these are no longer a v a i l a b l e . Much confusion surrounding the d e s c r i p t i o n of Protogonyaulax species has arisen due to the f a c t that Lebour's iconotype of the o r i g i n a l "tamarensis" was erroneously drawn with the epi t h e c a l plates o p t i c a l l y reversed. This point was recognized independently and corrected simultaneously by Loeblich and Loeblich (1975) and Taylor (1975). Loeblich and Loeblich (1975) attempted to resolve the d i f f i c u l t i e s surrounding the separation of tamarensoid morphotypes by adding new c r i t e r i a to the diagnosis of "tamarensis", "excavata", and "acatenella". They a r b i t r a r i l y selected one of two i s o l a t e s (Plymouth 173) from the type l o c a l i t y of G. tamarensis Lebour as t y p i c a l of the o r i g i n a l l y described species. Plymouth 173 was non-toxic, non-bioluminescent, and possessed a v e n t r a l pore; Plymouth 173a was bioluminescent but did not possess a v e n t r a l pore. The f a c t that Lebour's (1925) diagnosis makes no reference to a v e n t r a l pore was dismissed as an oversight (Loeblich and Loeblich, 1975). The. persistence of a stable d i f f e r e n c e i n a major biochemical feature ( t o x i c i t y ) , as well as a morphological c h a r a c t e r i s t i c was interpreted as an i n d i c a t i o n that these i s o l a t e s may not be c o n s p e c i f i c . Loeblich and Loeblich (1975) rejected Balech's (1971) diagnosis of G. excavata (Braarud) Balech, based on small t r o p i c a l specimens, i n favour of a d e s c r i p t i o n of New England i s o l a t e s , which were t o x i c , bioluminescent, and without a v e n t r a l pore. The degree of excavation i n the s u l c a l region, which was c r i t i c a l to Braarud's d e s c r i p t i o n , was discarded as d i a g n o s t i c a l l y u n r e l i a b l e . Nevertheless, these i s o l a t e s from the A t l a n t i c coast of North America were considered to represent the "true" excavata sensu Braarud 45 (1945). This was congruent with Dale's (1977) observations on t o x i c bioluminescent "excavata" from the Norwegian type l o c a l i t y which lacked a v e n t r a l pore. The morphological d i s t i n c t i o n between G. acatenella Whedon and Kofoid and G. tamarensis Lebour, once Lebour's inadvertent o p t i c a l r o t a t i o n of the epit h e c a l plates was recognized, r e s t s s o l e l y upon the presence of a more highly domed cone-shaped epitheca i n acatenella. This has considerably narrowed the morphological gap between these species. To r e - e s t a b l i s h the d i s t i n c t i o n between acatenella and tamarensis, the d e s c r i p t i o n of acatenella was emended (Loeblich and Loeblich, 1975) to include the presence of prominent hypothecal flanges along the plate margins, not i d e n t i f i e d i n the o r i g i n a l d e s c r i p t i o n (Whedon and Kofoid, 1936). Unfortunately, these redescriptions did not serve to a l l e v i a t e the problems associated with species d i s t i n c t i o n s within the tamarensoid group. The c r i t e r i a of t o x i c i t y , bioluminescence, and the lack of a v e n t r a l pore used to separate "tamarensis" from "excavata" have proven to be unworkable. In a d e t a i l e d examination of tamarensoid i s o l a t e s (Schmidt and Loeblich, 1979a and b), almost a l l possible combinations of the v e n t r a l pore, bioluminescence, and t o x i c i t y were observed. Turpin et a l . (1978) reported on two i s o l a t e s from B r i t i s h Columbia; one (NEPCC 71) resembled G. tamarensis Lebour, but had a c l e a r l y defined v e n t r a l pore and was determined to be weakly t o x i c by the mouse bioassay, while the other (NEPCC 254), resembled G. excavata Balech but did not possess the c h a r a c t e r i s t i c pore reported i n Balech's (1971) d e s c r i p t i o n . In Massachusetts, t o x i c s t r a i n s with and without the v e n t r a l pore were i s o l a t e d from nearby bays (Anderson and Wall, 1978; Alam et a l . , 1979). In Japan, s t r a i n s from Ofunato Bay (Fukuyo, 1979; Toriumi and Takano, 1979) conformed generally to 46 the d e s c r i p t i o n of var. excavata Braarud, but d i f f e r e d from G. excavata  sensu Loeblich and Loeblich (1975) i n that they possessed v e n t r a l pores. A recent re-examination of Braarud's (1945) drawings and micrographs of var. excavata and var. globosa, as well as d e t a i l e d observations of specimens conforming to h i s descriptions from the Norwegian type l o c a l i t y has revealed v e n t r a l pores i n both forms (Balech and Tangen, 1985). According to Balech and Tangen (1985), some t o x i c , bioluminescent specimens from Oslofjorden are i n accord with the d e s c r i p t i o n of G. tamarensis var. excavata Braarud (1945), which they have renamed Alexandrium excavatum; other more rotund specimens of unknown t o x i c i t y , conforming to Braarud's var. globosa, were transferred to another new species, A. o s t e n f e l d i i . The f a c t that, h i s t o r i c a l l y , A. o s t e n f e l d i i Balech and Tangen has had at l e a s t ten proposed 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 involved i n assigning i t a stable taxonomic p o s i t i o n . S h e l l f i s h t o x i c i t y i n the Bay of Fundy and the St. Lawrence estuary of eastern Canada was reportedly caused by a non-bioluminescent form of "tamarensis" (Prakash, 1963). Both bioluminescent and non-bioluminescent t o x i c s t r a i n s ( r e f e r r e d to as "excavata") were i s o l a t e d from the 1972 New England bloom and found to be morphologically i n d i s t i n g u i s h a b l e (Schmidt et a l . , 1978). S i m i l a r l y , Yentsch et a l . (1978) found both t o x i c and non-toxic forms of "tamarensis" from the coast of Maine. The r e l a t i o n s h i p between t o x i c i t y and bioluminescence i s obscure at best, and probably nonexistent. Fukuyo (1980; 1985; Fukuyo et a l . , 1985), working with Japanese specimens, has attempted to discriminate between "tamarensis" and " c a t e n e l l a " by using features not present i n the o r i g i n a l d e scriptions. These include the shape of the APC ( d o r s a l l y wide and t r i a n g u l a r i n 47 " c a t e n e l l a " ; narrow t r i a n g u l a r or rectangular i n "tamarensis"), the v e n t r a l pore (present only i n " c a t e n e l l a " ) , the shape of the p o s t e r i o r s u l c a l p late, and the p o s i t i o n of the posterior attachment pore (located c l o s e r to the plate margin i n "tamarensis"). Fukuyo did not regard the c l a s s i c antero-posterior compression c h a r a c t e r i s t i c of the c a t e n e l l o i d morphotype sensu Whedon and Kofoid (1936) as p a r t i c u l a r l y s i g n i f i c a n t , as h i s re-descriptions of both "tamarensis" and " c a t e n e l l a " are of approximately i s o d i a m e t r i c a l c e l l s . Unfortunately, chain length and the p o s t e r i o r attachment pore are not always d i a g n o s t i c a l l y u s e f u l , since these are not constant features i n mature c e l l s . Thus, to date, the frequent emendments to the diagnosis of members of the "tamarensis/catenella" complex have not yielded an unequivocal separation between morphotypic varian t s . E. Rationale for Experimental Studies 1. Geographical D i s t r i b u t i o n and Environmental S i g n i f i c a n c e of Protogonyaulax spp. Blooms P a r a l y t i c s h e l l f i s h poisoning (PSP), a t t r i b u t a b l e i n most cases to d i n o f l a g e l l a t e s of the genus Protogonyaulax, has been the cause of at l e a s t 300 deaths throughout the world. Based on h i s t o r i c a l records, the highest number of f a t a l i t i e s associated with a s i n g l e outbreak of PSP occurred i n 1799, i n the channel known as P e r i l S t r a i t s near Sit k a , Alaska (Fortune, 1975). I t i s reported that more than 100 Aleut hunters succumbed to PSP i n t o x i c a t i o n and died a f t e r eating t o x i c mussels. Blooms of Protogonyaulax spp. responsible for PSP are widely 48 d i s t r i b u t e d i n coastal waters, p a r t i c u l a r l y i n the temperate zones ( F i g . 5). There are occasional records of Protogonyaulax from the A r c t i c and the t r o p i c s , including Venezuela (Ferraz-Reyes et a l . , 1985), and recently from Thailand (Tamiyavanich et a l . , 1985), but there i s a strong tendency f o r blooms to be more prevalent between 30-60° N and S l a t i t u d e s (Taylor, 1984). In the north A t l a n t i c , Protogonyaulax blooms are implicated d i r e c t l y or are the prime suspects as causative agents for PSP i n the B r i t i s h I s l e s (Gemmill and Manderson, 1960; Wood, 1968), Belgium (Woloszynska and Conrad, 1939), Portugal ( S i l v a , 1963), Spain (Blanco et a l . , 1985; Fraga and Sanchez, 1985), Norway (Braarud, 1945; Dale, 1977; Balech and Tangen, 1985), A t l a n t i c Canada (Needier, 1949; Prakash, 1963; Prakash et a l . , 1971; White, 1982; White and White, 1985) and along the New England coast of North America (Anderson and Wall, 1978; Yentsch et a l . , 1978; Alam et a l . , 1979; Anderson and Morel, 1979; Anderson et a l . , 1982). On north P a c i f i c coasts, t o x i c Protogonyaulax spp. have been recorded from C a l i f o r n i a (Whedon and Kofoid, 1936; Sommer et a l . , 1937), Washington State (Norris and Chew, 1975), B r i t i s h Columbia (Prakash and Taylor, 1966; Taylor, 1975), and from Alaska ( H a l l et a l . , 1980; H a l l , 1982), extending through the Aleutian chain to the coast of Japan (Oshima, 1976; Oshima et a l . , 1978; 1982a, b and c; Oshima and Yasumoto, 1979; Nishihama, 1980; Ogata et a l . , 1982). In the southern hemisphere, Protogonyaulax blooms causing PSP i n temperate waters are known from southern Ch i l e (Lembeye et a l . , 1975), Uruguay (Davison and Yentsch, 1985) and Argentina (Benavides et a l . , 1983; Carreto et a l . , 1985), as well as the coast of South A f r i c a (Grindley and Nel, 1968; 1970; Grindley and Sapeika, 1969; Taylor, 1984). The present study i s based l a r g e l y upon tox i c i s o l a t e s from the coast of southwestern B r i t i s h Columbia and Washington State ( F i g . 6). In the 49 F i g . 5 Global d i s t r i b u t i o n of some members of the genus Protogonyaulax. A- P. acatenella; C- P. cat e n e l l a ; F- P. f r a t e r c u l a ; H- P. cohorticula; K- "Gonyaulax" kutnerae; P- P. peruviana; T- P. tamarensis; N- Protogonyaulax sp.: New Zealand tamarensoid i s o l a t e . Revised a f t e r Taylor, 1984. 51 F i g . 6 Map of the northeast P a c i f i c coast showing o r i g i n of Protogonyaulax i s o l a t e s . Morphotype at time of i s o l a t i o n : C = c a t e n e l l o i d ; I = intermediate; T = tamarensoid. 130 125 53 southwestern B r i t i s h Columbia region, sporadic red t i d e outbreaks a t t r i b u t a b l e to Protogonyaulax have resulted i n i l l n e s s , and, less frequently, i n f a t a l i t i e s . Three deaths were reported a f t e r the consumption of poisonous clams from Barkley Sound, Vancouver Island, i n 1942, and an incident at Malaspina I n l e t i n Geogia S t r a i t i n 1965 res u l t e d i n four 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 cockles were eaten (Prakash and Taylor, 1966). A poorly documented report of red water i n Georgia S t r a i t i n 1957 was associated with PSP i n t o x i c a t i o n i n more than 60 people, although no deaths occurred during t h i s outbreak. The most recent death i n B r i t i s h Columbia due to PSP was that of a native Indian at Health Harbour, G i l f o r d Island i n 1980. The frequency, widespread d i s t r i b u t i o n , and s e v e r i t y of PSP red ti d e s caused by Protogonyaulax underscores the need f o r continuing intensive research, as well as the maintenance of a v i g i l a n t and e f f e c t i v e monitoring program for t o x i c i t y . I t i s p a r t i c u l a r l y worrisome that i n some areas, outbreaks of PSP are increasing i n i n t e n s i t y and extending i n geographical range (Prakash et a l . , 1971; Anderson and Morel, 1979; Anderson et a l . , 1982; White, 1982; Anderson, 1984), p a r t i c u l a r l y i n southern New England. In general, PSP t o x i c i t y has not shown a consistent increase on the coast of B r i t i s h Columbia i n the recent years since routine s h e l l f i s h monitoring was i n i t i a t e d i n 1942; instead, a p e r i o d i c pattern of t o x i c i t y , f l u c t u a t i n g on an approximate seven year cycle, has been noted (Gaines and Taylor, 1985). Local conditions appeared to have a marked e f f e c t on t o x i c i t y i n s p e c i f i c locations on the B r i t i s h Columbian coast, making the long-term t o x i c i t y trends d i f f i c u l t to p r e d i c t . In Washington State, t o x i c blooms have shown recent tendencies to encroach into the lower extent of Puget Sound (Anderson, 1984), but t h i s has been somewhat o f f s e t by decreasing 5 4 t o x i c i t y i n some of the t r a d i t i o n a l l y more t o x i c northern areas (Erickson and N i s h i t a n i , 1985). Obviously, the ingestion of the potent neurotoxins produced by Protogonyaulax through the consumption of contaminated s h e l l f i s h , f o r which medical science has, as yet, f a i l e d to produce an e f f e c t i v e antidote, poses a s u b s t a n t i a l p u b l i c health hazard. In addition, the presence, or even suspected presence, of these d i n o f l a g e l l a t e toxins has had a markedly i n h i b i t o r y e f f e c t on both the commercial and r e c r e a t i o n a l harvest of s h e l l f i s h . The state of knowledge regarding bloom i n i t i a t i o n and dynamics has not yet provided r e l i a b l e p r e d i c t i v e models to account f o r t o x i c i t y d i s t r i b u t i o n . In many countries, the extent of the problem has required the implementation of an expensive and often i n e f f i c i e n t monitoring system, which i s only marginally adequate to deal with the immediate p u b l i c health concerns, much less to search for causal r e l a t i o n s h i p s and to pursue p o t e n t i a l s o l u t i o n s . 2. The Problem of Morphotypic Gradients and S t a b i l i t y i n Protogonyaulax In c e r t a i n areas of the northeast P a c i f i c - i n Japan, on the southwest coast of B r i t i s h Columbia, and i n Washington State and Alaska - the d i f f i c u l t i e s i n r e l i a b l y d i s t i n g u i s h i n g " c a t e n e l l a " from "tamarensis" are p a r t i c u l a r l y acute. Both the c a t e n e l l o i d and the tamarensoid morphotype have been found i n these waters. In southwestern B r i t i s h Columbia and Washington State there i s a tendency f o r s p a t i a l separation of the morphotypes, with the tamarensoid form generally predominating i n more 55 estuarine waters and the c a t e n e l l o i d form dominating on open coasts (Taylor, 1984). Nevertheless, there i s an apparent overlap i n the range of these alternate morphotypes, with intermediate forms (short chains of i s o d i a m e t r i c a l c e l l s ) often predominating i n boundary areas (Taylor, 1984; Cembella and Taylor, 1985a and b). In Japan, blooms of the tamarensoid and c a t e n e l l o i d morphotype often tend to be temporally as well as s p a t i a l l y separated, but they can occur i n the same l o c a t i o n (Kodama et a l . , 1982; Ogata et a l . , 1982; Oshima et a l . , 1982c; Fukuyo, 1985). Morphological intermediates have also been noted i n Japan, although i t must be recognized that the species descriptors currently used by prominent Japanese taxonomists (Fukuyo, 1979; 1980; 1985; Fukuyo et a l . , 1985) to discriminate "tamarensis" from " c a t e n e l l a " have res u l t e d i n considerable r e d e f i n i t i o n of these species. In addition to the morphological intergradation which can occur i n f i e l d populations of Protogonyaulax, there i s the problem of apparent morphological conversion from one morphotype to the other, e i t h e r p a r t i a l or complete, that can occur when i s o l a t e s are brought into c u l t u r e . An i s o l a t e from Hokkaido, Japan, which appeared as a " c a t e n e l l a " i n the f i e l d , except for the lack of chain formation, became rounder and approached the tamarensoid form i n culture (Nishihama, 1980). Similar transformations have also been observed (F.J.R. Taylor and A. Cembella, pers. obs.) for i s o l a t e s taken from B r i t i s h Columbian and Washington State populations. The v a r i a t i o n within the genus Protogonyaulax, expressed p a r t i c u l a r l y with respect to c e l l proportions, chain length and features of the thecal pla t e s , including pores, tends to blur the d i s t i n c t i o n between morphotypes and c a l l s into question conventionally used species descriptors. At a prosaic l e v e l , the r e l i a b i l i t y of these features involves the narrow 5 6 taxonomic question: "To what species does t h i s belong?" - which i s nonetheless important f o r the i d e n t i f i c a t i o n of blooms associated with PSP t o x i c i t y . There are also wider implications concerning more fundamental b i o l o g i c a l questions, such as: "What are species?", "How are good species defined?" and "What i s the basis f o r v a r i a t i o n among and within species?" -as they may be applied to d i n o f l a g e l l a t e s . 3. Research Objectives The research presented i n t h i s t hesis i s an attempt to examine some aspects of the biology of the organisms causing PSP red t i d e s , focusing on the r e l a t i o n s h i p between morphological and biochemical characters used to discriminate among members of the Protogonyaulax species complex. The general objectives are to address questions regarding v a r i a b i l i t y and polymorphism among Protogonyaulax i s o l a t e s , by applying chemotaxonomic approaches. In p a r t i c u l a r , biochemical techniques, including g e l electrophoresis of soluble isozymes, quantitative DNA analysis by epifluorescence microphotometry and toxin analysis by high-pressure (performance) l i q u i d chromatography are used to study chemotaxonomic a f f i n i t i e s and population v a r i a t i o n within t h i s group. The r e s u l t s of the biochemical analyses are compared with microscopic observations using morphological c r i t e r i a . P h y s i o l o g i c a l growth studies under a constant regime are used to estimate genotypic v a r i a t i o n within and among populations. Answers to the following s p e c i f i c questions are sought: 1) are i s o l a t e s from one l o c a t i o n t y p i c a l of the geographical population, morphotype or assigned species, e i t h e r morphologically or biochemically?; 57 2) what i s the degree of d i f f e r e n t i a t i o n (phenotypic and genotypic) within and among populations of the same morphospecies?; 3) i s the degree of morphological v a r i a t i o n r e f l e c t e d i n the amount of biochemical v a r i a t i o n ? ; 4) do d i f f e r e n t biochemical characters ind i c a t e the same l e v e l of 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 for t h i s group based upon phenetic or phylogenetic c r i t e r i a from the biochemical analyses be i n accord with one constructed from morphological characters, or from d i f f e r e n t biochemical characters?; 6) can P. catenella/P. tamarensis be discriminated as "good" species?; and 7) are there any c l e a r l y definable r e l a t i o n s h i p s between the natural environment, morphotype, the degree of genetic v a r i a t i o n and the biochemical characters investigated? Answers to these questions w i l l contribute to the understanding of the mechanisms underlying the processes of d i f f e r e n t i a t i o n and speciation i n d i n o f l a g e l l a t e s . 58 CHAPTER II MATERIALS AND METHODS A. Culture and Maintenance 1. I s o l a t e Origins and Growth Conditions Protogonyaulax cultures maintained f o r several years on ESNW medium (Harrison et a l . , 1980) (-Tris, - S i , +0.5uM Mo) were obtained from the North East P a c i f i c Culture C o l l e c t i o n (NEPCC), U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. 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 from other c o l l e c t i o n s , including an i s o l a t e from the Bay of Fundy y A t l a n t i c Canada (Clone //7 = NEPCC 545) provided through the courtesy of Dr. A. White (Biolog i c a l Station, St. Andrews, N.B., Dept. of F i s h e r i e s and Oceans, Canada). Twenty-three Protogonyaulax clones, i s o l a t e d i n June 1984 from Bamfield, B.C., were supplied by G. Gaines (Oceanography Dept., Un i v e r s i t y of B r i t i s h Columbia). Other cultures were i n i t i a t e d from c e l l s i s o l a t e d by the author from natural phytoplankton assemblages on the northeast P a c i f i c coast ( F i g . 6), p a r t i c u l a r l y those i n English Bay, at Vancouver. Inocula from the NEPCC reference cultures were used to i n i t i a t e stock cultures i n 125 ml Erlenmeyer f l a s k s . To produce s u f f i c i e n t c e l l s f o r experimental purposes, the stock cultures were added at approximately the maximum c e l l density to two l i t r e s of culture medium i n 2800 ml Fernbach f l a s k s . 59 A l l stock and experimental cultures were incubated i n a growth chamber at 16 °C on a 14:10 h light/dark cycle, at an irradiance of 120 uEin m~2 s ~ l (measured with a submerged Bi o s p h e r i c a l Instruments QSL-100P probe; B i o s p h e r i c a l Instruments, San Diego, CA) provided by cool-white fluorescent l i g h t s . The cultures were not s t i r r e d , but were p e r i o d i c a l l y shaken gently to r e - e s t a b l i s h homogeneous c e l l d i s t r i b u t i o n . Natural seawater f o r a l l experiments was obtained from Burrard I n l e t o f f the p i e r at the Westwater Laboratory, Canadian Department of Fi s h e r i e s and Oceans, West Vancouver, B.C., at a depth of 15 m. Af t e r f i l t r a t i o n through a 0.45 Mm M i l l i p o r e f i l t e r , seawater was stored i n darkness i n 20 l i t r e polyethylene carboys for at l e a s t a month before being used for c u l t u r i n g . The 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 not adjusted. 2. Development and Formulation of NWSP-7 Growth Medium A search of the l i t e r a t u r e revealed no lack of growth media used for the culture of d i n o f l a g e l l a t e s (Provasoli et a l . , 1957; Provasoli, 1963; 1968; McLachlin, 1973; Loeblich, 1975; Harrison et a l . , 1980; G u i l l a r d and K e l l e r , 1984). For t h i s reason, i t was not necessary to formulate a t o t a l l y new medium to culture Protogonyaulax spp., but rather to modify e x i s t i n g recipes to achieve the most e f f e c t i v e combination of rapid growth rate and high c e l l y i e l d f o r experimental requirements. Stock and experimental cultures were grown on NWSP-7 (Table 1), a newly formulated enrichment f o r natural seawater. This formulation was highly modified from the a r t i f i c i a l seawater medium ASP-7 (Provasoli, 1963), which has 6 0 Table 1. Formulation of NWSP-7 natural seawater enrichment medium. I. Nutrient Stock (add 1 ml l " 1 ) M.W. 8 1" [ f i n a l ] uM NaN03 Na 2gIycerophosphate•5H2O NaH 2P0 4-H 20 85.0 306.1 138.0 85.00 19.90 4.83 1000.0 65.0 35.0 I I . Trace Metals Stock (add 1 ml l " 1 ) Chelator:Trace Metal r a t i o 2.5:1 Na2EDTA-2H20 FeCl 3'6H 20 H3BO3 MnCl 2-4H 20 ZnS0 4-7H 20 CoCl 2'6H 20 Na 2Mo0 4•2H 20 .2 ,3 372. 270. 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 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 ) 191.1 19.11 100.0 IV. Vitamins Stock (add 1 ml l " 1 ) Cyanocobalamin (B^ 2) Thiamine HC1 N i c o t i n i c a c i d Ca pantothenate p-aminobenzoic acid F o l i c a c i d B i o t i n I n o s i t o l Thymine pH 7.8 mg 1" 1.0 500.0 100.0 100.0 10.0 2 1.0 5000.0 3000.0 0 Adjust pH of seawater to 6.0 a f t e r the addition of macronutrients and trace metals. Af t e r autoclaving, add s t e r i l i z e d NaHCC^ to y i e l d a f i n a l C-concentration of 2.0 mM. 61 been extensively used i n the culture of d i n o f l a g e l l a t e s (Prakash, 1967; Prakash et a l . , 1971; Tomas, 1974; Norris and Chew, 1975), but which gave unacceptably low growth rates and a high percentage of morphological aberrants i n the present study (see Appendix I ) . In addition to the replacement of the a r t i f i c i a l seawater s a l t s with a natural seawater base, NWSP-7 d i f f e r s from ASP-7 i n several other important ways. T r i s (hyroxymethyl aminomethane), added to ASP-7 as a buffer to minimize the increase i n pH occurring during autoclaving and as a r e s u l t of photosynthetic a c t i v i t y , was omitted a f t e r i t was shown to be i n h i b i t o r y to growth (see Appendix I ) . S i i s often added to broad-spectrum a l g a l growth media, but i t was deleted from NWSP-7, since t h i s element i s not usu a l l y considered a required nu t r i e n t f o r the culture of d i n o f l a g e l l a t e s . In any event, a trace requirement f o r S i would be r e a d i l y s a t i s f i e d by background contributions from the natural seawater base or by leaching from the culture glassware. The r a t i o of N:P i n the medium was adjusted to 10:1 to accurately r e f l e c t t y p i c a l i n t r a c e l l u l a r N:P r a t i o s found i n marine phytoplankton (Cembella et a l . , 1984b). The concentrations of N (as NaNOj) and P were increased to 1000 uM and 100 uM, res p e c t i v e l y , to maximize c e l l y i e l d . Glycerophosphate i s often added to a l g a l culture media as the only P-source (Provasoli, 1963; Harrison et a l . , 1980), due to i t s l e s s e r tendency to form coprecipitates with metals during autoclaving (Provasoli, 1968). However, the u t i l i z a t i o n of glycerophosphate, a phosphate ester, as the sole P-nutrient could p o t e n t i a l l y be l i m i t e d by the rate of enzymatic cleavage of P^ from the organic moiety by phosphomonoesterase a c t i v i t y (Cembella et a l . , 1984a). Phosphomonoesterase a c t i v i t y was 62 undetectable i n non-P-limited Protogonyaulax i s o l a t e 255, and low i n comparison to that detected i n the diatom Skeletonema costatum (NEPCC 18c) and the d i n o f l a g e l l a t e Prorocentrum minimum (NEPCC 96) i n P-limited culture (A. Cembella, unpublished obs.). Presumably, some free P^ may be released through thermal degradation of the ester linkage during autoclaving, but t h i s has never been established. In NWSP-7, both organic (as Na2~glycerophosphate*5H20) and inorganic P (P^) were supplied (2:1 molar r a t i o ) . Since B i s normally present i n excess of p o t e n t i a l growth requirements i n natural seawater (McLachlin, 1973), i t can s a f e l y be reduced to a f r a c t i o n of that t y p i c a l l y added to synthetic seawater base. The concentration of B added to NWSP-7 was decreased by more than 90% r e l a t i v e to ASP-7. The P II metals s o l u t i o n of ASP-7 was replaced by the formulation given i n Table 1. The modified metals mix included Mo (as Na2MoO^* 2 H 2 O ) , which was not present i n Provasoli's (1963) P II mixture. Compared to ASP-7, NWSP-7 contained a two-fold increase i n Fe (as FeCl3), while the excessive Mn concentration was reduced s u b s t a n t i a l l y . In the progress of experiments on the a c t i v i t y of Fe-binding siderophores (with G. Boyer, unpublished experiments), i t was noted that F e - l i m i t a t i o n can be more r e a d i l y induced i n Protogonyaulax than i n diatoms and other d i n o f l a g e l l a t e s ( T r i c k et a l . , 1983; Mueller, 1985). This suggests that t h i s species may have a comparatively high Fe requirement. Indeed, Iwasaki (1979) c l a s s i f i e d "tamarensis" as a "Type 2" red t i d e f l a g e l l a t e ; such species display accelerated growth i n the presence of high concentrations of dissolved Fe and/or Mn. Measurements of minimal c e l l Fe quota ( Q m i n Fe^» calculated per unit c e l l volume for 6 3 Fe-depleted batch cultures of Protogonyaulax i s o l a t e NEPCC 255, gave a value approximately twice as high 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  pseudonana, i n a s i m i l a r l y Fe-depleted condition (Mueller, 1985; A. Cembella and G. Boyer, unpublished obs.). The chelator: trace metal r a t i o i s a c r i t i c a l f a c t o r governing trace metal a v a i l a b i l i t y and t o x i c i t y . Since optimum r a t i o s are usually considered to be i n the range of 1.5-3.0: 1 (Provasoli et a l . , 1957; McLachlin, 1973; Harrison et a l . , 1980), the trace metals s o l u t i o n of NWSP-7 was formulated to y i e l d an EDTA (ethylenediamine t e t r a a c e t a t e ) : metals r a t i o of 2.5: 1. Although i t i s not as e f f e c t i v e a chelator as EDTA, NTA ( n i t r i l o -t r i a c e t i c acid) possesses considerable metal complexing capacity. Reducing the f i n a l concentration of NTA i n NWSP-7 from 366 LIM, as i s present i n ASP-7, to 100 MM, simultaneously acknowledges the e f f e c t of NTA i n stimulating the growth rate of Protogonyaulax (Yentsch et a l . , 1975), and the p o t e n t i a l f o r i n h i b i t i o n at excessive concentrations. This growth i n h i b i t i o n may be due ei t h e r to d i r e c t t o x i c i t y or to over-chelation of required metals. Even with the lowered NTA concentration, the nominal t o t a l r a t i o of chelator binding s i t e s to trace metal ions, assuming metal complexation only with the added synthetic chelators, was a r e l a t i v e l y high 27: 1. The NTA stock s o l u t i o n was prepared by adding the free acid at 1,000X f i n a l concentration to d i s t i l l e d water and t i t r a t i n g to n e u t r a l i t y with NaOH to enhance s o l u b i l i t y . The trace metals stock s o l u t i o n was prepared by d i s s o l v i n g the EDTA (Na 2) i n warm d i s t i l l e d water (~60 °C), then adding the trace metals at 1.000X f i n a l concentration. The pH was adjusted to ~2, af t e r which 64 the chelator-trace metal s o l u t i o n was heated to b o i l i n g f o r several minutes to ensure complexation. Af t e r cooling, the so l u t i o n was adjusted to a mi l d l y a c i d i c pH 5.0 and stored r e f r i g e r a t e d i n one l i t r e polycarbonate b o t t l e s . Nutrients were added (1.0 ml 1"^) from the macronutrient, chelated trace metal and NTA stock solutions p r i o r to autoclaving. To minimize p r e c i p i t a t i o n i n the medium, due to an increase i n pH while autoclaving, the pH was adjusted to 6.0 using 1.0 N HC1 a f t e r the addition of nutrients . Protogonyaulax cultures do not respond well to constant a g i t a t i o n (White, 1976) which might serve to a l l e v i a t e C - l i m i t a t i o n . Although C - l i m i t a t i o n may not be a s i g n i f i c a n t factor i n small volume cultures, p a r t i c u l a r l y those with large surface areas f o r gas exchange, i n large volume u n s t i r r e d cultures reduced growth rate and c e l l y i e l d may r e s u l t . To restore pH balance to 8.2, and to eliminate the p o t e n t i a l f o r C- l i m i t a t i o n i n large volume cultures, inorganic C (as Na2HC03) was added to NWSP-7. Anhydrous NaHCC^ was autoclaved f o r 15 min i n screw-cap v i a l s (8 ml) and then a s e p t i c a l l y added to the medium (2.0 mM f i n a l C-concentration) a f t e r autoclaving. Although i t i s l i k e l y that Protogonyaulax has an obligatory vitamin requirement only for ^>\2> t n e complex vitamin mixture i n NWSP-7 may stimulate growth by providing precursors f o r the biosynthesis of other metabolites. The vitamin mix for NWSP-7 was adopted d i r e c t l y from Provasoli's S3 s o l u t i o n (Provasoli, 1963), supplemented with 1.0 Mg 1~1 ^>12> a s s p e c i f i e d f o r ASP-7 medium. The vitamins were s t e r i l e -f i l t e r e d through a syringe equipped with a di s p o s i b l e 0.22 Mm M i l l i p o r e f i l t e r and stored frozen i n 50 ml polycarbonate b o t t l e s . The so 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 autoclaving (1 ml l " 1 ) . As a "semi-defined" medium f o r d i n o f l a g e l l a t e culture, based upon enrichment of natural seawater, NWSP-7 avoids some of the problems associated with c e r t a i n other natural seawater media. Natural media to which s o i l extract i s added, such as Erd-Schreiber (Fjflyn, 1934) and GPM (Loeblich, 1975), s u f f e r a severe disadvantage i n that the n u t r i t i o n a l q u a l i t y of the enrichment i s not only unknown, but cannot be co n s i s t e n t l y duplicated between preparations. NWSP-7 may be prepared i n large volumes for mass culture, while avoiding the time-consuming and expensive process of preparing t o t a l l y a r t i f i c i a l seawater base. 3. I s o l a t i o n and Cloning Protogonyaulax cultures were i n i t i a t e d from natural phytoplankton samples using a glass micropipette to i s o l a t e c e l l s from a two ml counting chamber under low power (160X) of the inverted microscope. Isolated c e l l s were passed s e q u e n t i a l l y through a se r i e s of four to s i x s t e r i l e washes i n autoclaved i s o l a t i o n medium ( f i l t e r e d seawater enriched with 10% ESNW nutrients) i n glass spot plates u n t i l v i s i b l y free of associated species. Individual c e l l s were then transferred to the wells of s t e r i l i z e d p l a s t i c i s o l a t i o n trays, with each well containing approximately one ml of i s o l a t i o n medium. Growth was monitored d a i l y by observation through the l i d of the p l a s t i c trays using a d i s s e c t i n g microscope. Af t e r approximately 20 c e l l d i v i s i o n s , the cultures were transferred to three ml of s t e r i l e i s o l a t i o n medium i n 15 ml screw-cap t e s t tubes. The cultures were allowed to reach l a t e exponential phase, as determined by following 66 the i n vivo fluorescence curve (see Chapter II C.l) to a maximum value. Then they were transferred to 75 ml of f u l l - s t r e n g t h ESNW i n 125 ml Erlenmeyer f l a s k s and maintained as reference i s o l a t e s within the NEPCC through routine t r a n s f e r s at the end of each culture cycle. 4. Preparation and Testing of Axenic Cultures Two Protogonyaulax i s o l a t e s , NEPCC 253 and 255, were treated with a n t i b i o t i c s and rendered axenic by the method of Droop (1967). The s t e r i l e - f i l t e r e d a n t i b i o t i c s o l u t i o n (Table 2) was prepared i n NWSP-7 medium and mixed a s e p t i c a l l y i n a 1:1 r a t i o with 6 ml of exponentially growing culture i n 15 ml screw-cap t e s t tubes. The a n t i b i o t i c concentration was progressively reduced by a two-fold s e r i a l d i l u t i o n (mixed 1:1) of the a n t i b i o t i c - t r e a t e d culture i n untreated cultures. Thus, the f i r s t c ulture tube contained h a l f the o r i g i n a l a n t i b i o t i c concentration, and so on through a sequence of s i x tubes. A f t e r mixing the a n t i b i o t i c s o l u t i o n with the cultures, one drop of s t e r i l i t y t e s t medium (Table 3), modified from STP medium (Provasoli et a l . , 1957), was added to each tube. A n t i b i o t i c - t r e a t e d cultures were incubated f or 24 h, then 1.0 ml from each was transferred into duplicate 15 ml screw-cap t e s t tubes containing eight ml of a n t i b i o t i c - f r e e medium. A f t e r two weeks, one drop of each of these subcultures was added to three ml of autoclaved s t e r i l i t y medium and kept i n the dark at room temperature to t e s t f o r b a c t e r i a l growth. Any increase i n t u r b i d i t y over a period of three weeks was noted by v i s u a l inspection of the tubes, and by following increases i n o p t i c a l density (Spectronic 20 spectrophotometer; Bausch and Lomb, Table 2. A n t i b i o t i c mix for Protogonyaulax p u r i f i c a t i o n . Concentration (mg ml Benzyl p e n i c i l l i n s u l f a t e Streptomycin s u l f a t e Chloramphenicol 8.0 1.6 0.2 Table 3. Modified STP s t e r i l i t y t e s t medium for the monitoring of possible 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 Autoclaved NWSP-7* , 95.0 ml Vitamin mix f o r NWSP-7"' 0.1 ml Autoclaved s o i l extract 5.0 ml Yeast autolysate 20 mg Sucrose 100 mg Na-glutamate 50 mg DL-alanine 10 mg Trypticase 20 mg Glycine 20 mg pH 7.5 "see Table 1. Rochester, NY) at 620 nm. Cultures were judged to be axenic i f the r e s u l t s of the s t e r i l i t y tests were negative ( i . e . no increase i n t u r b i d i t y ) , and i f no bacteria were detected by phase-contrast microscopic examination under high power (1,000X) using a Zeiss oil-immersion objective. In the two l i t r e u n i a l g a l Protogonyaulax cultures, p e r i o d i c b a c t e r i a l counts were c a r r i e d out using the epifluorescence technique (Hobbie et a l . , 1977), to ensure that the cultures were not highly contaminated. Two ml of u n i a l g a l culture were f i l t e r e d onto a Nuclepore f i l t e r (0.22 urn) pre-treated with Irgalan Black (C.I., acid black 107; Ciba-Geigy Corp., Greensboro, NC). The cultures were incubated with 0.01% ( f i n a l concentration) a c r i d i n e orange (3,6-bis [dimethylamino] acridinium chloride; Sigma, St. Louis, MO) for two minutes p r i o r to f i l t r a t i o n . At lea s t ten high power (1,000X) microscope f i e l d s and at l e a s t 200 bacteria for each sample were counted using a Zeiss epifluorescence microscope. The concentration of bacteria per ml and the 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 to that of the d i n o f l a g e l l a t e s were estimated. B. Morphological Examination of Protogonyaulax Isolates 1. Isolate I d e n t i f i c a t i o n and Morphological C h a r a c t e r i s t i c s Cultures f o r morphological examination were grown under standard conditions (Chapter II A.l) i n natural seawater enriched with NWSP-7 medium. Only cultures that were v e r i f i e d to be i n exponential growth phase, by following the growth curve f l u o r o m e t r i c a l l y , were used f or morphological studies. 69 Protogonyaulax c e l l s i n culture do not divide i n absolute synchrony, however they do display a tendency f o r phased d i v i s i o n ; d i v i s i o n u sually occurs toward the end of the dark cycle (Tomas, 1974; pers. obs.). C e l l s for morphological examination were c o l l e c t e d near the middle of the l i g h t period of the light/dark cycle, when the majority of the c e l l s were i n mi t o t i c interphase. The c e l l s examined were randomly selected, although obvious aberrants and those which appeared to be undergoing d i v i s i o n were rejected. In the case of the c a t e n e l l o i d i s o l a t e s which formed chains, a s i n g l e c e l l near the middle of each chain was taken to be a representative i n d i v i d u a l f o r c e l l dimension measurements. The c e l l s were immobilized i n 0.5% formalin and measured immediately. The a p i c a l dimension was measured as the maximum diameter between the a p i c a l and antapical poles; the tr a n s a p i c a l dimension was determined as the maximum equatorial diameter p a r a l l e l to the cingulum, when c e l l was oriented i n v e n t r a l view. The volume was calculated using the a p i c a l and tr a n s a p i c a l dimensions as the major (2a) and minor (2b) axes i n the formula f o r a prolate spheroid, where V = A/3irab 2. Epifluorescence microscopic observations of i n t a c t c e l l s and discarded thecae treated with 0.1% c a l c o f l u o r white M2R (Sigma, St. Louis, MO) were made to confirm the presence or absence of a v e n t r a l pore at the margin of the f i r s t a p i c a l ( l 1 ) plate. The s i z e and shape of the a p i c a l pore complex (APC) and the posterior s u l c a l plate, as well as the presence and p o s i t i o n of the anterior and pos t e r i o r attachment pores (when present) were also noted. Isolates designated by the NEPCC number were assigned a p r o v i s i o n a l taxonomic status within the Protogonyaulax species complex (P. ca t e n e l l a , Table 4. Location of Origin and Morphological Characteristics of Isolates of the Protogonyaulax tamarensis/catenella species complex. Isolate Origin of Isolate Ventral Apical Transapical Ratio Mean Shape of Morphotype*** # c e l l s per pore Diameter (um) Diameter (im) Apical: Volume epicone and chain**** 3? ± s.d. X" ± s.d. Transapical (urn-*) hypocone Diameter (X10 3) 71* Patricia Bay, Present 35.84 1 3.40 32.34 1 1.96 1.11 B.C. Canada, Aug., 1973 19.63 high domed Tamarensoid epicone; rounded hypocone SI 180 Brentwood Bay, Absent 37.96 t 3.94 37.25 i 3.05 1.02 B.C. Canada, Aug., 1973 183* Tamar estuary, Present 36.21 t 2.17 33.84 ± 1.89 1.07 Plymouth, England, June, 1957 27.60 Rounded Tamarensoid 21.72 Rounded Tamarensoid S2 52 253* Laguna Obldos Absent 24.88 ± 1.75 22.38 ± 2.09 1.11 Portugal, 1962 255 Lummi Island, Present 37.09 i 1.41 33.34 t 3.08 1.11 HA, USA. Aug., 1976 6.53 Rounded Tamarensoid 21.60 Rounded Tamarensoid $2 52 Isolate Origin of Isolate Ventral Apical Transapical Ratio Mean Shape of Morphotype* pore Diameter (|ia) Diameter (urn) Apicalt Volume epicone and Jf t a.d. X t s.d. Transapical (MB') hypocone Diameter (X10 3) I c e l l s per chain**** 400 English Bay, Absent 33.25 t 2.34 31.66 ± 2.58 1.05 B.C., Canada, June, 1981 17.46 Usually Tamarensoid rounded; few flattened S2 401 English Bay, B.C., Canada, June, 1981 403* English Bay, B.C., Canada June, 1981 404* English Bay, B.C., Canada June, 1981 Absent 36.04 i 2.80 32.00 t 1.88 1.13 19.33 Rounded Tamarensoid Absent 38.09 t 3.53 35.13 i 2.89 1.08 24.64 Rounded Tamarensoid Present 33.54 ± 3.13 33.41 1 2.88 1.00 19.62 Usually Tamarensoid rounded; few flattened S2 52 32 405* English Bay. B.C., Canada June, 1981 406* English Bay. B.C.. Canada June, 1981 407* English Bay, B.C., Canada, June, 1981 Absent 28.42 ± 2.80 25.91 t 2.48 1.10 10.00 Rounded Tamarensoid Absent 27.50 t 3.38 26.38 t 2.34 1.04 10.02 Rounded Tamarensoid Absent 28.33 ± 2.35 26.25 ± 2.28 1.08 10.24 Rounded Tamarensoid 22 52 Isolate Origin of Isolate Ventral Apical Transapical Ratio Mean Shape of Morphotype* pore Diameter (tan) Diameter (MB) Apical: Volume eplcone and X 1 s.d. JT t s.d. Transapical (ml hypocone Diameter (X10 3) # c e l l s per chain**** 409 English Bay, B.C., Canada, June, 1981 412 English Bay, B.C., Canada, June, 1981 508 Whangarei, North Island, New Zealand, Feb.. 1983 545* Bay of Fundy, N.B., Canada July, 1976 402 English Bay, B.C., Canada, June, 1981 Absent 30.09 t 2.36 27.21 ± 2.36 1.11 11.68 Rounded Tamarensoid Present 28.66 i 2.75 25.96 t 2.06 1.10 10.12 Rounded Tamarensoid Absent 29.38 ± 2.68 26.71 t 3.02 1.10 10.99 Rounded Tamarensoid Absent 35.95 t 3.09 32.00 ± 3.35 1.12 19.06 Rounded Tamarensoid Absent 32.91 t 2.58 33.46 t 3.34 0.98 18.99 Epicones/ Intermediate hypocones rounded or flattened 52 52 52 52 52 516 English Bay, Present 30.84 ± 6.73 29.66 i 3.19 1.04 B.C., Canada, June, 1982 14.21 Epicone Intermediate egg- or b e l l -shaped; hypocone tapered** 52 Isolate Origin of Isolate Ventral Apical Transapical Ratio Mean Shape of Morphotype*** # c e l l s per pore Diameter (um) Diameter (um) Apicali Volume epicone and chain**** JT ± s.d. x"± s.d. Transapical (um3) hypocone Diameter (X10 3) 254 Hidden Basin. Absent 28.33 t 3.05 30.21 t 3.20 0.94 Nelson Island B.C., Canada Oct., 1976 355* Penn Cove, Absent 29.34 ± 2.45 32.50 t 2.13 0.90 Whldbey Island, WA. USA, 1980 356* Quartermaster Absent 29.17 t 4.60 30.92 t 2.02 0.94 Harbor, Vashon Island. WA, USA 1980 12.70 Rounded Catenelloid 14.65 Flattened Catenelloid 13.78 Flattened Catenelloid S2 i4 £4 435 Friday Harbor, Absent 33.91 t 3.33 36.46 i 3.43 0.93 San Juan Island, WA, USA, Aug., 1982 529 Friday Harbor, Absent 32.13 t 2.66 32.50 t 2.23 0.99 San Juan Island, WA, USA, Aug., 1983 543 Friday Harbor, Absent 30.29 t 2.88 33.50 t 2.04 0.90 San Juan Island, WA, USA Aug., 1983 21.97 Flattened Catenelloid 17.58 Flattened Catenelloid 16.10 Flattened Catenelloid 24 £4 £4 •clonal Isolate| **some specimens have flattened epicones and hypocones; ***aorphotype as presently in culture; ****typical number of c e l l s per chain at time of o r i g i n a l isolation. 74 P. tamarensis, or Protogonyaulax intermediate) on the basis of general morphological c h a r a c t e r i s t i c s (Table 4). C e l l s of the " c a t e n e l l a " type were those o r i g i n a l l y i s o l a t e d i n chains t y p i c a l l y c o n s i s t i n g of four or more c e l l s and which remained somewhat antero-posteriorly compressed i n culture ( r a t i o a p i c a l : t r a n s a p i c a l diameter <1:1). The "tamarensis" type embraced those i s o l a t e s which did not o r i g i n a l l y form chains of greater than a few c e l l s , and i n which the a p i c a l : t r a n s a p i c a l diameter r a t i o was u s u a l l y >1:1. Isolates that c h a r a c t e r i s t i c a l l y made short chains of is o d i a m e t r i c a l c e l l s while i n exponential growth phase were considered to represent the "intermediate" morphotype. 2. Chain Length Experiments The e f f e c t s of low l e v e l s of p a r t i c u l a r nutrients (N, P, and Fe) on the length of chains formed in cultures of the c a t e n e l l o i d i s o l a t e NEPCC 355 (Penn Cove, Whidbey Island, WA) were investigated throughout a culture cycle. The c o n t r o l cultures contained n u t r i t i o n a l l y complete ESNW medium (Harrison et a l . , 1980) (-Tris, - S i , +0.5 pM Mo), with Fe added e x c l u s i v e l y i n the form of FeCl^ (6.6 uM). Low N, P, and Fe cultures were prepared by reducing the added concentrations of the prospective l i m i t i n g n u t r i e n t to 1% of the c o n t r o l concentration. For the low N (5.5 uM) medium, the N:P r a t i o was 0.25:1, while the low P (0.2 pM) medium had an N:P r a t i o of 2500:1. The low Fe medium contained 0.07 pM Fe (as FeCl3). The concentration of EDTA i n the metals mix was reduced proportionately, to keep the chelator: metal r a t i o equivalent i n the low Fe and c o n t r o l cultures (1.6:1). 75 Ten ml of NEPCC 355 maintained on ESNW were transferred at the end of exponential growth phase into 125 ml b o r o s i l i c a t e glass Erlenmeyer f l a s k s containing 50 ml of medium. T r i p l i c a t e cultures f o r each treatment were maintained under standard conditions (Chapter II A . l ) , and sampled at in t e r v a l s i n mid-exponential, l a t e exponential, e a r l y stationary and advanced senescence phases of the culture cycle. The fl a s k s were gently swirled to ensure homogeneity before the samples were removed. Four ml of each culture were withdrawn using a 7 mm diameter length of s t e r i l e glass tubing, to minimize non-random s e l e c t i o n against longer chains, a r i s i n g from the use of a narrow bore pipette. A f t e r f i x a t i o n (0.5% formalin), chain length was examined under phase-contrast microscopy and counts were made of 25 random f i e l d s (125X) i n duplicate 2 ml inverted microscope counting chambers. C. V a r i a t i o n i n Growth Rates 1. Determination of Acclimated Growth Rates For the determination of acclimated growth rates, u n i a l g a l stock cultures from the NEPCC were maintained i n 125 ml Erlenmeyer f l a s k s on NWSP-7 medium under standard conditions (see Chapter II A . l ) . To minimize the possible e f f e c t s of water q u a l i t y differences on growth, the growth rates f o r a l l i s o l a t e s were determined simultaneously, using the same natural seawater ( c o l l e c t e d from Burrard I n l e t , Vancouver, July 15, 1983 at a depth of 15 m) for the preparation of the medium. In l a t e exponential phase, 1.0 ml of each culture was transferred into t r i p l i c a t e 25 X 150 mm screw cap b o r o s i l i c a t e glass culture tubes each containing 25 ml of medium. The tubes were placed at a 45° angle with caps loosened to allow for gas exchange. The cultures were incubated without modification of the standard environmental regime and were allowed to proceed through e a r l y exponential phase u n t i l approximately 10% of the maximal c e l l density was achieved. Then they were re-inoculated into tubes containing fresh medium. Growth rates were compared during t h i s second trans f e r s e r i e s . Growth i n experimental and c o n t r o l cultures was monitored f l u o r o m e t r i c a l l y , e s s e n t i a l l y according to the method of Brand et a l . (1981a) for the determination of acclimated growth rates. At three day i n t e r v a l s through the growth cycle, the tubes were removed from the incubation chamber and held for 15 min i n a 16 °C water bath at ambient room l i g h t before the fluorometric measurements were made. The fluorescence was always determined during the l a t e afternoon, which corresponded approximately to the middle of the l i g h t period of the light/dark cycle. Protogonyaulax i s unusually susceptible to turbulence (White, 1976) that can cause the formation of p e l l i c u l a r cysts and a reduction i n growth rate. Rather than vortex mixing the samples to ensure homogeneity, the tubes were gently inverted three times immediately p r i o r to i n s e r t i o n into the fluorometer. In vivo fluorescence was determined using a Turner 10-000R fluorometer (Turner Designs, Mountain View, CA) equipped with an i n f r a r e d s e n s i t i v e photomultiplier, and the appropriate f i l t e r system ( e x c i t a t i o n f i l t e r : Corning 5-60; reference f i l t e r : Corning 3-66; emission f i l t e r : Corning 2-64) for the detection of i n vivo c h l o r o p h y l l a fluorescence. Acclimated maximal growth rates were determined from the semi-log pl o t s of 77 fluorescence versus time over the time interval (t j - t 0 ) indicated by the linear portion of the growth curve. As a calibration for departure from linearity in the relationship between cell number and in vivo fluorescence, which can arise due to self-absorption by cells, particularly in dense cultures, and at the lower detection limit of the fluorometer when the cell density is low, log-log plots of cell number versus in vivo fluorescence were constructed using a series of two-fold dilutions of a dense culture in exponential phase. Since cell densities in Protogonyaulax cultures rarely exceeded ~10^ ml"^ -, self-absorption effects were less than would be expected for many other phytoplankton species. For comparison, cell counts were made on several cultures, as previously described, and the growth rates calculated using the formula, k = lnCN-i/NQXl.AAS/t), where k (div d"1) is the growth rate, Nj_ and NQ are the cell densities at tj and t Q , respectively, and t is the time interval t ^ - t Q . Control experiments performed here and by others (Brand et al., 1981a; Watras et al., 1982) have shown that increases of in vivo fluorescence can be used to accurately represent increases in numbers of Protogonyaulax cells through division, provided sufficient time is allowed for acclimation in a constant environment. 78 D. DNA Analysis by Epifluorescence Microphotometry 1. Nuclear DNA Determination The DNA of Protogonyaulax c e l l s was stained by incubating c e l l s i n a buffered s o l u t i o n of the n u c l e i c a c i d fluorochrome DAPI (4,6-diamidino-2-phenyl-indole) (Sigma, St. Louis, MO). The stock s o l u t i o n was prepared by d i s s o l v i n g DAPI (0.1 mg ml " 1 ) , T r i s (0.60 mg m l - 1 ) , and EDTA (0.37 mg m l - 1 ) i n d i s t i l l e d deionized water. For the working s o l u t i o n , 0.2 ml of DAPI stock were added d i r e c t l y to 10 ml of exponentially growing culture (= 1:50 d i l u t i o n ) and the mixture was incubated at room temperature. The curve of resultant epifluorescence i n t e n s i t y versus time was followed over 48 h to determine saturation k i n e t i c s f o r fluorochrome binding. A standard incubation period of not les s than 2 h was adopted, as no further increase i n nuclear DNA epifluorescence was detected a f t e r t h i s elapsed time. C e l l s were examined using a Zeiss epifluorescence microscope (200X) connected to a computerized photomultiplier system programmed i n BASIC (J.D. Berger, Univ. B r i t i s h Columbia, Zoology Dept.), to y i e l d a d i g i t a l readout of r e l a t i v e epifluorescence. To minimize the e f f e c t s of extraneous fluorescence from background and non-nuclear DNA, the aperature was stopped down to circumscribe only the nuclear area. A blue b a r r i e r f i l t e r was interposed between the epifluorescent microscope stage and the detector to suppress the b r i l l i a n t red autofluorescence of the chlo r o p l a s t s . The detector 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 using the nucleated erythrocytes of the domestic chicken, Gallus gallus domesticus, which were determined to be uniform i n s i z e and nuclear fluorescence a f t e r DAPI 79 s t a i n i n g . The mean nuclear DNA content of chicken erythrocytes (2.55 ± 0.43 s.e.m.) was calculated from l i t e r a t u r e values obtained using various techniques (Fasman, 1976). Nuclear epifluorescence f o r i n d i v i d u a l Protogonyaulax c e l l s , and those of an i s o l a t e of Gonyaulax polyedra Stein (NEPCC 202a; ex. B. Sweeney, La J o l l a , CA), was measured i n quintuplicate (n=30 c e l l s per i s o l a t e ) . Epifluorescence of i n t a c t n u c l e i was measured from the v e n t r a l e q u a t o r i a l view of the c e l l s , a f t e r applying gentle pressure to the cover s l i p to l i n e a r l y extend the nucleus. In addition to vegetative c e l l s , the nuclear DNA content of zygotic c e l l s r e s u l t i n g from sexual fusion i n senescent cultures of NEPCC i s o l a t e s 183, 255, 403, 405 and 435 was determined. Zygotes were ope r a t i o n a l l y i d e n t i f i e d by t h e i r large diameter (50-70 nm) and lumpy morphology, r e l a t i v e to normal vegetative c e l l s . Planozygotes could be distinguished from excysted planomeiocytes by t h e i r darker brown pigmentation and lack of a dark reddish accumulation body (Anderson et a l . , 1983; Anderson, 1984). E. Method f or Polyacrylamide Gel Electrophoresis 1. Electrophoresis of Soluble Enzymes a. C e l l c u l t u r e , harvest and storage For the el e c t r o p h o r e t i c studies, Protogonyaulax i s o l a t e s were cultured under the standard conditions already s p e c i f i e d (Chapter II A . l ) . To y i e l d cultures f o r isozyme analysis that were subject to the minimal 80 accumulation of genotypic modifications i n long-term culture, two c l o n a l i s o l a t e s from English Bay, NEPCC 403 and 407, were transferred into progressively larger culture volumes- 1 ml, 8 ml, 75 ml, then 2000 ml- as soon as possible a f t e r the o r i g i n a l i s o l a t i o n . Their isozyme patterns were followed over a period of more than one year; multiple subcultures of these i s o l a t e s were cultured independently to compare any possible c l o n a l i n s t a b i l i t y i n enzyme patterns produced. C e l l c o l l e c t i o n was always confined to the middle of the light/dark cycle of the incubator. By harvesting at approximately the same time of day, when most c e l l s were i n interphase, the p o t e n t i a l f o r quantitative and q u a l i t a t i v e v a r i a t i o n i n enzyme a c t i v i t y within the c e l l c ycle, p a r t i c u l a r l y f o r those enzymes which may be photoinducible, was minimized. C e l l s were harvested at the end of exponential growth, determined by following the i n vivo fluorescence curve to a maximum value. By comparing o p t i c a l c e l l counts with i n vivo fluorescence, a conversion f a c t o r was calculated and applied to adjust the culture volume harvested, such that approximately equal numbers of c e l l s (~2 X 10^) were obtained f o r each enzyme extr a c t i o n . At harvest, c e l l d e n s i t i e s were t y p i c a l l y between 1.0-5.0 X 10^ per ml, depending upon the i s o l a t e . Cultures were centrifuged f o r 5 min at 5,000 X g i n a r e f r i g e r a t e d centrifuge ( S o r v a l l GSA rotor; Dupont Instruments - S o r v a l l , Wilmington, DE) at 4 °C; the loose p e l l e t s were rinsed and resuspended i n a 50-fold volume of s t e r i l e - f i l t e r e d washing buffer (100 mM T r i s , 1 mM EDTA, 10 mM Cleland's reagent, DTT [ d i t h i o t h r e i t o l ] ; Sigma, St. Louis, MO) at pH 7.5 (4 °C), then recentrifuged (3,000 X g, S o r v a l l SS-34 rotor; Dupont Instruments -S o r v a l l , Wilmington, DE) i n p r e - c h i l l e d 8 ml glass v i a l s . The supernatant wash buffer was removed 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 quick 81 frozen i n dry i c e and l y o p h i l i z e d overnight. V i a l s were stored i n a vacuum desiccator at -40 °C u n t i l extraction. , b. B a c t e r i a l contamination Although every e f f o r t was made to reduce b a c t e r i a l contamination and subsequent growth, low numbers of bacteria were noted under high-power (1,000X) phase-contrast microscopy i n a l l cultures. Periodic counts using the epifluorescence technique (Hobbie et a l . , 1977) (Chapter II A.3), were t y p i c a l l y < 1 x 10^ bacteria per ml. Tot a l c e l l volume c a l c u l a t i o n s indicated that bacteria were always < 1% of the t o t a l d i n o f l a g e l l a t e volume. As a co n t r o l , 1.0 l i t r e of u n i a l g a l culture was f i l t e r e d through a Whatman f i l t e r under gentle vacuum to remove the d i n o f l a g e l l a t e s , while allowing the bacteria to pass into the f i l t r a t e . The f i l t r a t e was r e f i l t e r e d through a 0.22 nm GSWP M i l l i p o r e membrane to r e t a i n the bacte r i a . The f i l t e r e d b a cteria were then c o l l e c t e d i n s t e r i l e e x traction buffer and electrophoresed along with d i n o f l a g e l l a t e extracts. As a further t e s t of possible b a c t e r i a l contamination of d i n o f l a g e l l a t e extracts, 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, were electrophoresed i n p a r a l l e l with extracts from u n i a l g a l culture. No supernumerary putative b a c t e r i a l bands were ever observed. c. Determination of t o t a l protein In order to ensure that s u f f i c i e n t q uantities of protein were co n s i s t e n t l y applied to the gels, t o t a l soluble protein from extracted samples was p e r i o d i c a l l y determined by the Bradford technique (Bradford, 82 1976). The method involves spectrophotometrically measuring the increase i n absorbance of the general protein s t a i n Coomassie B r i l l i a n t Blue G-250 (Sigma, St. Louis, MO), caused by non-specific binding to sample proteins. Protein samples were sonicated and c l a r i f i e d by c e n t r i f u g a t i o n according to the protocol given herein for enzyme extraction. Sample protein values were determined from the c a l i b r a t i o n curve p l o t t e d using bovine serum albumin standards i n the range 0.1-1.0 mg m l - 1 . Reagent blanks were prepared i n the buffer used for enzyme extraction. Due to the high blank absorbance ,of Coomassie B r i l l i a n t Blue G-250, sample absorbance was measured . both before and a f t e r the addition of the s t a i n , and the sample blank was subtracted. Absorbance was measured i n 1 cm pathlength cuvettes i n a Spectronic 21 spectrophotometer (Bausch and Lomb, Rochester, NY) at 595 nm. d. Enzyme ext r a c t i o n C e l l ' p e l l e t s containing approximately 2 X 10^ c e l l s were suspended i n 0.35 ml of i c e - c o l d e xtraction buffer (100 mM T r i s , 1 mM EDTA, 10 mM DTT, 50 •uM NAD+ [nicotinamide adenine dinucleotide] and 50 nM NADP+ [nicotinamide adenine dinucleotide phosphate], pH 7.5 at 4 °C), and sonicated (Bronwill Biosonik Model I I I , W i l l S c i e n t i f i c , Rochester, NY) i n an ice-ETOH bath at 60% of maximum output (= 210 W) f o r 90 s i n 10 s bursts. This procedure minimized enzyme denaturation. Microscopic examination of sonicated extracts indicated that only minute membrane fragments, and v i r t u a l l y no i n t a c t c e l l s or major organelles remained. Sonicated extracts were centrifuged at 27,000 X g ( S o r v a l l SS-34 rotor) at 4 °C for 15 min and the supernatant was d i r e c t l y applied to the gels. 83 e. Electrophoretic separation Electrophoretic separation of Isozymes was performed i n 1.5 mm thick 7% polyacrylamide gel (PAG) (T=7.2$; C=2.6$) (Table 5), using a dual-slab vertical gel apparatus (Model VS-14, Proteus Technology, Richmond, B.C.), with plate dimensions of 180 X 140 nun. Table 5. Polyacrylamide Gel Formulation Separating gel (7$) 7.0 ml acrylamide: Bis-acrylamide (30$: 0.8?) 15.4 ml d i s t i l l e d H20 7.4 ml 1.67 M Tris, pH 8.8 at 4°C 0.15 ml ammonium persulfate (10$) 15 f l TEMED A l l electrophoresis reagents were obtained from Sigma (St. Louis, M0). The gel buffer was 0.4 M Tris, pH 8.8 at 4 °C; the electrode buffer for a l l enzyme systems was 0.19 M glycine - 0.05 M Tris, pH 8.3 at 4 °C. Electrophoresis was carried out in a refrigerator compartment at 4 °C, with additional cooling provided by a internally circulating ice-water bath driven from a two l i t r e external reservoir by a peristaltic pump. Prior to the application of enzyme extracts, gels were pre-electro-phoresed for 50 Vh at 2 W per gel constant power (voltage max.: 100 V; current max.: 40 mA per gel). Glycerol was added to equal 10$ of the total sample volume, after which 84 80 n l of extract (= ~120 Mg protein) from each i s o l a t e , were loaded into p a r a l l e l wells i n random order. Bromophenol blue (0.01%) was added to 10% g l y c e r o l i n the extraction buffer and applied to the end wells to serve as the tracking dye. To standardize minor discrepancies between runs, a reference s t r a i n , NEPCC 255, of known electrophoretic c h a r a c t e r i s t i c s was always run i n the same gel p o s i t i o n . Electrophoretic m o b i l i t i e s were normalized to a primary band from t h i s s t r a i n . A f t e r extract loading, the power was maintained at 2 W per gel for 50 Vh to allow for protein alignment within the g e l . The power, was then increased to 8 W per gel (voltage max.: 400 V; current max.: 75 mA per gel) u n t i l a t o t a l of 750 Vh had elapsed. Electrophoretic runs normally required approximately 4.5 h, at which time the tracking dye migrated a distance of 105 mm from the o r i g i n . f. Gel s t a i n i n g and band scoring Gels were stained for the following enzymes, with minor modifications of standard techniques (Brewer and Sing, 1970; Harris and Hopkinson, 1976) (see Table 6): Alanine dehydrogenase (AlaDH, E.C. 1.4.1.1), glutamate dehydrogenase (GDH, E.C. 1.4.1.2), glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49), B-hydroxybutyrate dehydrogenase (HBDH, E.C. 1.1.1.30), NADP-dependent i s o c i t r a t e dehydrogenase (IDH, E.C. 1.1.1.42), NAD-dependent malate dehydrogenase (MDH, E.C. 1.1.1.37), NADP-dependent malic enzyme (ME, E.C. 1.1.1.40), and succinate dehydrogenase (SucDH, E.C. 1.3.99.1). A l l gels were incubated i n the dark at 37 °C for one hour, then at room temperature u n t i l maximum r e s o l u t i o n was achieved. Duplicate gels containing extracts of each i s o l a t e were TABLE 6. Stain protocols for dehydrogenase Isozymes Components added to 60 ml buffer Enzyme Alanine dehydrogenase AlaDH (B.C. .1.4.1.1) Glucose-6-phosphate dehydrogenase G6PDH (E.C. 1.1.1.49) Glutamate dehydrogenase GDH (E.C. 1.4.1.2) B-hydroxybutyrate dehydrogenase HBDH (E.C. 1.1.1.30) Isocitrate dehydrogenase IDH (E.C. 1.1.1.42) Malate dehydrogenase MDH (E.C. 1.1.1.37) Malic enzyme ME (E.C. 1.1.1.40) Succinate dehydrogenase SucDH (E.C. 1.3.99.1) Stain buffer pH at 37°C Substrate Chelator Metal cofactor Nucleotide cofactor Stain 0.1 M phosphate 8.0 DL-alanine 500 mg 0.1 M Tris 0.1 M Tris 8.0 8.0 0.1 M Tris 0.1 M Tris 0.1 M Tricine 8.0 8.0 D-glucose-6-phosphate 400 mg L-glutamate 1000 mg 0.1 M phosphate 7.5 3-DL-hydroxy-butyrate(Na) 500 mg DL-isocitric acid(Na) 500 mg L-malate(Na) 500 mg 8.0 L-malate 500 mg MgCl2(0.5 M) 1.0 ml MgCl2(0.5M) 1.0 ml MgCl,(0.5 M) 1.0 ml 0.1 M phosphate 7.0 succinic acid (Na 2) 400 mg EDTA(Na2) 200 mg NAD 10 mg NADP 10 mg NAD 10 mg NAD 10 mg NADP 10 mg NAD 10 mg NADP 10 mg NAD 20 mg ATP 30 mg MTT (10 mg ml" 1) 1.5 ml PMS (5 mg ml" 1) 0.5 ml MTT (10 mg ml" 1) 1.5 ml PMS (5 mg ml" 1) 0.5 ml MTT (10 mg ml" 1) 1.0 ml PMS (5 mg ml" 1) 0.5 ml MTT (10 mg ml* 1) 1.5 ml PMS (5 mg ml" 1) 1.0 ml MTT (10 mg ml" 1) 1.0 ml PMS (5 mg ml" 1) 0.5 ml MTT (10 mg ml" 1) 1.0 ml PMS (5 mg ml" 1) 0.5 ml MTT (10 mg ml" 1) 1.0 ml PMS (5 mg ml" 1) 0.5 ml MTT (10 mg ml" 1) 2.0 ml . PMS (5 mg ml" 1) 1.0 ml 00 86 electrophoresed simultaneously for each enzyme, and the r e s u l t s of at l e a s t two separate runs were used to c a l c u l a t e mean migration distance and band i n t e n s i t y . Band migration was measured from the o r i g i n with a r c h i t e c t ' s d i v i d e r s , and s t a i n i n g i n t e n s i t y was recorded. In cases involving the absence of s t a i n i n g a c t i v i t y , the questionable appearance of f a i n t bands or obvious migration anomalies for isozymes of an i s o l a t e within or between runs, samples were re-electophoresed u n t i l congruent r e s u l t s were obtained. A photographic record (Kodak Ektachrome EPY Professional f i l m , ASA 50) of c e r t a i n stained gels was made on a l i g h t table illuminated by an incandescent tungsten floodlamp (3,200°K). Gels were f i x e d and preserved i n isopropanol:glycerol:acetic acid (50%:20%:7%) mixed with an equal volume of 0.2 M s t a i n i n g buffer. F. Toxin Analysis by High-Pressure (Performance) L i q u i d Chromatography 1. HPLC Analysis of Toxins a. C e l l culture and harvest Unialgal Protogonyaulax i s o l a t e s from the NEPCC, as well as cultures of two species, Gonyaulax polyedra (NEPCC 202a) and G. g r i n d l e y i (=Protoceratium reticulatum; NEPCC 535, G. Gaines i s o l a t e , Friday Harbor, San Juan Island, WA, 1983), from a r e l a t e d gonyaulacoid genus, were examined f o r the presence of PSP toxins. For the determination of toxin p r o f i l e s and t o x i c i t y by HPLC, i s o l a t e s were cultured i n two l i t r e s of NWSP-7 medium i n 2800 ml Fernbach f l a s k s under standard conditions 87 (Chapter II A . l ) . C e l l s were harvested i n l a t e exponential growth phase, which was determined by monitoring i n vivo fluorescence (Chapter II C . l ) . To determine the e f f e c t of culture age on toxin content and composition, samples of seven i s o l a t e s , NEPCC 183, 253, 401, 402, 409, 508, and 516, were c o l l e c t e d at three d i f f e r e n t times during the exponential phase of growth, and a f t e r 10 days i n stationary phase. Since the growth rates and the length of the lag phase varied markedly among i s o l a t e s , samples representing e a r l y - , mid- and late-exponential phase cultures were taken at approximately one-quarter, one-half, and three-quarters of the time elapsed between the end of the lag period and the time corresponding to maximal c e l l density, r e s p e c t i v e l y . The samples c o l l e c t e d at various times were used to confirm that the toxin p r o f i l e s were stable enough throughout the culture cycle to be used for chemotaxonomic an a l y s i s . S u f f i c i e n t culture volume was removed to y i e l d 1.0 x 10^ c e l l s f or toxin extraction, as determined by phase-contrast microscope counts (160X) of at l e a s t 400 c e l l s i n duplicate Palmer-Maloney counting chambers. C e l l s were c o l l e c t e d by c e n t r i f u g a t i o n (5 min at 5,000 X g, 5 °C, S o r v a l l GSA r o t o r ) , and the p e l l e t was suspended i n 5 ml of dR^O. Each suspended p e l l e t was transferred to an 8 ml v i a l and recentrifuged (5 min at 2,000 X g) i n a c l i n i c a l centrifuge. The supernatant was removed by a s p i r a t i o n and the p e l l e t resuspended i n 3 ml 0.03 N a c e t i c a c i d for toxin e x t r a c t i o n . b. Toxin e x t r a c t i o n Toxins were extracted from the c e l l s by sonication (Bronwill Biosonik Model I I I ) , at 60% of maximum output (= 210 W), using f i v e 10 s bursts 88 with the v i a l immersed i n an ice-ETOH bath. Complete c e l l d i s r u p t i o n was confirmed by examination under phase-contrast microscopy (400X). Sonicated samples were recentrifuged (10 min at 2,000 X g) to remove p a r t i c u l a t e debris, and the supernatant acid extract was f i l t e r e d through a syringe-mounted 0.45 \Jia M i l l i p o r e f i l t e r p r i o r to d i r e c t i n j e c t i o n (20 ul) into the HPLC. c. A n a l y t i c a l method The HPLC method f o r the determination of toxins from Protogonyaulax was developed by Dr. J . J . S u l l i v a n , who collaborated a c t i v e l y i n the experiments by performing the toxin analysis at the Seafood Products Research Centre, USFDA, Seattle, WA. The method has been previously described i n d e t a i l ( S u l l i v a n and Iwaoka, 1983; S u l l i v a n and Wekell, 1984; S u l l i v a n et a l . , 1985). In the most recent modification ( S u l l i v a n et a l . , 1985), toxin separation was achieved by reverse phase HPLC on a polystyrene divinylbenzene r e s i n column (Hamilton PRP-1, Reno, NV), with PIC B6 (hexane sulfonate, Na) and PIC B7 (heptane sulfonate, Na) (Waters Assoc., M i l f o r d , MA) as ion-pair reagents. Since the PSP toxins lack native fluorescence, they were oxidized to fluorescent d e r i v a t i v e s , according to the basic method of Bates and Rapoport (1975). The mobile phases were as follows: Phase A: H 20, with 1.5 mM PIC B6 and 1.5 mM PIC B7, 1.0 mM (as PO4) ammonium phosphate (pH 7.00); Phase B: as Phase A, but with 20% a c e t o n i t r i l e substituted for H2O. Following post-column oxidation (Kratos URS-051 post-column reaction system; Kratos A n a l y t i c a l , Ramsey, NJ) with 5.0 mM a l k a l i n e periodate i n 50 mM Na 3PO A (pH 7.80), to y i e l d 89 fluorescent d e r i v a t i v e s , and a c i d i f i c a t i o n (0.75 M n i t r i c a c i d ) , toxins were detected by a spectrofluorometer (Perkin-Elmer LS-4; Perkin-Elmer, Norwalk, CT) ( e x c i t a t i o n : 340 nm; emission: 400 nm). The current HPLC method employed a combination of lower periodate concentration and higher reaction temperature, r e l a t i v e to that previously recommended ( S u l l i v a n and Iwaoka, 1983). These conditions were a compromise for the detection of toxins, y i e l d i n g adequate s e n s i t i v i t y for detecting a l l fluorescent d e r i v a t i v e s by favouring oxidation of the N-1 hydroxy toxins (Table 7), NEO, GTX-^  and G T X 4 , which produce weakly fl 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, 1984). A Spectra Physics Model 4270 integrator (Spectra Physics, San Jose, CA) was used to p l o t peak areas. The integrator o c c a s i o n a l l y displayed a tendency to miss the peak corresponding to G T X 4 , as i t often appeared as a shoulder on the more prominent B 2 peak. Substantial baseline d r i f t was evident i n the region of the column c h a r a c t e r i s t i c for NEO and STX, the PSP toxins having the longest retention times. For these reasons, chromatograms p l o t t e d by the integrator were checked manually using the Rf values and toxin response factors from the most recently run standards. Baselines were redrawn, and peaks areas were recalculated as deemed appropriate. The HPLC was r e c a l i b r a t e d a f t e r every f i v e samples by chromatographing a standard mixture of toxins d i l u t e d i n 0.03 M a c e t i c a c i d . P u r i f i e d t o x i n standards were obtained from Dr. Sherwood H a l l (U.S. Food and Drug Administration, Washington, D . C ) . Toxin p r o f i l e s were run i n duplicate, using samples from approximately the same phase of the growth curve of consecutive culture t r a n s f e r s , f or a t o t a l of four determinations. The C - l l s u l f a t e d C-13 carbamyl-N-sulfo-compounds (C^-C^) (Table 7) were not adequately separated by HPLC, as Table 7. Abbreviations and names of PSP toxins from Protogonyaulax spp.; sax i t o x i n and i t s nat 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 STX sa x i t o x i n NEO neosaxitoxin GTX : gonyautoxin 1 GTX 2 gonyautoxin 2 GTX 3 gonyautoxin 3 GTX 4 gonyautoxin 4 »1 gonyautoxin 5 B 2 gonyautoxin 6 C l epigonyautoxin 8 c 2 gonyautoxin 8 C3 c4 Semi-systematic Name saxit o x i n N-1-hydroxysaxitoxin lla-hydroxyneosaxitoxin s u l f a t e lla-hydroxysaxitoxin s u l f a t e HB-hydroxysaxitoxin s u l f a t e HB-hydroxyneosaxitoxin s u l f a t e 2 1-sulfosaxitoxin 21-sulfoneosaxitoxin 21 -sulfo-1 lct-hydroxysaxitoxin s u l f a t e 21-sulfo-113-hydroxysaxitoxin s u l f a t e 21-sulf o-1 loc-hydroxyneosaxitoxin s u l f a t e 21-sulf0-113-hydroxyneosaxitoxin s u l f a t e 91 they co-eluted near the solvent front ( S u l l i v a n and Wekell, 1984; Boyer et a l . , 1985). Consequently, they were amalgamated as the C x toxin complex fo r data a n a l y s i s . Although GTX^ and GTX2 were c l e a r l y separated from t h e i r respective epimers, GTX4 and GTX3, the epimers were pooled to avoid the d i f f i c u l t y i n e s t a b l i s h i n g the equilibrium r a t i o , and the problem of f a c i l e epimerization at C - l l (Boyer, 1980; H a l l et a l . , 1980; H a l l , 1982; H a l l and Reichardt, 1984; Boyer et a l . , 1985). T o x i c i t y f o r each i s o l a t e was calculated from unhydrolyzed samples not subjected to hot 0.1 N HC1 treatment, which would convert B^ and B 2 to STX and NEO, r e s p e c t i v e l y , and C x to GTX^GTX,^. The conversion of the lower t o x i c i t y sulfamate compounds to higher t o x i c i t y carbamate products by hot a c i d hydrolysis i s the basis of the Proctor enhancement (Proctor et a l . , 1975; H a l l , 1982) often used to report PSP t o x i c i t y values. Conversion factors f or s p e c i f i c toxin a c t i v i t y ( i n Mouse units [MU] umol--'-) for the t o x i c i t y c a l c u l a t i o n s (MMU c e l l - 1 ) were as follows (Boyer et a l . , 1985): C 1/C 2 (250), B : (150), B 2 (180), GTX 1/GTX 4 (1700), GTX 2/GTX 3 (1400), NEO (2100) and STX (2050). Previous work comparing mouse bioassay data with values calculated from HPLC p r o f i l e s of NEPCC 255 (Boyer et a l . , 1985) indicated that calculated values agree quite well with bioassay r e s u l t s , considering the assumptions made regarding the s p e c i f i c t o x i c i t y of the various toxin analogues and the lower l e v e l of r e p l i c a b i l i t y of mouse assays (±20%; H a l l , 1982; S u l l i v a n and Iwaoka, 1983). I f anything, the calculated values from HPLC tend to underestimate t o x i c i t y as determined by the bioassay. 92 CHAPTER I I I EXPERIMENTAL RESULTS AND DISCUSSION A. Morphological C h a r a c t e r i s t i c s 1. V a r i a t i o n i n C e l l Size Protogonyaulax c e l l s cultured under uniform conditions varied widely i n s i z e among i s o l a t e s (Table A), yet v a r i a t i o n i n l i n e a r dimensions within a given i s o l a t e , when measurements were made on interphase c e l l s during exponential growth, was l i m i t e d to ~ ±10% C.V. ( c o e f f i c i e n t of v a r i a t i o n ) . Within a chain con s i s t i n g of c e l l s i n interphase, there was considerably less s i z e v a r i a t i o n . The c h a r a c t e r i s t i c c e l l s i z e within an i s o l a t e has been well conserved over time, even a f t e r a period of many years i n cult u r e . The a p i c a l and tr a n s a p i c a l diameters within Protogonyaulax i s o l a t e s followed a frequency d i s t r i b u t i o n which was approximately normal ( F i g . 7). The only notable exception was the d i s t r i b u t i o n of t r a n s a p i c a l diameters of NEPCC 529, that showed a pronounced skewness to the r i g h t . Morphometric measurements of contemporaneous English Bay i s o l a t e s (NEPCC A00-A12) c l e a r l y revealed the p o t e n t i a l f o r c l o n a l v a r i a b i l i t y i n c e l l s i z e among i s o l a t e s from the same geographical l o c a t i o n (Table A). The 1981 i s o l a t e s from English Bay were divided into two s i z e classes: smaller forms with a mean volume ~1 X 10^ unP, including NEPCC A05-A12, and those, such as NEPCC A00-A0A, with a volume approximately 93 F i g . 7 Frequency d i s t r i b u t i o n of a p i c a l and t r a n s a p i c a l diameters (um) of Protogonyaulax i s o l a t e s (n=30). APICAL TRANSAPICAL APICAL TRANSAPICAL 20-u ID-S' O1 NEPCC 183 301 34|36|42 30 34 38 32 36 40 32 36 APICAL TRANSAPICAL NEPCC 2 5 3 20 24|28 22 26 30 APICAL TRANSAPICAL APICAL TRANSAPICAL APICAL TRANSAPICAL 24 26 30 34 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 value. The New Zealand i s o l a t e , NEPCC 508, corresponded i n s i z e to the smaller s i z e c l a s s from English Bay. The smallest i s o l a t e , NEPCC 253 (Laguna Obidos, Portugal) was approximately h a l f the volume of i s o l a t e s i n t h i s s i z e c l a s s . A one-way analysis of variance (ANOVA) using Fisher's F - s t a t i s t i c (Sokal and Rohlf, 1981) was performed to compare the v a r i a t i o n i n c e l l volume between groups of Protogonyaulax i s o l a t e s (Table 8). The analysis showed that s i z e v a r i a t i o n s among i s o l a t e s from English Bay were not s i g n i f i c a n t l y less (a=0.05) than among a l l i s o l a t e s considered together. Variance among c a t e n e l l o i d i s o l a t e s was not s i g n i f i c a n t l y d i f f e r e n t from that among tamarensoid forms. F i n a l l y , i s o l a t e s i n i t i a t e d as clones did not e x h i b i t less s i z e v a r i a t i o n than those begun as mu l t i c l o n a l i s o l a t e s , e i t h e r within a culture or among a l l the 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 C h a r a c t e r i s t i c Features Among Protogonyaulax i s o l a t e s maintained i n culture, the a p i c a l : t r a n s a p i c a l diameter r a t i o s ranged from 0.90-1.13: 1, a maximum deviation from i s o d i a m e t r i c a l proportions of about 10% (Table 4). Certain i s o l a t e s , p a r t i c u l a r l y among the chain-forming c a t e n e l l o i d s , varied more from the 1:1 diameter r a t i o when o r i g i n a l l y i s o l a t e d . A f t e r the f i r s t few culture t r a n s f e r cycles, the production of markedly antero-posteriorly compressed c e l l s u s u a l l y ceased, i n favour of forms which more c l o s e l y approached Table 8. ANOVA comparing variance in c e l l volume between Protogonyaulax i s o l a t e s grouped by location of o r i g i n , present morphotype i n culture and c l o n a l i t y the time of o r i g i n a l culture i n i t i a t i o n , n = number of i s o l a t e s i n each group. Mean Volume d.f. F g S i g n i f i c a n c e of ± s.e.m. (n-1) variance diffe r e n c e Isolates (um 3X10 3) oc=0.05 F a ( 2 ) > F s 5 n s ' F a ( 2 ) > F s 5 n s >  F a ( 2 ) > F s 5 n s ' English Bay/ 15. .12 + 1. .54 10 1, .05 a l l i s o l a t e s 16. .26 + 1. .07 23 Catenelloid/ 16. ,13 + 1, .36 5 3, .55 tamarensoid 16. ,26 + 1, .57 15 Clonal/ 15. ,44 + 1. .70 10 1. .33 non-clonal 16. ,95 + 1. .40 12 "not s i g n i f i c a n t 102 isodiametric proportions. This trend was observed f o r c a t e n e l l o i d i s o l a t e s 435, 529 and 543 from Friday Harbor (San Juan Island, WA), as well as for the group of clones recently i s o l a t e d from Bamfield, B.C. Nevertheless, Protogonyaulax i s o l a t e s derived from compressed c a t e n e l l o i d morphotypes did r e t a i n a v e s t i g i a l tendency to produce s l i g h t l y f l a t t e n e d c e l l s , even i n long-term culture. Once established i n culture, the modified c a t e n e l l o i d morphotypes appeared to be stable and were never observed to revert to the highly compressed forms o r i g i n a l l y obtained from natural populations. The use of the ' fluorescent thecal s t a i n c a l c o f l u o r white M2R, which binds s e l e c t i v e l y to c e l l u l o s e , rather than formulations of c h l o r a l hydrate-iodine-hydriodic acid (Y. Fukuyo, pers. comm., based on the Von Stosch, 1969 technique), made possible the re s o l u t i o n of d e t a i l s at the plate margins, such as the presence of the l 1 v e n t r a l pore (Figs. 1 and 3), from i n t a c t c e l l s . When c a l c o f l u o r M2R was used on empty thecae ( F i g . 8) or thecal squashes ( F i g . 9), d e t a i l s of the APC, attachment pores and plate margins were r e a d i l y v i s i b l e . S i g n i f i c a n t l y , the v e n t r a l 1' pore was absent from a l l the c a t e n e l l o i d i s o l a t e s examined under epifluorescence and phase-contrast microscopy. Among the tamarensoid and intermediate i s o l a t e s , the occurrence of the ve n t r a l 1" pore was not geographically consistent. Three i s o l a t e s from English Bay, 404 and 412 from the 1981 bloom, and 516, from the same st a t i o n i n 1982, displayed t h i s feature, while the re s t d i d not (Table 4). Thus, i s o l a t e s possessing the pore may not simply be considered as geographically i s o l a t e d v a r i a n t s . Yet, t h i s feature ( i f present) i s stable within a given i s o l a t e i n culture over time, and appears not to be acquired by an i s o l a t e i f not o r i g i n a l l y present. For example, the existence of a 103 F i g . 8 A p i c a l view of empty theca of NEPCC 403 stained with 0.1% c a l c o f l u o r showing epithecal plates under epifluorescence microscopy (400X). Exposure: 1 min; Ektachrome ASA 160. F i g . 9 A p i c a l view of epith e c a l plates of NEPCC 516 stained with 0.1% c a l c o f l u o r showing v e n t r a l pore on the f i r s t a p i c a l ( l 1 ) p l ate under epifluorescence microscopy (800X). Exposure: 1 min; Ektachrome ASA 160. 105 v e n t r a l pore i n NEPCC 71, f i r s t shown by Turpin et a l . (1978) i n a SEM micrograph, was independently confirmed i n 1979 (Schmidt and Loeblich, 1979a) by examination of iodine-hydriodic acid stained thecae under phase-contrast microscopy. The present epifluorescence microscopic observations, made almost a decade a f t e r the o r i g i n a l i s o l a t i o n , continued to show the presence of the v e n t r a l pore. Nevertheless, t h i s feature varied from a c l e a r l y defined pore i n some specimens, to a v e s t i g i a l notch at the 1' plate margin, even among c e l l s from the same culture. Although NEPCC 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 "tamarensis" type, on the basis of the diameter r a t i o s and i t s i n a b i l i t y to form chains i n culture, i t possesses the prominent hypothecal flange and highly domed epicone c h a r a c t e r i s t i c of Gonyaulax acatenella sensu Loeblich and Loeblich (1975), and as previously described by Prakash and Taylor (1966) from Malaspina I n l e t , B.C. 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 recognized immediately upon i s o l a t i o n by R. Waters and were noted i n a b r i e f record i n the NEPCC species l i s t . The general form has been conserved i n cul t u r e since 1973, and continues to be i n accord with the de s c r i p t i o n l a t e r given by Turpin et a l . (1978). The major discrepancies between the o r i g i n a l d e s c r i p t i o n of acatenella (Whedon and Kofoid, 1936) and NEPCC 71 i s the consistent dark brown pigmentation and the v e n t r a l l 1 pore i n the cultured NEPCC i s o l a t e . The acatenella type specimens were almost without pigmentation, although t h i s was not thought to be highly s i g n i f i c a n t . I s o l a t e 180 forms large egg-shaped c e l l s with rounded epicones and hypocones ( F i g . 10). The c e l l s of t h i s i s o l a t e are usu a l l y heavily pigmented. In NEPCC 181(b) ( F i g . 11), i s o l a t e d as P. ca t e n e l l a from Pat Bay, B.C. by R. Waters i n 1977, the formation of chains has ceased completely i n 106 F i g . 10 Photomicrograph of 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 equatorial view (250X). Exposure: f l a s h 1/125 s; Ektachrome EPY ASA 50. F i g . 11 Photomicrograph of NEPCC 181(b), i s o l a t e d as a chain-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 equatorial view (400X). Exposure: f l a s h 1/125 s; Ektachrome EPY ASA 50. 108 culture. The epicone has become high-domed and bell-shaped, while the hypocone remains rather f l a t t e n e d . This i s o l a t e could be only poorly maintained i n culture on seawater from Burrard I n l e t , B.C. enriched with ESNW or NWSP-7. Since the d i v i s i o n rate was <0.1 div d - 1 , i t was omitted from further experimentation. NEPCC 183 (= Plymouth i s o l a t e 173) from the Tamar estuary, the type l o c a l i t y of tamarensis, has been maintained without s u b s t a n t i a l morphological change i n culture since 1957. I t continues to conform well with the 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 lack of chain formation. The Portuguese i s o l a t e NEPCC 253, while tamarensoid i n o v e r a l l morphology and plate patterns, i s s i g n i f i c a n t l y smaller than the t y p i c a l "tamarensis". This i s o l a t e i s capable of achieving unusually high c e l l d e n s i t i e s i n cult u r e (>1 X 10^ m l - 1 ) and i t always maintains a greenish-hued pigmentation u n c h a r a c t e r i s t i c of P. tamarensis. Thin-layer chromatography of i t s pigments on c e l l u l o s e plates revealed a low r a t i o of accessory pigments: c h l o r o p h y l l , with undetectable l e v e l s of p e r i d i n i n (A. Cembella, unpublished r e s u l t s ) . I s olate 254 (Nelson Island, B.C.), o r i g i n a l l y derived from excysted i n d i v i d u a l s , was p r o v i s i o n a l l y c l a s s i f i e d i n a previous p u b l i c a t i o n (Turpin et a l . , 1978) as a s t r a i n of Gonyaulax tamarensis resembling the t r o p i c a l form G. excavata sensu Balech (1971), except f o r the absence of a ve n t r a l pore. In long-term culture, t h i s i s o l a t e has assumed a fl a t t e n e d morphology and the a b i l i t y to form chains of c e l l s . I t would now be more appropriately designated as a "c a t e n e l l a " , by v i r t u e of i t s angularly f l a t t e n e d apex and antapex, with c e l l s wider than long. Nevertheless, NEPCC 254 has not varied appreciably i n o v e r a l l s i z e from that reported by 109 Turpin et a l . (1978), two years a f t e r i t was i s o l a t e d into culture, to the present time. The i s o l a t e from New Zealand, (NEPCC 508), l i k e NEPCC 253 from Portugal, i s a small tamarensoid form lacking a v e n t r a l pore that never forms chains i n culture, yet i s s i m i l a r l y capable of a t t a i n i n g high c e l l d e n s i t i e s . The c e l l s of NEPCC 508 also have a tendency to appear greenish i n colour, rather than brownish, but the dif f e r e n c e i s less s t r i k i n g than f o r the Portuguese i s o l a t e . The New Zealand i s o l a t e possesses a very narrow s i x t h precingular plate, which may serve to separate i t from "tamarensis" as a new morphospecies (Taylor, 1984). Isolate 516 (English Bay, B.C.) i s more morphologically v a r i a b l e i n culture than the other Protogonyaulax i s o l a t e s . Many c e l l s are of isodiametric proportions and are intermediate i n morphology between ca t e n e l l a and tamarensis. For most specimens, the epicone i s egg- or bell-shaped, although c e l l s with f l a t t e n e d epicones are also observed. The hypocone us u a l l y tapers toward the antapex, yet f l a t t e n e d antapices also occur. This i s o l a t e appears to lack the a b i l i t y to form chains longer than four c e l l s i n cult u r e . NEPCC 545, a tamarensis morphotype from the Bay of Fundy, conforms to the d e s c r i p t i o n of Gonyaulax excavata sensu Loeblich and Loeblich (1975) i n that i t i s t o x i c , bioluminescent and lacks a v e n t r a l pore. The shape of the APC among Protogonyaulax i s o l a t e s varied from narrow or wide subrectangular, to regular ovoid, tear-drop shaped, t r i a n g u l a r or lamb-chop shaped, depending upon the i s o l a t e ( F i g . 12). In general, the shape of the APC was conserved within 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 were examined at the same point of the culture cycle. However, there were some notable exceptions. In NEPCC 508 (New Zealand), the shape of the APC 110 F i g . 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 posterior s u l c a l plate of Protogonyaulax i s o l a t e s i n culture. N E P C C 71 o NEPCC 180 a NEPCC 183 o NEPCC 2 5 3 a NEPCC 255 o NEPCC 355 D NEPCC 4 0 0 & o N E P C C 401 ® D NEPCC 402 0 NEPCC 403 NEPCC 404 ® NEPCC 405 NEPCC 406 o NEPCC 407 O NEPCC 409 NEPCC 412 M K3 NEPCC 435 o NEPCC 508 NEPCC 516 a NEPCC 529 NEPCC 543 a NEPCC 545 (3) o 114 v a r i e d from c l e a r l y rectangular (most common), to an ovoid form tapering toward the base that contacted the f i r s t a p i c a l plate. The APC i n NEPCC 516 was u s u a l l y lamb-chop shaped, but occasionally regular ovoid forms were observed. In NEPCC 529 (San Juan Island, WA), a chain-forming " c a t e n e l l a " , the APC showed an intergradation between a round-contoured and an angular form, with c h a r a c t e r i s t i c differences i n the shape of the associated a p i c a l pore. In another c a t e n e l l o i d morphotype from the same l o c a t i o n , NEPCC 543, the APC varied from lamb-chop to tear-drop shaped i n exponential phase cultures. Occasionally, an anterior attachment pore, adjacent to the hook-shaped a p i c a l pore of the APC, and a posterior attachment pore on the p o s t e r i o r s u l c a l p l a t e , were observed. A p o s t e r i o r pore i s evident i n the photomicrograph of the hypothecal plates of a tamarensoid i s o l a t e from the New England region ( F i g . 13). These corresponding attachment pores were not consistent features within an i s o l a t e . The pores were indicated i n the drawings of the APC and p o s t e r i o r s u l c a l plate of the i s o l a t e s ( F i g . 12) i f they were commonly present. There was no c l e a r r e l a t i o n s h i p between the p o s i t i o n of the p o s t e r i o r pore r e l a t i v e to the posterior s u l c a l plate margin and morphotype. The shape of the p o s t e r i o r s u l c a l plate i n cultured i s o l a t e s could not be c l o s e l y linked with the assigned morphotype. In c a t e n e l l o i d specimens from natural populations from Bamfield, B.C. (A. Cembella, pers. obs.) and Japan (Y. Fukuyo, pers. comm. regarding Japanese Protogonyaulax), the p o s t e r i o r s u l c a l p l a t e appeared unusually wide i n c e l l s that were markedly compressed and i n chains, compared to that of tamarensoids. 115 F i g . 13 Antapical view of hypothecal plates from tamarensoid specimens stained with c h l o r a l hydrate-iodine-hydriodic acid, examined under phase-contrast microscopy (250X). The po s t e r i o r s u l c a l plate i s shown with a pore near the plate margin. Massachusetts specimens from D.M. Anderson; courtesy of F.J.R. Taylor. F i g . IA Photomicrograph of chain-forming P. c a t e n e l l a i n a phytoplankton sample from Puget Sound, WA (250X). Courtesy of F.J.R. Taylor. 116 117 3. Chain Length The tendency for c a t e n e l l a - l i k e i s o l a t e s to form long chains of antero-posteriorly f l a t t e n e d c e l l s , a c h a r a c t e r i s t i c t r a i t i n natural populations ( F i g . 14), was l o s t following i n i t i a t i o n of laboratory cultures, u s u a l l y a f t e r several t r a n s f e r s . There was an apparent inverse c o r r e l a t i o n between chain length and the progression from exponential through stationary growth phase i n cultures of NEPCC 355 ( F i g . 15 and 16a-d). At the time of the t r a n s f e r to the experimental medium (Day 0), c e l l s of the stock culture inoculum had reached the boundary between exponential and stationary phase. Two hours a f t e r inoculation, >90% of the c e l l s existed as s i n g l e t s - the remainder were present as duplets; no longer chains were observed. During mid-exponential growth (by Day 13), the proportion of s i n g l e t s declined to ~60%, concomitant with the appearance of chains longer than two c e l l s i n the c o n t r o l culture ( F i g . 16a). S t a t i s t i c a l l y i n s i g n i f i c a n t numbers of chains of up to 12 c e l l s were observed i n t h i s phase. The percentage of s i n g l e t s and duplets remained approximately constant from mid-exponential through stationary phase, yet there was an apparent s l i g h t decrease i n the r e l a t i v e number of chains of four c e l l s or more a f t e r Day 13. From the growth curves ( F i g . 15), i t was evident that growth under low Fe conditions resulted i n a lower i n i t i a l growth rate, r e l a t i v e to the c o n t r o l , probably through prolongation of the lag phase. Nevertheless, by Day 13, the growth rate increased i n the low Fe cultures to give a maximal c e l l y i e l d at Day 28 approximately equal to the control cultures. The low Fe cultures were apparently able to scavenge s u f f i c i e n t r e s i d u a l Fe from the medium, or to draw on i n t e r n a l reserves, to eventually achieve high 118 F i g . 15 Growth curves of NEPCC 355 on control ESNW medium ( • ) , low N ( A ) , low P (•) and low Fe ( x ) . Error bars: ±1.0 s.d. 6TT 120 F i g . 16 Histograms of the percentage of c e l l s present i n chains of various lengths throughout the growth cycle of NEPCC 355. 16a, standard ESNW medium; 16b, low Fe; 16c, low P; 16d, low N. Number of c e l l s per chain: l c - s i n g l e t s ; 2 C- duplets; 3 C- t r i p l e t s ; A c- quadruplets, etc. 121 122 O >-< I I I ! I • 1 i 1 t 5 o o o D O) 00 N O CD O IO o o 10 o CM o o SIT30 TV101 JO % 124 S 1 1 3 0 1 V 1 0 1 JO % 125 growth rates. Between Day 13 and 17, when the growth rates were approximately equal i n the low Fe and con t r o l cultures, the percentage of c e l l s i n multiples increased s i g n i f i c a n t l y i n the low Fe cultures, while remaining approximately constant i n the controls ( F i g . 16a and b). The decline i n the percentage of s i n g l e t s i n the low Fe cultures was mirrored i n a r e l a t i v e increase i n chains of more than two c e l l s during exponential phase ( F i g . 16b). As the c e l l s entered stationary phase, and then progressed through senescence (approximately Day 24-36), the percentage of s i n g l e t s r e l a t i v e to c e l l s i n chains rose. C e l l s i n the low N and low P cultures divided at a markedly lower rate than those i n the low Fe and con t r o l cultures, and reached a much lower maximal c e l l density ( F i g . 15). Both growth rate and c e l l y i e l d l i m i t a t i o n were c l e a r l y evident f o r the low N and low P cultures. In the P-limited cultures, the end of exponential growth was approached around Day 13. By t h i s time, the percentage of c e l l s i n multiples had increased s l i g h t l y from i n i t i a l values ( F i g . 16c). In stationary phase, the r a t i o of s i n g l e t s to multiples i n the P-limited cultures rose marginally. Singlets always comprised >75% of the t o t a l c e l l s throughout the growth curve. The growth rate i n low N cultures was lower than that of low P cultures, but c e l l numbers i n the low N medium, continued to increase slowly u n t i l Day 28 (Fi g . 15), to give approximately the same c e l l y i e l d . Although the growth rates f o r the low N and low P cultures were s i m i l a r (close to 0) between Day 13 and 36, the chain length histograms were s u b s t a n t i a l l y d i f f e r e n t ( F i g . 16c and d). During t h i s period, the percentage of c e l l s i n chains decreased dramatically i n the low N cultures, while i t remained roughly constant i n the low P cultures. In general, the se r i e s of percentage histograms showing trends i n chain 126 length f o r the low N cultures resembles that f o r the low Fe cultures ( F i g . 16b and d). In both cases, the greatest proportion of chains occurred i n exponential phase, while chain formation decreased i n senescence. However, a greater percentage of c e l l s i n chains longer than two i n d i v i d u a l s was found i n low Fe than i n low N cultures. No chains of greater than f i v e c e l l s were ever observed i n low N cultures. 4. Discussion In Protogonyaulax, m i t o t i c d i v i s i o n r e s u l t s i n the formation of daughter c e l l s which are smaller than the parent c e l l immediately following cytokinesis. However, the c e l l s r a p i d l y achieve the s i z e c h a r a c t e r i s t i c of the p a r t i c u l a r i s o l a t e i n post-mitotic growth. This s i z e i s constant throughout interphase, u n t i l the next m i t o t i c d i v i s i o n i s i n i t i a t e d . Since chains represent clones of c e l l s d i v i d i n g approximately i n synchrony (Tomas, 1974), the observed uniformity i n the s i z e of interphase c e l l s within a chain i s not unexpected. The f a c t that c e l l s i z e i s e s s e n t i a l l y constant during exponential phase within a given i s o l a t e , and that the marked differences among i s o l a t e s are maintained when the culture regime i s alt e r e d , strongly suggests that the c h a r a c t e r i s t i c s i z e of Protogonyaulax i s o l a t e s i s fundamentally determined by genetic, rather than by environmental f a c t o r s . The accumulated evidence from DNA analysis and breeding a f f i n i t y studies i n d i n o f l a g e l l a t e s also indicates that such s i z e v a r i a t i o n has a genetic basis. Within the Crypthecodinium cohnii (Beam and Himes, 1982) and Symbiodinium microadriaticum (Schoenberg, 1976; Schoenberg and Trench, 127 1980b) species complexes, g e n e t i c a l l y d i f f e r e n t i s o l a t e s that were morphologically d i s t i n g u i s h a b l e only on the basis of s i z e differences could be segregated into d i s t i n c t s i z e classes. Some Protogonyaulax i s o l a t e s , such as NEPCC 253, and perhaps i s o l a t e s 508 and the sma l l e r - c e l l e d forms from English Bay, may be analogous to the "small forms" of d i n o f l a g e l l a t e s reported from European waters by S i l v a (1971; 1977). There i s evidence that some environmental factors which can a f f e c t growth rate have l i t t l e influence on c e l l s i z e . For example, White (1978b) found that c e l l s i z e of a P. tamarensis i s o l a t e was constant within the s a l i n i t y range from 6-43°/oo, although the growth rate varied s u b s t a n t i a l l y . 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 nutrient l i m i t a t i o n , do have a marked e f f e c t on mean c e l l s i z e . The t y p i c a l response of Protogonyaulax c e l l s to N - l i m i t a t i o n i s the production of abundant p e l l i c u l a r cysts and a reduction i n vegetative c e l l s i z e (A. Cembella and G. Boyer, pers. obs.). On the other hand, P - l i m i t a t i o n r e s u l t s i n the formation of large lumpy c e l l s that appear unable to complete mitosis. I t i s d i f f i c u l t to morphologically d i s t i n g u i s h between the large immotile vegetative c e l l s formed i n c l o n a l cultures, under P-depletion, and those which appear i n m u l t i c l o n a l cultures following sexual fusion of gametes p a r t i c u l a r l y when the q u a d r i f l a g e l l a t e stage has passed (A. Cembella, pers. obs.; Anderson and Lindquist, 1985). The d i f f e r e n t i a l e f f e c t of N- and P - l i m i t a t i o n suggests that changes i n c e l l s i z e are more l i k e l y a d i r e c t metabolic response to l i m i t a t i o n by a s p e c i f i c n u t r i e n t , than a non-specific function of the attenuated growth rate. According to volume measurements made with the Coulter counter (Prakash, 1967; Watras et a l . , 1982), i t was previously found that c e l l s d i v i d i n g r a p i d l y during e a r l y exponential phase were smaller than those 128 formed i n mature cultures. Yet, a decrease i n the s i z e of P. tamarensis c e l l s as the cultures approached stationary phase has also been previously observed (White and Maranda, 1978). Unfortunately, the s p e c i f i c f a c t o r ( s ) l i m i t i n g growth i n these experiments cannot be c l e a r l y deduced. As a cautionary note, i t should be recognized that as Protogonyaulax cultures age, the percentage of aberrant morphotypes, p e l l i c u l a r cysts (~12-20 nm), thecal fragments from ecdysed c e l l s , b a c t e r i a and gametes (possibly anisogamous), increases. Furthermore, chain length i n those i s o l a t e s forming chains varies throughout the culture cycle. Use of the Coulter counter to determine s i z e d i s t r i b u t i o n s i n Protogonyaulax cultures (Prakash, 1967; Watras et a l . , 1982), may lead to severe misinterpretations i f the r e s u l t s are considered as s t r i c t l y i n d i c a t i v e of " c e l l s i z e " . The Coulter counter y i e l d s only indiscriminant measurements of s p h e r i c a l volume of undefined p a r t i c l e s , which may not c o r r e l a t e well with the s i z e of d i s t r i b u t i o n vegetative c e l l s , p a r t i c u l a r l y for mature and senescent cultures. In an e f f o r t to integrate morphological c h a r a c t e r i s t i c s f o r use i n taxonomic an a l y s i s , i t i s tempting to speculate upon the possible linkages between the occurrence of p a r t i c u l a r thecal plate features and general morphology i n Protogonyaulax. The presence or absence of a v e n t r a l pore i s a u s e f u l diagnostic character to d i s t i n g u i s h between i s o l a t e s , i n that the t r a i t i s c o n s i s t e n t l y maintained within a given s t r a i n . I t s stable persistence over many years, regardless of the environmental regime or growth stage i n culture, leaves l i t t l e doubt that t h i s character i s g e n e t i c a l l y f i x e d . However, i t i s of l i t t l e use to discriminate unequivocally between "tamarensis" and " c a t e n e l l a " , since although the pore i s apparently lacking i n " c a t e n e l l a " , i t may be present or absent i n 129 "tamarensis". With the notable exception of the v e n t r a l 1' pore v i s i b l e i n the SEM micrographs of c e l l s i d e n t i f i e d as " c a t e n e l l a " from Washington State (Postek and Cox, 1976), v e n t r a l pores have not been associated with specimens conforming to the c a t e n e l l o i d morphotype. This leaves open the p o s s i b i l i t y that such pores may be f i l l e d i n and c r y p t i c under l i g h t microscope observation i n some Protogonyaulax specimens (Taylor, 1984). I t i s also conceivable that the species was o r i g i n a l l y m i s i d e n t i f i e d - the general morphology of the c e l l s depicted i n the micrographs of Postek and Cox (1976) i s rather "tamarensis"-like. The pore on the posterior s u l c a l plate i s c h a r a c t e r i s t i c a l l y associated with c e l l s e x i s t i n g i n pai r s or chains. The pos t e r i o r pore i s t y p i c a l l y absent i n mature i n d i v i d u a l c e l l s and the terminal i n d i v i d u a l i n duplets and longer chains. Both the anterior and posterior attachment pores are often sealed and become i n v i s i b l e as c e l l s mature and cease chain formation. Since most of the NEPCC Protogonyaulax i s o l a t e s existed predominantly as s i n g l e t s i n culture, the r e l a t i v e l y uncommon appearance of the attachment pores i s not unexpected. The occurrence of chains i n culture i s a function of the growth stage, i s affe c t e d by environmental fa c t o r s , such as nutrient l i m i t a t i o n , and i s perhaps, d i r e c t l y or i n d i r e c t l y , linked to the d i v i s i o n rate. As a consequence, the attachment pores are transient features and are 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 species descriptors (Loeblich and Loeblich, 1975; Schmidt and Loeblich, 1979a; Taylor, 1984). The po s t e r i o r s u l c a l plate and the dorsal end of the APC may be wider i n the compressed c e l l s of the c a t e n e l l o i d morphotype than i n c e l l s which are not f l a t t e n e d . The greater distance from the pos t e r i o r attachment pore to the margin of the pos t e r i o r s u l c a l plate, as i n " c a t e n e l l a " sensu Fukuyo 130 (1985), may be due to the widening of the plate i t s e l f . That t h i s c h a r a c t e r i s t i c was not evident i n the cultured " c a t e n e l l a " i s o l a t e s examined, may r e f l e c t the tendency f o r formation of extremely wide c e l l s , with correspondingly broad posterior s u l c a l plates, to cease i n culture. The decrease i n chain length when Protogonyaulax i s o l a t e s from natural populations are brought into culture i s true also of diatoms (Taylor, 1980b). The reason(s) f o r the loss of chain formation i n culture remain speculative. Possible explanations to be considered include: p h y s i c a l / mechanical f a c t o r s , genetic s e l e c t i o n i n culture, environmentally induced phenotypic changes and the e f f e c t of growth rate per se. Cursory observations of r a p i d l y s t i r r e d and air-bubbled Protogonyaulax cultures has shown that mechanical a g i t a t i o n r e s u l t s i n the breaking of chains of c e l l s . The impact of chains against the f l a s k walls may also contribute somewhat to the reduction i n chain length, even i n u n s t i r r e d cultures. However, t h i s explanation f o r the f a i l u r e to produce long chains i n culture i s weakened somewhat by the observations of long chains i n natural populations, even i n those found i n highly turbulent waters, such as i n the surf zone. Furthermore, mechanical breakage does not account f o r the stable differences i n chain-forming tendency among i s o l a t e s , nor between d i f f e r e n t stages i n the culture cycle. The genetic p r e d i s p o s i t i o n possessed by c e r t a i n s t r a i n s f o r the formation of long chains, nevertheless requires the proper combination of favourable environmental conditions f o r expression. When i s o l a t e d c e l l s are brought into culture, they are subject to short-term shock e f f e c t s , u s u a l l y r e s u l t i n g i n an extended lag phase before rapid c e l l d i v i s i o n i s resumed. During t h i s i n i t i a l acclimation period there may be genetic s e l e c t i v e pressures against the long-chain forming phenotypes. In 131 m u l t i c l o n a l c ultures, t h i s could r e s u l t i n the rapid dominance of a short-chain forming clone with a high growth rate, within a few tr a n s f e r cycles a f t e r i s o l a t i o n . In c l o n a l cultures, the switch from the i n i t i a l production of long-chains to short-chains and s i n g l e t s may be the r e s u l t of the f i x a t i o n of a random favourable or neutral mutation f o r shorter chains. I f the formation of long chains i n natural populations i s an adaptation f o r increasing buoyancy, as has been hypothesized f o r diatoms (Taylor, 1980b), i t may be less advantageous as a s u r v i v a l strategy i n small volume homogeneous cultures where sinking i s les s s i g n i f i c a n t . These genetic in t e r p r e t a t i o n s hypothesize that the lack of chain-forming phenotypes i n culture r e s u l t s from the loss of capacity f o r chain formation and represents the "genetics of survivors". As any a l t e r n a t i v e hypothesis, perhaps i t i s not the genetic p o t e n t i a l f o r the expression of the long-chain phenotype that i s l o s t , but only that the conditions f o r the expression of the genotype are unfavourable. Protogonyaulax forming long chains have never been observed i n phytoplankton samples from Burrard I n l e t , and i s o l a t e s from t h i s area have always conformed to the tamarensoid or intermediate morphotypes (Table A). Furthermore, i s o l a t e s from elsewhere grown on enriched seawater from Burrard I n l e t r a r e l y formed long chains, even when they were i s o l a t e d as chain-forming c a t e n e l l o i d s . I t may be s i g n i f i c a n t that NEPCC 181(b), i s o l a t e d as a " c a t e n e l l a " , resumed the formation of chains when subcultured i n the U n i v e r s i t y of Washington (Seattle) culture c o l l e c t i o n on enriched seawater from Puget Sound, and when grown by Dr. Maria Ross (University of Southern C a l i f o r n i a ) on water from the Los Angeles region, where the c a t e n e l l o i d form i s dominant i n natural populations. The expression of the "c a t e n e l l a " morphotype may be i n h i b i t e d by growth on "tamarensis water". 132 However, since i s o l a t e 181(b) survives only marginally on Burrard I n l e t water, i t i s d i f f i c u l t to factor out the e f f e c t of growth rate 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 chain production. The higher percentage of c e l l s of P. c a t e n e l l a occurring i n chains during exponential growth than i n stationary phase has been previously noted (Tomas, 1974). The i n t e r p r e t a t i o n of increasing chain length as merely due to the f a i l u r e of c e l l s to separate when d i v i d i n g r a p i d l y , focuses on the c e l l d i v i s i o n rate per se, rather than on s p e c i f i c environmental and genetic factors that can a f f e c t growth rate. This would imply that growth rates i n cultures where long chains are not formed are well below those i n natural " c a t e n e l l a " populations. Although r e l i a b l e estimates of i n s i t u growth rates are d i f f i c u l t to obtain, there i s no evidence that t h i s i s the case (Norris and Chew, 1975). Another implication, that tamarensoid morphotypes have a lower growth rate i n natural populations than c a t e n e l l o i d s , has also not been supported. The evidence from acclimated growth rate studies i n culture (Chapter III B.l) indicated that the growth rates of " c a t e n e l l a " and "tamarensis" were not s i g n i f i c a n t l y d i f f e r e n t . Since the progressive trends i n chain length throughout the culture cycle for the low Fe and low N cultures were s i m i l a r , yet the growth rates and c e l l y i e l d s were markedly d i f f e r e n t , chain length appeared to be more a function of the phase of the culture cycle, than the absolute growth rate. On the other hand, the influence of higher growth rate on chain length was expressed i n the longer chains formed i n exponential phase i n the low Fe than the low N culture. Nutrient l i m i t a t i o n may a f f e c t chain length by d i r e c t a f f e c t s on the growth rate or by i n d i r e c t a f f e c t s on the morphology. In the former case, 133 for example, N- or P - l i m i t a t i o n may r e s u l t i n the i n a b i l i t y of c e l l s to undergo rapid mitosis, due to the lack of these required nutrients f o r nuc l e i c acid and protein synthesis. In the l a t t e r case, nutrient l i m i t a t i o n could lead to s t r u c t u r a l d e f i c i e n c i e s causing thecal p l a t e d i s t o r t i o n s and aberrant morphotypes, which are incompatible with chain formation. The use of morphological features, such as the presence of the v e n t r a l 1' pore, the presence and p o s i t i o n of the anterior and po s t e r i o r attachment pores, and the shape of the APC and pos t e r i o r s u l c a l plate, as advocated by Fukuyo and co-workers (Fukuyo, 1980; 1985; Fukuyo et a l . , 1985) to separate " c a t e n e l l a " from "tamarensis", cannot be viewed as s t r i c t l y diagnostic f o r specimens from a l l environments. Although these c r i t e r i a may be narrowly applicable to Protogonyaulax spp. within the r e s t r i c t e d geographical range of the Japanese coast, severe problems a r i s e when they are extended to other regions. At le a s t within and among populations from the northeast P a c i f i c , as i n cultu r e , i t i s not cl e a r that these features do not represent a rather continuous morphological spectrum i n f i e l d populations. There are other d i f f i c u l t i e s involved i n the a p p l i c a t i o n of the revised descriptions f o r "tamarenis" and " c a t e n e l l a " . For example, by Fukuyo's diagnosis, the presence of the v e n t r a l pore i n only some contemporaneous i s o l a t e s from English Bay, would assign the pore-bearing i s o l a t e s to "tamarensis", while those without the pore, present even i n the same water sample, would s h i f t to "c a t e n e l l a " . The shape of the posterior s u l c a l p l a t e and the lack of a v e n t r a l pore i n some Massachusetts "tamarensis" i s o l a t e s would reassign them to " c a t e n e l l a " (Taylor, 1984). Even those "tamarensis" i s o l a t e s from the type l o c a l i t y i n the Tamar estuary, England which do not have a v e n t r a l pore (e.g., Plymouth 173a), and some of the 134 non-pore bearing tamarensoid morphotypes from the Gulf of Maine, the Bay of Fundy, and O s l o f j o r d , Norway ("excavata" sensu Loeblich and Loeblich, 1975) would move to " c a t e n e l l a " . This r e i n t e r p r e t a t i o n requires invoking a spec i a t i o n mechanism whereby stable species d i f f e r e n c e s , expressed morphologically, would a r i s e within sympatrically d i s t r i b u t e d populations. At present, the r e d e s c r i p t i o n appears to introduce an unacceptable l e v e l of taxonomic r e v i s i o n and d i s t o r t i o n , while r e l y i n g too heavily upon minor morphological differences of unknown e c o l o g i c a l and genetic s i g n i f i c a n c e . Unfortunately, the more conventionally used morphological c r i t e r i a , c e l l shape and chain formation, are apparently even more subject to v a r i a t i o n and intergradation. B. Growth Rates 1. Acclimated Growth Rates of Protogonyaulax i s o l a t e s a. Results Protogonyaulax i s o l a t e s varied widely i n acclimated growth rate (X = 0.66 ± 0.42 s.d.; range: 0.06 - 1.42 div d" 1) ( F i g . 17). The data were analyzed by a one-way ANOVA using Fisher's F-test (Sokal and Rolhf, 1981), to compare variance i n growth rates between i s o l a t e s grouped by l o c a t i o n of o r i g i n , present morphotype i n culture and c l o n a l i t y at the time of i s o l a t i o n (Table 9). Since the number of i s o l a t e s examined varied considerably among groups, and the variances i n growth rates between groups were sometimes s i g n i f i c a n t l y d i f f e r e n t , the nonparametric Mann-Whitney 135 F i g . 17 Histogram of acclimated growth rates of Protogonyaulax i s o l a t e s from Bamfield and English Bay i n B r i t i s h Columbia, and other regions. Table 9. ANOVA comparing variance i n acclimated growth rates between Protogonyaulax i s o l a t e s grouped by lo c a t i o n of o r i g i n , present morphotype i n culture and c l o n a l i t y at the time of i s o l a t i o n , n = number of is o l a t e s i n each group. Variance ( s 2 ) d.f. F s in k (div d" 1) (n-1) Isolates A l l i s o l a t e s / 0.18 43 7.29 Bamfield 0.02 22 A l l i s o l a t e s / 0.18 43 1.05 English Bay 0.17 10 English Bay/ 0.17 10 8.50 Bamfield 0.02 22 Bamfield/ 0.02 22 9.54 non-Bamfield 0.23 20 English Bay/ 0.17 10 1.55 non-English Bay 0.11 32 A l l tamarensoid/ 0.17 12 2.43 a l l c a t e n e l l o i d 0.07 28 Non-EB/BF tamarensoid/, 0.16 3 1.63 non-EB/BF c a t e n e l l o i d " 0.26 5 Si g n i f i c a n c e of variance d i f f e r e n c e a=0.05 F s > F a ( 2 ) ; s * * F s < F a ( 2 ) 5 ns*** Fs> Fa(2)5 s * * F s > F a ( 2 ) ; s * * Fs< Fa(2)5 n s * * * F s < F a ( 2 ) 5 ns^ s ~ a ( 2 ) Variance (s~") in k (div d , Isolates A l l c l o n a l / 0.18 a l l non-clonal 0.19 English Bay c l o n a l / 0.12 English Bay non-clonal 0.18 d.f. (n-1) 32 10 4 5 1.06 1.50 Si g n i f i c a n c e of variance d i f f e r e n c e <x=0.05 Fs< Fcx(2); ^ * * * F s < F a ( 2 ) ; " s * * * ''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 either English Bay or Bamfield were compared. " " s i g n i f i c a n t not s i g n i f i c a n t 139 U-test (Zar, 1974) was used to tes t f o r s i g n i f i c a n t d ifferences i n mean acclimated growth rate between groups (Table 10). When grouped by geographical l o c a t i o n of o r i g i n , the growth rates among recent contemporaneous i s o l a t e s from Bamfield, B.C. were shown to be s i g n i f i c a n t l y l e s s v a r i a b l e than among populations not from Bamfield, and among a l l i s o l a t e s considered together (a=0.05). In p a r t i c u l a r , the Bamfield group was 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 growth rate than the i s o l a t e s from English Bay, B.C. The mean growth rate of the Bamfield c a t e n e l l o i d i s o l a t e s was also s i g n i f i c a n t l y lower than that of i s o l a t e s o r i g i n a t i n g elsewhere (a=0.05). Within the group of i s o l a t e s o r i g i n a t i n g from English Bay, the variance i n growth rates was not s i g n i f i c a n t l y d i f f e r e n t from that of i s o l a t e s from other regions nor among a l l i s o l a t e s . However, the mean growth rate of English Bay i s o l a t e s was notably higher than f o r Bamfield i s o l a t e s and among a l l i s o l a t e s . When grouped by morphotype, there were no apparent differences i n growth rate v a r i a t i o n between the c a t e n e l l o i d and tamarensoid forms, neither when a l l i s o l a t e s were considered together, nor when only those i s o l a t e s which did not come from e i t h e r E n g l i s h Bay or Bamfield were compared. Among a l l i s o l a t e s , the mean growth rate of tamarensoid forms was higher than that of 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 longer apparent when the English Bay and Bamfield populations were excluded. Isolates derived from a si n g l e c e l l exhibited no marked differences i n mean growth rates or genotypic v a r i a t i o n compared with those o r i g i n a t i n g as mu l t i c l o n a l i s o l a t e s . This was true among a l l the i s o l a t e s , and when only i s o l a t e s from English Bay were considered. The maximal growth rates presented i n Table 11 were c o l l e c t e d from the Table 1 0 . Nonparametric Mann-Whitney U-test for the s i g n i f i c a n c e of the d i f f e r e n c e i n mean acclimated growth rates for 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 , present morphotype i n culture and c l o n a l i t y at the 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. Mean growth rate Isolates k (div d - 1 ) X + s.d. English Bay/ 1 . 0 6 + 0 . 4 1 11 Bamfield 0 . 4 4 + 0 . 1 5 23 Bamfield/ 0 . 4 4 + 0 . 1 5 23 non-Bamfield 0 . 9 1 + 0 . 4 7 21 English Bay/ 1 . 0 6 + 0 . 4 1 11 non-English Bay 0 . 5 4 + 0 . 3 3 33 A l l tamarensoid/ 1 . 0 5 + 0 . 4 1 13 a l l c a t e n e l l o i d 0 . 4 6 + 0 . 2 6 29 Non-EB/BF tamarensoid/ 1 . 0 5 + 0 . 4 0 4 non-EB/BF c a t e n e l l o i d * 0 . 5 6 + 0 . 5 1 6 A l l c l o n a l / 0 . 5 8 + 0 . 3 8 33 a l l non-clonal 0 . 9 0 + 0 . 4 2 11 English Bay cl o n a l / 1 . 2 4 + 0 . 3 4 5 English Bay non-clonal 0 . 9 1 + 0 . 4 3 6 Significance of difference i n means u o . o 5 ( 2 ) < u < o r u ' ) ; s * * u o . o 5 ( 2 ) < u < o r u ' > ; s * * u o . o 5 ( 2 ) < u < o r u ' ) ; s * * U 0 . 0 5 ( 2 ) < U ( ° r u ' > ; s * * U 0 > 0 5 ( 2)>U(and U 1); ns*** U 0 > 0 5 ( 2 ) > U ( a n d U 1 ) ; ns*** U 0 - 0 5 ( 2 ) > U ( a n d U'); ns*** 'only tamarensoid and ca 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 ei t h e r English Bay or Bamfield were compared. > % x s i g n i f i c a n t ; '''"''not s i g n i f i c a n t Table 11. Maximum growth rates of Protogonyaulax spp batch culture experiments. Species Origin of i s o l a t e k (div d" 1) P. tamarensis Cape Ann, MA, USAb 0.29 0.36 0.29 0.22 0.30-1.05c 0.10-2.00° 0.48 P. tamarensis Ipswich Bay, Gulf of Maine, USA 0.45 P. tamarensis Bay of Fundy, N.B., Canada 0.41 0.36 P. tamarensis P. tamarensis Bay of Fundy, 0.21 N.B., Canada or 0.32 Plymouth, England (?) 0.36 0.52 Perch Pond, Falmouth, 0.55d MA, USA 0.19-0.66e P. tamarensis M i l l Pond, Orleans, MA, USA 0.60 0.73 0.53 in exponential phase from Growth medium3 Reference f/2 f/2 f/2 f Fogel and Hastings A q u i l GPM White, 1976 White, 1978b White and Maranda, 1978 Shimizu et a l . , 1975a Yentsch and Mague, 1980 Yentsch and Mague, 1980 Schmidt and Loeblich, 1979b Fogel and Hastings Cole et a l . , 1975 Yentsch et a l . , 1975 Erd-Schreiber ASP-7 Prakash, 1967 Prakash, 1967 ASW ASW+humic acid ESWA ESWA+humic acid Prakash and Rashid, 1968 Prakash and Rashid, 1968 Prakash and Rashid, 1968 Prakash and Rashid, 1968 f/2 f/2 f/2 f/2 f/2 Brand et a l . , 1981a Brand, 1981 Watras et a l . , 1982 Anderson and Lindquist, 1985 Anderson et a l . , 1984 Species Origin of is o l a t e k (div d" 1) P. tamarensis P. tamarensis P. tamarensis P. cat e n e l l a P. catenella P. ca t e n e l l a Ofunato Bay, 0.48 Honshu, Japan Lummi Island, WA, 0.68 USA Tamar estuary, 0.50 Plymouth, England Provasoli i s o l a t e 1 0.28 0.26 Sequim Bay, WA, 0.50 USA 0.52 Whidbey Island, 0.49 WA, USA Protogonyaulax Porpoise Island, 0.41 sp. Alaska, USA Growth medium3 Reference Singh et a l . , 1982 ESNW Boyer et a l . , 1985 ESNW Boyer et a l . , 1985 Erd-Schreiber organic^ ASP-7 (N0 3") ASP-7 (NH 4 +) ESNW Prakash, 1967 Proctor et a l . , 1975 Norris and Chew, 1975 Norris and Chew, 1975 Boyer et a l . , 1985 H a l l , 1982 References for nutrient media: f,f/2 ( G u i l l a r d and Ryther, 1962); GPM (Loeblich, 1975); Erd-Schreiber (Foyn, 1934); Fogel and Hastings medium (Fogel and Hastings, 1971); Aquil (Morel et a l . , 1979); ASP-7 (Provasoli, 1963); ASW (Prakash and Rashid, 1968); ESWA (Prakash and Rashid, 1968); ESNW (Harrison et a l . , 1980) ^believed to be i s o l a t e 429 (Loeblich and Loeblich, 1975) i s o l a t e d by C. Martin, Gloucester Marine Station, Gloucester, MA. This s t r a i n has been designated a l t e r n a t i v e l y as G. excavata (Loeblich and Loeblich, 1975; Yentsch and Mague, 1980; White, 1976 and 1978b; Schmidt et a l . , 1978; White and Maranda, 1978), G. tamarensis (Shimizu et a l . , 1975a) and G. tamarensis var. excavata (Schmidt and Loeblich, 1979a and cseasonal o s c i l l a t i o n s i n growth rate ^mean growth rate among i s o l a t e s (n=83) erange of growth rates among i s o l a t e s (n=75) fgeographical o r i g i n of i s o l a t e ( s ) not given Shigh concentrations of organic nutrients, no inorganic nutrients added. 144 l i t e r a t u r e , - as given by the o r i g i n a l authors, or were calculated from the growth curves i n d i c a t i n g changes i n c e l l density or i n vivo fluorescence with time. These values cannot be s t r i c t l y compared with those measured i n the current study, since, with the exception of the growth rates f o r P. tamarensis from Falmouth, MA (Brand, 1981; Brand et a l . , 1981a), they do not n e c e s s a r i l y represent acclimated growth. The experimental regimes varied i n irradiance l e v e l , temperature, s a l i n i t y , nutrient enrichment medium, and culture volume. Nevertheless, these data give an impression of the range of growth rates a n t i c i p a t e d f o r " c a t e n e l l a " and "tamarensis" i s o l a t e s . When the values are segregated into two groups s o l e l y on the basis of morphotype, there are no cl e a r differences i n the growth rates between c a t e n e l l o i d and tamarensoid i s o l a t e s . b. Discussion As used i n the present study to determine genetic v a r i a t i o n i n growth rates within and among phytoplankton populations, the i n vivo fluorescence technique s a t i s f i e s the necessary c r i t e r i a of r e p l i c a b i l i t y , s t a b i l i t y and pr e c i s i o n . The studies of Brand and co-workers on several species of phytoplankton (Brand, 1980; 1981; 1982; Brand et a l . , 1981a and b), indicated that acclimated growth rates of haploid species, including d i n o f l a g e l l a t e s , such as Protogonyaulax, and coccolithophores, were constant with time, although those of d i p l o i d diatoms were not. The r e s u l t s of the current investigations on Protogonyaulax supported the view that the acclimated growth rates are g e n e t i c a l l y f i x e d . Yentsch and Mague (1980) reported an annual p e r i o d i c i t y i n the growth rate of an i s o l a t e of P. tamarensis from Cape Ann, MA, but no evidence of such c y c l i c a l events 145 was observed among the NEPCC Protogonyaulax i s o l a t e s . I t might be argued that the use of u n i a l g a l rather than axenic i s o l a t e s to determine acclimated growth rates i s i n v a l i d , because of the p o s s i b i l i t y of d i f f e r e n t i a l conditioning e f f e c t s on the medium caused by bact e r i a . Protogonyaulax cultures from the NEPCC are known to have dominant b a c t e r i a l f l o r a which d i f f e r d i s t i n c t i v e l y from each other (Dimanlig and Taylor, 1985). However, Brand (1980; 1981) e f f e c t i v e l y countered t h i s argument when he found that the acclimated growth rates of axenic and non-axenic i s o l a t e s were v i r t u a l l y i d e n t i c a l , and that bacteria i n low concentrations had l i t t l e e f f e c t on growth. Since the growth rates were determined i n exponential growth phase, when dissolved organic nutrients and b a c t e r i a l numbers were lower than the l a t e r growth stages, the p o t e n t i a l influence of bact e r i a on d i n o f l a g e l l a t e growth was minimized. In any case, the r i s k of genotypic changes and c l o n a l s e l e c t i o n associated with the use of a n t i b i o t i c s to p u r i f y phytoplankton cultures, which could bias "natural" growth rates, more than outweighs the possible benefits of preparing axenic cultures. Yentsch et a l . (1975) found that although axenic and non-axenic P. tamarensis cultures of the same i s o l a t e had the same maximal growth rate, the axenic cultures displayed a s i g n i f i c a n t l y extended lag phase. This i s evidence that such a n t i b i o t i c -treated cultures were experiencing d i f f i c u l t i e s i n acclimation. The v a r i a t i o n i n acclimated growth rates within Protogonyaulax populations from English Bay (C.V.: 38.7%) and Bamfield (C.V.: 34.1%) was much greater than among i s o l a t e s from Falmouth, MA (C.V.: 10.4%) (Brand, 1981), i n d i c a t i n g that the B r i t i s h Columbian populations were more genotypically heterogeneous. The Protogonyaulax i s o l a t e s from Massachusetts (Brand, 1981; Brand et a l . , 1981a) were derived from benthic hypnocysts, 146 whereas a l l i s o l a t e s from the two locations i n B r i t i s h Columbia came o r i g i n a l l y from i s o l a t e d vegetative c e l l s . The lower genotypic heterogeneity i n the cyst-derived cultures i s somewhat s u r p r i s i n g , given that hypnocysts contain two mating types (= d i f f e r e n t genotypes). Cultures i n i t i a t e d from s i n g l e c e l l s and i n d i v i d u a l chains are c l o n a l , and would be expected to e x h i b i t less p o t e n t i a l for heterogeneity. I t i s conceivable that such cyst-derived i s o l a t e s may have already passed through a p r e - s e l e c t i v e " f i l t e r " i n the natural environment. Under unfavourable environmental conditions, only a f r a c t i o n of the vegetative c e l l s i n the population survive and undergo the sexual fusion required f o r the formation of hypnocysts. I t i s not c l e a r that c e l l s which form sexual cysts represent an unbiased estimator of the t o t a l range of genotypes i n the population. I f t h i s i s not the case, and hypnocysts represent the product of genotypes hi g h l y selected f o r s u r v i v a l , only a r e s t r i c t e d range of a l t e r n a t i v e genotypes may be expressed i n cyst-derived cultures. This may 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 rates among cultured i s o l a t e s . For cultures i n i t i a t e d from vegetative c e l l s , the "genetics of survivors" problem may also have a s i g n i f i c a n t impact on estimates of genetic v a r i a t i o n . I f the loss of i s o l a t e s due to i n v i a b i l i t y through transf e r from natural samples to culture i s s u f f i c i e n t l y high, the c u l t u r i n g process i t s e l f may be viewed as a genetic sieve, n e c e s s a r i l y l i m i t i n g the range of genotypes a v a i l a b l e f o r experimentation to those which can be s u c c e s s f u l l y cultured. In the case of the Bamfield i s o l a t e s , the rate of successful transfers from s i n g l e chains i s o l a t e d from the natural environment to culture was 100%; of the E n g l i s h Bay i s o l a t e s , only two i s o l a t e s of 12 attempts to obtain cultures by i s o l a t i n g s i n g l e c e l l s were l o s t i n the i n i t i a l sub-147 c u l t u r i n g . The percent success i n c u l t u r i n g Protogonyaulax i s o l a t e s from germinated hypnocysts was not given i n the Massachusetts study (Brand, 1981; Brand et a l . , 1981a), however Brand (1982) l a t e r reported that only 10-50% of the si n g l e c e l l i s o l a t i o n s of other phytoplankton species were successful - mostly due to contamination. Another i n t e r p r e t a t i o n of the differences i n genotypic v a r i a t i o n i n growth rates among samples from natural populations tre a t s the differences observed i n culture as r e a l , and as unbiased estimators of the degree of i n s i t u d i v e r s i t y . In t h i s context, i t i s i n s t r u c t i v e to compare the r e s u l t s of t h i s study on acclimated growth rates with the findings f o r other phytoplankton species from diverse habitats. For the coccolithophores, Emiliana huxleyi and Gephyrocapsa oceanica, no i n t r a s p e c i f i c genetic d i f f e r e n t i a t i o n was found among oceanic i s o l a t e s , but n e r i t i c and oceanic populations were c l e a r l y d i s t i n c t (Brand et a l . , 1982). There were also s i g n i f i c a n t differences i n the growth rate of E. huxleyi i s o l a t e s from the Gulf of Maine and those from other n e r i t i c populations from warmer waters to the south. In contrast to these observations, i s o l a t e s of the diatom T h a l a s s i o s i r a pseudonana varied only s l i g h t l y , i f at a l l , among i s o l a t e s from geographically separated n e r i t i c habitats (Brand et a l . , 1981b). Regardless of t h e i r geographical o r i g i n , a l l of the Protogonyaulax i s o l a t e s examined i n the present study were representatives from n e r i t i c waters, yet the v a r i a t i o n i n acclimated growth rates within and among geographical regions was s u b s t a n t i a l . A high growth rate might be less important as a s u r v i v a l strategy f o r Protogonyaulax than other mechanisms, such as a l t e r n a t i v e l i f e cycle stages, including encystment, and may not be selected f o r as r i g i d l y . In the terminology of e c o l o g i c a l s u r v i v a l s t r a t e g i e s , Protogonyaulax spp. may be less " r - s e l e c t e d " than n e r i t i c 148 diatoms ( G u i l l a r d and Kilham, 1977). The paired-group comparisons (Table 9 and 10) make i t possible to analyze f a c t o r s , including geographical l o c a t i o n of o r i g i n , age and morphotype of the i s o l a t e s i n cul t u r e , and c l o n a l i t y at the time of i s o l a t i o n , to account f o r the differences i n variance and mean growth rates between groups. In some cases, these factors may be g e n e t i c a l l y linked, but the data must be viewed cautiously to minimize d i s t o r t i o n s due to coincident sampling bias. For example, although Table 9 indicates that c a t e n e l l o i d i s o l a t e s are not 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 rates than tamarensoid i s o l a t e s , an incautious look at the data i n Table 10 appears to 4 show that the mean growth rate of tamarensoid i s o l a t e s i s s i g n i f i c a n t l y higher. However, the influence of factors associated with geographical o r i g i n , rather than morphotype per se, may be more s i g n i f i c a n t l y r e l a t e d to genotypic d i v e r s i t y i n growth rate components. One must bear i n mind that most of the c a t e n e l l o i d i s o l a t e s were from a s i n g l e l o c a t i o n , Bamfield, while the majority of the tamarensoid i s o l a t e s as well as the natural water f o r the growth medium were from English Bay. When i s o l a t e s conforming to e i t h e r morphotype from other locations were compared, t h i s apparent connection between morphotype and mean growth rate i s no longer s i g n i f i c a n t . Brand (1980) noted that recent c l o n a l i s o l a t e s of P. tamarensis us u a l l y had higher growth rates than those i n i t i a t e d as non-clonal i s o l a t e s and maintained for a longer time. I t was suggested (Brand et a l . , 1981b) that the decline i n growth rate with time i n culture was due to inbreeding depression among a few genotypes. This may be true, but i t i s necessary to separate the e f f e c t s of time, as measured by asexual generations, and the e f f e c t s of c l o n a l i t y on the p o t e n t i a l f o r sexual recombination. In the 149 present study, there were no s i g n i f i c a n t differences between a l l c l o n a l versus non-clonal i s o l a t e s from d i f f e r e n t geographical locations which have been retained i n culture f o r varying periods of time. However, i t i s important to consider that the most recent i s o l a t e s - a l l from Bamfield i n the summer of 1984 - also comprised the majority 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 i s o l a t e s from English Bay allows one to factor out the e f f e c t of the length of time i n culture and geographical o r i g i n on growth rate, since, with the exception of NEPCC 516, these were contemporaneous i s o l a t e s . Again there was no s i g n i f i c a n t d i f f e r e n c e . This suggests that the p o t e n t i a l f o r sexual recombination i n m u l t i c l o n a l cultures does not have a large impact on genotypic modifications that could r e s u l t i n increased growth rates as a form of "hybrid vigor". The issue of how to account f o r the s i g n i f i c a n t l y lower variance and mean growth rate i n the Bamfield i s o l a t e s i s problematic, since, i n addition to t h e i r geographical i s o l a t i o n from other populations, a l l of these i s o l a t e s were c l o n a l and belonged to the c a t e n e l l o i d morphotype. Given the above comments on the e f f e c t s of c l o n a l i t y on the growth rates of contemporaneous i s o l a t e s , i t i s u n l i k e l y that the lower growth rates observed were s t r i c t l y due to the lack of opportunity f o r growth rate enhancement through genetic recombination. Furthermore, since these i s o l a t e s were those most recently brought into culture, i t i s not reasonable to postulate that the slower growth can be a t t r i b u t e d to the accumulation of m i t o t i c DNA r e p l i c a t i o n e r r o r s . I t i s not l i k e l y that these cultures were not completely acclimated to existence i n culture before the growth rate experiments, since they were not u t i l i z e d u n t i l several months, and several t r a n s f e r s , a f t e r i s o l a t i o n . The s i g n i f i c a n t l y lower variance i n growth rates among i s o l a t e s from t h i s population may be 150 evidence that the time elapsed since i s o l a t i o n was i n s u f f i c i e n t f o r large genetic divergence to have occurred i n these c l o n a l cultures. I t i s tempting to conclude that the i n s i t u Bamfield p o p u l a t i o n s ) i s / a r e g e n e t i c a l l y l e s s heterogeneous than the populations from elsewhere, but without a large number of contemporaneous i s o l a t e s from other locations f o r comparison, t h i s cannot be conclusively demonstrated or disproved. Nevertheless, i t i s reasonable to suggest that differences i n reproductive rates 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 populations are based upon genetic differences a r i s i n g from the evolution of populations i n d i f f e r e n t habitats. I t may be s i g n i f i c a n t that the highest growth rates were attained by i s o l a t e s from the same natural m i l i e u from which the seawater base f o r the culture medium was obtained. Crossovers and inversions i n the pattern of growth rates among i s o l a t e s could possibly occur i f the culture regime was selected to favour conditions more c l o s e l y representative of other environments. C. Quantitative Nuclear DNA Content 1. Results and Discussion Epifluorescence microphotometry of i n d i v i d u a l c e l l s i s an important t o o l i n the estimation 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 nuclear DNA content and the c l a r i f i c a t i o n of stages i n the l i f e cycle of d i n o f l a g e l l a t e s . Although the a b i l i t y to r a p i d l y measure the nuclear DNA content of many c e l l s , as with flow cytometry (Yentsch et a l . , 1983; Hayhome, 1985), i s s a c r i f i c e d , epifluorescence microphotometry does o f f e r several advantages. 151 S e l e c t i o n of the correct aperature s i z e f or nuclear measurement and the appropriate b a r r i e r f i l t e r eliminates the need for the photo-oxidation of pigments and p r e - f i x a t i o n required for flow cytometry of nuclear DNA from unextracted n u c l e i . This i s necessary to minimize the interference of c h l o r o p h y l l autofluorescence caused by exposure of the stained c e l l s to e x c i t a t i o n wavelengths (Yentsch et a l . , 1983). The measurement of DNA content from extracted n u c l e i by flow cytometry (Hayhome, 1985) undoubtedly y i e l d s values with a high degree of p r e c i s i o n and accuracy, but the procedure i s rather more involved than that employed for epifluorescent microscope determinations. D i r e c t microscopic observations allow for the v i s u a l r e j e c t i o n of obvious aberrants and lysed (presumably non-viable) c e l l s . The epifluorescent microscope technique also permits the separation of p e l l i c u l a r cysts from small vegetative c e l l s , and of planozygotes and fusing gametes from abnormally large vegetative c e l l s . Furthermore, the i n t e r p r e t a t i o n of DNA values from i n d i v i d u a l c e l l s i n a chain configuration i s possible. When viewed a p i c a l l y under the epifluorescence microscope, the DAPI-stained n u c l e i of a l l Protogonyaulax i s o l a t e s were C-shaped ( F i g . 18), and were morphologically i n d i s t i n g u i s h a b l e . This observation upon the nuclear shape agrees with previous l i g h t and electron microscopic studies of n u c l e i i n Protogonyaulax (Dodge, 1964; S i l v a , 1971; 1977; Tomas, 1974; G a v r i l a , 1977; H a l l , 1982; Balech and Tangen, 1985; Fukuyo, 1985). In v e n t r a l view, the nucleus appeared as a thick blue-fluorescing l i n e a r band along the c e l l equator ( F i g . 19). The p o t e n t i a l f o r self-shading of the nucleus, p a r t i c u l a r l y of the nuclear apices, which curved back below the plane of observation, or for fluorescence absorption by c e l l u l a r components in i n t a c t c e l l s , was estimated by comparing DNA values obtained from 152 F i g . 18 A p i c a l view of NEPCC 516 stained with DAPI showing crescent-shaped nucleus and red autofluorescence under epifluorescence microscopy (800X). Exposure: 1 min; Ektachrome ASA 160. F i g . 19 Equatorial view of NEPCC 516 stained with DAPI showing fluorescent nuclear bar at the cingulum under epifluorescence microscopy (500X). Red autofluorescence has been suppressed 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 from compressed versus non-compressed c e l l s . These factors did not appear to be s i g n i f i c a n t . Only when the crescent-shaped nucleus was viewed with the t i p s projecting toward the observer at right-angles to the equatorial plane was there a marked decrease i n emitted fluorescence, e s p e c i a l l y from the c e n t r a l portion of the nucleus. Nuclear squashes, produced by applying pressure to the overlying cover s l i p , increased the variance of measurements due to uneven disruption of the chromosomes, but did not increase the mean DNA values. DNA measurements made from a polar view were not usu 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 those from v e n t r a l l y oriented c e l l s . Nevertheless, since c e l l s n a t u r a l l y tended to orient under the cover s l i p with the v e n t r a l surface exposed, t h i s perspective was adopted f o r obtaining highly consistent DNA measurements. The shape of the nucleus of Gonyaulax polyedra was v i r t u a l l y i d e n t i c a l to that of Protogonyaulax spp., as was previously reported (Dodge, 1964). The nuclear DNA content of G. polyedra (36.13 ± 2.68 s.d. pg c e l l - ! ) lay close to the mean value f o r a l l Protogonyaulax i s o l a t e s (45.76 ± 2.35 s.e.m. pg cell"-'-), and was well within the range of values (21.42-63.29 pg c e l l - 1 ) . The quantity of nuclear DNA content i n G. polyedra from these experiments contrasted sharply with the older report i n the l i t e r a t u r e (Holm-Hansen, 1969), where DNA, measured by an ex t r a c t i v e fluorometric technique as t o t a l DNA, was given as 200 pg c e l l " 1 - the highest DNA content ever reported f o r a d i n o f l a g e l l a t e . That the DNA content of Amphidinium carterae, given i n the same pu b l i c a t i o n , was 1.6 times greater than that determined subsequently by other investigators (Galleron and Durrand, 1978) lends credence to the suggestion that these values may be i n error. This discrepancy cannot be accounted f o r by assuming that i t represents the contribution of non-nuclear DNA, since t h i s 155 would t y p i c a l l y comprise <5% of the t o t a l c e l l u l a r DNA. I t i s of course conceivable that the e a r l i e r measurement i n G. polyedra (Holm-Hansen, 1969) was based upon a highly p o l y p l o i d i s o l a t e . The mean DNA content (= 41.20 pg c e l l " 1 ) of 11 i s o l a t e s of nine other d i n o f l a g e l l a t e species given by Spector (1984) i s remarkably close to the mean values obtained f o r Protogonyaulax and G. polyedra. A previous mean value of ~20 pg c e l l - 1 found f o r P. tamarensis, determined by an e x t r a c t i v e technique (Mickelson and Yentsch, 1979; Yentsch et a l . , 1983), i s reasonably consistent with the range of values found i n the present study, when probable losses through extraction are considered. In the study by Mickelson and Yentsch (1979), the DNA content per c e l l v aried markedly with the stage i n culture, and even exhibited a f o u r - f o l d decrease with culture age during exponential phase. If t h i s phenomenon i s r e a l , i t requires further explanation. No apparent changes i n mean DNA content per c e l l throughout exponential phase were observed i n the current study among multiple i s o l a t e s . Regression analysis ( F i g . 20) revealed no apparent c o r r e l a t i o n between nuclear DNA content i n Protogonyaulax and time elapsed since the o r i g i n a l i s o l a t i o n s ( r c = -0.15; slope = -0.25). This may be s i g n i f i c a n t i n view of previous observations (Loper et a l . , 1980; Holt and P f i e s t e r , 1982) which suggested that the appearance of chromosomal increases i n long-term d i n o f l a g e l l a t e cultures may be i n d i c a t i v e of aneuploid increase or autodiploidy. On the other hand, morphologically s i m i l a r i s o l a t e s of the Symbiodinium microadriaticum species complex were s i m i l a r to Protogonyaulax i n that no r e l a t i o n s h i p between chromosome number and the length of time that an i s o l a t e had been maintained i n culture was found (Blank and Trench, 1985). 156 F i g . 20 Linear regression analysis of the r e l a t i o n s h i p of mean nuclear DNA content to time elapsed since the o r i g i n of i s o l a t e s i n cult u r e . DNA values f o r contemporaneous i s o l a t e s NEPCC 400-412 from English Bay (1981) were combined as a s i n g l e point. (X ± s.e.m.). Regression c o e f f i c i e n t r c = -0.15, slope = -0.25. Error bars = ±1.0 s.d.; n=30. 70.0r 60.0\ 400-412 i <D 50.0 O Q. 40.01 30.0 O zo.o\ O 3 , x5 355 529J j $543 ^508 516 435 lOJOf 255 • 545 ,180 71 i 253 183, J L. J ' ' I I I I L 10 15 ' L 20 _ J 1 i _ 25 Years in culture 158 Although the vegetative c e l l s of Protogonyaulax are considered to be at lea s t f u n c t i o n a l l y haploid, the chromosome s i z e ( G a v r i l a , 1977) and number. (Dodge, 1963) are v a r i a b l e . In P. tamarensis, the s p l i t t i n g of chromosomes i s asynchronous ( G a v r i l a , 1977), which may explain some of the v a r i a t i o n i n chromosome number and DNA content found even among c e l l s from the same population. Unequal d i s t r i b u t i o n of chromatin during m i t o t i c d i v i s i o n may lead to uneven chromosome numbers i n daughter n u c l e i (Dodge, 1963) and the formation of n u c l e i of unequal s i z e ( S i l v a , 1977). The smaller nuclear fragment can give r i s e to a smaller, yet v i a b l e , daughter c e l l with reduced DNA content. The only a v a i l a b l e chromosome count from a member of the Protogonyaulax species complex i s the value of 134-152 chromosomes given by Dodge (1963) for the haploid genome of the Plymouth i s o l a t e of P. tamarensis (= NEPCC 183). Assuming that no changes i n e i t h e r chromosome number or mean DNA content per chromosome have occurred i n t h i s i s o l a t e , each chromosome contained approximately 0.3 pg DNA. Using the estimate that 1 pg DNA = 2.01 X 10^ nucleotide base pai r s (Haapala and Soyer, 1973), t h i s i s equivalent to ~6 X 10^ base pai r s per chromosome. Without the evidence from accurate chromosome counts, i t was not possible to determine whether the v a r i a t i o n i n nuclear DNA content among i s o l a t e s r e f l e c t e d differences i n the number of chromosomes per nucleus, or va r i a t i o n s i n the quantity of DNA contained i n equivalent numbers of chromosomes. In Symbiodinium microadriaticum, not only did the t o t a l chromosomal volume vary i r r e g u l a r l y among i s o l a t e s , but there was no apparent r e l a t i o n s h i p between chromosome number and volume (Blank and Trench, 1985). In the Crypthecodinium cohnii species complex, the DNA content was us u a l l y well correlated with chromosome number, but not ne c e s s a r i l y with c e l l volume (Beam and Himes, 1984). Among Peridinium 159 i s o l a t e s examined by flow cytometry (Hayhome, 1985), the d i s t r i b u t i o n of nuclear DNA content indicated two s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n t groups, but these differences were not correlated with c e l l volume, which remained constant. The nuclear DNA content among Protogonyaulax i s o l a t e s did not vary with mean c e l l volume ( r c = 0.19, slope = 0.00) ( F i g . 21), calculated as a prolate spheroid (Table A). In a previous analysis of one i s o l a t e of P. tamarensis by flow cytometry (Yentsch et a l . , 1983), DNA per c e l l also f a i l e d to c o r r e l a t e c l o s e l y with c e l l s i z e . Nevertheless, i s o l a t e s 253 and 508 were noteworthy i n that they lay at the lower end of the s i z e spectrum, while t h e i r mean DNA content, 25.55 ± 2.58 s.d. pg c e l l " 1 and 21.A2 ± 2.60 s.d. pg c e l l - 1 , r e s p e c t i v e l y , was judged to be s i g n i f i c a n t l y lower than the mean value obtained from a l l Protogonyaulax i s o l a t e s (Tables 12 and 13). The DNA content of i s o l a t e s 253 and 508 was almost exactly h a l f the mean value f o r Protogonyaulax. These "small form" d i n o f l a g e l l a t e s may r e s u l t from reduction aneuploidy as reported by S i l v a (1971; 1977). The quantity of nuclear DNA i n Protogonyaulax i s o l a t e s was not obviously r e l a t e d to morphotype or geographical l o c a t i o n of o r i g i n . Mean values of nuclear DNA from c a t e n e l l o i d i s o l a t e s were not s i g n i f i c a n t l y d i f f e r e n t from those obtained f o r tamarensoid i s o l a t e s (Student's t - t e s t , two-tailed; a=0.05) (Tables 12 and 13). The mean DNA content of the ten contemporaneous i s o l a t e s from the 1981 English Bay bloom did not d i f f e r s i g n i f i c a n t l y from the mean of a l l i s o l a t e s combined. The amount of nuclear DNA contained i n the large darkly pigmented c e l l s >50 nm i n diameter i n cultures of 183, 255, A03, A05, and A35 during stationary phase, was approximately double that of the vegetative c e l l s (Table IA). This appears to confirm 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 zygotes. 160 F i g . 21 Nuclear DNA content of Protogonyaulax i s o l a t e s i n r e l a t i o n to 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 c = 0.19; slope = 0.00. Error bars = ±1.0 s.d.; n=30. Q) O 0)80.0 a § 6 0 . 0 £40 .0 o z 20.0 405 {253 •412 [406 I J409 ^29 1508 255 J180 4aa_ ± 5.0 10.0 15.0 20.0 25.0 Mean cell volume (um 3x 103) 162 Table 12. Mean nuclear DNA content morphotype and location each group. Isolates Catenelloid Tamarensoid English Bay (1981) NEPCC 253 NEPCC 508 All isolates of Protogonyaulax isolates by f origin, n = number of isolates in Mean nuclear DNA content (pg c e l l - 1 ) 39. 49 + 4. 97 s . e.m. > n=4 47. 16 + 2. 92 s .e.m. 5 n=16 51. 81 + 2. 70 s .e.m. 9 n=10 25. 55 + 2. 58 s .d.; n; =30 21. 42 + 2. 60 s .d.; n =30 45. 76 + 2. 35 s .e.m. J n=22 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; HQ: Mi= n = number of isolates in each group. Catenelloid/ 1.20 tamarensoid English Bay (1981)/ 1.54 a l l isolates NEPCC 253/ 8.39 al l isolates NEPCC 508/ 10.10 al l isolates d.f. (ni+^-2) 18 30 21 21 ter[.05(2)] Hypothesis 2.10 HQ accepted' 2.05 2.08 Hn accepted* 2.08 HQ rejected-HQ rejected' ns": the population means are not significantly different s'°': the population means are significantly different Table 14. Nuclear DNA content of vegetative c e l l s of Protogonyaulax i s o l a t e s r e l a t i v e to that of zygotes. Number of i s o l a t e s , n = 30. Nuclear DNA content (X ± s. d.; n=30) (pg c e l l " 1 ) I solate Vegetative c e l l s Zygotes DNA Ratio 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. General El e c t r o p h o r e t i c Theory In e l e c t r o p h o r e t i c separation, the migration of proteins i n an e l e c t r i c a l f i e l d i s e f f e c t e d i n a porous medium using an i o n i c buffer system. The migration rate of the protein molecules within the medium to the electrode of opposite charge depends p r i m a r i l y upon t h e i r net e l e c t r i c a l charge. The molecular s i z e and shape are also important determinants of the net migration rate, due to s i e v i n g and retardation e f f e c t s ; the rate i s proportional to the e l e c t r i c a l charge:mass r a t i o m u l t i p l i e d by the f r i c t i o n a l resistance. In the discontinuous polyacrylamide gel electrophoresis (DISC-PAGE) system pioneered by Ornstein (1964) and Davis (1964), el e c t r o p h o r e t i c techniques are combined with gel f i l t r a t i o n . The DISC-PAGE technique i s s o - c a l l e d since separation i s achieved using a gel buffer which d i f f e r s i n composition and pH from the electrode buffer. Usually a sample and/or stacking gel of higher porosity i s superposed above the separating g e l . Coincidently, the well-defined separated protein bands frequently resemble f l a t t e n e d " d i s c s " when DISC-PAGE i s performed i n the narrow tubes o r i g i n a l l y recommended for t h i s method. DISC-PAGE i s capable of y i e l d i n g sharper r e s o l u t i o n than continuous buffer systems, since the sample proteins remain bracketed within a narrow zone formed between the "leading" ion and the " t r a i l i n g " ion, r e s u l t i n g i n a moving front proceeding through the g e l . Acrylamide (CH^CH'CONI^) i s polymerized and cross-linked by the a d d i t i o n of the b i f u n c t i o n a l reagent N,N'-methylenebisacrylamide ("Bis") 165 [(CH2=CH-C0NH)2,CH2]• Persulfate i s often used as the c a t a l y s t , and N,N,N',N'-tetramethylethylenediamine (TEMED) serves to accelerate polymerization. Band r e s o l u t i o n by the DISC-PAGE technique i s superior to that achieved i n starch gels due to the a b i l i t y of acrylamide to polymerize to form gel pores of a more d i s c r e t e and reproducible s i z e , and the greater f l e x i b i l i t y i n regulating pore s i z e , accomplished by adjusting the concentration of acrylamide and c r o s s - l i n k e r . 2. The Use of Electrophoresis i n Chemosystematics, Taxonomy and Population Genetics The morphological features of taxa are phenotypic expressions of the genome, but are presumably coded for by complex i n t e r a c t i o n s between several genes, and may be highly susceptible to environmental modifications. Isozyme electrophoresis i s an attempt to e s t a b l i s h relatedness based upon biochemical characters - primary gene products -which are more d i r e c t l y linked to the genome. The phenotypes expressed on e l e c t r o p h o r e t i c gels may be interpreted as i n d i c a t i v e of r e l a t i v e genetic d i v e r s i t y , since there i s a d i r e c t correspondence between enzyme l o c i and the polypeptides f o r which they code. With the discovery that many enzymes e x i s t i n multiple isozymic forms, varying i n e l e c t r i c a l charge and molecular weight (Markert and M i l l e r , 1959), isozyme electrophoresis became an important t o o l i n determining enzyme variant s . E l e c t r o p h o r e t i c separation of proteins has been combined with histochemical s t a i n i n g techniques to i d e n t i f y l o c a l i z e d enzyme bands i n a gel matrix (Hunter and Markert, 1957). The technique was adopted by 166 population b i o l o g i s t s and g e n e t i c i s t s (Lewontin and Hubby, 1966; Prakash et a l . , 1969; King et a l . , 1974) to measure the degree of genetic v a r i a t i o n i n natural populations, by screening for the multiple molecular forms of enzymes coded for by v a r i a n t a l l e l e s . When i t was recognized that c o r r e l a t i o n s among species groups based upon isozyme data generally corresponded to phenetic s i m i l a r i t y and phylogenetic r e l a t i o n s h i p s previously recognized on the basis of c l a s s i c a l taxonomic c r i t e r i a (Avise, 1975), enzyme electrophoretic techniques become extensively used i n systematics. Enzyme electrophoretic data are p a r t i c u l a r l y amenable to phylogenetic analysis (at l e a s t among c l o s e l y r e l a t e d taxa), since the data on a large number of homologous l o c i from various taxa can be r e a d i l y c o l l e c t e d , analyzed and interpreted. From s t r i c t l y systematic point of view, the r e l a t i v e s e l e c t i v e n e u t r a l i t y of a l l e l e s i s not a major concern; i n f a c t , a low degree of s e l e c t i v i t y w i l l tend to minimize convergence, and thus can a i d i n systematic reconstruction (Selander and Johnson, 1973). Using d i f f e r e n t algorithms ( F i t c h and Margoliash, 1967; F a r r i s , 1970; 1972; Prager and Wilson, 1978), i t has been possible to generate phylogenetic trees based on isozyme data f o r a wide v a r i e t y of organisms (Avise and Ayala, 1976; Mickevitch and Johnson, 1976; Prager and Wilson, 1978; Baverstock et a l . , 1979). Isozyme electrophoresis has been frequently employed to discriminate between and within assemblages of a wide v a r i e t y of lower eukaryotic organisms. Systematic and population genetic studies using electrophoresis are p a r t i c u l a r l y w ell represented f o r the protozoa ( T a i t , 1970; A l l e n and Gibson, 1971; 1975; A l l e n and Weremiuk, 1971; Borden et a l . , 1973a and b; Adams and A l l e n , 1975; Goncalves de Lima, 1979; Nanney et a l . , 1980; Beam et a l . , 1982; Machelon and Demar, 1984). Enzyme electrophoretic studies 1 6 7 involving macroalgae (Thomas and Brown, 1970a; Cheney and Babbel, 1978; Grant and Proctor, 1980; B l a i r et a l . , 1982), and microalgae (Thomas and Brown, 1970b; Thomas and Delcarpio, 1971; Schoenberg, 1976; Schoenberg and Trench, 1980a; Hayhome and P f i e s t e r , 1983; Hayhome, 1985; Whitten and Hayhome, 1985), e s p e c i a l l y diatoms (Murphy and G u i l l a r d , 1976; Murphy, 1978; Gallagher, 1980; 1982; Soudek and Robinson, 1983), are becoming in c r e a s i n g l y common. Among d i n o f l a g e l l a t e s , e l ectrophoretic v a r i a b i l i t y and host s p e c i f i c i t y r e l a t i o n s h i p s have been examined for the symbiotic d i n o f l a g e l l a t e Symbiodinium (=Gymnodinium) microadriaticum species complex, based upon four enzymes and t o t a l soluble protein (Schoenberg, 1976; Schoenberg and Trench, 1980a and b). Watson and Loeblich (1983) used electrophoretic isozyme analysis to i n f e r evolutionary r e l a t i o n s h i p s between p o t e n t i a l s i b l i n g species within the marine genus Heterocapsa. This technique was s i m i l a r l y applied to the study of the c l o s e l y a l l i e d s i b l i n g species of the marine heterotrophic d i n o f l a g e l l a t e Crypthecodinium  co h n i i (Daggett and Nerad, 1980; Beam and Himes, 1982; Beam et a l . , 1982). However, with the exception of work by Hayhome and co-workers (Hayhome and P f i e s t e r , 1983; Hayhome, 1985; Whitten and Hayhome, 1985), separation of isozymes from d i n o f l a g e l l a t e s has been conventionally performed on starch gels, and has t y p i c a l l y involved only a few enzyme systems. Using PAG-electrophoresis, the general c o r r e l a t i o n between conventional morphological features used to discriminate among Peridinium spp. and isozyme patterns was established (Hayhome and P f i e s t e r , 1983; Hayhome, 1985). Evidence from isozyme analysis by PAG-electrophoresis was also employed to taxonomically separate c l o n a l i s o l a t e s of two binucleate d i n o f l a g e l l a t e s , Peridinium balticum (Levander) Lemmermann and Glenodinium  foliaceum Stein into d i f f e r e n t genera (Whitten and Hayhome, 1985). 168 Enzyme electrophoresis i s e s p e c i a l l y u s e f u l i n discriminating among i n c i p i e n t and s i b l i n g species (Ayala, 1983). However, as has been pointed out (Avise, 1975; Ferguson, 1980; G o t t l i e b , 1984), t h i s technique may be less useful i n e s t a b l i s h i n g systematic r e l a t i o n s h i p s at higher taxonomic l e v e l s . The p o s s i b i l i t y f o r the misinterpretation of coincident electrophoretic bands as representative of genetic i d e n t i t y increases with the degree of phylogenetic divergence, as does the p r o b a b i l i t y that information regarding mutational differences w i l l be l o s t i n the expression of electrophoretic non-identity. This r e s t r i c t i o n on the use of electrophoresis to the lower taxonomic l e v e l s of c l o s e l y r e l a t e d organisms may be even more stringent f o r d i n o f l a g e l l a t e s (and other p r o t i s t s ) than for higher organisms, given the higher genetic d i v e r s i t y and v a r i a t i o n i n electrophoretic patterns among i s o l a t e s assigned to the same morphospecies. In s p i t e of the above l i m i t a t i o n s and drawbacks to the use of elec t r o p h o r e t i c data as a probe of the genome in population genetics and systematics, c l e a r advantages over c e r t a i n other character data are also demonstrable. The mobility of isozyme bands can be o b j e c t i v e l y measured and reproduced within a taxon with a high degree of p r e c i s i o n , and the data can be converted to q u a n t i f i a b l e s i m i l a r i t i e s among taxa. The existence of gene products i d e n t i c a l i n ele c t r o p h o r e t i c mobility can usu a l l y be interpreted to imply homology and common o r i g i n (Avise, 1975). 169 3. Interpretation of Electrophoretic Data Isozymes are defined as multiple molecular forms of a given enzyme capable of catalyzing the same reaction, and which possess at l e a s t one common substrate. These multiple molecular forms may occur within a s i n g l e species, organism, c e l l , or organelle, and are u s u a l l y operationally defined by t h e i r differences i n r e l a t i v e e l e ctrophoretic m ob il it y (Brewer and Sing, 1970). To eliminate ambiguity i n the term "isozyme", enzyme variants r e s u l t i n g from multiple a l l e l l i s m at a s i n g l e locus are usually c a l l e d "allozymes", while "isozymes" are considered to be enzymatic products of d i f f e r e n t l o c i (Prakash et a l . , 1969). Differences i n electrophoretic m o b i l i t y may be due to the presence of d i f f e r e n t polypeptide chains coded for by multiple gene l o c i . Both simple amino acid s u b s t i t u t i o n s and chromosomal modifications such as deletions and duplications of portions of the DNA chain may be responsible for a l t e r i n g net protein charge. A d d i t i o n a l e l e c t r o p h o r e t i c variants can be produced by p o s t - t r a n s l a t i o n a l modification of the protein structure, or may r e s u l t from a r t i f a c t s introduced by the electrophoretic separation i t s e l f . Since the p r i n c i p l e of e l e c t r o p h o r e t i c separation in non-denaturing gels depends p r i m a r i l y upon charge differences between isozymes, only mutations which can cause changes i n net charge can a f f e c t the enzyme migration rate. The majority of amino acids possess non-ionizable side chains and are e l e c t r i c a l l y neutral at the t y p i c a l running pH of most ele c t r o p h o r e t i c buffer systems. Only the s u b s t i t u t i o n of the a c i d i c (+ charged) amino acids glutamate and aspartate, and the basic (- charged) amino acids arginine and l y s i n e can induce charge differences i n the 170 primary protein structure. Furthermore, s i n g l e substitutions of amino acids of the same charge, or of two amino acids of opposite charge, are also e l e c t r o p h o r e t i c a l l y neutral. For these reasons, i d e n t i c a l band patterns detected on electrophoretic gels are not n e c e s s a r i l y i n d i c a t i v e of i d e n t i c a l genomes. , Due to the above f a c t o r s , and the redundancy i n the genetic code, i t has been calculated that only 27% of DNA base su b s t i t u t i o n s r e s u l t i n charge differences that are e l e c t r o p h o r e t i c a l l y detectable (Shaw, 1965). Electrophoretic studies sample only the products of s t r u c t u r a l genes, yet <10% of the t y p i c a l eukaryotic genome i s devoted to genes producing f u n c t i o n a l enzymes and other proteins (Selander, 1976). Thus, estimates of genetic v a r i a b i l i t y based upon isozyme data must be considered to be gross underestimates of the t o t a l v a r i a t i o n . There are many underlying assumptions involved i n the i n t e r p r e t a t i o n of electrophoretic banding patterns which are not always s t r i c t l y v a l i d (Hubby and Throckmorton, 1968; Ayala et a l . , 1974). Although a s i n g l e s i t e of enzyme a c t i v i t y on a gel was often conventionally interpreted as the product of a s i n g l e gene locus - the "one-gene, one isozyme" hypothesis, there i s always the p o s s i b i l i t y that the s i n g l e band represents a polymer whose substituents are derived from multiple gene l o c i . A more recent paradigm - the "one c i s t r o n , one polypeptide" hypothesis, i s now more frequently used to i n t e r p r e t electrophoretic bands. Enzyme ele c t r o p h o r e t i c studies t y p i c a l l y involve only a few enzyme systems within the t o t a l enzyme complement, i . e . those f o r which good st a i n i n g protocols have been developed. For operational reasons, the enzymes selected are often common enzymes of intermediary metabolism, which are ubiquitous i n d i s t r i b u t i o n and well characterized. A further 171 l i m i t a t i o n i s imposed by the e l e c t r o p h o r e t i c requirement that the enzymes be soluble, thereby eliminating from consideration those which are p r e c i p i t a t e d or strongly bound to c e l l fragments or membranes. I t i s assumed that t h i s enzymatic subset represents an unbiased estimator of genetic v a r i a t i o n among a l l s t r u c t u r a l genes. In turn, the subset of s t r u c t u r a l genes i s considered to be an unbiased sample of the t o t a l genome.. In view of these assumptions, i t i s c l e a r how sampling bias may a r i s e . The production and subsequent electrophoretic detection of enzymatically a c t i v e s t r u c t u r a l gene products may be a function not only of the s t r u c t u r a l gene i t s e l f , but may involve the i n t e r a c t i v e functioning of one or more regulatory genes ( A l l e n and Weremiuk, 1971). Enzyme systems that are inducible rather than c o n s t i t u t i v e may be subject to induction/repression e f f e c t s r e s u l t i n g from environmental influences which cannot always be t i g h t l y c o n t r o l l e d . This phenotypic i n s t a b i l i t y makes such enzymes u n r e l i a b l e indicators of genetic v a r i a t i o n . A d d i t i o n a l problems i n the electrophoretic detection and i n t e r p r e t a t i o n of isozyme banding patterns are e s s e n t i a l l y operational and a r t i f a c t u a l . The necessity for standardization i n extraction, storage and electrophoretic separation of protein samples cannot be overemphasized. Each of these conditions must be optimized to minimize changes i n the p h y s i c a l and chemical properties of the enzymes. V a r i a t i o n i n electrophoretic migration of enzymes has been observed i n response to sample treatment involving changes i n pH and temperature during storage and electrophoresis (see Appendix I I ) . Enzymes systems, or p a r t i c u l a r isozyme v a r i a n t s , may be d i f f e r e n t i a l l y s e n s i t i v e to changes i n pH and composition of the various buffers employed i n electrophoresis. 172 A l t e r n a t i v e storage and extraction techniques, including freezing and thawing, u l t r a s o n i c a t i o n and l y o p h i l i z a t i o n can also e f f e c t band migration and r e s o l u t i o n (Harborne, 1980) (see Appendix I I ) . Enzyme i n a c t i v a t i o n or conformational changes can occur through thermal denaturation during sonication and electrophoresis, or by interference with acrylamide and i t s contaminants. The tendency for some enzymes to band more t i g h t l y than others poses problems i n the d i s c r i m i n a t i o n of d i s c r e t e bands. The enzymatic products of " s i l e n t a l l e l e s " or "quiet genes" may be represented on the g e l but not stainable by the s t a i n i n g protocol adopted. Conversely, "nothing-ases" may appear on gels, due to enzymatic cross-reactions and non-enzymatic s t a i n i n g a r t i f a c t s . For example, "nothing dehydrogenases" frequently appear as a r e s u l t of the non-specific formation of formazans i n the presence of excess electron t r a n s f e r agents, p a r t i c u l a r l y at elevated pH (Harris and Hopkinson, 1976; Gaal et a l . , 1980). An excess of p e r s u l f a t e , used to catalyze polymerization, can cause rapid oxidation of enzymes (King, 1970). E f f o r t s to counter t h i s by the addition of s u l f h y d r y l reducing reagents, may introduce other a r t i f a c t s , since the reduction of tetrazolium dyes used i n the detection of dehydrogenases by t h i o l groups can proceed r a p i d l y i n the presence of the electron t r a n s f e r reagents, such as phenazine methosulfate (PMS). F i n a l l y , i t i s not always clear that extracted enzymes are derived from the same i n t r a c e l l u l a r compartment. D i f f e r e n t forms of the same enzyme, i n the sense that they a l l react with the same i n v i t r o substrate under the s p e c i f i e d s t a i n i n g conditions, may be present i n the cytoplasm, mitochondria, or c h l o r o p l a s t s . As detected i n the gel on the basis of r e l a t i v e m o b i l i t y , they may be d i f f e r e n t i a t e d as "isozymes" of the same 173 enzyme, yet t h e i r i n vivo functions, substrates and genomic o r i g i n s may be markedly d i f f e r e n t . 4. Enzyme Reactions: P y r i d i n e - l i n k e d Dehydrogenases The p y r i d i n e - l i n k e d (NAD- and NADRj dependent) dehydrogenases catalyze c r i t i c a l oxidation-reduction reactions of enzyme-mediated pathways and are important components of intermediary metabolism. The enzymes surveyed e l e c t r o p h o r e t i c a l l y i n the current study are representative of a wide v a r i e t y of intermediary pathways, including the oxidative Krebs cycle and pentose phosphate pathways, as well as those of amino acid and l i p i d metabolism. Pyrid i n e - l i n k e d dehydrogenases are considered to be " s p e c i f i c " enzymes, since i n most cases the i n vivo substrates are known. However, i t must be recognized that the d i s t i n c t i o n between " s p e c i f i c " and "non-specific" enzyme systems i s l a r g e l y an operational one (Shaw, 1965). The evidence that the substrates used to detect " s p e c i f i c " isozymes on electrophoretic gels are i d e n t i c a l to those metabolized i n vivo i s usually not conclusive. The following i n vivo enzymatic reactions have been previously i d e n t i f i e d from a v a r i e t y of organisms: Alanine dehydrogenase AlaDH L-alanine + H 20 + NAD+ pyruvate + NADH + NH 4 + Glucose-6-phosphate dehydrogenase G6PDH p-D-glucose-6-phosphate + NADP+ " A 6-phosphogluconate + NADPH + H+ Glutamate dehydrogenase GDH L-glutamate + NAD+ + H 20 _ i a-oxoglutarate + NADH + NH 4 + Hydroxybutyrate dehydrogenase HBDH D-B-hydroxybutyrate + NAD+ s - acetoacetate + NADH + H+ I s o c i t r a t e dehydrogenase IDH D L - i s o c i t r a t e + NADP+ 2-oxoglutarate + C0 2 + NADPH + H + Malate dehydrogenase MDH L-malate + NAD+ ""* %- oxaloacetate + NADH + H + Malic enzyme (NADP-dependent malate dehydrogenase: decarboxylating) ME L-malate + NADP+ "* pyruvate + C0 2 + NADPH + H + 175 SucDH Succinate + NAD+ i -ATP The dehydrogenases were detected on the electrophoretic gels by the enzymatic t r a n s f e r of electrons from the pyridine cofactor to a tetrazolium s a l t v i a an electron t r a n s f e r intermediate. The f i n a l product was formed by the reduction of the tetrazolium s a l t to an insoluble purple-coloured formazan (Harris and Hopkinson, 1976) which marks the s i t e of enzyme a c t i v i t y . The basic reactions were as follows: Substrate + NAD(P) + = Product + NAD(P)H + H + NAD(P)H + H + + PMS + Tetrazolium s a l t = Formazan ( i n s o l u b l e , purple) + NAD(P) + Succinate dehydrogenase fumarate + NADH + H+ The electron transport systems involved i n the st a i n i n g reaction at the s i t e of dehydrogenase a c t i v i t y are given i n the following scheme: s u b s t r a t e r e d v / NAD(P) + > enzyme substrate ox \- , NAD(P)H / t P M S r e d \ \ PMS o x i I Tetrazolium s a l t ox I Tetrazolium s a l t r e c j P r e c i p i t a t e d formazan 176 For a l l dehydrogenases, MTT (3-[4,5-dimethylthiazolyl-2]- 2,5 diphenyl-tetrazolium bromide) was selected as the tetrazolium s a l t . M T T ( T h i o z o l y l Blue) PMS (N-methyl-phenazinium methylsulfate = phenazine methosulfate) served as the electron t r a n s f e r agent. Phenazine Methosulf ate ( P M S ) 5. Ele c t r o p h o r e t i c P r o f i l e s of Pyri d i n e - l i n k e d Dehydrogenases The eight p y r i d i n e - l i n k e d dehydrogenase systems exhibited consistent s t a i n i n g patterns within an i s o l a t e , even when r e p l i c a t e s were compared over a period of more than a year i n culture. With the exception of NAD +-linked GDH, which showed more intensely stained primary bands and, occasionally, a d d i t i o n a l f a i n t l y stained bands i n response to high ammonium 177 l e v e l s (>25 LIM) i n the culture, the isozyme bands produced were not notably affected by the n u t r i t i o n a l status of the c e l l s . M u l t i p l e molecular forms, i d e n t i f i e d as non-coincident mobility v a r i a n t s , were detected on the zymograms for a l l the Protogonyaulax dehydrogenases examined. The zymograms ( F i g . 22) reveal that a high degree of enzymatic polymorphism e x i s t s within t h i s group. Using the s i m i l a r i t y c o e f f i c i e n t of Jaccard ( i n Sneath and Sokal, 1973), S j = a/(a + u), where a i s the number of band matches, and u i s the number of mismatches, when zymogram band patterns for i s o l a t e s are compared pairwise, a s i m i l a r i t y matrix (Table 15) was generated. An S j value of 1.00 indicates complete ele c t r o p h o r e t i c i d e n t i t y ; a value of 0.00 indicates complete d i s s i m i l a r i t y . The l e v e l of enzymatic polymorphism for dehydrogenases within a given i s o l a t e was generally high; monomorphism, as i n the GDH p r o f i l e s of i s o l a t e s 71, 255, 407, 412, and 435, was rather exceptional. This high degree of isozymic d i v e r s i t y was apparent both within and between morphotypes, and among morphologically s i m i l a r contemporaneous i s o l a t e s from English Bay (NEPCC 400-412). The mean genetic s i m i l a r i t y values among a l l Protogonyaulax i s o l a t e s ( S j = 0.22) and for the English Bay group ( S j = 0.33) (Table 16) were much lower than would be a n t i c i p a t e d for congeneric higher organisms. At l e a s t two bands were detected f o r AlaDH from each Protogonyaulax i s o l a t e ; a maximum of eight bands was found for i s o l a t e 180 ( F i g . 22). A comparison of s i m i l a r i t y c o e f f i c i e n t s (Sj) (Table 16) f o r AlaDH showed that the i s o l a t e s did not group c l o s e l y by geographical l o c a t i o n or morphotype, although a s l i g h t l y higher l e v e l of relatedness was expressed within the c a t e n e l l o i d group and the English Bay i s o l a t e s , than when the isozyme data were pooled for a l l i s o l a t e s . 178 F i g . 22 Zymograms of dehydrogenases extracted from Protogonyaulax i s o l a t e s . AlaDH, alanine dehydrogenase; GDH, glutamate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; HBDH, hydroxybutyrate dehydrogenase; IDH, i s o c i t r a t e dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; SucDH, succinate dehydrogenase. Staining i n t e n s i t y : •£Bj| intense; ]/////\ moderate; |v,y:.;| weak. V AlaDH 71 180 183 253 255 400 401 403 404 405 406 407409 412 402 516 355 435 529 508 GDH 71 180 183 253 255400401 403 404 405 406 407409412 402 516 355 435 529 508 fi'i 'J G6PDH 71 180183 253 255 400 401 403 404 405 406407 40? 412 402516 355435 528 SOfl HBDH 71 180 183 253 255 400 401 403 404 405 4(18 407 4llB 11? iH7 Stfi 3SS 435 ~J9Q 5n« IDH 71 180 183 253 255 400 401 403 404 405 406407 409 412 402 516 355 435 529 508 MDH 71 180 183 253 255 400 401 403 404 405 406407 409 412 402 516 355 435 529 508 ME 71 180183 253 255400 401403 404 405408 407 409 412 402 516 355 435 529 508 SucDH 71 180 183 253 255 400 401 403 404 405 406 407 409 412 402 516 355 435 529 508 180 183 253 255 355 400 401 402 403 404 405 406 407 409 412 435 508 516 529 0.17 0.14 0.22 0.15 0.22 0.35 0.28 0.16 0.15 0.09 0.09 0.20 0.11 0.17 0.09 0.14 0.14 0.16 0.17 0.24 0.10 0.46 0.23 0.18 0.22 0.31 0.13 0.60 0.23 0.25 0.24 0.19 0.32 0.16 0.44 0.49 0.09 0.16 0.22 0.14 0.23 0.15 0.26 0.32 0.38 0.16 0.17 0.13 0.09 0.27 0.13 0.31 0.32 0.46 0.60 0.21 0.17 0.16 0.12 0.24 0.10 0.18 0.20 0.30 0.42 0.31 0.12 0.19 0.22 0.13 0.29 0.10 0.27 0.27 0.38 0.53 0.52 0.30 0.17 0.11 0.14 0.12 0.20 0.17 0.19 0.19 0.21 0.36 0.39 0.29 0.20 0.17 0.16 0.24 0.18 0.22 0.13 0.26 0.23 0.33 0.38 0.36 0.23 0.32 0.22 0.20 0.14 0.21 0.14 0.18 0.18 0.20 0.17 0.26 0.17 0.18 0.16 0.19 0.19 0.36 0.15 0.19 0.22 0.24 0.21 0.13 0.12 0.18 0.18 0.23 0.19 0.21 0.13 0.19 0.16 0.10 0.19 0.10 0.19 0.26 0.10 0.14 0.16 0.16 0.23 0.16 0.23 0.13 0.12 0.13 0.16 0.15 0.07 0.27 0.25 0.23 0.30 0.38 0.18 0.30 0.38 0.32 0.33 0.38 0.28 0.26 0.22 0.18 0.17 0.24 0.21 0.28 0.26 0.42 0.33 0.16 0.21 0.30 0.30 0.35 0.18 0.16 0.21 0.19 0.14 0.21 0.11 0.21 0.22 0.25 71 180 183 253 255 355 400 401 402 403 404 405 406 407 409 412 435 508 516 Table 15. Similarity coefficients (Sj) of Protogonyaulax isolates, based upon dehydrogenase band patterns. Sj » a/(a + u), where a - number of matches and u • number of unmatched bands for a l l enzyme bands compared pairwise. Table 16. Mean s i m i l a r i t y c o e f f i c i e n t s (Sj) of Protogonyaulax i s o l a t e s by geographical o r i g i n and morphotype for i n d i v i d u a l dehydrogenases. ENZYME Isolate o r i g i n Combined or Morphotype AlaDH GDH G6PDH HBDH IDH MDH ME SucDH dehydrogenases A l l i s o l a t e s 0.18 0.23 0.18 0.13 0.14 0.44 0.24 0.11 0.22 English Bay 0.24 0.31 0.19 0.21 0.13 0.78 0.47 0.14 0.33 Catenelloid 0.25 0.25 0.25 0.14 0.09 0.22 0.06 0.08 0.17 Tamarensoid 0.18 0.22 0.12 0.14 0.17 0.41 0.25 0.07 0.21 185 For GDH, 14 e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e forms were evident, with polymorphism apparent for most i s o l a t e s ( F i g . 22 and 23). Clonal i s o l a t e s 403, 404 and 406, representing a contemporaneous population from English Bay, revealed an e l e c t r o p h o r e t i c a l l y i d e n t i c a l triple-banded pattern. S j values f or GDH (Table 16) indicated l i t t l e apparent c o r r e l a t i o n with morphotype, but the English Bay i s o l a t e s were s u b s t a n t i a l l y more c l o s e l y r e l a t e d to each other than when a l l i s o l a t e s were amalgamated. The G6PDH zymograms exhibited polymorphism for most i s o l a t e s , but NEPCC 180, 255, 355 and 407 were monomorphic. I n t e r e s t i n g l y , the two European tamarensoid i s o l a t e s , NEPCC 183 (Plymouth, England) and NEPCC 253, the small form from Laguna Obidos, Portugal, were e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e f o r G6PDH isozymes. Nevertheless, neither the e n t i r e group of tamarensoid morphotypes, nor the English Bay i s o l a t e s were strongly c o r r e l a t e d f or t h i s enzyme (Table 16). Twenty di s t i n g u i s h a b l e bands for HBDH were expressed among a l l the Protogonyaulax i s o l a t e s ( F i g . 22). Four i s o l a t e s , NEPCC 355, a c a t e n e l l o i d morphotype, and the tamarensoids 400, 401 and 403, showed a monomorphic pattern. As for AlaDH and GDH, the l e v e l of relatedness i n HBDH p r o f i l e s was greater within the English Bay group than among a l l i s o l a t e s . For IDH, a t o t a l of 14 unique bands were produced, with monomorphic i d e n t i c a l bands exhibited f or English Bay i s o l a t e s 403, 404 and 407 ( F i g . 22). Two tamarensoid i s o l a t e s from Vancouver Island, NEPCC 71 and 180, yielded the same quadruple-banded pattern f or t h i s enzyme. In general, however, electrophoretic variants of IDH did not appear to be re l a t e d geographically or according to morphotype (Table 16). In contrast to IDH from many other p r o t i s t s , such as Paramecium ( T a i t , 1970) and 186 F i g . 23 Zymogram of NAD-dependent glutamate dehydrogenase (GDH) isozymes of some Protogonyaulax i s o l a t e s from English Bay, B.C. Numbers r e f e r to NEPCC i s o l a t e designations. The two p a r a l l e l tracks BAC show the absence of bands produced by b a c t e r i a l extracts from u n i a l g a l cultures of i s o l a t e NEPCC 255. Exposure: Ektachrome EPY ASA 50; f l l at 1/30 s. F i g . 24 Zymogram of NAD-dependent malate dehydrogenase (MDH) isozymes of Protogonyaulax i s o l a t e s . Numbers r e f e r to NEPCC i s o l a t e designations. Exposure: Ektachrome EPY ASA 50; f l l at 1/30 s. 187 01 CO — i 00 Or cn CO - A cn 00 o > . o to cn U l O 00 MDH 188 Tetrahymena (Nanney et a l . , 1980), isozymes from Protogonyaulax were apparently non-functional when Mn"*-*" was substituted f o r Mg*"1" as the divalent cation cofactor. IDH isozymes from Protogonyaulax were s t r i c t l y NADP-dependent; attempts to substitute NAD f o r NADP as the nucleotide cofactor i n the extraction and st a i n i n g preparations resulted i n no a c t i v i t y . Although banding patterns f o r MDH were highly v a r i a b l e among the i s o l a t e s , producing 13 unique m o b i l i t y v a r i a n t s , t h i s enzyme system appeared to be the most g e n e t i c a l l y conservative of the dehydrogenases investigated (Figs. 22, 24 and 25). The mean S j value pooled f o r a l l i s o l a t e s was s u b s t a n t i a l l y higher f o r MDH than f o r any of the other enzymes (Table 16). Several p a i r s of i s o l a t e s , 400/401 (English Bay), 403/404 (English Bay), and 255 (Lummi Island, WA)/405 (English Bay), formed, e l e c t r o p h o r e t i c a l l y equivalent polymorphic groups f o r MDH. Two i s o l a t e s from the Bay of Fundy, NEPCC 544 and 545, displayed an i d e n t i c a l quintuple-banded pattern (data not shown). The MDH patterns correlated rather highly among the tamarensoid i s o l a t e s from English Bay (mean S j = 0.78), but less well i f the d i s t i n g u i s h i n g c r i t e r i o n was morphotype alone (Table 16). The multiple forms of ME comprised 20 mo b i l i t y variants ( F i g . 22 and 26). A l l i s o l a t e s exhibited polymorphism f o r ME, except 508 (New Zealand); contemporaneous c l o n a l i s o l a t e s 403 and 407 showed i d e n t i c a l t r i p l e t bands. As with MDH, the ME p r o f i l e s among i s o l a t e s from English Bay appeared to be more s i m i l a r than when the i s o l a t e s were grouped s t r i c t l y by morphotype (Table 16). Notably, the mean s i m i l a r i t y among ME variants within the c a t e n e l l a - l i k e group was extremely low (S j = .06), although a l l such i s o l a t e s o r i g i n a t e d from Washington State waters. 189 F i g . 25 Zymogram of NAD-dependent malate dehydrogenase (MDH) isozymes of some Protogonyaulax i s o l a t e s from English Bay, B.C. Numbers r e f e r to NEPCC i s o l a t e designations. Exposure: Ektachrome EPY ASA 50; f l l at 1/15 s. F i g . 26 Zymogram of NADP-dependent malic enzyme (ME) isozymes of some Protogonyaulax i s o l a t e s from English Bay, B.C. Numbers r e f e r to NEPCC i s o l a t e designations. The reference i s o l a t e NEPCC 255 i s shown at the l e f t f o r comparison. Exposure: Ektachrome EPY ASA 50; f l l at 1/30 s. 191 For SucDH, sixteen m o b i l i t y variants were i d e n t i f i e d ; i s o l a t e s 71 ( P a t r i c i a Bay) and 516 (English Bay) expressed the same triple-banded enzyme pattern; i s o l a t e s 255 (Lummi Island) and 400, 401, 402 and 407 (Engl i s h Bay) showed a s i n g l e i d e n t i c a l r a p i d l y migrating band ( F i g . 22). The l e v e l of relatedness among SucDH isozymes was very low and did not seem to be linked to geographical o r i g i n or morphotype (Table 16). Comparison of the integrated S j values f o r a l l dehydrogenases suggested that enzyme el e c t r o p h o r e t i c patterns were not t i g h t l y coupled with the p r o v i s i o n a l l y assigned morphotypes. However, there appeared to be a stronger r e l a t i o n s h i p between tamarensoid i s o l a t e s from a given geographical region (English Bay), than when el e c t r o p h o r e t i c data were pooled f o r a l l i s o l a t e s conforming to t h i s morphotype. 6. Isozyme-Based Chemotaxonomic Relationships among Protogonyaulax Isolates a. Phenetic c l u s t e r a nalysis The term "phenon" (Sneath and Sokal, 1962) has been used to r e f e r to a group of p h e n e t i c a l l y s i m i l a r taxa revealed by c l u s t e r or s i m i l a r i t y a n a l y s i s , without reference to an pre-established h i e r a r c h i c a l taxon l e v e l . A more s p e c i f i c term, "zymogen", i s proposed to apply to such groupings established on the basis of s i m i l a r i t y i n e l e c t r o p h o r e t i c a l l y generated zymogram patterns. The boundaries of the zymogen are set by a r b i t r a r i l y s e l e c t i n g the s i m i l a r i t y l e v e l , and may vary depending upon the choice of c l u s t e r i n g algorithm or s i m i l a r i t y measure. 192 A UPGMA linkage dendrogram, a c l u s t e r analysis based upon unweighed pair-group arithmetic average values of S j (Table 16), i s presented i n F i g . 27. Isolates which j o i n the c l u s t e r at a higher s i m i l a r i t y value are considered to be more c l o s e l y r e l a t e d . This phenetic analysis l i n k s an i s o l a t e to previously joined i s o l a t e s i n a stepwise fashion, using the average value of the s i m i l a r i t y c o e f f i c i e n t r e l a t i v e to that of i s o l a t e s already linked i n the c l u s t e r . The "goodness of f i t " of the UPGMA dendrogram to the o r i g i n a l S j data matrix (Table 16) was compared by constructing the cophenetic c o r r e l a t i o n matrix (Table 17) from the dendrogram, and then c a l c u l a t i n g the product-moment c o r r e l a t i o n c o e f f i c i e n t ( r C O p n ) (Sneath and Sokal, 1973) f o r the two matrices. The cophenetic value between any two OTUs (= i n d i v i d u a l i s o l a t e s ) j and k, i s the maximal s i m i l a r i t y S j ^ between the two OTUs implied by the dendrogram. The cophenetic c o r r e l a t i o n c o e f f i c i e n t was calculated according to the formula: Eyiy2 rcoph = ( E y i 2 E y 2 2 ) * where 2y^ and 2y 2 a r e t n e s u m °f squares of and Y2> and £yjy 2 i s the sum of the products; Y^ and Y 2 are the i n d i v i d u a l S j values from the o r i g i n a l data matrix and the cophenetic c o r r e l a t i o n matrix, r e s p e c t i v e l y . The good c o r r e l a t i o n between the cophenetic matrix and the o r i g i n a l character matrix ( r C O p n = 0.74) indicates that the dendrogram i s an acceptably accurate representation of the s i m i l a r i t y r e l a t i o n s h i p s among the Protogonyaulax i s o l a t e s . Interpreted i n the s t r i c t e s t sense, t h i s 1 9 3 F i g . 27 UPGMA linkage 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 , constructed from S j values. Location of i s o l a t e o r i g i n : BC, B r i t i s h Columbia; WA, Washington State; PL, Plymouth, England; PO, Portugal; NZ, New Zealand. A, B and C r e f e r to zymogens clustered on the basis of o v e r a l l e l e c t r o p h o r e t i c s i m i l a r i t y . U P G M A t •C - 4 0 3 • 4 0 4 - 4 0 6 - 4 0 5 4 0 7 4 0 0 - 4 0 1 4 0 2 . 2 5 5 . • 5 1 6 • 7 1 1 8 3 . 5 2 9 . 2 5 3 . 1 8 0 . 5 0 8 . 4 3 5 3 5 5 . 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0 180 0.18 183 0.18 0.23 253 0.18 0.23 0. 34 255 0.33 0.18 0. 18 355 0.14 0.14 0. 14 400 0.30 0.18 0. 18 401 0.30 0.18 0. 18 402 0.30 0.18 0. 18 403 0.26 0.18 0. 18 404 0.26 0.18 0. 18 405 0.26 0.18 0. 18 406 0.26 0.18 0. 18 407 0.26 0.18 0. 18 409 0.23 0.18 0. 18 412 0.23 0.18 0. 18 435 0.18 0.20 0 20 508 0.18 0.22 0 22 516 0.33 0.18 0 18 529 0.18 0.23 0.42 71 180 183 253 255 355 400 401 402 403 404 405 406 407 409 412 435 508 516 Table 17. Cophenetlc correlation matrix constructed from UPGMA dendrogram (Fig. 27) based on dehydrogenase banding patterns. Cophenetic correlation coefficient ( r C O p n ) = 0.74. 1 9 6 phenetic c l u s t e r analysis makes no assumptions or e x p l i c i t statements regarding evolutionary d i r e c t i o n or phylogenetic r e l a t i o n s h i p s , although such inferences have been made before from UPGMA dendrograms (Nei, 1975). Three zymogens may be distinguished from the UPGMA s i m i l a r i t y dendrogram ( F i g . 27) at two a r b i t r a r i l y chosen l e v e l s of s i m i l a r i t y (Sj>0.18 and >0.23). The i s o l a t e s of zymogen A a l l or i g i n a t e from the northeast P a c i f i c , within a radius of less than 100 km from each other. This group comprises a l l the English Bay i s o l a t e s , plus two other i s o l a t e s from the v i c i n i t y , NEPCC 71 from P a t r i c i a Bay, B.C. and 255 from Lummi Island, WA. The second c l u s t e r , zymogen B, includes the i s o l a t e s from Plymouth, England (183), Portugal (253), New Zealand (508), a lone B r i t i s h Columbian i s o l a t e (180), and two i s o l a t e s , NEPCC 435 and 529, from the southern part of the S t r a i t of Georgia i n Washington State. Only the ca t e n e l l o i d NEPCC 355, from the protected waters of Penn Cove, Whidbey Island, at the northern extent of Puget Sound, belongs to the t h i r d zymogen. According to the UPGMA c l u s t e r a n a l y s i s , t h i s i s o l a t e was the most e l e c t r o p h o r e t i c a l l y d i s s i m i l a r from the English Bay tamarensoid group. As might be expected, contemporaneous i s o l a t e s from English Bay, including NEPCC 403/404 and 400/401, were most c l o s e l y paired. However, the Eng l i s h Bay i s o l a t e s did not form a si n g l e uniform c l u s t e r . For example, i s o l a t e s 71 and 255, from the northeast P a c i f i c region, but from outside English Bay, clustered with t h i s group before English Bay i s o l a t e s 409/412. Ca t e n e l l o i d i s o l a t e 529 (Friday Harbor, San Juan Island, WA) was most c l o s e l y grouped with the two European tamarensoid forms, NEPCC 183 and 253. The most geographically d i s t a n t i s o l a t e , NEPCC 508 (New Zealand) was also clustered very l a t e i n the s e r i e s . Nei's genetic i d e n t i t y s t a t i s t i c I (Nei, 1972) has frequently been used 197 to estimate the degree of genetic d i f f e r e n t i a t i o n among populations at the i n t r a s p e c i f i c and intrageneric l e v e l s . This c a l c u l a t i o n gives the p r o b a b i l i t y that two a l l e l e s , one selected from each population, are i d e n t i c a l . The I s t a t i s t i c i s calculated according to the formulae: where x^ i s the frequency of the i t n a l l e l e at locus j i n population X, and y^ i s the frequency of the i t n a l l e l e at locus j i n population Y. The o v e r a l l genetic i d e n t i t y of populations X and Y i s defined as: 1 x y I = --7 ( l X I y ) * where I i s the mean genetic i d e n t i t y over a l l l o c i , I x v i s the mean of ^ xiYi» -'-x i s t n e mean of Sx^ , and I y i s the mean of £y^ 2, f o r populations 1 through n. When a l l a l l e l e frequencies i n the two populations compared are equal, the value of I = 1; i n the case when the populations have no common a l l e l e s 1 = 0 . For the c a l c u l a t i o n of genetic i d e n t i t y , the binary scored isozyme bands i n the character matrix (Table 18) were assumed to represent the monomeric gene products of independent l o c i . The i s o l a t e s of zymogen A were considered as one "population", while the i s o l a t e s of zymogen B, were treated as a second "population". The calculated I value between these two zymogens was 0.47. The genetic distance D (Nei, 1972) between two populations, given as the accumulated number of codon substitutions per locus since the time of Table 18. Binary character state matrix f o r dehydrogenase isozymes coded as presence(l)-absence(0) data. Number of taxa: 20; number of characters: 130. PATBAY71 BRENB180 PLYM0183 LAG0B253 LUMMI255 PENNC355 ENGLB400 ENGLB401 ENGLB402 ENGLB403 1 2 3 4 5 6 7 8 9 IO 1 0. 0 0. 0 0. 0 0. 0 1. 000 0. 0 0. 0 0. 0 0.0 0.0 2 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 1. 000 0. 0 0.0 0.0 3 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 1. 000 1. OOO 1 .000 0.0 4 1. 000 1. 000 0. 0 0, .0 0, ,0 0. 0 0. 0 0. 0 1 .000 O . O 5 0. 0 1. 000 0. 0 0 .0 1. 000 1. 000 0. 0 0. 0 0.0 1 .000 6 0. 0 1. 000 0. 0 1. 000 0. 0 0. 0 0. 0 0. 0 0.0 0.0 7 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 1 .OOO 1 .OOO e 0. 0 1. 000 1. 000 1. 000 0, 0 0. 0 1. OOO 1. OOO 1 .OOO 0.0 9 0. 0 1, .000 0. 0 0. .0 0. 0 0. 0 1. 000 0. o O . O O . O 10 0. 0 0. 0 0. 0 0 .0 0. .0 0. 0 0. 0 0. 0 0.0 0.0 1 1 0. 0 1. 000 0. 0 0. .0 0. .0 1. OOO 0. .0 0, 0 0.0 0.0 12 0. 0 1. 000 ' 0. 0 0 .0 0. .0 1. 000 0. .0 0 .0 0.0 0.0 13 0. 0 1. OOO 0. .0 1 .000 0 .0 0 .0 0 .0 0 .0 0.0 0.0 14 0. 0 0. 0 0. .0 1 OOO 0 .0 0. .0 o. .0 0 .0 0.0 0.0 15 0 .0 0. .0 1. OOO 0 .0 0 .0 0 .0 0 .0 0 .0 0.0 0.0 16 0. .0 0. .0 0. .0 0 .0 1 .000 0. .0 o .0 0 .0 0.0 0.0 17 0 .0 0 .0 0 .0 0 .0 0 .0 0. .0 0 .0 0 .0 0.0 0.0 18 1 .000 : o .0 1 .000 1 .000 0 .0 0 0 o .0 1 OOO 1 OOO 1 .OOO 19 0 .0 1, .000 0. .0 0 .0 0 .0 0. ,0 0. .0 0 .0 0.0 0.0 20 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 1 OOO 1 .OOO 0.0 1 OOO 21 0. .0 0, ,0 0 .0 1 .000 0 .0 1 .000 0 .0 0 .0 0.0 0.0 22 0, .0 0 .0 0 .0 0 .0 0 .0 1 .000 1 .000 1 OOO 1 OOO 0.0 23 0, .0 1. .000 1 .000 1 .000 0 .0 0 .0 1 .000 0 .0 0.0 0.0 24 0 .0 0. .0 0 .0 0 .0 0 .0 0 .0 0 .0 1 OOO 1 .OOO 1 .OOO 25 1. .000 0, .0 0. .0 0 .0 1 .000 0. ,0 1. OOO 1 .OOO 1 OOO 1 .OOO 26 0 .0 o. .0 0 .0 1 .000 0 .0 0 .0 0 .0 1 .OOO 0.0 0.0 27 0 .0 o, .0 0. .0 0 0 0 .0 1. 000 1. .000 0 .0 0.0 O . O 28 0 .0 0. .0 1 .000 0 .0 0 .0 0 .0 0 .0 0 .0 0.0 0.0 29 0 .0 0. .0 0 .0 0 .0 0 .0 0. .0 0 .0 0 .0 0.0 0.0 30 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0. .0 0 .0 0.0 0.0 31 0 .0 0 .0 0 .0 0 .0 0 .0 0. .0 0 0 0 .0 0.0 0.0 32 0 .0 0. .0 0 .0 0 .0 0 .0 0. .0 0 0 0 .0 0.0 0.0 33 1 .000 1 .000 0 .0 0 .0 1 .000 0 .0 0. .0 0, .0 0.0 0.0 34 0 .0 0 .0 0. .0 0 .0 0 .0 0. ,0 0. .0 0. .0 0.0 0.0 35 0 .0 0 .0 1 .000 1 .000 0 .0 0. .0 1. OOO 0 0 O . O 1 .OOO 36 0 .0 0. .0 0. .0 0 .0 0 .0 0. 0 0. 0 0. .0 0.0 O . O 37 0 .0 0 .0 0 .0 0 .0 0 .0 0. .0 0. .0 0. 0 O.O O . O 38 0 .0 0. .0 0 0 0 .0 0 .0 • 0. 0 0. 0 0. 0 1 .OOO 0.0 199 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O i " O o o o o d ' - d o d o o o o o o O ' O o o o o o o o o O ' - O ' o o o o o o o o o o o o 666666666^66 O O O O O O O O O O O O O O O O O l 6*- 6 6 6^66*- 66666006' O O O O O O O O O O O O O O i d ' o o d o o d o o d o - d ' o o o o o 6 -6 6 -o o o o o o '-OOOOO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 6 6 0 0 0 0 0 0 6 0 0 0 0 1 O O ' - ' - O O O O O O O O O O O O O ' - O O O O O O O O O O O O ' - O ' - ' - ' - O O ' - O O ' - ' - O O O O ' 8 8 8 8 888 8 88 88 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O d ' d o « - O O 0 0 0 O 0 0 0 O 0 O ' O O O O 0 0 0 0 O 0 O O « - O < - " - O O ' - O 0 ' - » O O O " -O O Q O O o O 6 O O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 • • ' d d d d o d o o d o o o O ' - o o o o o o o o d d o o o o O ' O O O ' - ' - o o o o o o o o o o o § Q O O O O O Q O O O O O O O O • O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O ' - ' - O O O O O O O — 0 0 0 - " 0 * - - - ' ' - O O O o 6 o — O O O O - -O O O O O O O O O O O O O O O O O O O O O O O O O O O O O i O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O ' - O O O O O O O O O O O O O O O O ' - O O ' - ' - O - ^ O O ' - ' - ' - O O O O ' - O O O O O § 8 8 8 ° ° ° 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 8 8 0 0 0 0 8 0 0 8 0 0 O O O O O O O O ' - O O O O O O O p O O O ' - O O O O O O O ' - O O O O ' - O ' - ' - O O O O ' - O O ' - O O O O O O o O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 * - O 0 0 * - O 0 0 0 0 0 0 0 » - C > 0 0 0 0 0 0 0 0 0 0 0 6 0 6 0 0 b - 6 " : 88 .! O O O O O O O O ' ' 6 0 6 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 6 0 6 0 0 0 0 0 0 ' O ' - o o ' - o o o ' - ' - o o o o ' - ' - o o ' - o o d d o ' - O ' - ' - d d ' - d ' - d d d d o d » 0 ' ^ c < c o n m i * r - e o » 0 ^ c N n ^ i n ( p t ^ « o ) 0 ^ c > i T O v i n u 5 t ~ e o w o ^ c N O « i n u 5 t ~ t t O ) 0 ' - w » n i D n n v « 9 « V T t « 9 « l n l f ) l I l l n l f l l f l l f l l l ) l l l l C l D 0 W U l O w o u l l l > u > ^ ^ ^ ^ ^ ^ s ^ ^ ^ e D l t l B O B l ( l U U U U M U H U M U M - ' - . - . - ' - > - > - ' - ' - ' - » O O O O O O O O O O I 0 (0(0(0(0(0(0(0(0(0000809(8 0 0 - - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - - 0 0 0 0 - 0 0 0 0 0 O O O O - O - O - O O O O b b ^ o b b b b b b b b b b b b b b b b o ^ b b b b ^ b b b b b b b b b ^ b ^ b g b b b b — o o o o o o o o o - - o a o o - o - - o o o — - o — o — - 0 - 0 0 0 0 - - - 0 — o o o o i b b b b b b b b b b b b b b b b b b b b b b b b 8 o o o o o o o o o g g o o o o o o g g o o o g o o g o o o o g o o o o g c n o o g o o 0 5 o g o o g o i o o o o o o o o o o o o O O i - O O O O O O O O O - - O O - O - O O - O O - O O O - O O O O O O O O O - O O O O - O - O o g o o o b o o b b b b o b b o o o o o b o b b o b b o b o o o o o o o o o o o o o o o g 8 0 0 o 0 0 o ' O O O O O O O O O ' 0 0 o O O o - O O O O O O O O - O - O O O - O O O O O O O O O — O O O O — O O O O O - O - O O — - O O 8 o o o g o o b o o b o o o b o o o o g o o b o o o o o o o g b o o 5 o g g g g g 0 0 o o O O O O O O O O O O O O O o o 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 - 0 0 0 - 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 O O O O O O O O O O O O O O O O O O O O O ' O O o o I O O O O O O O O O O 0 0 0 0 0 0 0 0 0 0 0 0 0 : 8 . 8 0 0 - 0 0 0 0 0 0 0 0 - 0 0 b b b b o b b b b b b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 - 0 0 0 0 - - 0 0 0 0 0 o o o o o 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o g o o 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 - 0 0 0 - 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 o o o ^ o o b b o b o o o o o o b b o ^ o o ^ o o o o ^ o o b o o o o b o o o b o o o o o o o o o 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 - 0 0 0 -o o g b b o o o b b o o o o b o g o o g o b o o 0 0 0 0 0 0 0 - 0 0 0 0 -o o o o o o o g o o o o g - 0 0 0 0 0 0 b o o b b b b 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 - 0 - -b b b b b o b b b b b b b b b b b b b b b b b - - 0 0 - 0 0 0 0 - 0 0 0 0 0 0 0 - 0 0 0 0 b b b b b b o o b b o o o b b b o g b b b b 0 0 0 0 0 0 0 - 0 0 0 0 0 - 0 0 - 0 0 0 0 0 0 0 0 0 0 - 0 - 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 o o o o b o b b b b b o b b b b b b b b b b b b b b b b b b b b b b b b b b b 003 o o o o o o o o o o o o o -O O O O O O O O O O O O O Q i O O O — O — O O O O O O O O O O -O O O O O Q O O O O O O O O O O O m z - o - O O O O O - r -CD § 0 0 0 0 0 to o z - a O O O - O O O O — O O O O - O O O O O O — O O O — O O O O O O — — — O — O O M R • CD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Q O O O O TO O 0 O O O o o o o o O 5 O O O O O O O O l z - a O O O O O O O O O O O O O — — O O O — O O O O O O O O O O O O - — O O O O O u r CD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O to O O O O o o O O O O O CD m z — o O O O - O O — O O O O O O O O O O O O O — O O O O O — O — O O O O O O O O O t o i -CD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O to O O O O O o o o o o O z - o O O O - O O O O O - O O O — O O O O O O - - O O - O O O O - - O — O O O O O c n r -CD O O O O O O O O O Q O O O Q O O O O O O O O O O O O O O O O O O O O O O O O to O O O O O O O O O o O O O O O O O O O CD m z — o — 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 - - 0 0 < J ) i -CD 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 to o O O 0 0 -O O O O O ro O O - - O O O O O O O O O - O - O O O O - O O O O - - O O O O O O O O O O O - J O O O O Q O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O to O O O O O O O CJ O O O O O O O tn 0 0 - 0 0 0 0 0 0 0 0 -o b g o o o o o o o o g - o o - o o o o o o o o o - o - o o o - o o o o o o » > r~ o oo O O O O O O O O O O O O ' j O O O g O O O O O O 1 o 0 0 0 0 - 0 0 0 0 0 0 0 0 - 0 -b o b o b o b o b o o b b b o o z - o - o o o - o o o o o o o o - o - o o o - o o l o r -CD j ^ O O O ^ O O O O O O O O ^ O ^ O O O ^ O O Ul O - O - O O O - - O O O O - O - - O O O - O O O O O O - O -o o o o o o o o o o b o o o o o o o o o o o o o o o o o o b i 0 0 0 0 0 0 0 0 a b b b b b b b tn T03 202 O o o o o o o o 6 o o o o o o 6 o o o o o o o o o o 6 o 6 o o o o 6 6 6 o 6 o o 6 6 o o o o o 6-666666-6666-6-6666666666-6-0666---6-00--66666 0 0 0 0 0 0 0 6 0 6 0 0 0 0 6 0 6 0 0 0 0 0 6 0 0 6 0 0 0 0 6 6 6666666-6-6666-6-66666-66-6666--6 6 0 0 0 0 0 0 6 0 0 0 0 0 --666666-66666 0 6 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 6-666666-666666-6-66-0 0 0 0 0 0 0 0 0 0 6 ' b b b b b b d b b b -6 0 0 6 6 0 6 0 0 0 0 0 -66--6-66606 o o 0 8 8 8 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 8 0 0 0 0 0 0 0 0 0 0 0 0 1 O O O O O O O O — O O O O O O O O O — O O O O O O O O — — O O O O — O O O O O O O — O O O O — 8 o o o o o 8 0 8 8 8 8 0 0 8 8 8 ° ° 8 0 0 0 0 0 0 0 0 0 0 0 8 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 8 8 0 0 8 8 0 0 0 0 8 8 8 — O O O O O O O O O O — O O — O O — O O O O — O O O O O O O O O — — — — O O — — O O O O — — — O O O O o o o o o 0 0 § 0 0 0 6 0 0 0 6 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 - — O O O O O O O O O O O O O O O — O— O O O O O O O O O O O O — — — — 0 — 0 0 0 0 0 0 — 0 — 8 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 6 0 6 6 8 0 0 0 0 0 0 0 0 0 0 0 0 O O O O O O O O O O O O - O O O O O O O O O O O O O — O O O - O - - - - O O O O O O O O — O O O O 1 8 0 8 ! 0 5 o o 0 8 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 8 0 8 0 0 0 0 0 0 0 0 0 0 8 0 8 0 8 0 0 0 0 0 0 6 0 0 0 0 0 O ' - o d ' - o o o o d o o o o o o d ' - O ' - o o o o o o o o o o ' - O ' O ' O O O o o o ' O O ' - o o 0 0 0 0 0 0 0 0 6 0 0 0 6 0 6 0 0 0 0 0 0 0 0 6 0 0 6 0 0 0 6 0 6 o o d o d o o o ' - d o d - d ' d d o o d d d d ' - o d ' - d o d ^ d ' -0 0 0 0 0 0 6 6 6 6 6 6 6-§ 0 8 0 0 0 - - b o b 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 6 0 0 0 0 0 0 0 0 0 0 6 0 6 6-666666666666066-6-6666606666-6-6 0 0 0 0 0 0 0 0 0 6 0 0 - 0 0 0 0 0 0 0 0 0 - 0 0 oiO - MOvmioMsmO'- oir)«iniDr-eoo>0 — cMnvwior-eooo- cMCTniipr^oowo — n n v in U M M M U M M U I O M M - > - - - . - . - - . - - . - . O O O O O O O O O g < < l a ) « l l l ) I O < 0 < l l l ' I I O g i » 2 » 2 O i o a i - 4 0 ) i i i i » u u - ' O i o a i ^ » o i * u » - O i o o > i i » u i * u M - ' 0 « i ( o - J o u i f c U M - ' O i o o i > i i » o o — — o o o o o o o o o o o o o o o o o o o o o o o — o — o o — o o o o o o o o o o o o b b o b o b b b o b b b o b b b b o b o b b b o b b o g o Q O o g o o o o o o o o o o o o l O O O O O O O O O O O O O O O O O O O O O O O O O ' i O i i O ' 8 O O O — O O O O O O O O — — O O — O O O O O O O O O O — O — O O O O O — O O O O O O O O O o o o g o o o o o b o o g o o o g b o o o o o b o o o g o o o o o o o o o o o o o o o o o o o o o 0 8 8 O O O O O O — — O O O O O — O O — O O O O O O O O O O — O — O O — O O — — O O O O O O o o o b o b o o o o b o o o o b b o b o b g b o 0 0 0 0 0 0 0 o o O O O O O O O O O O O O O O O ' O O ' O O ' 8 0 0 0 0 0 0 0 0 0 0 0 0 o o o O O O o 0 0 — 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 — 0 — 0 0 — 0 0 0 0 0 0 0 0 0 0 0 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O ' 8 8 : O O O O O O O O O O O O O O O o 1 o 0 0 0 0 0 0 0 0 — 0 0 0 0 0 0 0 — 0 0 0 0 0 0 0 0 0 0 - 0 — 0 0 0 0 0 0 0 0 0 0 - 0 0 0 -b b o b o o b b b o b b b o o o g o o o o o o b o b o g o o o 8 O O O O O O O O O O O O O O O O O o o 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 - 0 0 0 0 0 0 0 0 — — - 0 - 0 0 0 0 - 0 0 0 0 0 - 0 0 — o b o o b o o b o o b o b b o b o o o o o o o b b o o o o o b o o o o o o o o o o g o o o o 8 8 8 8 8 8 8 8 8 0 0 - 0 0 0 0 0 0 - 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 - 0 0 - 0 0 0 0 0 o o g o o o o o o g b b b o o o g o o b o o o b o o o o o g o o o o o o g o o g o o o o o 0 0 0 0 - 0 - 0 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 - - 0 0 o o b b g b g o o o b g o o o o o o o b o o o o o b o b o o b g o o o o o o o o o g g o o 0 0 - - 0 - 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 - 0 0 0 0 - 0 0 0 0 -b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b - 0 0 0 0 0 0 b b b b b b b - - 0 0 0 0 - 0 0 0 0 0 0 0 0 0 - 0 0 - 0 0 0 - 0 0 - 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 - 0 204 divergence, was estimated as: D = -In I where I was obtained from the c a l c u l a t i o n of genetic i d e n t i t y . In t h i s formula, D can vary from 0 to i n f i n i t y . Zymogen A and B were separated by a genetic distance corresponding to 0.76. The divergence time T was calculated from the genetic distance D as: T = D/2a where a i s the rate of e l e c t r o p h o r e t i c a l l y detectable codon sub s t i t u t i o n s per year. D i f f e r e n t estimates f o r a (1.0 X 10"^, Nei, 1972; 2.5 X IO" 8, Sarich, 1977; 1.7 X 10" 8, Thorpe, 1983) gave v a s t l y d i f f e r e n t values f o r the time since divergence of zymogens A and B ranging from 4-22 m i l l i o n years. b. Phylogenetic Linkage Analysis For phylogenetic a n a l y s i s , trees were constructed on the basis of derived characters (apomorphies), rather than shared s i m i l a r i t i e s . Phylogenetic i n t e r p r e t a t i o n of the isozyme data followed the "independent a l l e l e " model (Mickevitch and Johnson, 1976), which assumes, i n the absence of a p r i o r i evidence to the contrary, that the occurrence and/or non-expression of a l l e l e s are independent events. Acquired isozymes were regarded as highly s i g n i f i c a n t , and shared absences, as well as unshared and shared (synapomorphic) isozyme band presences, were noted. 205 To construct the character (Table 18) and Manhattan distance (Table 19) matrices, isozyme m o b i l i t y variants were considered as d i s c r e t e binary coded character states, with equal s i g n i f i c a n c e given to the presence and absence of shared bands ("homologous a l l e l e s ) . The presence-absence coding d i f f e r e d from the Jaccard s i m i l a r i t y comparison, which omitted consideration of negative character matches between taxa. A Prim network ( F i g . 28), an undirected minimum spanning tree constructed using OTUs, rather than hypothetical intermediates, as nodes, was generated from the Manhattan distance matrix with the FORTRAN program PHYSIS (J.S. F a r r i s and M.F. Mickevitch, 1983). The Manhattan distance i s defined as the length of the internode between nodes j and k: n d x ( j , k ) = E | X i r X i k | i=l where X^j and X ^ are the character states of a given character i at nodes j and k, r e s p e c t i v e l y . The Prim algorithm ( F a r r i s , 1970) allows f o r the r e v e r s i b i l i t y of character states, and makes no i m p l i c i t assumptions regarding the phenotype of the ancestral (plesiomorphic) taxon. The b i f u r c a t i o n at English Bay i s o l a t e 407 divided the Prim network ( F i g . 28) into two major branches, one comprising the 1981 i s o l a t e s from English Bay, and an alternate fork which included both c a t e n e l l o i d and tamarensoid morphotypes. The Lummi Island i s o l a t e 255 was c l o s e l y a l l i e d with the intermediate morphotype from the 1982 English Bay bloom, NEPCC 516, and i s o l a t e 71, from adjacent south Vancouver Island waters. B r i t i s h Columbian i s o l a t e 180 (Brentwood Bay) was separated from i t s northeast P a c i f i c contemporaries v i a the two c l o s e l y linked European forms, NEPCC 183 206 Table 19. Manhattan distance matrix computed from the character matrix (Table 18) showing distance 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). PATBAY7 1 BRENB180 PLYM0183 LAG0B253 LUMMI255 1 2 3 4 5 BRENB180 0 354 PLYM0183 0 346 0 269 LAG0B253 O 354 0 308 0 208 LUMMI255 0 223 0 315 0 277 0 300 PENNC355 0 292 0 277 0 285 0 277 0 223 ENGLB400 0 331 0 346 0 308 0 346 0 215 ENGLB401 0 285 0 315 0 308 0 331 0 200 ENGLB402 0 277 0 354 0 300 0 369 0 238 ENGLB403 0 323 0 354 0 269 0 323 0 208 ENGLB404 0 254 0 346 0 292 0 315 0 185 ENGLB405 0 308 0 385 0 315 0 323 0 223 ENGLB406 0 323 0 354 0 300 0 338 0 208 ENGLB407 0 238 0 331 0 246 0 285 0 185 ENGLB409 0 308 0 323 0 285 0 308 0 238 ENGLB412 0 292 0 354 0 300 0 338 0 254 FRIDH435 0 308 0 277 0 208 0 231 0 177 NZEAL508 0 338 0 354 0 269 0 262 0 285 ENGLB516 0 254 0 331 0 292 0 315 0 215 FRIDH529 o 354 0 308 0 223 0 292 0 300 PENNC355 ENGLB400 ENGLB401 ENGLB402 ENGLB403 6 7 8 9 10 ENGLB400 0 269 ENGLB401 0 269 0 138 ENGLB402 0 308 0 192 0 146 ENGLB403 0 246 0 238 0 223 0 200 ENGLB404 0 238 0 200 0 185 0 177 0 085 ENGLB405 0 292 0 300 0 285 0 277 O 169 ENGLB406 0 292 0 238 0 238 0 231 0 108 ENGLB407 0 192 0 231 0 231 0 238 0 146 ENGLB409 0 292 0 238 0 254 0 231 0 169 ENGLB412 0 262 0 315 0 315 0 292 0 262 FRIDH435 0 246 0 254 0 254 0 292 0 215 NZEAL508 o 246 o 300 0 331 0 323 0 292 ENGLB516 0 269 0 262 0 231 0 269 0 254 FRIDH529 0 323 0. 238 O 269 0 262 0. 292 ENGLB404 ENGLB405 ENGLB406 ENGLB407 ENGLB409 1 1 12 13 14 15 ENGLB405 0 192 ENGLB406 o 100 O. 215 ENGLB407 0 123 0. 192 0 208 ENGLB409 0 162 0. 246 0 215 0. 192 ENGLB412 0 254 0. 292 0 277 0. 238 0. 215 FRIDH435 0 223 0. 246 0 262 0. 192 0. 246 NZEAL508 o 269 0. 338 O 292 0. 285 O. 323 ENGLB516 0 231 0. 238 0 285 0. 262 0. 285 FRIDH529 0 315 O. 308 0 323 0. 285 O. 277 ENGLB412 FRIDH435 NZEAL508 ENGLB516 16 17 18 19 FRIDH435 0 308 NZEAL508 0 292 0. 262 ENGLB516 0 315 0. 238 0. 285 FRIDH529 O 369 0. 262 O. 292 0. 269 208 F i g . 28 Unsealed Prim network i n d i c a t i n g minimum spanning distances between Protogonyaulax i s o l a t e s , based upon electrophoretic p r o f i l e s of dehydrogenase isozymes. Numbers are sequential Manhattan distance i n t e r v a l s between nodes ( i s o l a t e s ) . O . 138 E N G L B 4 0 0 " 0 . 1 4 6 E N G L B 4 0 1 ' PENNC355—-| NZEAL508 210 (Plymouth, England), and the Portuguese i s o l a t e , NEPCC 253. Notably, the Friday Harbor (San Juan Island, WA) c a t e n e l l o i d i s o l a t e s 435 and 529 were connected through the c l a s s i c "tamarensis" from Plymouth, while catenella-morphotype 355 from Penn Cove (Whidbey Island, WA) was most c l o s e l y associated with the New Zealand tamarensoid i s o l a t e . Phylogenetic r e l a t i o n s h i p s among i s o l a t e s were also diagramatically expressed as a Wagner tree ( F a r r i s , 1970), which permits the r e v e r s i b i l i t y of character states and allows considerable heterogeneity i n evolutionary rates without serious d i s t o r t i o n s . An optimized Wagner tree ( F i g . 29), with the nodes representing intermediate hypothetical taxonomic units (HTUs), was constructed from the binary coded character data matrix (Table 18). Since the tree was rooted at the hypothetical midpoint, the character states of the most ancestral HTU were assumed to be close to the centroid of extant OTUs. This rooting procedure requires only the assumption that the homogeneity i n evolutionary rates i s approximately equivalent for large amounts of divergence, a l e s s stringent c r i t e r i o n than for i n f e r r i n g phylogeny from phenetic c l u s t e r a n a l y s i s . The "goodness of f i t " of a l t e r n a t i v e phylogenetic trees to the input data matrix was compared by various f i t s t a t i s t i c s ( F i t c h and Margoliash, 1967; Kluge and F a r r i s , 1969; F a r r i s , 1972; Sneath and Sokal, 1973; Prager and Wilson, 1976). The F - s t a t i s t i c of Prager and Wilson (1976) i s given by: n 100 S ( I ± - 0 ±) F = i=l n S I i i-1 211 F i g . 29 Optimized midpoint rooted Wagner tree showing phylogenetic 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 elec t r o p h o r e t i c p r o f i l e s of dehydrogenase isozymes. Numbers on internodes and terminal branches represent branch lengths corresponding to the number of character changes (apomorphies). F i t s t a t i s t i c s : Length=323; 01=40.25; F-ratio=34.29. 212 ENGLB 406 5 ^ ENGLB 403 EN GLB 402 ENGLB 400 9 10 19 ±9 ENGLB 407 ENGLB 412 LUMMI 255 ENGLB 405 19 PATBAY 71 ENGLB 516 FRIDH 435 17 IS FRIDH 529 PLYMO 183 LAGOB 253 BRENB 180 PEN NC 355 NZEAL 508 213 where f o r n pairwise comparisons of taxa, I and 0 are input (observed) and output ( p a t r i s t i c ) distances, r e s p e c t i v e l y . A better f i t , i n d i c a t i n g less homoplasy i n the data, i s given by smaller F values. The consistency index CI (Kluge and F a r r i s , 1969) assesses the f i t of the character d i s t r i b u t i o n s given by the phylogenetic tree to the character data matrix. The CI for character i i s computed according to: C I i = S i / P i where S^ i s the number of changes for character i implied from the observed data and i s the number of steps given by the c l a s s i f i c a t i o n scheme. The o v e r a l l consistency value i s the sum of a l l the character ranges, or the number of steps implied by the data set, divided by the t o t a l length of the tree. The most d i a g n o s t i c a l l y e f f i c i e n t (parsimonious) tree was given by the WAG.S option of the PHYSIS program, which invoked global branch swapping to improve parsimony. The aim of t h i s procedure i s to minimize P^, the number of steps i n the tree. This was accomplished a f t e r optimization using the command LFIT to generate phylogenetic f i t s t a t i s t i c s f o r a l t e r n a t i v e t rees. Only a s i n g l e minimum length tree (Length=323; CI=A0.25; F-ratio=34.29) ( F i g . 29) was obtained that described the data. The r e l a t i v e l y low CI value i s i n d i c a t i v e of abundant homoplasy i n the data represented by the tree. From the hypothetical root, the i s o l a t e s were d i s t r i b u t e d along two major branches. The i s o l a t e s i n the upper f u r c a t i o n were e x c l u s i v e l y tamarensoid or intermediate morphotypes from the southern Georgia S t r a i t region. The lower f u r c a t i o n consisted of a l l the c a t e n e l l o i d i s o l a t e s , plus tamarensoid i s o l a t e s from diverse locations (England, New Zealand, 214 Portugal), but also included NEPCC 180, an i s o l a t e geographically a f f i l i a t e d with those on the other branch. Another anomaly regarding the phylogenetic p o s i t i o n of i s o l a t e 180 was r e f l e c t e d i n i t s highly derived state; of a l l the OTUs examined, i t was aligned along the longest s i n g l e branch (23 apomorphies) from a terminal node to the nearest ancestral node. The i s o l a t e s from English Bay (1981) were c l e a r l y d i f f e r e n t i a t e d from the r e s t of the i s o l a t e s at a node located close to the root. As i n the UPGMA c l u s t e r analysis ( F i g . 27) and the Prim network ( F i g . 28), the Plymouth "tamarensis" i s o l a t e was c l o s e l y linked with the c a t e n e l l o i d 529 from Friday Harbor. Both the Wagner analysis and the Prim network indicated that the c l o s e s t r e l a t i o n s h i p between the New Zealand i s o l a t e and an extant OTU was with the Washington State c a t e n e l l o i d NEPCC 355. However, according to the Wagner ana l y s i s , both i s o l a t e s are highly derived; NEPCC 355 and 508 exhibited 15 and 17 apomorphies, r e s p e c t i v e l y , from the nearest a n c e s t r a l node. 7. Discussion i . The dehydrogenase isozymes and genotype As a group, the " s p e c i f i c " dehydrogenases of primary intermediary metabolism are considered to be rather genotypically conservative (Johnson, 1973; King et a l . , 1974; G i l l e s p i e and Langley, 1974; G o t t l i e b , 1981). By monitoring the r e p r o d u c i b i l i t y of c o n s t i t u t i v e l y produced isozyme p r o f i l e s within a given i s o l a t e i n long-term culture, i t was p o s s i b l e to "type" i s o l a t e s according to t h e i r enzyme complement and to 215 compare e l e c t r o p h o r e t i c a l l y c l a s s i f i e d s t r a i n s with fresh i s o l a t e s from natural populations. Isozyme banding patterns were stable enough to serve as chemotaxonomic characters to discriminate between i s o l a t e s , morphotypes and geographical populations. The increased GDH a c t i v i t y and the induction of formerly undetected isozyme bands, associated with high ammonium l e v e l s i n two i s o l a t e s , p a r t i c u l a r l y i n older cultures, appeared to be correlated with higher b a c t e r i a l numbers. The d i r e c t contribution of bacteria to GDH isozyme bands was discounted by the d i n o f l a g e l l a t e - f r e e b a c t e r i a l controls. The most l i k e l y explanation for t h i s enzyme induction i s that s u b s t a n t i a l ammonium was released into the medium by increased b a c t e r i a l a c t i v i t y and senescent d i n o f l a g e l l a t e s . The increase i n ambient ammonium concentration may have favoured a switch i n d i n o f l a g e l l a t e metabolism from the higher energy, higher a f f i n i t y GS/GOGAT (glutamine synthetase: glutamine [amide]: 2-oxoglutarate aminotransferase) pathway of ammonium a s s i m i l a t i o n to the lower energy r e q u i r i n g (but lower a f f i n i t y ) system based on GDH. The temptation to assign dehydrogenase isozymes to s p e c i f i c gene l o c i was d e l i b e r a t e l y r e s i s t e d , i n . the absence of information on inheritance patterns. Without d e t a i l e d knowledge regarding the chromosome number of the i s o l a t e s , the chromosomal events of sexual recombination, and the p o s s i b i l i t y of p o s t - t r a n s l a t i o n a l modification and e l e c t r o p h o r e t i c a r t i f a c t s , the observed v a r i a t i o n was interpreted phenetically, even though the basis f o r the differences i n isozyme mobility were recognized to be fundamentally genetic. In most organisms, the occurrence of monomers among the p y r i d i n e - l i n k e d dehydrogenases i s r e l a t i v e l y infrequent (Harris and Hopkinson, 1976). I t i s l i k e l y that many, i f not most, of the multiple dehydrogenase bands 216 expressed i n Protogonyaulax i s o l a t e s represent multimeric enzymes with isozyme substituents derived from d i f f e r e n t gene l o c i . The p o s s i b i l i t y that the multiple isozyme expression observed i n Protogonyaulax may r e s u l t from gene d u p l i c a t i o n a r i s i n g through interchromosomal events such as overlapping r e c i p r o c a l translocations and unequal crossing over ( G o t t l i e b , 1977) could not be confirmed or disproved. I t i s also possible that eupolyploid or aneuploid increase i n chromosome number may be responsible for the abundant isozymic heterogeneity i n Protogonyaulax. P o l y p l o i d i z a t i o n produces an enlarged genome and, i n theory (Sneath and Sokal, 1973; Ferguson, 1980; G o t t l i e b , 1981), and u s u a l l y i n p r a c t i c e ( G o t t l i e b , 1976; 1984; Roose and G o t t l i e b , 1976), increased enzymatic d i v e r s i t y r e s u l t s from the consequent increase i n the numbers of s t r u c t u r a l gene l o c i . Increased isozymic heterogeneity due to gene d u p l i c a t i o n and polyploidy has been credited with extending the tolerance range of organisms by providing enhanced metabolic f l e x i b i l i t y ( G o t t l i e b , 1976; Roose and G o t t l i e b , 1976). I t i s s i g n i f i c a n t to note that there was no c o r r e l a t i o n between the number of isozyme bands produced by Protogonyaulax i s o l a t e s and the amount of DNA per c e l l . The two i s o l a t e s , which contained h a l f the DNA t y p i c a l l y found i n Protogonyaulax NEPCC 253 and 508, displayed a number of ele c t r o p h o r e t i c bands approximately equal to the mean among a l l i s o l a t e s . Although i t was not possible to a t t r i b u t e i n d i v i d u a l isozyme bands to s p e c i f i c i n t r a c e l l u l a r compartments, the enzyme extraction procedure adopted was apparently able to s o l u b i l i z e both cytoplasmic and organellar constituents (see Appendix I I ) . For enzyme systems, such as NAD-dependent MDH, which may be l o c a l i z e d within separate s u b c e l l u l a r compartments - i n mitochondria, microbodies, and the cytoplasm ( G o t t l i e b , 1981) - c e l l u l a r 217 f r a c t i o n a t i o n techniques are required to d i s t i n g u i s h allozyme variants of the same l o c i from isozymes derived from d i f f e r e n t compartments. The f a c t that dehydrogenases, such as SucDH and IDH, which are u s u a l l y considered to be mitochondrial enzymes, at l e a s t i n higher organisms, and ME, which occurs i n the chloroplasts of higher plants ( G o t t l i e b , 1981), were e l e c t r o p h o r e t i c a l l y detectable, supports the view that the organelles containing these enzymes were completely disrupted by sonication. Examination of c e l l fragments by high power phase-contrast microscopy (1,000X), following intensive sonication, revealed few i n t a c t chloroplasts or mitochondria. The evidence from fungi, protozoa, and higher animals indicates great i n t e r s p e c i f i c and i n t r a s p e c i f i c d i v e r s i t y i n the mitochondrial genetic system (Mahler, 1981). R e s t r i c t i o n endonuclease digests of the mitochondrial DNA i n one syngen of the Paramecium a u r e l i a complex, P. t e t r a u r e l i a , d i f f e r e d in one-quarter of the stocks examined (Fin d l y and G a l l , 1980), yet isozyme homogeneity was strongly conserved within the syngen. I t i s i n t e r e s t i n g to note that the dehydrogenases which are presumed to be mitochondrial, also showed the lowest l e v e l of genotypic s i m i l a r i t y among the Protogonyaylax i s o l a t e s . Current studies have suggested that the mitochondrial genome of higher vertebrates has evolved more r a p i d l y than the nuclear genome (Brown et a l . , 1979). I t remains to be determined whether mitochondrial and c h l o r o p l a s t i c isozymes i n Protogonyaulax are coded for by organellar or nuclear genes, or a combination of the two. An answer to t h i s question may have evolutionary s i g n i f i c a n c e with respect to the rate of change occurring i n the genes coding f o r such organellar proteins. 218 i i . Isozyme v a r i a t i o n and genetic i d e n t i t y Almost i n v a r i a b l y , genetic i d e n t i t y values among co n s p e c i f i c populations determined by enzyme electrophoresis are higher than among congeneric populations (Avise, 1975; Avise et a l . , 1975). For sexually reproducing higher plants, genetic i d e n t i t y values calculated as Nei's I s t a t i s t i c (Nei, 1972) for co n s p e c i f i c populations were t y p i c a l l y >0.90, while values f o r congeneric plant species ranged from 0.28-0.94, with a mean of 0.67 ( G o t t l i e b 1977; 1981). For vertebrate populations, mean values of I c a l c u l a t e d from Ayala (1983) yielded comparable estimates for l o c a l populations (0.96), subspecies (0.80), and between species and c l o s e l y r e l a t e d genera (0.53), Recently compiled l i t e r a t u r e values f o r thousands of co n s p e c i f i c populations and hundreds of congeneric and c o n f a m i l i a l groupings of higher plants and animals revealed that only about 2% of the I values between cons p e c i f i c populations were lower than 0.90; approximately 85% of the congeneric associations were above 0.35, while 80% of the values between c o n f a m i l i a l genera f e l l below 0.35 (Thorpe, 1983). I t i s s t r i k i n g l y obvious that i n comparison with o b l i g a t e l y sexual d i p l o i d higher plants ( G o t t l i e b , 1981) and animals (Shaw, 1965), and with d i p l o i d algae (Murphy and G u i l l a r d , 1976; Cheney and Babbel, 1978; Gallagher, 1980), the dehydrogenases of the Protogonyaulax species complex are extremely polymorphic. Similar large numbers of electrophoretic variants were reported f o r GDH and MDH i n the haploid d i n o f l a g e l l a t e s , Peridinium spp. (Hayhome and P f i e s t e r , 1983; Hayhome, 1985; Whitten and -Hayhome, 1985) and Glenodinium foliaceum (Whitten and Hayhome, 1985). The remarkably high degree of enzymatic d i v e r s i t y and heterogeneity observed within the Protogonyaulax tamarensis/catenella species complex 219 was evident both between and within morphotypes and among geographical groupings of morphologically s i m i l a r i s o l a t e s . There was su b s t a n t i a l v a r i a b i l i t y even among contemporaneous i s o l a t e s from the same l o c a l e (English Bay). This i s consistent with the observations that, i n general, the proportion of polymorphic l o c i (P) i n invertebrate populations i s s i g n i f i c a n t l y higher than f o r vertebrates and plants (Avise, 1975; Nevo, 1978); marine invertebrates are more polymorphic than t e r r e s t r i a l forms (Nevo, 1978). Electrophoretic studies on c e r t a i n a l g a l species have often indicated a high degree of genetic homogeneity. For the diatoms T h a l a s s i o s i r a spp. (Murphy and G u i l l a r d , 1976) and A s t e r i o n e l l a formosa (Soudek and Robinson, 1983), there was l i t t l e apparent electrophoretic v a r i a t i o n among c l o n a l i s o l a t e s from a given geographical l o c a t i o n , even when i s o l a t e s were obtained during d i f f e r e n t seasons or years. However, a l e v e l of electrophoretic s i m i l a r i t y congruent with that observed within the Protogonyaulax species group has been noted within other a l g a l morphospecies and among congeneric populations. E l e c t r o p h o r e t i c v a r i a b i l i t y between seasonal blooms of the d i p l o i d marine diatom Skeletonema costatum exceeded that between s i b l i n g species of Drosophila and higher plants (Gallagher, 1980), while among species placed i n the red a l g a l genus Eucheuma (Cheney and Babbel, 1978), genetic i d e n t i t y values as low as 0.37 were recorded between one pa i r of species. The mean s i m i l a r i t y c o e f f i c i e n t (Sj) determined i n t h i s study (0.22), was comparable to values reported f o r other d i n o f l a g e l l a t e s (Schoenberg, 1976; Schoenberg and Trench, 1980a; Hayhome and P f i e s t e r , 1983; Hayhome, 1985) and for s i b l i n g species (syngens) of c i l i a t e s i n the Paramecium and Tetrahymena pyriformis complexes (Borden et a l . , 1973b; data recalculated 220 from Adams and A l l e n , 1975; Landis, 1982). Hayhome and P f i e s t e r ' s (1983) study on congeneric Peridinium i s o l a t e s gave a mean Sj value of 0.34 (range 0.11-0.75). Calculations based upon the data given by Schoenberg (1976) for morphologically s i m i l a r s t r a i n s of Symbiodinium microadriaticum yielded a mean S j value of 0.19 (range 0.00-0.72). The genetic i d e n t i t y (I) found between the two major zymogens of Protogonyaulax was within the range of values f o r pairwise comparisons of zymogen groups of the S. microadriaticum species complex. The high l e v e l of e lectrophoretic d i v e r s i t y i n Protogonyaulax i s cl o s e r to that observed among outbreeding syngens of the bursaria complex of Paramecium, than to that found among the P. a u r e l i a group, which displays a more patchy e c o l o g i c a l d i s t r i b u t i o n and l e s s e r associated isozyme v a r i a t i o n (Landis, 1982). The nearly randomly d i s t r i b u t e d P. bursaria complex i s considered to represent the more g e n e r a l i s t approach to resource u t i l i z a t i o n . An analysis of genetic v a r i a t i o n within more than 200 species (Nevo, 1978) has shown that higher l e v e l s of genetic v a r i a t i o n , as determined from isozyme data, were us u a l l y associated with cosmopolitan species with a g e n e r a l i s t strategy for resource u t i l i z a t i o n . This i n t e r p r e t a t i o n of e c o l o g i c a l strategy and d i s t r i b u t i o n may account for the high v a r i a t i o n observed i n Protogonyaulax populations. The e l e c t r o p h o r e t i c v a r i a t i o n observed i n Protogonyaulax could not be co r r e l a t e d with s p e c i f i c habitat d i f f e r e n c e s . This i s i n contrast to the s i t u a t i o n i n the diatom T h a l a s s i o s i r a pseudonana, for which gross v a r i a t i o n i n e l e c t r o p h o r e t i c phenotypes was associated with habitat differences between oceanic and n e r i t i c populations; populations from boundary waters exhibited a mixture of the phenotypes c h a r a c t e r i s t i c of other regions (Murphy and G u i l l a r d , 1976; Brand et a l . , 1981b). The absence of apparent 221 c o r r e l a t i o n i n Protogonyaulax may be due to a low l e v e l of habitat d i f f e r e n t i a t i o n among areas where bloom populations are observed, or to a lack of knowledge regarding the features which characterize the mic r o s t r u c t u r a l d i v e r s i t y of these habitats. Two important points must be noted i n the i n t e r p r e t a t i o n of the implications of these r e s u l t s . F i r s t , the sample s i z e (number of i s o l a t e s examined) was rather small; t h i s may allow some degree of s t a t i s t i c a l bias i n the determination of "gene frequencies", as i n f e r r e d from ele c t r o p h o r e t i c patterns, due to unrepresentative sampling. I t may also r e s u l t i n an underestimate of v a r i a t i o n i n the "population". No one c l o n a l i s o l a t e of Protogonyaulax can be considered to represent the zymogen "type" of a l o c a l bloom population; thus i n d i v i d u a l S j values are properly regarded as r e f l e c t i n g s i m i l a r i t y between pai r s of i s o l a t e s , not n e c e s s a r i l y between geographical populations. With t h i s i s mind, e f f o r t s to determine e l e c t r o p h o r e t i c v a r i a t i o n and genetic i d e n t i t y among geographically d i s t i n c t populations require the sampling of multiple i s o l a t e s from each l o c a l e . Nevertheless, an empirical analysis of the e f f e c t of small sample s i z e on e l e c t r o p h o r e t i c a l l y determined estimates of genetic i d e n t i t y and divergence within and among populations (Gorman and Renzi, 1979) has shown that sample s i z e has r e l a t i v e l y l i t t l e e f f e c t on these c o e f f i c i e n t s , provided the number of l o c i sampled i s s u f f i c i e n t l y large. Assuming that each m o b i l i t y v a r i a n t represents the product of a s i n g l e independent locus i n Protogonyaulax, the number of l o c i sampled (n=130) i n the present study i s large enough to minimize random sampling error, even though geographical populations are very diverse. A second, perhaps more serious, possible source of error i s due from the f a c t that these d i n o f l a g e l l a t e s do not always represent a "population" i n the 222 c l a s s i c a l Mendelian sense, since they are not n e c e s s a r i l y f r e e l y interbreeding nor sympatrically d i s t r i b u t e d . i i i . Evolutionary divergence and phylogeny For d i p l o i d outbreeding populations, estimates of genetic i d e n t i t y and divergence times based upon ele c t r o p h o r e t i c data are i n reasonable agreement with values derived by other techniques (Nei, 1975). Nei's genetic i d e n t i t y I and genetic distance D c o e f f i c i e n t s were designed to apply to d i p l o i d sexual organisms, yet nothing precludes t h e i r t h e o r e t i c a l a p p l i c a t i o n to p r i m a r i l y or e x c l u s i v e l y asexual haploid species (Tibayrenc, 1980). Nevertheless, estimates of divergence time calculated from Nei's index must be viewed with several caveats i n mind. Such c a l c u l a t i o n s make the i m p l i c i t assumptions than evolution proceeds by the most parsimonious route and that rates of gene s u b s t i t u t i o n are constant, not only among d i f f e r e n t l o c i , but across p h y l e t i c l i n e s over time, as w e l l . Although primary protein sequence data often ind i c a t e that amino a c i d s u b s t i t u t i o n rates are roughly constant, serving as a "molecular clock", and immunological distances are u s u a l l y a reasonable r e f l e c t i o n of divergence time, exceptions have also been noted (De Haen and Neurath, 1976; Ferguson, 1980; F a r r i s , 1981). I f s e l e c t i o n pressures are not constant i n time and among d i f f e r e n t f u n c t i o n a l proteins, n o n - l i n e a r i t y i n s u b s t i t u t i o n rates i s anticipated. Serious d i s t o r t i o n could a r i s e when the estimates are used for i n t r a - and i n t e r s p e c i f i c comparisons of genetic distance and phylogenetic reconstruction. In s p i t e of the above cautionary notes on the p o t e n t i a l n o n - l i n e a r i t y 223 of genetic divergence time, several attempts to c a l i b r a t e the "molecular clock" have been made. Nei's (1972) o v e r a l l estimate of the rate of protein evolution suggested that D = 1 corresponded to a divergence time of about 5 X 10^ years. Sarich (1977) c r i t i c i z e d t h i s value, when applied to e l e c t r o p h o r e t i c data, claiming that the l o c i of the enzymes involved i n complex metabolism u s u a l l y sampled i n electrophoresis evolve more slowly than other proteins, by an order of magnitude (T = 30 X 10^ years f o r D = 1). An integrated average value f o r various taxa approximately i n agreement with divergence data from other sources, suggested that D = 1 corresponded to a divergence time i n the region of 18-20 x 10^ years (Thorpe, 1983). The lack of congruity i n these estimates makes ca l c u l a t i o n s of divergence time f o r Protogonyaulax zymogens A and B based upon such values h i g h l y speculative at t h i s time. Wagner analysis of electrophoretic patterns was previously shown to y i e l d representations of phylogeny congruent with those based upon morphometric characters, even with s u b s t a n t i a l homoplasy i n the data (Mickevitch and Johnson, 1976). In any case, the assumption of uniform, i f not constant, evolutionary rates among s t r u c t u r a l genes of Protogonyaulax i s o l a t e s i s not unreasonable. Character evolution can include convergences, re v e r s a l s , as well as the p a r a l l e l retention of plesiomorphic states ( F a r r i s , 1981; Avise, 1983). Unfortunately, the e f f e c t s of many of these homoplasious events may be l o s t through conversion of the character data to a distance matrix used f o r the construction of Prim networks and distance Wagner trees. Since evolutionary changes occur i n the characters d i r e c t l y and not on the converted distances (Avise, 1983), more phylogenetic information can often be retained by inputting the character matrix d i r e c t l y to the tr e e - b u i l d i n g 224 algorithm. Nevertheless, i t i s not s u r p r i s i n g that the branching pattern given i n the Prim network was s i m i l a r to that generated by the character Wagner algorithm. The WAG algorithm e s s e n t i a l l y creates branching by constructing a network analogous to a Prim network and then roots i t at the designated terminal taxon. i v . E l e c t r o p h o r e t i c evidence and speciation The isozyme data f o r Protogonyaulax support the conclusion (Hayhome and P f i e s t e r , 1983; Hayhome, 1985) that enzyme electrophoresis o f f e r s a means of biochemically discriminating between c l o s e l y r e l a t e d thecate d i n o f l a g e l l a t e s f o r which the pl a t e patterns are e s s e n t i a l l y i d e n t i c a l . However, i n contrast to other e l e c t r o p h o r e t i c studies on the enzyme p r o f i l e s of d i n o f l a g e l l a t e s , that showed a rough c o r r e l a t i o n between electrophoretic relatedness and the conventionally assigned morphospecies (Daggett and Nerad, 1980; Beam et a l . , 1982; Hayhome and P f i e s t e r , 1983; Watson and Loeblich, 1983; Beam and Himes, 1984; Hayhome, 1985), t h i s was not the case f o r Protogonyaulax. S i g n i f i c a n t l y , the isozyme data do not support the establishment of a clear l i n e of demarcation between i s o l a t e s conforming to the tamarensoid versus the c a t e n e l l o i d morphotype. Correspondence between isozyme banding patterns indicates that, i n general, contemporaneous i s o l a t e s from the same l o c a t i o n were more c l o s e l y r e l a t e d to each other, than to i s o l a t e s obtained from outside the region. However, NEPCC 355, and to a l e s s e r extent NEPCC 180, were rather exceptional i n t h e i r e l e c trophoretic d i s t i n c t n e s s from other i s o l a t e s of the northeast P a c i f i c region. Only a s i n g l e i s o l a t e from Puget Sound (NEPCC 355) was subjected to electrophoresis, since the other a v a i l a b l e 225 i s o l a t e , NEPCC 356, grew extremely poorly on enriched seawater from Eng l i s h Bay - a possible i n d i c a t i o n of genotypic divergence. Mutational changes or random genetic d r i f t occurring e i t h e r within i n s i t u populations or during long-term maintenance i n culture may account f o r the large electrophoretic gap between i s o l a t e 355, the New Zealand i s o l a t e , and the re s t of the Protogonyaulax i s o l a t e s . Such divergence could represent i n c i p i e n t speciation i n these i s o l a t e s . This i n t e r p r e t a t i o n would not be unprecedented f o r d i n o f l a g e l l a t e s ; many reproductively i s o l a t e d s i b l i n g species of the Crypthecodinium cohnii complex are represented by s i n g l e e l e c t r o p h o r e t i c a l l y unique i s o l a t e s (Beam et a l . , 1982). Further i n v e s t i g a t i o n of Protogonyaulax i s o l a t e s from Puget Sound would be required to t e s t hypotheses regarding the uniqueness of t h e i r genotypic c h a r a c t e r i s t i c s . E. V a r i a t i o n i n Toxin Composition and T o x i c i t y 1. Introduction Investigations on the l i n k between the occurrence of P. ca t e n e l l a i n the water column and s h e l l f i s h t o x i c i t y on the P a c i f i c coast of North America (Sommer and Meyer, 1937; Sommer et a l . , 1937; Riegel et a l . , 1949; Schantz and Magnusson, 1964) led to the e a r l i e r assumption that a s i n g l e toxin was responsible f o r t o x i c i t y i n both the d i n o f l a g e l l a t e and i n s h e l l f i s h (Schantz et a l . , 1966; Proctor et a l . , 1975; Schantz et a l . , 1975a and b). The toxin, previously extracted p r i m a r i l y from C a l i f o r n i a n mussels and Alaskan butter clams, was eventually i s o l a t e d and characterized 226 from P. c a t e n e l l a i n culture (Schantz et a l . , 1966; Proctor et a l . , 1975). When the structure of the s h e l l f i s h toxin, named sax i t o x i n (STX) a f t e r the Alaskan butter clam Saxidomus giganteus, was compared with that extracted from the d i n o f l a g e l l a t e , i t was found to be i d e n t i c a l (Schantz, 1975a and b). Reports of the r e l a t i o n s h i p between s h e l l f i s h t o x i c i t y and the presence of P. tamarensis on the A t l a n t i c coast of North America (Needier, 1949; Prakash, 1963; Prakash et a l . , 1971) gave r i s e to s i m i l a r e f f o r t s to e s t a b l i s h the nature of "tamarensis" toxin(s) (Schantz et a l . , 1975b; Shimizu et a l . , 1975a and b). For P. tamarensis, the separation of previously unknown a d d i t i o n a l t o x i c f r a c t i o n s from STX by chromatographic methods (Shimizu et a l . , 1975a and b; 1977; Oshima et a l . , 1976; 1977; Oshima and Yasumoto, 1979) suggested the p o s s i b i l i t y that to x i n composition might be used chemotaxonomically to separate " c a t e n e l l a " from "tamarensis". As more re f i n e d methods were applied to the analysis of PSP toxins from P a c i f i c populations, H a l l et a l . (1979) succeeded i n i d e n t i f y i n g the presence of toxins other than STX i n P. c a t e n e l l a from Washington State waters. In Japan, other PSP toxins, i n addition to STX, were also detected i n c a t e n e l l o i d morphotypes from Senzaki Bay (Noguchi et a l . , 1983) and Owase Bay (Oshima et a l . , 1976; Shimizu et a l . , 1977; Shimizu, 1979), and from s h e l l f i s h contaminated by such organisms from Owase Bay (Hashimoto et a l . , 1976; Oshima et a l . , 1976; Shimizu et a l . , 1977; Shimizu, 1979) and the Inland Sea (Oshima et a l . , 1978; Shimizu, 1979). These a d d i t i o n a l toxins corresponded i n chemical properties to those found in "tamarensis" from the A t l a n t i c coast of North America, r a i s i n g doubts regarding the morphospecific and geographic uniqueness of the toxin 227 spectra. The toxins extractable from Protogonyaulax include STX and at l e a s t 11 n a t u r a l l y occurring water soluble d e r i v a t i v e s ( H a l l , 1982; H a l l and Reichardt, 1984; Boyer et a l . , 1985) (Table 7; F i g . 30), which vary i n t o x i c i t y , ease of h y d r o l y s i s , and chromatographic behavior. Although t h i s s u i t e of r e l a t e d compounds has been r e f e r r e d to as the "saxitoxins" ( H a l l , 1982; H a l l and Reichardt, 1984), sax i t o x i n i s the semi-systematic name for an i n d i v i d u a l compound. Thus the name applied c o l l e c t i v e l y seems inappropriate. More su i t a b l e i s the term "gonyautoxins", o r i g i n a l l y used to r e f e r to GTX^-GTX^, and which by l o g i c a l extension may be applied to the e n t i r e family of compounds (e.g., GTX5 = B]_, GTX5 = B2, e t c . ) . This term has the added benefit of r e f l e c t i n g the gonyaulacoid d i n o f l a g e l l a t e o r i g i n of the toxins, rather than emphasizing t h e i r l o c a l i z a t i o n within s h e l l f i s h . Toxin p r o f i l e s have been shown to vary among i s o l a t e s , even i f they conform to the same morphotype, p a r t i c u l a r l y among geographically d i s t a n t populations. Such toxin v a r i a t i o n , i f i t can be shown conclusively to be a stable c h a r a c t e r i s t i c within an i s o l a t e , presents the opportunity f o r chemotaxonomic analysis of i s o l a t e s and populations based upon toxin composition. There have been several previous e f f o r t s to quantify and compare t o x i c i t y among Protogonyaulax i s o l a t e s (Alam et a l . , 1979; Schmidt and Loeblich, 1979a and b; H a l l , 1982; Kodama et a l . , 1982; Oshima et a l . , 1982a; H a l l and Reichardt, 1984; Boyer et a l . , 1985), but a l l have been l i m i t e d by the s e n s i t i v i t y of the technique, the r e s t r i c t e d geographical range, and/or the low number of i s o l a t e s examined. Q u a l i t a t i v e attempts to chemotaxonomically i n t e r p r e t toxin composition of Protogonyaulax spp. (Oshima et a l . , 1976; 1982a and c; Alam et a l . , 1979; Oshima and Yasumoto, 228 F i g . 30 Structures and abbreviations f o r s a x i t o x i n (STX) and the eleven other PSP toxins obtained from Protogonyaulax spp. C x r e f e r s to the undetermined mixture of C^- C4. H R , R 2 R, R , STX H H H H neoSTX OH H H H GTX, OH H OS07 H GTX2 H H os 07 H GTX, H OSCT H H GTX„ OH os 07 H H B , H H H SO" 3 OH H H so; H H os 07 so; H 0S07 H so; c, OH H OSO-3 so; —c OH osoT H so~ 4 3 3 230 1979; 1985; Shimizu, 1979; Boyer, 1980; H a l l , 1982; Boyer et a l . , 1985; Maranda et a l . , 1985) have been s i m i l a r l y l i m i t e d i n scope. Three i s o l a t e s of P. tamarensis from Massachusetts d i f f e r e d widely i n t o x i c i t y , but had a s i m i l a r toxin composition (Alam et a l . , 1979). Oshima et a l . (1982a) used the t o x i c i t y p r o f i l e s of seven i s o l a t e s from the Tohuku and ft Hokkaido d i s t r i c t s of Northern Japan to c l a s s i f y them into three d i s t i n c t " s t r a i n s " . The most d e t a i l e d analysis of toxin p r o f i l e s of Protogonyaulax from the P a c i f i c coast of North America revealed v a r i a t i o n i n toxin composition among the 11 i s o l a t e s examined ( H a l l , 1982). A l l 12 gonyautoxins were not always present within a given i s o l a t e . From a chemotaxonomic viewpoint, i t i s important to e s t a b l i s h the d i s t r i b u t i o n of PSP toxins among other gonyaulacoid d i n o f l a g e l l a t e s . Although present i n abundance during an outbreak of "red water" causing high l e v e l s of mussel t o x i c i t y and faunal m o r t a l i t y i n i t s type l o c a l i t y o f f the the coast of South A f r i c a i n 1966 (Grindley and Nel, 1968), Gonyaulax g r i n d l e y i was ruled out as the source of PSP t o x i c i t y i n t h i s region. C e l l extracts of G. g r i n d l e y i from t h i s bloom were found to be non-toxic by the mouse bioassay (Grindley and Sapeika, 1969; Grindley and Nel, 1970). Low concentrations of P. c a t e n e l l a were simultaneously present and may have been responsible for the observed t o x i c i t y . Schmidt and Loeblich (1979b) tested crude extracts of G. g r i n d l e y i and three C a l i f o r n i a n i s o l a t e s of G. polyedra obtained i n d i f f e r e n t years and found no detectable PSP a c t i v i t y by mouse bioassay. Cultured i s o l a t e s of both G. g r i n d l e y i and G. polyedra from the Caribbean Sea were tested by mouse bioassay a f t e r e x t r a c t i v e procedures and solvent p a r t i t i o n i n g were applied to crude extracts ( T i n d a l l et a l . , 1984). No t o x i c i t y was recorded for G. g r i n d l e y i , but one f r a c t i o n from G. polyedra was reported to be t o x i c . 231 An e a r l i e r report (Schradie and B l i s s , 1962) noted a water-soluble J a f f e reagent p o s i t i v e toxin from cultured G. polyedra (ex. B. Sweeney, La J o l l a , CA) s i m i l a r i n chromatographic behavior and mouse t o x i c i t y to P. c a t e n e l l a toxin. This toxin was believed to be analogous, i f not i d e n t i c a l , to STX. These uncertainties warranted further i n v e s t i g a t i o n of both species by HPLC. E f f o r t s to characterize the gonyautoxins by combining TLC, ion exchange chromatography and electrophoretic techniques, have been superseded by the a p p l i c a t i o n of HPLC, which provides a rapid, highly s e n s i t i v e non-degradative method of toxin analysis ( S u l l i v a n and Iwaoka, 1983; S u l l i v a n and Wekell, 1984; Boyer et a l . , 1985; S u l l i v a n et a l . , 1985). The HPLC technique minimizes the p o s s i b i l i t y of e q u i l i b r a t i o n of epimeric pa i r s and hydrolysis of sulfocarbamates, while achieving a s e n s i t i v i t y of from three times ( f o r B 2) to four orders of magnitude ( f o r C^) better than that of the mouse bioassay, with higher p r e c i s i o n . 2. Toxin Concentrations and P r o f i l e s T o x i c i t y and toxi n composition varied widely among Protogonyaulax i s o l a t e s (Table 20; F i g . 31a-x). The uncorrected HPLC toxin p r o f i l e s f o r several i s o l a t e s with d i s t i n c t i v e l y d i f f e r e n t toxin composition are given i n F i g . 32-35. No detectable gonyautoxins were found during l a t e exponential growth phase i n three i s o l a t e s : G. g r i n d l e y i (NEPCC 535), G. polyedra (NEPCC 202a), and the Plymouth i s o l a t e of Protogonyaulax  tamarensis (NEPCC 183). In l a t e exponential phase, the most t o x i c i s o l a t e , NEPCC 545 (Bay of Fundy) contained approximately 4000 times as Table 20. PSP toxin composition of Protogonyaulax isolates expressed as the concentration of each toxin (fmol cell"1), percent of total toxin composition represented by each component (Z total) and toxicity (uMU cell - 1), determined in late exponential growth phase by HPLC of toxin extracts in 0.03 N acetic acid. X ± 1.0 s.d.; n-4. TOXIN COMPONENTS Isolate and Origin GTX 1+4 GTX-+3 52 NEO STX TOTAL TAMARENSOID 71* Patricia Bay,B.C., Canada Aug., 1973 Cone. 0.02 ±0.00 X total 100 Toxicity 0.01 ±0.00 0.02 ± 0.00 100 0.01 ±0.00 180 Brentwood Bay, B.C., Canada Aug., 1973 Cone. 0.02 ±0.00 X total 8.70 ±0.00 Toxicity 0.01 ±0.00 0.15 ±0.00 65.22 ± 0.00 0.02 ±0.00 0.06 ±0.00 0.23 ±0.00 26.09 ±0.00 100 0.12± 0.00 0.15 ±0.00 183* Tamar estuary, Plymouth, U.K. June, 1957 Cone. X total Toxicity 0.00 253* Laguna Obidos, Portugal, 1962 Cone. X total Toxicity 1.13±0.13 97.41t 2.24 1.92 ± 0.22 0.03 t 0.01 2.59 ± 2.24 0.04 t 0.01 1.16 * 0.06 100 1.96± 0.23 255 Lummi Island, WA, USA Aug., 1976 Cone. 6.06 ±3.48 X total 17.06 ±4.99 Toxicity 1.52 ±0.87 2.11-tl.Ol 0.20± 0.04 2.64± 0.76 19.65± 6.89 4.59* 1.76 0.26± 0.17 35.53 ± 12.74 5.94± 1.88 0.56± 0.13 7.43* 1.06 55.31 ± 2.73 12.92 ± 1.77 1.05±0.69 100 3.59± 1.72 0.28 ± 0.06 0.40 ±0.11 3.54± 1.24 9.64± 3.70 0.53 ± 0.35 19.50±8.05 TOXIN COMPONENTS Isolate and Origin GTX 1+4 GTX 2+3 »1 NEO STX TOTAL TAMARENSOID 400 English Bay, B.C., Canada June, 1981 401 English Bay, B.C., Canada June, 1981 403* English Bay, B.C., Canada June, 1981 404* English Bay, B.C., Canada June, 1981 405* English Bay, B.C., Canada June, 1981 406* English Bay, B.C., Canada June, 1981 Cone. 2.44 ±1.18 X total 31.77 ±9.07 Toxicity 0.61 ±0.30 Cone. 2.62 ±0.56 7. total 15.35 ±3.14 Toxicity 0.66 ±0.14 1.24 ±0.56 16.14 ±5.54 2.11 ±0.09 0.07 ±0.04 0.91 ±0.57 0.10 ±0.09 0.18 ±0.15 2.83 *0.71 2.34 ±1.55 36.85 ±3.73 0.03 ±0.02 0.51 ±0.13 0.75 ±0.55 9.77 ±1.71 1.58 ±1.16 0.19 ±0.18 2.47 ±1.26 0.39 ±0.37 7.68 ±2.76 100 5.33 ±3.03 1.72 ±0.76 0.11 ±0.02 2.10 ±0.90 9.41 ±3.62 0.86 ±0.48 10.08 ±3.45 0.64 ±0.12 12.30 ±4.92 55.16 ±6.59 5.04 ±2.81 2.92 ±1.29 0.15 ±0.03 0.32 *0.14 1.69 ±0.65 1.81 ±1.00 0.24 ±0.20' 17.06 ±1.57 1.41 ±1.16 0.49 ±0.41 100 8.04 ±3.66 Cone. 7.64 ±2.80 0.79 ±0.77 0.20 ±0.05 7. total 30.22 ±8.22 3.31 ±3.21 0.79 ±0.17 Toxicity 1.91 ±0.70 1.34 ±1.31 0.28 ±0.07 Cone. 1.54 *0.62 0.30 ±0.21 0.05 ±0.02 7. total 48.73 ± 11.46 9.49 ±8.91 1.58 ± 1.15 Toxicity 0.39 ±0.16 0.51 ±0.36 0.07 ±0.03 Cone. 2.41± 0.62 0.53 ± 0.05 0.04 ±0.03 X total 43.98 ± 16.63 9.67 ±1.01 0.74 ±0.41 Toxicity 0.60 ± 0.16 0.90 ± 0.09 0.06 ± 0.04 Cone. 2.90 ±1.13 1.97 ± 1.23 0.06 ±0.02 X total 19.15 ± 5.71 13.01 ± 5.84 0.40±0;15 Toxicity 0.73 ± 0.28 3.35 ± 2.09 0.08 ±0.03 1.46 ±0.5 12.28 ±2.40 2.81 ±0.62 5.78 ±2.22 48.58 ±7.04 11.12 ±3.86 0.22 +0.08 2.21 ±0.43 5.90 ±1.30 0.10 ± 0.07 25.28 ±4.27 0.40 ±0.30 100 0.21 ±0.14 12.07 ±4.03 0.09 ±0.04 0.72 ±0.16 0.42 ±0.19 0.03 ±0.02 3.16 ±0.79 2.85±0.78 22.78 ± 3.83 13.29 ±3.83 0.95 ±0.12 100 0.01 ±0.00 0.13 ± 0.03 0.88 ± 0.40 0.06 ±0.05 2.05 ±1.04 0.10 ± 0.02 1.56 ± 0.71 0.68 ± 0.64 0.16 ± 0.03 1.82 ± 0.87 28.47 ±9.32 12.41 ±11.61 2.92 ±0.98 0.02 ± 0.00 0.28 ± 0.13 1.41±0.61 7.49*1.81 9.31 * 5.02 49.41 * 3.35 1.43 * 1.34 1.26* 1.22 0.33 ± 0.06 5.48 t 1.19 100 3.62± 2.67 0.05±0.04 15.1414.34 0.21 * 0.09 1.35 * 0.33 8.32 ± 7.28 0.33 ±0.26 100 2.65± 2.57 0.10± 0.09 8.47±5.48 M U> TOXIN COMPONENTS Isolate and Origin GTX 1+4 GTX 2+3 22 NEO STX TOTAL TAMARENSOID 407* English Bay, B.C., Canada June, 1981 Cone. 2.94 + 2.44 0.84 ± 0.53 0.04 ±0.02 X total 47.73 ± 13.30 13.64 ±8.21 0.65±0.31 Toxicity 0.74 + 0.61 1.43 ±0.90 0.06 ±0.03 0.24± 0.09 1.69 * 1.13 3.90 ±