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UBC Theses and Dissertations

A survey of zoospore and sperm ultrastructure in the Laminariales (Phaeophyceae) Henry, Eric Charles 1980

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c l A SUBVEI OF ZOOSPORE AND SPERM ULTBASTBUCTUBE IN THE LAHINASIALES (PHAEOPHYCE AE) by EBIC CHARLES HENRY B.A,, U n i v e r s i t y Of C o n n e c t i c u t , 1971 M.S., Oregon S t a t e U n i v e r s i t y , 1976 A THESIS SUBMITTED IS PABTIAL FULFILLMENT OF THE BEQOIBEMENTS FOB THE DEGPEE OF DOCTOB OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES {Department of Botany) He accept the t h e s i s as conforming to the r e q u i r e d standard THE UNI7EBSITY OF BBITISH COLUMBIA J u l y 1980 © E r i c C. Henry, 1980 In present ing t h i s t he s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t ha 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 re fe rence and study. I f u r t h e r agree that permiss ion f o r ex tens i ve copying of t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n permi s s ion . Department The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 Date ///fyuff /fro D E - 6 B P 75-5 1 I E &BSX.B ACT Zoospores of 17 s p e c i e s i n 14 genera and sperm of 13 s p e c i e s i n 11 genera of L a m i n a r i a l e s were s t u d i e d by e l e c t r o n microscopy. The zoospores are unique i n the brown algae i n l a c k i n g both an eyespot i n the s i n g l e c h l o r o p l a s t and an a s s o c i a t e d s w e l l i n g at the base o f the s h o r t e r , p o s t e r i o r f l a g e i l u m . , Spores of a l l s p e c i e s possess a d i s t a l whiplash on the l o n g e r , mastigoneme-bearing a n t e r i o r f l a g e i l u m ; although i t i s only seldom preserved f o r e l e c t r o n microscopy, t h i s appendage may sometimes be as l o n g as the mastigoneme-bearing p o r t i o n of the f l a g e i l u m . A m i c r o t u b u l a r c y t o s k e l e t o n w i t h c o n n e c t i o n s to the f l a g e l l a r b a s a l bodies i s r e s p o n s i b l e f o r m a i n t a i n i n g the shape of the zoospore. The sperm are a l s o unique i n the brown algae. They are elongate and possess two to three p l a s t i d s and s e v e r a l mitochondria, but l a c k an eyespot.. T h e i r most d i s t i n c t i v e f e a t u r e i s the long p o s t e r i o r f l a g e i l u m which t a p e r s d i s t a l i y as the doublet microtubules of the axoneme ar e transformed i n t o s i n g l e t s and then decrease i n number., The sperm a l s o bear a d i s t a l whiplash on the mastigoneme-bearing a n t e r i o r f l a g e i l u m . These l a m i n a r i a l e a n zoospores and sperm are u l t r a s t r u c t u r a l l y d i s t i n c t from those known i n other o r d e r s o f brown a l g a e , and from Chorda o f the L a m i n a r i a l e s , which confirms that Chorda i s p r i m i t i v e . The l o n g e r p o s t e r i o r f l a g e i l u m of the sperm r e c a l l s s i m i l a r m o d i f i c a t i o n s i n sperm of other oogamous brown a l g a e , and suggests a common i i i f u n c t i o n a l s i g n i f i c a n c e . i v TABLE OF CONTESTS LIST OP TABLES .......................... . . . . v . . . . . . . . . . . . v i LIST OP FIGURES v i i ACKNOWLEDGEMENTS • - x i i i CHAPTER I. INTRODUCTION ................................. 1 I. , Importance of c y t o l o g i c a l c h a r a c t e r i s t i c s i n a l g a l taxonomy .......... ....... ......... 1 I I . M o t i l e c e l l s of hrown algae ...................... 2 I I I . S i g n i f i c a n c e Of H o t i l e C e l l S p e c i a l i z a t i o n 4 A. The " P r i m i t i v e F u c o i d " Of Manton ............... 4 IV. Choice of L a m i n a r i a l e s As Subject Of I n v e s t i g a t i o n ....... , , ... . . ........... ...... 6 CHAPTER 2. MATERIALS AHD METHODS 9 I . C o l l e c t i o n s • »•.• »«*-• •• *••••• «• • • v . . •»>• . . . • > 9 I I . Gametophyte c u l t u r e s ................. 11 I I I . L i g h t flicroscopy ........ ....... 12 IV. E l e c t r o n Microscopy .. ..... .,.. • v , , y . : . : , v 13 CHAPTER 3. (RESULTS ................................ ...... 16 I . , Zoospores . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 A. Sporangia ••........,.. ...... ................... 16 B. Zoospore S t r u c t u r e ............................. 18 C. Zoospore F u n c t i o n ............................. 29 I I . ,S per m ....................... %..,,,.«•*•>• - • ,• 32 A. . A n t h e r i d i a ................. .... ............ .... 32 B. . Sperm S t r u c t u r e ................................ 32 C. Sperm Fu n c t i o n ............... 36 CHAPTER 4. /DISCUSSION ........................... ........ 37 I.,Sporangia ................................*........ 37 V A. u n i l o c u l a r Sporangia ................ . 37 B. P l u r i l o c u l a r Sporangia ......................... 39 I I . Spores ...... ...... ... 39 A. , V a r i a b i l i t y Among Spores ....................... 39 B. The Mi s s i n g Eyespot ............................ 40 C. . flow Many P l a s t i d s ? .. 42 D. ,Mitochondria ................................... 43 E. .The Cytoskeleton .......................... 43 F. The F l a g e l l a ................................... 44 G. The Adhesion V e s i c l e s ................... 47 H. Other M o d i f i e d Spores 48 I I I . Garnet an g i a 49 A. P l u r i l o c u l a r Gametangia ........................ 49 B. , A n t h e r i d i a Of L a m i n a r i a l e s . 50 IV. Gametes ................... 51 A. V a r i a t i o n Among Gametes ........................ 51 B. Sperm Of L a m i n a r i a l e s .......................... 54 C. Comparison With Other Brown A l g a l Sperm 56 V. I m p l i c a t i o n s f o r p h y l e t i c s o f L a m i n a r i a l e s ........ 58 VI. I m p l i c a t i o n s f o r s w a r i e r f u n c t i o n and l i f e h i s t o r i e s . . • ..........••...................... 59 BEFEBENCES 146 APPENDIX .....• ..... - . . . . . . . . . . . . . . . 156 U l t r a s t r u c t u r e of Sperm of D e s a a r e s t i a V i r i d i s ....... 156 LIST OF TABLES Table I Taxa S t u d i e d . ... 10 v i i LIST OF FIGURES Fig. 1 .... ....... ........................ .... ............ ,66 F i g . 2 66 Fig. 3 . . ........ .. .. . 66 Fig. ,. 4 . . . . ........ . ...... ...... . . ...... . . ...... . . . . . . . . , 6 6 F i g . ,-5- . , ......... ... ....... ...... .... ........... 66 Fig. ,6 . . . 66 Fig. ,7 ......... ... ... ....... ....... .. ....... . 6 8 F i g . 8 .... 68 Fig. 9 . .... 68 Fig. ,10 .... . ................. ............. .... ... 68 Fi g . 11 ....... . .. 68 F i g . 1 2 ......................, . . . . . . . . . . . . 7 0 Fig. 13 ... ..............................................70 Fig. 14 . ... ...... . .. ... . .. ... .. . v. . . ....... . .... .... 70 F i g . ,15. ............... 70 • Fig. 16 . ......... . . ............ . . . . . .... . . . . .....70 Fig.,, 1.7 ... * ... . 70 Fig. „ 1.8 . .... 72 Fi g . 19 72 Fig. ,2 0 • , W ^ . r « ' M « . V V V * f * . ; v * « * : . ^ * ^ * * » t . . 72 F i g . 21 . • , 72 F i g . 22 .... . ........ . . . i . ...... 72 Figs. „• 23a-23E ...... ..... .... , . . . - ...... . . . . . * . . .... ... > 74 Fig. 24 .......... ............. 74 . Figs.251-256 ..... . ... ... ............................... 76 Fig. ,26 . .... ... .-. . . . . . . - • - . . • 76 v i i i Fig. 2 8 ..................... ..,80 Fig. 29 ........ ., 8 0 Fig. 3 0 . ......... . ....... ........... .. 80 Fig. 31 .............................. ...w...............82 Fig. 32 ... . . ..... . ...... ........ ... . . . . 8 2 Fig. 33 ............. . ..... w. . ... . . . , ....... 8.2 Fig. 34 . ..... . ... ...... ............... 82 Figs.., 35-36 . .......... ... 82 Fig. 37 ........... . . .,w,,.^ .*.^ %V*'.*. ... 84 Fig. 38 ..... ........ * i . . . . . . ...................,84 Fig. 39 84 Fig. 4 0 ..... ... . .... . 86 Fig. 4 1 ................................................. 86 Fig. 42 86 Fig. 43 ....... ..... . , ,86 Fig. ,44 ....... ...... ......... •..•..•*v*>»w...i.,vv.*»**»-..-^ 88 Fig. , 4 5 . . .>,...>*. , ., • . .... .... ,88 Fig. ,4 6 ,. .......... . .......... ... ......... ,, ,88 Fi g . .. 47 . .-. ....... . . .... . . ..... . . . . ...... ........ . ...... 88 Fig. 48 ................ 90 Fig. .4 9 . . . . . . . . . . . . . ............ ,r,*,4,%^W»:***^^<. v;«'90 Figs. 50-51 . ....... ......... ....... .. . . . 90 Fig. 52 . ,. v. .... .... 90 Fig. 53 .. ... ........ . ... . . . 90 Fig..,54 . ..... ......... ..... .... . . ....... . . .,... 4.. ... . ..... . 9 2 Figs. „. 55-56 . .... ..... , , ,* . ..v, . . .,9 2 Fig. 57 ... .... ...... 92 Fig. ,58 .... ..... ... ...... ......... . . . .... . ......w. ....... . 92 i x F i g . ,59 ....... ..... ... .... ... . 92 F i g . 60 i ............................... ........ 94 F i g s . 61-63 .., ....-»..,,• 9 4 F i g . 64 .... ...*,.-. ... . ..... . . 96 F i g . 65 ............. ..... . ......... . , ..... ... ... .... ... ... . 9 6 F i g . , 66 F i g . .,67 ... 96 F i g . 6 8 ....... . .... 96 F i g . 69 ,. ......... „ ...96 F i g . ,7 0 . . .... ... . .. ... . .. ..... . 98 F i g . 7 1 . ... . . ... ... . ... . . ... . . ..... . .... .. ... ....... ..... 98 F i g . 72 ........ ... ............. . 98 F i g s . 73-74 . ... ....... , ........... . 9 8 F i g s . 75-76 ...............100 F i g s . ,77-78 . .........>...... .... ........ ..w , < . . . . .w . . ... 100 F i g s . ., 79-8 0 „ . .... ..... , ....... . .. . . ... . v»^**---*-^v* .*•»*< 102 F i g . 81 ............... ... ... . . . . .;.. .... .. .... ........ .... J 0 2 F i g . 82 . .. ... .... .... .... ....... ... .... . . .102 F i g . . 83 • ... . .... . .................... ......... 102 F i g . 84 .102 F i g . ,85 ................................ F i g . 86 ........ ... 104 F i g . •, 87 ;.. 104 F i g . ,88 .,............... ..........104 F i g . 89 104 F i g . 90 .......... . ..... ....,. .... ... . . . . .. .« ... 104 F i g . 91 ............ ..... 104 F i g . ,92 -,.106 X F i g . ,-93 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • 106 F i g . 94 . . . . . . . . . . , . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 F i g . , 95 . . . . . . • . ... . . , ,:. . . . . . . . . . . . . . . . . . . . . 106 F i g . ,96 . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 6 F i g . 97 . . . . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 6 F i g . 98 . .108 F i g . 99 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 8 F i g . „. 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 F i g . 101 . . ... . . • • • • • • 108 F i g . ., 102 . . . . . . . . . . . . . . . . . . . . . . . . . . 108 F i g . 1 03 . 108 F i g . 1 0 4 - . . . F i g . ,105 v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 F i g . , 106 110 F i g s . 107-109 . v. .......... , , ......... , J10 F i g . . 1 1 0 ^ , . . , . . . , . v v , » * . . . , . . . . , . . . . . . . . . . . . . . . . . . . . . . . 110 F i g . .,-111. 112 F i g . 11.2 112 F i g . 113 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2 F i g . 114 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 F i g . 115 112 F i g . , 116 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-.v.- . .112 Figs..., 117-120 114 F i g . 121 . . . . . . . . . . . . . . . . . . . . . . , . . . . - ,4«. 116 F i g s . 122-123 . . . . . . . . . . i . . . . . . . . . . . . . . . . . . 4 . . . * . . . . . . . . . 1 1 6 F i g . , 124 . . . . . . . • . . ........44>,;:i . . . 116 F i g . 1 2 5 - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f 116 F i g . ,126A . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 x i F i g . F i g . : F i g . F i g . , F i g . F i g s . F i g . F i g . / F i g . F i g . ., F i g . F i g . , F i g . / F i g . F i g . F i g s . F i g . F i g . „ F i g . , F i g . F i g . F i g s . Fig.\, F i g . F i g . ,/ Fig. fig*... 26B .... 27 ..... 28 ...• • < 29 ..... 30 131-132 33 34 35 36 37 38 39 40 . 41 118 120 120 120 120 120 122 122 122 122 124 124 124 124 124 142&-D . ... .. . . ........ .. 126 43 44 45 46 47 128 128 128 128 130 148-149 ... . . . . . . . . . . . . . . . . . . ... . . . . . . i i . . . . ... ....... 13 0 50 51 52 53 54 55 130 130 130 .332 132 132 x i i F i g . 156 132 F i g s . , 157-158 ... ..... .. ...... .... ... ... ....... . . ..... .. .134 F i g s . , 159-160- 134 Fig.., 161 .............................. ..... ... ........ 134 F i g . ,,-162 . ... ..... ... ,. .... ........ 134 F i g . ,163 ... 134 F i g . 164 136 F i g . ,-165- . ... .. . , . ... ............. . 136 F i g . 166 136 F i g . 167 ... 136 Fig.,168 ......... ........... ....,.......w 136 F i g . 169 .. ....... ................ . . J36 F i g . , 1 70 . ...... . . ... ........... ...V. ... 138 F i g . , 171.. . .. ... 138 Fig.„172 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v * * . * ..138 F i g . 173 . ....... ...... ..... ................. 138 F i g . 174 ....... ..... .... ... . ... ... ....... ...... ..... ... .. * . 140 F i g s . 175-177 . ....... .v..... . ................. .... ,...... 140 F i g s . 178-179 142 F i g s . , 180-182- . . . . . . . . . . . . . . . 1 4 2 F i g . .. 183 . ....... .<. ....... ... ................. . 144 F i g . 184 ......................................v.........146 x i i i ACKNOWLEDGEMENTS The a s s i s t a n c e of many people made t h i s work p o s s i b l e . Thanks a r e due to Dr. Kathleen Cole f o r p r o v i d i n g r e s e a r c h f a c i l i t i e s , thorough c r i t i c i s m of the manuscript, and u n f a i l i n g encouragement; Dr. a. F. Seagel f o r access t o r e s e a r c h f a c i l i t i e s and f o r c r i t i c i s m of the manuscript; Dr. G. .Hughes f o r c r i t i c i s m o f the manuscript; and Dr. fi. Dewreede f o r c r i t i c i s m of the manuscript, encouragement, and f o r i n v a l u a b l e s u b t i d a l c o l l e c t i o n s . Equally v i t a l s u b t i d a l c o l l e c t i o n s uere a l s o made by H.,Hawkes, M. Haylock, H . Nelson, A. Mackinnon, c . Tanner and H. Vandermeulen. D. Z i t t i n advised on manuscript p r e p a r a t i o n by computer, and Caro l e Henry a s s i s t e d with p r e p a r a t i o n of the f i g u r e s . 1 CHAPTER I . , INTRODUCTION I.„ Importance Of C y t o l o g i c a l C h a r a c t e r i s t i c s In A l g a l Taxonomy In the many d i v i s i o n s and c l a s s e s of algae r e c o g n i z e d i n v a r i o u s c l a s s i f i c a t i o n schemes, the nature of the f l a g e l l a r apparatus i s of primary importance., The Bhodophyta are c h a r a c t e r i z e d by complete absence of f l a g e l l a or r e l a t e d s t r u c t u r e s such as c e n t r i o i e s . , Pyrrophyta and Euglenophyta possess many unique c y t o l o g i c a l f e a t u r e s i n t h e i r n u c l e a r o r g a n i z a t i o n and c e l l armor but can be j u s t a s e a s i l y separated by t h e i r d i s t i n c t i v e f l a g e l l a r arrangements. Among the Chlorophyta, the Prasinophyceae and Charophyceae a r e r e a d i l y s e parated from each other and other C h l o r o p y t a by p e c u l i a r f e a t u r e s of t h e i r f l a g e l l a . The r e l a t i o n s h i p of the c l a s s e s comprising the Chromophyta of C h r i s t e n s e n <I962, c i t e d i n Hanton 1965) i s based on f l a g e l l a as much as pigmentation, and the r e c e n t r e c o g n i t i o n of the c l a s s e s Eustigmatophyceae (Hibberd and Leedale 1970) and Prymnesiophyceae {Hibberd 1976) are based l a r g e l y on f l a g e l l a r c h a r a c t e r i s t i c s . I t i s o f t e n suggested t h a t the Oomycetes are descended from a heterokont a l g a l ancestor ( T a y l o r 1978); and recent work (Barr and Hadland-Hartmann 1978) attempts t o c o n s t r u c t a taxonomy and phylogeny o f C h y t r i d i o m y c e t e s on the b a s i s of zoospore u l t r a s t r u c t u r e , i n c l u d i n g c o n s i d e r a t i o n of the f l a g e l l a r apparatus., In a r e c e n t t e x t (van den Hoek 1978) the Chrysophyceae, Xanthophyceae, B a c i l l a r i o p h y c e a e , 2 Chloromonadophyceae, and Phaeophyceae are grouped i n t o the Heterokontophyta, based on the po s s e s s i o n of heterokont f l a g e l l a {one with mastigonemes, one without) o r an obvious m o d i f i c a t i o n of the heterokont plan (e.g., u n i f l a g e l l a t e sperm of c e n t r i c diatoms). I I . H o t i l e £ells Of Brown Algae Within the Phaeophyceae the only f l a g e l l a t e d c e l l s are zoospores and gametes ( c o l l e c t i v e l y termed "swarmers"), and they have r e c e i v e d r e l a t i v e l y l i t t l e a t t e n t i o n from taxonomists; c l a s s i f i c a t i o n has been based l a r g e l y on v e g e t a t i v e c o n s t r u c t i o n and l i f e h i s t o r y . B e f o r e the advent of e l e c t r o n microscopy i t was known ( F r i t s c h 1945) t h a t swarmers of brown algae {with the e x c e p t i o n o f the U n i f l a g e l l a t e sperm of a t l e a s t some D i c t y o t a l e s ) possess two l a t e r a l l y a t t a c h e d f l a g e l l a unequal i n l e n g t h , the l o n g e r d i r e c t e d forward and be a r i n g mastigonemes {Longest 1946), the s h o r t e r d i r e c t e d backward and bearing only a t e r m i n a l "whiplash? {except i n some F u c a l e s the p o s t e r i o r f l a g e l l u m i s the longer) . An eyespot was known t o be l o c a t e d near the attachment of the f l a g e l l a , a s s o c i a t e d with a p l a s t i d ; one or more p l a s t i d s and one nucleu s were known to be present. The s i z e of the nucleus was a s u b j e c t o f c o n t r o v e r s y among e a r l i e r l i g h t m i c r o s c o p i s t s {Kylin 1920). „ To da t e , approximately t h i r t y - f i v e p u b l i c a t i o n s have r e p o r t e d on e l e c t r o n m i c r o s c o p i c a l s t u d i e s of phaeophycean r e p r o d u c t i v e c e l l s , c o n f i n e d t o o b s e r v a t i o n s of the o r d e r s E c t o c a r p a l e s , C h o r d a r i a l e s , C u t l e r i a l e s , D i c t y o t a l e s , 3 L a m i n a r i a l e s and F u c a l e s . Some examine only one s p e c i e s but i n d e t a i l (Ectocarpus; Baker and Evans 1973a, 1973b; Lofthouse and Capon 1975; P i l a y e l l a : Markey and Wilce 1976a, 1976b; C u t l e r i a : Caram 1975, La C l a i r e and West 1978, 1979; Chorda; l o t h 1 9 7 4 , 1976a; Focus; Manton and C l a r k e 1951a, 1951b, 1956, B e r k a l o f f and Rousseau 1979). Others t r e a t only one aspect i n s e v e r a l s p e c i e s (Loiseaux 1973: u n i l o c u l a r s p o r a n g i a of P i l a y e l l a , E l a c h i s t a , Hecatonema: Manton e t a l . . 1953; f l a g e l l a of sperm °f D i c t y o t a , H i m a n t h a l i a . P e l v e t i a , Ascophyllum, Facus; Loiseaux and West 1970: mastigonemes of Compspnema, G i f f o r d i a . Hecatonema, Leptonemate11a. Myrionema, B a l f s i a ) . Many s t u d i e s are only concerned with one aspect of one s p e c i e s {e.g., Bouck 1970, on eyespots of Fucus sperm; C a s s e l l and P o l l o c k 1978 on FUGus sperm mitochondria) or only s u p e r f i c i a l l y examine s e v e r a l a s p e c t s . „, S e v e r a l o f the e a r l i e r papers used techniques t h a t have become so outmoded t h a t the work needs t o be repeated with modern methods, as the r e s u l t s cannot be compared u s e f u l l y with r e c e n t f i n d i n g s . There has been no attempt at a comprehensive survey of phaeophycean m o t i l e c e i l s s i n c e Manton*s (1964) comparison of the Scytosiphon zoospore with the sperm... of Fucales and D i c t y o t a . but i t i s now p o s s i b l e t o make some f u r t h e r g e n e r a l i z a t i o n s based on a d d i t i o n a l a v a i l a b l e i n f o r m a t i o n . I t appears t h a t the p r i m i t i v e phaeophycean m o t i l e c e l l always l a c k s a c e l l w a l l , but possesses a nucleus with a c l o s e l y a s s o c i a t e d dictyosome, s e v e r a l mitochondria, one or more c h l o r o p l a s t s , and two f l a g e l l a i n s e r t e d l a t e r a l l y . The l o n g e r , a n t e r i o r l y d i r e c t e d f l a g e l l u m bears mastigonemes, whereas the 4 s h o r t e r , t r a i l i n g f l a g e i l u m has no l a t e r a l appendages but possesses a b a s a l s w e l l i n g i n t i m a t e l y appressed to the plasmalemma d i r e c t l y o v e r l y i n g an eyespot c o n t a i n e d w i t h i n a c h l o r o p l a s t . The b a s a l bodies of the f l a g e l l a are a s s o c i a t e d with a group of microtubules t h a t appears to serve as a c y t o s k e l e t o n , m a i n t a i n i n g the shape of the swarmer. S i g n i f i c a n c e Of M o t i l e C e l l S p e c i a l i z a t i o n A. The " P r i m i t i v e F u c o i d " Of Manton Manton (1964) proposed t h a t the m o d i f i c a t i o n s she observed i n the sperm of Fucus sere evidence that Fucus was l e s s p r i m i t i v e than C y s t o s e i r a . whose sperm resembles the zoospore °f Scy t o s i p h o n . Formerly, g r e a t emphasis had been given to the r e d u c t i o n of the number of eggs produced i n the oogonium of these genera, e i g h t i n Fucus c o n s i d e r e d more p r i m i t i v e than the one (with seven aborted n u c l e i ) i n C y s t o s e i r a ( F r i t s c h 1945)« Before flanton, K y l i n (1940) had s t r e s s e d the d i f f e r e n c e between the sperm of Fucus (with i t s l o n g e r p o s t e r i o r f l a g e i l u m ) and zoospores of non-fucalean brown algae {with t h e i r s h o r t e r p o s t e r i o r f l a g e l l a ) . He proposed the s e g r e g a t i o n of the Order F u c a l e s i n t o a separate C l a s s Cyclosporeae, which he c o n s i d e r e d to have o r i g i n a t e d from a p h y l e t i c l i n e of d i p l o i d a n c e s t o r s e n t i r e l y removed from the other brown algae t h a t show a l t e r n a t i o n of h a p l o i d and d i p l o i d g e n e r a t i o n s . He f a i l e d to note t h a t o n l y sperm of the f a m i l y Fucaceae (indeed only those of the Northern Hemisphere) have t h i s f l a g e l l a r p a t t e r n . 5 Although F r i t s c h (1945) s t a t e d with regard to the r e l a t i v e l e n g t h s of the two f l a g e l l a i n the F u c a l e s "The evidence....is c o n f l i c t i n g , " many subsequent authors (Papenfuss 1951, 1955; Smith-1955; S c a g e l e t a l . 1965; Scagel 1966; Dawson 1966; Bold and Wynne 1978; Boney 1978) have accepted K y l i n ' s c o n t e n t i o n . However Manton, who d i d not seem to be aware of the c o n t r o v e r s y , had pointed out t h a t i n s e v e r a l B r i t i s h C y s t o s e i r a c e a e the sperm "In shape...are a l l e x a c t l y comparable to the average brown a l g a l zoospore...As i n the zoospore...the s h o r t h i n d f l a g e i l u m being a p p a r e n t l y stuck to the s u r f a c e o f the eyespot and the l o n g e r f r o n t f l a g e i l u m being a • F l i m m e r g e i s s e l ' of the o r d i n a r y k i n d " (Manton 1964, p. 247). Other s i g n i f i c a n t m o d i f i c a t i o n s of brown a l g a l sperm are the " p r o b o s c i s " of the Fucaceae with the longer hind f l a g e i l u m , s p i n y f l a g e l l a , and the l a c k of eyespots., A l l these s p e c i a l i z a t i o n s are c o n f i n e d t o gametes of oogamous taxa, where the r a d i c a l t r a n s f o r m a t i o n of the female gamete from a swimming isogamete to a non-motile egg as much as 20,000 times the mass of the sperm (e.g. Fucus, Ja e n i c k e 1977) has proceeded hand i n hand with m o d i f i c a t i o n of the male gamete. The taxonomy of the brown algae has t r a d i t i o n a l l y been based on c o n s i d e r a t i o n of both v e g e t a t i v e s t r u c t u r e and r e p r o d u c t i v e c h a r a c t e r i s t i c s , and among the l a t t e r the type of l i f e h i s t o r y and the form of the sporangia are paramount. I t i s obvious t h a t l i f e h i s t o r y and r e p r o d u c t i v e s t r u c t u r e s a r e f u n c t i o n a l l y r e l a t e d , so one would a n t i c i p a t e t h a t those o r d e r s p o s s e s s i n g the more s p e c i a l i z e d , oogamous l i f e h i s t o r i e s ( F u c a l e s , D i c t y o t a l e s , L a m i n a r i a l e s , D e s m a r e s t i a l e s , 6 Sporochnales) would have modified sperm. I f one wished to determine the v a r i a t i o n i n sperm s t r u c t u r e throughout the brown algae, the f i r s t p l a c e t o look would be a t the oogamous taxa t h a t are s t i l l poorly known. The means of i n v e s t i g a t i o n must be e l e c t r o n microscopy, s i n c e c o n t r a d i c t o r y d e s c r i p t i o n s have r e s u l t e d from l i g h t microscopy of these s m a l l c e l l s . The v a r i a t i o n i n sperm s t r u c t u r e among the f a m i l i e s o f the F u c a l e s suggests t h a t sperm u l t r a s t r n c t u r e may be u s e f u l f o r d i s t i n g u i s h i n g taxa a t the l e v e l o f f a m i l y as w e l l as o r d e r . V a r i a t i o n i n the occurrence of eyespots (Sauvageau 1911, Roberts 1978) might be a good i n d i c a t o r of p h y l e t i c s e r i e s w i t h i n f a m i l i e s , as l o s s i s presumably an advanced c h a r a c t e r i s t i c . S p e c u l a t i o n has been made (Manton 1964) about the p o s s i b l e f u n c t i o n a l s i g n i f i c a n c e of sperm m o d i f i c a t i o n s . While only e x p e r i m e n t a l s t u d i e s ( c f . Jaenicke 1977) can r e v e a l the t r u e importance o f these s t r u c t u r a l a l t e r a t i o n s , a more complete knowledge of the v a r i a t i o n s among the d i f f e r e n t p h y l e t i c l i n e s with oogamous r e p r o d u c t i o n may f a c i l i t a t e our comprehension by d i s c o v e r i n g u n i v e r s a l changes r e l a t i n g t o oogamy per se. I V ./ Choice Of L a m i n a r i a l e s As S u b j e c t Of I n v e s t i q a t i o n I chose t o survey the u l t r a s t r u c t u r e of zoospores and sperm of L a m i n a r i a l e s f o r s e v e r a l reasons. The L a m i n a r i a l e s are not w e l l known u l t r a s t r u c t u r a l l y , and the g r e a t d i f f e r e n c e between the sporophyte and gametophyte i n s i z e and c o n s t r u c t i o n suggests that the l i f e h i s t o r y i s h i g h l y d e r i v e d ( F r i t s c h 1945)., A l l L a m i n a r i a l e s are oogamous, so a s p e c i a l i z e d sperm 7 may be expected, e s p e c i a l l y as many r e p o r t s (Sauvageau 1918, Kanda 1936, 1938, F r i t s c h 1945, Kemp and Cole 1961) suggest that the zoospores of some s p e c i e s l a c k eyespots and so are unigue i n the brown a l g a e . The L a m i n a r i a l e s are a l s o very d i v e r s e , with 16 genera and perhaps 30 s p e c i e s i n the B r i t i s h Columbia f l o r a . Nine of these genera are monotypic, but two, Laminaria (9 species) and A l a r i a (5 species) have enough r e p r e s e n t a t i v e s i n our waters to permit the study o f both i n t e r - and i n t r a r g e n e r i c v a r i a t i o n , Because zoosporangia i n L a m i n a r i a l e s are borne i n s o r i whose presence and degree of maturity are evident i n the f i e l d , and c u l t u r e methods f o r growing the gametophytes are w e l l e s t a b l i s h e d , t h i s order i s e s p e c i a l l y s u i t a b l e f o r an u l t r a s t r u c t u r a l study of i t s r e p r o d u c t i o n * Furthermore, there i s an e x t e n s i v e l i t e r a t u r e c o v e r i n g r e p r o d u c t i o n i n the L a m i n a r i a l e s ( F r i t s c h 1945). Since F r i t s c h p u b l i s h e d h i s review of a l g a l s t r u c t u r e and r e p r o d u c t i o n , there have been numerous f u r t h e r l i g h t - m i c r o s c o p e s t u d i e s ( e s p e c i a l l y those of Cole et a l . and v a r i o u s Japanese and European workers)., U l t r a s t r u c t u r a l s t u d i e s have been l i m i t e d to those on L a m i n a r i a (Manton and C l a r k e 1951, whole mounts of spores only; B i s a l p u t r a e t a l . 1971, oogenesis o n l y ) , Chorda s p o r o g e n s i s , r e l e a s e and germination (Toth 1974, 1976a), M a c r o c y s t i s sporogenesis (Chi and Neushul 1972, G h e r a r d i n i and North 1972, C h i 1973) and a very incomplete account of M a c r o c y s t i s spermatogenesis ( G h e r a r d i n i and North 1972).,/ T h i s body of l i t e r a t u r e shows t h a t c e r t a i n i s s u e s should be addressed, such as presence or absence o f eyespot, number of 8 p l a s t i d s , and presence or absence of the a n t e r i o r "whiplash" i n both zoospores and sperm. The d i v e r s i t y , o f s p e c i e s ensured t h a t a good account of v a r i a t i o n w i t h i n the order c o u l d be made from the i n f o r m a t i o n gathered, and common f e a t u r e s would be seen o f t e n enough so that g e n e r a l i z a t i o n s about the whole order would be j u s t i f i e d , such as mechanisms of spor o g e n e s i s and spermatogenesis. With the r e s u l t s o f such a survey i n hand i t would be p o s s i b l e t o make more informed approaches to i n v e s t i g a t i o n o f other brown a l g a e , as w e l l as t o gain a b e t t e r e v a l u a t i o n of r e l a t i o n s h i p s between the L a m i n a r i a l e s and the r e s t of the Phaeophyta. . 9 CHAPTEB 2. MATERIALS AND METHODS i» C o l l e c t i o n s Host sporophytes were c o l l e c t e d i n t h e e n v i r o n s of the Bamfield Marine S t a t i o n , on Ba r k l e y Sound, Vancouver I s l a n d . A d d i t i o n a l Vancouver I s l a n d c o l l e c t i o n s were made a t Port Hardy, B o t a n i c a l Beach (near Pt. ; Benfrew), Sombrio B i v e r , Sooke and V i c t o r i a . Other c o l l e c t i o n s were made a t Bath I s l a n d , o f f G a b r i o l a I s l a n d i n the S t r a i t of Georgia; S t a n l e y Park, Vancouver; Whidbey I s l a n d , Washington; and Marine Gardens and B o i l e r Bay, Oregon. Table I l i s t s the taxa c o l l e c t e d , and shows whether zoospores, sperm, or both were s t u d i e d , and by which techniques. 10 Table I Taxa S t u d i e d S p e c i e s Zoospores Sperm Whole mount S e c t i o n Whole mount S e c t i o n 1» Agarum cribosum + • • • • Bory 2. A l a r i a marginata * • P o s t e l s et Buprecht 3. A. nana + + + Schrader 4. A. t a e n i a t a + • K j e l l man 5. A. t e n u i f o l i a + + Setche 1 1 6. C o s t a r i a c o s t a t a • + + * (Turner) Saunders 7. ,cymathere t r i p l i c a t a + + (P. et B.-) J• Agar dh 8* Dietyoneurum c a l i f o r n i c u m • • + Buprecht 9. E i s e n i a arborea + + + Areschoug 10. Hedophyllum s e s s i l e • • • . • (C. Ag.) Steche 1 1 11. Laminaria q r o e n l a n d i c a • Bosenvinge 12. L. s a c c h a r i n a • (L.) Lamouroux 13. L e s s o n i o p s i s l i t t o r a l i s • • * "(Tilden) Beinke 14. M a c r o c y s t i s i n t e g r i f o l i a • • • • Bory 15. N e r e q c y s t i s luetkeana • * + (Bert.) P..et~B. 16. Pleurophycus q a r d n e r i • • • • S e t c h e l l e t Saunders 17. P o s t e l s i a palmaeformis + • i u p r e c h t 18. Pterygophora c a l i f o r n i c a + • • • Buprecht 11 I I . Gametophyte C u l t u r e s Gametophyte c u l t u r e s were e s t a b l i s h e d from f i e l d - c o l l e c t e d sporophytes by c u t t i n g p i e c e s of f e r t i l e s o r i from p l a n t s kept under r e f r i g e r a t i o n f o r one t o f o u r days a f t e r c o l l e c t i o n , washing the p i e c e s i n c o l d running tap water t o remove contaminating organisms, and l e a v i n g the p i e c e s i n 0.4 urn-f i l t e r e d seawater i n p e t r i d i s h e s at 10 C u n t i l spore r e l e a s e occurred ( w i t h i n a few minutes to s e v e r a l days)• The sorus p i e c e s were then removed, and a f t e r spore s e t t l e m e n t the seawater was r e p l a c e d with c u l t u r e medium. A l l c u l t u r e s were maintained i n 15 x 90 mm p l a s t i c p e t r i d i s h e s , i n SMH-3 medium (Chen e t a l . 1969), without s o i l e x t r a c t , l i v e r e x t r a c t , or TRIS b u f f e r , a t 5, 10, or 15 C. L i g h t was provided by c o o l - w h i t e f l u o r e s c e n t tubes at i n t e n s i t i e s of 7.7-45 uE/sq. m/sec with a 12:12 or 18:6 -hour l i g h t ; d a r k photoperiod. Stock c u l t u r e s were maintained with changes of medium at one- t o three-month i n t e r v a l s ; these c u l t u r e s g e n e r a l l y remained v e g e t a t i v e . S u b c u l t u r e s were s t a r t e d by g e n t l y s e p a r a t i n g the entangled gametophytes by hand with a t i s s u e homogeaizer, and growing these gametophytes at a low d e n s i t y (1-5/sq. mm) under red l i g h t {"cinemoid" No. 14 "ruby" f i l t e r ) at a l i g h t i n t e n s i t y of 1.1-5.5 uE/sq. m/sec or e l e v a t e d temperature (15-19 C) under white l i g h t f o r one t o four months. Hhen gametophytes were c l e a r l y d i f f e r e n t i a t e d as to sex, and were l a r g e enough to be i n d i v i d u a l l y p icked up by p i p e t t e or f o r c e p s , s e p a r a t e male and female c u l t u r e s were s t a r t e d and grown under r e d l i g h t at 10 C f o r one to four months or under 12 white l i g h t a t 10 C f o r 12 months but without change of medium. When brought i n t o white l i g h t and f r e s h medium, these c u l t u r e s became f e r t i l e . ., Sperm r e l e a s e was obtained by p l a c i n g the f e r t i l e gametophytes i n drops o f f i l t e r e d seawater i n i n d i v i d u a l d e p r e s s i o n s of T e r a s a k i d i s h e s (Falcon P l a s t i c s ) . The sperm were thus r e l e a s e d i n t o a smal l volume of water so t h a t high c o n c e n t r a t i o n s were a v a i l a b l e f o r e l e c t r o n microscopy. I U . M a M Microscopy An Olympus CK i n v e r t e d microscope p r o v i d i n g m a g n i f i c a t i o n s up to 40Ox was used t o monitor the c o n d i t i o n of c u l t u r e s and to observe spore and sperm r e l e a s e . A L e i t z c o o l i n g stage was f i t t e d t o a Wild M20 microscope f o r long-term ( s e v e r a l hours) o b s e r v a t i o n s and photography of spores and sperm at h i g h e r m a g n i f i c a t i o n . T h i s microscope was f i t t e d with Wild phase-c o n t r a s t o p t i c s ; a d d i t i o n a l l y , an Olympus "Hoffman modulation c o n t r a s t " condenser and o b j e c t i v e s were adapted t o t h i s instrument t o take advantage of the c o l d s t a g e . During prolonged o b s e r v a t i o n , spores and sperm were maintained i n an o b s e r v a t i o n c e l l made by removing a r e c t a n g l e from the c e n t r a l a rea o f a p i e c e of a c e t a t e adhesive tape stuck t o a microscope s l i d e . A drop o f c e l l suspension was placed i n t h i s r e c t a n g l e and covered with a c o v e r s l i p , forming a w e l l of reasonably l a r g e volume, shallow enough t h a t i t s f u l l depth_ c o u l d be focused under o i l immersion, and t h a t c o u l d be e a s i l y s e a l e d to prevent e v a p o r a t i o n . , Photographs were taken with a L e i c a M1 or Wild 13 Microphotoautomat 35 mo camera using Kodak PAH-X, TBI-X, Ektachrome or Kodachrcme fi l m s . IV. Electron Microscopy For electron microscopy of zoospores before release, pieces of mature s o r i were cut up and placed i n f i x a t i v e . For pre-release sperm, gametophytes were transferred from culture medium to f i x a t i v e . Swimming sperm and spores were c o l l e c t e d on M i l l i p o r e f i l t e r s (Bisalputra et a l . ; 1973); 3 um pore s i z e was used for very dense spore suspensions, 0.45 um for sparse spore and sperm suspensions. After f i x a t i v e was passed through the f i l t e r , the membrane with adierent c e l l s was removed from the f i l t r a t i o n apparatus and processed as i f i t were a th i n piece of tissue. F i l t e r s with very sparse c e l l aggregations were cut into convenient sized pieces and embedded i n agar after osmication to prevent l o s s of c e l l s . Several f i x a t i v e recipes were employed. I n i t i a l l y , the f i x a t i v e mixture contained 0,1M cacodylate buffer diluted 1:1 with f i l t e r e d seawater, 2.51 glutaraldehyde and 5% sucrose, at p8 7.|. , Other combinations t r i e d were 2% glutaraldehyde in 0.115 cacodylate with 1 0 1 sucrose; and 2% glutaraldehyde and 2% paraformaldehyde i n 0.1M cacodylate buffer with 6.8% sucrose; The best r e s u l t s were obtained with the use of 0.38 PIPES buffer (Salema and Brandao 1973), usually with 2.5% glutaraldehyde and 6..6X sucrose (PIPES buffer i s apparently non-toxic and i s therefore safer to handle than cacodylate). Other additives used from time to time were 0.II osmium tetroxide (Barr and Hadland-Hartmann 1978) or 2% 14 paraformaldehyde. A f t e r aldehyde f i x a t i o n , a graded s e r i e s of r i n s e s served to g r a d u a l l y lower the o s m o l a l i t y of the specimen environment p r i o r t o p o s t f i x a t i o n with I X osmium t e t r o x i d e : f i r s t r i n s e i n 0.313 PIPES with 6.6% sucrose; second r i n s e i n 0. 1515 PIPES i n 5% sucrose; t h i r d r i n s e i n 0.15H PIPES without sucrose, then o s m i c a t i o n i n 0.Q7H PIPES., Osmication proceeded a t room temperature f o r 2-3 h or o v e r n i g h t under r e f r i g e r a t i o n f o l l o w e d by 1-2 a at room temperature . O c c a s i o n a l l y 0,1% ruthenium red was added to the p o s t f i x to enhance p o l y s a c c h a r i d e s t a i n i n g , or t h i o c a r b o h y d r a z i d e treatment (OTOTO of Postek and Tucker 1977) t o enhance l i p i d r e t e n t i o n . fiinsing i n d i s t i l l e d water was f o l l o w e d by dehydration i n a methanol s e r i e s of T O * , 251, 50%, 10%, 90% and 1005S methanol with .0.5 H a d added ( H i l l o n i g 1966), s e v e r a l changes i n 1008 methanol without NaCl, 1:1 methanol:propylene oxide, and 100% propylene oxide, followed by gradual i n f i l t r a t i o n with Epon, Spurr's r e s i n or the u l t r a low v i s c o s i t y r e s i n o f Hascorro e t a l . (1976). Eesin was polymerized a t 70 C (or 85 C f o r Mascorro r e s i n ) , f o r 8-24 h. S e c t i o n s were cut with g l a s s or diamond k n i v e s on a fieichert OH-3 ultramicrotome, mounted on c a r b o n - s t a b i l i z e d , c o l l o i d i n - c o a t e d g r i d s , s t a i n e d r o u t i n e l y i n u r a n y l a c e t a t e and l e a d c i t r a t e , and viewed i n a Z e i s s EH-10 e l e c t r o n microscope. For whole-mounts of spores and sperm, the swimming c e l l s were f i x e d by a d d i t i o n of g l u t a r a l d e h y d e to a \% c o n c e n t r a t i o n , or by osmium t e t r o x i d e to a 0,1% c o n c e n t r a t i o n , or both f i x a t i v e s t o g e t h e r . The suspension of f i x e d c e l l s was c e n t r i f u g e d a t 1000 X 6 f o r f i v e minutes, the c e l l p e l l e t 1 5 r i n s e d i n d i s t i l l e d water, c e n t r i f u g e d and resuspended, then drops of c e l l s were placed on c o l l o i d o n - c a r b o n coated g r i d s and allowed t o a i r dry. a l t e r n a t i v e l y , e s p e c i a l l y when only s m a l l numbers of c e l l s were a v a i l a b l e , i n d i v i d u a l drops of c e l l s uspension were p l a c e d on c o v e r s l i p s , f i x e d i n vapor from 2% osmium t e t r o x i d e f o r one minute and t r a n s f e r r e d to g r i d s a f t e r 15 min, or placed d i r e c t l y on g r i d s and vapor f i x e d . To observe spore s e t t l e m e n t , drops of spore suspension were p l a c e d on g o l d g r i d s (to avoid copper t o x i c i t y t h a t might a f f e c t spore behavior) coated with c o l l o i d o n - c a r b o n . These were stuck at a t i n y p o r t i o n of the margin to the edge of masking tape glued s t i c k y - s i d e up on a microscope s l i d e , with the g r i d f i l m f a c i n g upward. The s l i d e was i n v e r t e d and put on the c o o l i n g stage over a water chamber to prevent e v a p o r a t i o n , and observed u n t i l spores c o u l d be seen s e t t l i n g , whereupon the i n v e r t e d s l i d e was placed over an osmium vapor chamber t o f i x the spores i n the a c t of s e t t l i n g . .,• A f t e r vapor f i x a t i o n , the seawater was allowed t o evaporate dry and the s a l t s were r i n s e d away by a p p l i c a t i o n and removal of i n d i v i d u a l drops of d i s t i l l e d water, a f t e r f i n a l d r y i n g , the c e l l s were observed i n the e l e c t r o n microscope, o r were f i r s t shadowed with platinum-palladium or n e g a t i v e - s t a i n e d with phosphotungstic a c i d . 16 CHAPTER 3. / RESULTS These r e s u l t s r e p r e s e n t a composite o f a l l data d e r i v e d from the study of zoospores o f 14 genera and sperm of 11 genera of L a m i n a r i a l e s . I . ,• Zoospores A. , Sporangia A l l L a m i n a r i a l e s produce t h e i r s porangia i n aggregations termed s o r i . The sorus i s d i s t i n g u i s h a b l e m a c r o s c o p i c a l l y as a r a i s e d r e g i o n , u s u a l l y darker than or a d i f f e r e n t c o l o r from the n e i g h b o r i n g v e g e t a t i v e t i s s u e . , I t i s comprised of s p o r a n g i a and accompanying s t e r i l e c e l l s , the paraphyses, which are l o n g e r than the sporangia ( F i g s . 1, 2 ) . The a b u t t i n g d i s t a l t i p s of paraphyses provide a p r o t e c t i v e cover f o r s p o r a n g i a . Sporangia and paraphyses are produced (at l e a s t i n i t i a l l y ) i n a 1:1 r a t i o , as each paraphysis i s produced by a meristoderm c e l l which next produces a sporangium. Paraphyses c o n t a i n l a r g e o s m i o p h i l i c g l o b u l e s , which are absent i n the sporangium ( F i g s . 3, 4) and may r e p r e s e n t t a n n i c substances t h a t serve to d e t e r g r a z i n g (Conover and S i e b u r t h 1965). Often an outer l a y e r , which has been termed a " c u t i c l e " (Hanic and C r a i g i e 1969), i s produced at the t i p s o f the paraphyses ( F i g s . 1 , 2, 3 ) . / The u l t r a s t r u c t u r e o f t h i s l a y e r i s s i m i l a r t o t h a t of the u n d e r l y i n g paraphysis w a l l . I t c o u l d be 17 an a r t i f a c t of d i f f e r e n t i a l shrinkage during p r e p a r a t i o n f o r microscopy, which r e s u l t s i n a p u l l i n g away of the o l d e s t l a y e r of w a l l m a t e r i a l . In p r e p a r a t i o n s made from s o r i that have a l r e a d y r e l e a s e d most of t h e i r s p o r a n g i a l c o n t e n t s , the t i p s of paraphyses are o f t e n swollen and no l o n g e r c o n f l u e n t ( F i g . 6) , i m p l y i n g e i t h e r t h a t p r e - e x i s t i n g l i n e s of weakness allow t i p s to s e p a r a t e , or t h a t an enzyme d i s s o l v e s the substance h o l d i n g the t i p s together. D i s s o l u t i o n o f h y d r o p h i l i c p o l y s a c c h a r i d e s i n the w a l l would r e s u l t i n s w e l l i n g of t i p s of paraphyses. Sporangia are c l a v a t e and very elongate, t y p i c a l l y 100 urn long and 15 um i n diameter., The w a l l s are t h i n n e r than the w a l l s of paraphyses ( F i g . 7 ) , and the d i s t a l t i p s are l i n e d with a "cap" of m a t e r i a l that appears g r a n u l a r i n the e l e c t r o n microscope, i n c o n t r a s t to the f i b r i l l a r appearance of the w a l l proper ( F i g s . 4, 5). T h i s cap u n d e r l i e s the s i t e of dehiscence o f the sporangium, and so presumably serves some f u n c t i o n r e l a t i n g to b u r s t i n g of the c e l l w a l l . Sporangia o f t e n develop with v a r y i n g degrees of synchrony (cf., Angst 1929), f o r uncleaved spor a n g i a are found next t o apparently mature ones ( F i g . 7). Even the uncleaved sporangia show c o n s i d e r a b l e o r g a n i z a t i o n i n the a s s o c i a t i o n of o r g a n e l l e s ; a g i v e n plane of s e c t i o n f r e q u e n t l y shows many basal b o d i e s , n u c l e i or other o r g a n e l l e s i n s i m i l a r c o n f i g u r a t i o n s i n d i f f e r e n t a reas o f the s e c t i o n ( F i g . 8 ) . T h i s i n d i c a t e s t h a t the development of spores w i t h i n a sporangium i s very t i g n t l y c o o r d i n a t e d . 18 fi« Zoospore S t r u c t u r e as cleavage of the c y t o p l a s m i c mass of the sporangium o c c u r s , the s p o r e s begin to take shape. T h e i r p r e - r e l e a s e form i s l a r g e l y determined by the c l o s e packing t h a t p r e v a i l s b e f o r e the sporangium i s mature (Pigs. 9-11), although i n l a t e r stages some l o n g i t u d i n a l e xtension i s e v i d e n t (Figs.,12, 13). They measure from 4 x 4 um t o 4 x 8 um, depending on the o r i e n t a t i o n of the s e c t i o n through the spore. a f t e r r e l e a s e , the spores are f r e e t o adopt a more el o n g a t e form and measure about 5 x 7 um i n s e c t i o n s . , Whole mounts t h a t r e t a i n t h e i r shape, and (presumably) are shrunken minimally d u r i n g a i r ^ - d r y i n g , measure about 4 x 8 um. In some specimens the a n t e r i o r "nose" of the spore i s s t i l l apparent ( F i g . 15). I t i s p o s s i b l e f o r the mass of the uncleaved spores to be prematurely r e l e a s e d from the sporangium, presumably with the p r o t r u d i n g f l a g e l l a f u n c t i o n a l ( F i g . 15); the f a t e of such a spore mass i s unknown. C y t o l o g i c a l Components Of The Zoospore The m i c r o t u b u l a r apparatus c o n s i s t s of the c y t o s k e l e t o n and the f l a g e l l a r axonemes. The f l a g e l l a are produced p r i o r to cleavage of the s p o r a n g i a l r c y t o p l a s m ; they a l l protrude from the cytoplasm of the sporangium, the b a s a l body of each a s s o c i a t e d with a c h l o r o p l a s t ( F i g s . 8, 9). a f t e r cleavage, elements of the c y t o s k e l e t o n o c c a s i o n a l l y , become v i s i b l e . "Bands" of p a r a l l e l m i c r o t u b u l e s ( F i g s . 14, 17), as w e l l as apparent s i n g l e t u b u l e s ( F i g . , 1 3 ) , are present. I t i s the c y t o s k e l e t o n which permits the zoospore to have a form other 19 than the lowest-energy shape of a sphere. There i s a d e f i n i t e a s s o c i a t i o n , and perhaps c o n n e c t i o n , between the c y t o s k e l e t o n and the b a s a l bodies o f the f l a g e l l a . C l e a r e s t i s the a s s o c i a t i o n o f a band of 7-9 microtubules with the a n t e r i o r b a s a l body (Figs..,17, 20-24). Immediately adjacent t o l o n g i t u d i n a l s e c t i o n s of b a s a l , bodies a dense g r a n u l a r i t y can be seen (Figs.,18, 19 , 21> 23C); t h i s may re p r e s e n t the t e r m i n a t i o n of a t u b u l a r band ( F i g . 24) , although i t i s d i f f i c u l t t o determine whether or not i t i s t u b u l a r . , I t i s seen o n l y i n c e r t a i n s e c t i o n s , so i t does n o t surround the basa l body. In s e c t i o n s where the t u b u l a r c o n s t r u c t i o n of the bands i s c l e a r (Figs. 17, 22, 23B) , the t u b u l e s can sometimes form an a r c , which e x p l a i n s why a band i s r a r e l y caught e n t i r e l y i n the plane of a l o n g i t u d i n a l s e c t i o n . A s h o r t d i s t a n c e from the b a s a l bodies some of the microtubules spread out i n t o the cytoplasm and are l e s s conspicuous, e s p e c i a l l y i n o b l i q u e s e c t i o n . The a n t e r i o r band (or a t l e a s t some t u b u l e s , perhaps separated) a r c s around the a n t e r i o r p e r i p h e r y o f the c e l l ( F i g s . 13, 25). T h i s p r o v i d e s the necessary support f o r the prominent "nose" of the spore. Other t u b u l e s converge at the p o i n t of the nose ( F i g . 26) ; t h e i r r e l a t i o n s h i p with the microtubules of the bands or the b a s a l bodies i s not c l e a r . O c c a s i o n a l l y , m i c r o t u b u l es can be seen i n the p o s t e r i o r p o r t i o n of the spore ( F i g . 26), but the presence of l a r g e o r g a n e l l e s such as the nucleus and p l a s t i d probably p r o v i d e s most of the r i g i d i t y f o r t h i s p a r t o f the c e l l . The c y t o s k e l e t a l c o n f i g u r a t i o n i s bes t v i s u a l i z e d with the help o f "exploded" c e l l s , i n which only the microtubular 20 apparatus i s preserved, the r e s t of the spore having been removed by osmotic and s u r f a c e t e n s i o n shocks d u r i n g p r e p a r a t i o n f o r e l e c t r o n microscopy ( F i g s . 2 7-30; a l l these specimens are of A l a r i a t e n u i f o l i a ) . E i g h t microtubules can be counted i n the a n t e r i o r band ( F i g . 28) ; i n some p r e p a r a t i o n s the a n t e r i o r and p o s t e r i o r b a s a l bodies were connected by a s h o r t band ( F i g s . 27, 29, 39, 43) o f about 10 microtubules ( F i g . 30). In some specimens ( F i g s . 28, 29) the a n t e r i o r band extends a t l e a s t 8.3 um; i t may loop around the c e l l p e r i p h e r y t o connect with the p o s t e r i o r b a s a l body ( F i g s . 24, 32), forming a continuous s t r a n d ( F i g . 27). The f l a g e l l a c o n s t i t u t e the remainder of the m i c r o t u b u l a r apparatus., They are s i m i l a r to those t h a t have been d e s c r i b e d f o r zoospores of other brown algae by some workers, although they a r e not formed wi t h i n c y t o p l a s m i c v e s i c l e s as i s the case i n many s p e c i e s . They are i n s e r t e d l a t e r a l l y ( F i g . 31) , and i t i s convenient t o designate the s i d e of the spore b e a r i n g the f l a g e l l a as the v e n t r a l s u r f a c e . Before r e l e a s e the b a s a l bodies viewed i n s e c t i o n were sometimes p a r a l l e l to each other ( F i g s . 33, 34), but a f t e r r e l e a s e they are i n s e r t e d p e r p e n d i c u l a r t o each other ( F i g . 32) , so r e o r i e n t a t i o n must occur as the r e l e a s e d spore changes t o a swimming posture. In t h i s c o n f i g u r a t i o n the spore becomes almost b u l l e t - s h a p e d and the a n t e r i o r f l a g e i l u m i s d i r e c t e d forward, while the p o s t e r i o r f l a g e i l u m t r a i l s to the rear ( F i g . 37). The a n t e r i o r f l a g e i l u m c o n s i s t s o f two p a r t s ( F i g . 37). The proximal p o r t i o n i s up to 22 um l o n g , bears mastigonemes, and i n c r o s s - s e c t i o n shows the us u a l "9*2 H microtubule 21 arrangement of the axoneme. The d i s t a l p o r t i o n , which may be as much as 17.5 urn l o n g , bears no appendages and i s merely an e x t e n s i o n of the two c e n t r a l microtubules of the axoneme ( F i g . 38). Although t h i s d i s t a l " whiplash" was r a r e l y p r e s e r v e d , i t s presence was e v i d e n t i n a l l s p e c i e s examined. I t may be t r a n s i t o r y or very l a b i l e , which would account f o r i t s r a r i t y . Before r e l e a s e o f the spore the mastigonemes are c o n f i n e d t o 1/2 t o 1/3 of the c i r c u m f e r e n c e of the f l a g e l l u m ( F i g s . 34-36). In the swimming spore they are probably arranged i n two rows on o p p o s i t e s i d e s o f the f l a g e l l u m , because whole mounts i n v a r i a b l y show uniform d i s t r i b u t i o n of the mastigonemes on both s i d e s ( F i g s . 37, 42, 45) . In d i s r u p t e d f l a g e l l a where the mastigonemes remain attached, t h i s same arrangement i s found ( F i g s . 39, 42, 43). The most p l a u s i b l e e x p l a n a t i o n f o r t h i s i s t h a t the rows are attached t o o p p o s i t e microtubules o f the axoneme, r a t h e r than t o the f l a g e l l a r membrane. In i n t a c t mastigonemes that have been removed from the f l a g e l l u m , a b a s a l p i e c e may be preserved t h a t i s s p h e r i c a l ( F i g . 41), presumably the remnant of the attachment to the f l a g e l l u m . The proximal p o r t i o n o f such detached mastigonemes i s 1.4 urn long and i n n e g a t i v e - s t a i n e d p r e p a r a t i o n s ( F i g s . 40, 91) and c r o s s - s e c t i o n s ( F i g . 75) shows a t u b u l a r c o n s t r u c t i o n with an e l e c t r o n - d e n s e c o r e . The d i s t a l p o r t i o n i s a f i n e f i l a m e n t up t o 0.8 urn l o n g ( F i g . 44). The mastigonemes u s u a l l y appear s t r a i g h t i n whole mounts, evidence of an i n h e r e n t s t i f f n e s s t h a t should be f u n c t i o n a l l y s i g n i f i c a n t . The p o s t e r i o r f l a g e l l u m i s up to 12 urn long and bears no 22 mastigoneaes ( F i g s . 15, 47). The t e r m i n a l attenuated t i p u s u a l l y i s v e i l preserved ( F i g . 46) and r e p r e s e n t s a c o n t i n u a t i o n of the two c e n t r a l m i c r o t u b u l e s as i n the a n t e r i o r f l a g e i l u m . Hovever, the t i p has a s w e l l i n g which i s not present on the a n t e r i o r whiplash. The usual "9*2** axoneme i s present, d e s p i t e the f a c t t h a t t h i s f l a g e i l u m does not appear to beat d u r i n g locomotion. "Exploded" f l a g e l l a show c l e a r l y the s t r u c t u r e o f the axonemes, i n c l u d i n g the independence of the two c e n t r a l m i c r o t u b u l e s , i n c o n t r a s t to the i n t e g r a l s t r u c t u r e o f the doublets ( F i g . 27). The coherence o f the two s i n g l e t s i n the t i p o f the p o s t e r i o r f l a g e i l u m (at the very end of the "9*2" region) i s s t r i k i n g ( F i g s . 27, 28) . S i m i l a r l y , i n the a n t e r i o r f l a g e i l u m the t e r m i n a l f i l a m e n t i s o f t e n not preserved except a t i t s very hase ( F i g s . 38, 42, 99, 103)./ The a n t e r i o r f l a g e l l a were found d i s r u p t e d much more o f t e n than the p o s t e r i o r , and they were always fragmented s t a r t i n g at the d i s t a l end, never a t the b a s a l end or p o i n t s i n between ( F i g s . 42, 45). T h i s , combined with the tendency f o r the d i s t a l f i l a m e n t or "whiplash" t o be l o s t , suggests t h a t the end of the a n t e r i o r f l a g e i l u m i s fundamentally d i f f e r e n t from the p o s t e r i o r f l a g e i l u m . O c c a s i o n a l l y c o n n e c t i o n s between ad j a c e n t microtubule d o u b l e t s were preserved i n the a n t e r i o r f l a g e i l u m , but they were not e v i d e n t i n the the p o s t e r i o r f l a g e i l u m ( F i g . 27). I f these c o n n e c t i o n s r e p r e s e n t dynein arms, t h e i r absence would be c o n s i s t e n t with the apparent absence of b e a t i n g i n the p o s t e r i o r f l a g e i l u m . 23 P l a s t i d s kt the time of r e l e a s e , t h e r e i s one p l a s t i d per spore, appressed to the p o s t e r i o r s i d e of the nucleus and f o l d e d around i t , g i v i n g the appearence of two p l a s t i d s i n many s e c t i o n s ( F i g . 48). The p l a s t i d t y p i c a l l y measures 3.5 x 1.25 um, although when wrapped around the nucleus i t may be 8.3 um l o n g and only 0.75 um t h i c k . T h i s i s s m a l l e r than p l a s t i d s i n paraphyses, which measure 7.75 x 2 um. P l a s t i d s possess the usual 3 - t h y l a k o i d bands with c o n n e c t i o n s between adjacent bands., O c c a s i o n a l l y membrane-bound i n c l u s i o n s , presumably t h y l a k o i d p r e c u r s o r s , were seen i n the stroma ( F i g s . 49, 50, 51). , The r i n g genophore i s e v i d e n t i n s e c t i o n s that are c u t i n a plane p e r p e n d i c u l a r t o the plane of f l a t t e n i n g of the " d i s k " of the p l a s t i d s ( F i g . 49)., Ho p y renoids were seen. I n the swimming zoospore, p a r t o f the p l a s t i d i s a d j a c e n t to the plasmalemma i n the r e g i o n where the p o s t e r i o r f l a g e l l u m passes p o s t e r i o r l y a f t e r emerging from the spore midway along the " v e n t r a l " s u r f a c e (Fig. 31). T h i s i s the l o c a t i o n of the eyespot i n the p l a s t i d i n a l l s p e c i e s of brown algae and Chrysophyceae i n which t h i s s t r u c t u r e i s known., However, there was no t r a c e i n any of the s p e c i e s examined of an eyespot i n the p l a s t i d s and no pronounced s w e l l i n g of the p o s t e r i o r f l a g e l l u m i n t h i s r e g i o n . P l a s t o g l o b u l i , when present, were s c a t t e r e d and never aggregated i n t o clumps t h a t resembled eyespots ( F i g s . ,49, 50) . , The p l a s t i d begins to d i v i d e a f t e r r e l e a s e ( F i g s . 25A, 52) , and by the time w a l l d e p o s i t i o n commences i n 24 the s e t t l e d spore, the two r e s u l t i n g p l a s t i d s are separated from each other ( F i g . 53). Mitochondria The mitochondria are t y p i c a l of brown a l g a e : o v o i d , l o b e d or cupped at times ( F i g . 54) (probably when d i v i d i n g ) , commonly measuring 1.7 x 0.4 um. There are s e v e r a l mitochondria per spore, not p r e c i s e l y l o c a t e d i n r e l a t i o n to oth e r o r g a n e l l e s , but g e n e r a l l y c o n f i n e d t o the a n t e r i o r o f the spore, e s p e c i a l l y i n the v e n t r a l p o r t i o n . The t u b u l a r c r i s t a e possess t u b u l a r i n c l u s i o n s l a c k i n g i n mitochondria o f v e g e t a t i v e c e l l s ( F i g s . 55, 56). These i n c l u s i o n s run p a r a l l e l t o the a x i s of the c r i s t a , number as many as f o u r per c r i s t a , and have a diameter of about 12 nm. They may extend n e a r l y the e n t i r e l e n g t h of the c r i s t a ( F i g . 56). They appear i n the c r i s t a e b efore c l e a v a g e of the s p o r a n g i a l cytoplasm and p e r s i s t at l e a s t u n t i l germination o f the s e t t l e d spore. Nucleus The m u l t i n u c l e a t e sporangium c l e a v e s t o form u n i n u c l e a t e s p o r e s . / The spore n u c l e i are roughly s p h e r i c a l and measure about 3 um i n diameter., N u c l e o l i are r a r e l y seen ( F i g . 57). Heterochromatin i s conspicuous i n pre-cleavage n u c l e i ( F i gs.,58, 59), and i t di s a p p e a r s a f t e r cleavage o n l y to reappear before r e l e a s e of the spores, remaining prominent u n t i l a f t e r spore settlement ( F i g s . 25, 52 # 53). Nuclear pores and e x t e n s i o n s o f the n u c l e a r envelope are e v i d e n t ( F i g s . 60-25 63, 65). , The nucleus and p l a s t i d are c l o s e l y a s s o c i a t e d , the outer membrane of the nuclear envelope being c o n t i n u o u s with the c h l o r o p l a s t endoplasmic r e t i c u l u m ( F i g . , 6 2 ) . The n u c l e u s t y p i c a l l y i s appressed t o the p l a s t i d on i t s p o s t e r i o r s i d e , c l o s e l y a s s o c i a t e d with both the g o l g i apparatus and a system of EB s t a c k s toward the a n t e r i o r ( F i g s . 64, 65). Endomembrane System The p e r i n u c l e a r g o l g i apparatus i s prominent i n a l l stages of spore development from pre-cleavage to the s e t t l e d spore. I t may be n e s t l e d i n a c o n c a v i t y of the n u c l e u s at times ( F i g . 66) . S i m i l a r s t a c k s of rough EE a r e f r e q u e n t l y present, and i n precleavage n u c l e i these s t a c k s o c c a s i o n a l l y surround the n u c l e u s completely i n one plane (Fig.,.67). Sometimes a r r a y s of ribosomes are conspicuous i n these s t a c k s ( F i g s . 68, 69). The products of both g o l g i v e s i c l e s and EB a r r a y s are not o f t e n obvious, d e s p i t e the presence of numerous membrane-bound i n c l u s i o n s i n zoospores.^ These v e s i c u l a t e i n c l u s i o n s are of s e v e r a l t y p e s . E l e c t r o n - d e n s e v e s i c l e s , presumably o s m i o p h i l i c although not n o t a b l y i n t e n s i f i e d by t h i o c a r b o h y d r a z i d e treatment, are present from the pre-cleavage stage to germination of the spore. They resemble t h e l a r g e r g l o b u l e s found i n the paraphyses, and o n l y o c c a s i o n a l l y i s t h e i r bounding membrane d i s c e r n i b l e ( F i g . 70). In the spore they measure up to 0.6 um i n diameter, and a r e u s u a l l y s c a t t e r e d around the c e l l , although o c c a s i o n a l aggregations may occur ( F i g s . ,14,71) . They most c l o s e l y resemble the "type A vacuole" 26 of Rawlence (1973). Another conspicuous v e s i c l e type, s i m i l a r t o the "type B vacuole" of Bawlence (1973) c o n t a i n s a homogeneous, f i n e l y g r a n u l a r m a t e r i a l ( F i g s . 9, 10) t h a t may be p a r t l y or wholly leached by some f i x a t i o n s ( F i g s . 5, 27, 48, 59, 99). These i n c l u s i o n s a r e o f t e n l a r g e (to 1.3 um) , f i l l i n g as much as 1/3 of the ar e a o f c e r t a i n spore s e c t i o n s (Pigs. 10, 73) • , They are c o n f l u e n t i n many specimens ( F i g s . 9, 25C, 109), e v i d e n t l y having been squeezed together. Often the bounding membrane i s i n d i s t i n c t , and i n r e l e a s e d spores the c o n t e n t s may break through the plasmalemma ( F i g s . 25C, 25F). They probably c o n s t i t u t e a p o i n t of weakness i n the s t r u c t u r e of the spore where deformation o r d i s r u p t i o n of membranes i s l i k e l y to occur.. T h e i r c o n t e n t s are r e t a i n e d and enhanced i n e l e c t r o n d e n s i t y by t h i o c a r b o h y d r a z i d e treatment ( F i g s . , 7 3 , 74), s u p p o r t i n g fiawlence*s (1973) c o n t e n t i o n t h a t they c o n t a i n l i p i d or p o l y p h e n o l i c m a t e r i a l , both of which should be o s m i o p h i l i c . These v e s i c l e s were a l s o present from pre-cleavage through spore germination, and a t times the p e r i n u c l e a r rough endoplasmic r e t i c u l u m (EB) s t a c k s o f the p r e - r e l e a s e spores appeared to be producing these v e s i c l e s ( F i g . 72) . A t h i r d k i n d of v e s i c l e , c o r r e s p o n d i n g to the "type C vacu o l e " of Rawlence (1973), has an e l e c t r o n - t r a n s p a r e n t matrix with s c a t t e r e d f i b r i l l a r o r g r a n u l a r m a t e r i a l . I n e a r l y s t a g e s these v e s i c l e s produce the e x t r a c y t o p l a s m i c matrix t h a t surrounds the cytoplasm i n the sporangium, presumably c o n s i s t i n g of a l g i n i c a c i d and fu c o i d a n (cf. McCully 1968a). I n d i s t i n c t v e s i c l e s such as these were o c c a s i o n a l l y seen 27 at the plasmalemma of pre-cleavage sporangia i n a s s o c i a t i o n with mastigonemes -emerging from the c y t o p l a s m i c mass ( F i g s , 75, 76), T h i s was the o n l y time t h a t s t r u c t u r e d contents were seen i n such v e s i c l e s , which have l a r g e l y disappeared by the time the s p o r e s are mature, When the r e l e a s e d spores s e t t l e and begin t o produce a, w a l l , v e s i c l e s c o n t a i n i n g t u b u l a r membranes appear, perhaps comparable to the "type C v a c u o l e " o f Bawlence (1973). Membrane s t a c k s , with o n l y l i m i t e d or i n some cases no a s s o c i a t i o n with the cytoplasm, were o c c a s i o n a l l y seen i n spo r a n g i a ( F i g s . 77-80)•. Sometimes they appeared t o be i n v o l v e d with formation of the f l a g e l l a ( F i g s . 79, 80) . They were o f t e n accompanied by membrane-bounded v e s i c l e s c o n t a i n i n g dense g r a n u l a r m a t e r i a l s i m i l a r to neighboring cytoplasm, but f r e e i n the e x t r a c y t o p l a s m i c space (Figs.,79, 81). No c o n s i s t e n t d i f f e r e n c e s among s p e c i e s were observed i n any of the preceding c e l l u l a r c o n t e n t s . However, i t i s s t r i k i n g t h a t d i f f e r e n c e s were observed i n a v e s i c l e type t h a t i s unigue to the mature zoospore: these a r e the presumptive adhesion v e s i c l e s . T h e i r c o n t e n t s are extruded from the cytoplasm when the swimming zoospore s e t t l e s , forming a l a y e r of m a t e r i a l t h a t p r o v i d e s f o r adhesion of the s e t t l e d spore to the s u b s t r a t e . , These v e s i c l e s are formed a f t e r cleavage of the s p o r a n g i a l p r o t o p l a s t , but w e l l before spore r e l e a s e . , The c o n t e n t s are u s u a l l y d i f f e r e n t i a t e d i n t o a core and p e r i p h e r y of d i f f e r i n g e l e c t r o n d e n s i t i e s . One type of adhesion v e s i c l e , observed i n Pterygpphora ( F i g . ,66) , L e s s o n l o p s i s ( F i g . 14) A l a r i a 28 ( F i g . 25C), and Hedophyllum ( F i g . 82) , i s s p h e r i c a l with a f i b r o u s core and a g r a n u l a r , more electron-dense p e r i p h e r y . / A second type, observed i n H a c r o c y s t i s i n t e g r i f o l i a ( F i g . 83) and La mi l i a r i a g r o e n i a n d i c a ( F i g . 84) , has a l i g h t , g r a n u l a r c o re and a much darker, granular p e r i p h e r y ; these v e s i c l e s are markedly elo n g a t e . A t h i r d kind i s a l s o e l o n g a t e , and o f t e n curved, with a core and periphery of a u n i f o r m l y g r a n u l a r , e l e c t r o n - d e n s e m a t e r i a l , separated by a l i g h t e r , very f i n e l i n e t h a t c o u l d not be r e s o l v e d as a membrane.,, T h i s k i n d was observed only i n Laminaria s a c c h a r i n a ( F i g . 85) f i x e d i n chromate b u f f e r . A f o u r t h k i n d o f adhesion v e s i c l e was a l s o elongated and curved, with a g r a n u l a r , e l e c t r o n - d e n s e core and r e t i c u l a t e p e r i p h e r y , observed i n Agarum ( F i g . ,861. P o s t e l s i a ( F i g . 87) , and E i s e n i a (Fig. 13). Perhaps the most d i s t i n c t i v e type of adhesion v e s i c l e was observed i n Cymathere t r i p l i c a t a ( F i g . 10, 52, 61, 65, 73, 74). T h i s form was c o n s i s t e n t l y present i n m a t e r i a l c o l l e c t e d from widely separated p o p u l a t i o n s f i x e d i n both c a c o d y l a t e and PIPES b u f f e r s . , These v e s i c l e s have an elongated o u t l i n e , an e l e c t r o n - d e n s e periphery> and a core of electron-dense m a t e r i a l forming s t r i a e i n a l i g h t e r , f i b r o u s matrix ( F i g . 89). C r o s s -s e c t i o n s of these v e s i c l e s show t h a t the dense core m a t e r i a l may be continuous w i t h i n the s t r i a t i o n , or may possess a l i g h t e r i n n e r core ( F i g . 88). F i g . 126A-B shows a g e n e r a l i z e d zoospore model based on my o b s e r v a t i o n s . 29 £• Zoospore Function The structure and a c t i v i t y of the f l a g e l l a are related to the two main functions of the released zoospore, d i s p e r s a l and settlement. The anterior flageilum i s held i n front of the swimming spore and heats s i n u s o i d a l l y . The posterior flageilum does not appear to beat, but presumably functions as a rudder. There i s no sign of phototaxis, such as aggregation of swimming spores or s e l e c t i o n of s i t e of settlement i n r e l a t i o n to l i g h t . Sor do the spores seem to prefer the bottom of a container to the sides when s e t t l i n g , and so show no geotaxis. In some species the spores w i l l swim f o r at least 24 hours i f suitable conditions are provided (crowding i n the presence of mucilages from the sorus), while they w i l l s e t t l e within minutes i f not crowded and given access to substrate. The spore attaches to the substrate by the t i p of the anterior flageilum, and may remain attached i n t h i s way and continue beating the flageilum f o r as long as 20 minutes without s e t t l i n g . , Besorption of the f l a g e l l a and attachment of the spore to the substrate were not observed with the l i g h t microscope, but spores fixed f o r electron microscopy have been found to exhibit a l l the stages from swimming to germination. There i s good agreement i n the l i t e r a t u r e that the anterior flageilum makes the f i r s t contact with the substrate, although i t i s rarely preserved i n electron microscope preparations (Figs. ,92-103), the long anterior whiplash i s implicated i n t h i s action, and that i t has adhesive properties i s indicated by occasional preservation of clumps of mastigonemes on i t s t i p (Figs. 90, 91, 92) . , That the 30 mastigonemes are not adhesive i s suggested by the f a c t t h a t they r a r e l y appear clumped together along the a n t e r i o r f l a g e l l u m , and clumps of mastigonemes have not been observed s t i c k i n g to the s u b s t r a t e . I f adhesion i s accompanied by i n c r e a s e d f l u i d i t y o f the membrane, t h i s might account f o r the l a b i l i t y of the whiplash. I t i s important to note t h a t s e c t i o n s of f l a g e l l a t h a t show only the two c e n t r a l m i c r o t u b u l e s are very r a r e w i t h i n the sporangium, and c o u l d be a t t r i b u t e d t o the whiplash of the p o s t e r i o r f l a g e l l u m ( F i g s . 104-106). There i s no s i g n t h a t the whiplash i s removed from the a n t e r i o r f l a g e l l u m , f o r I have never found f r e e whiplashes i n whole mount p r e p a r a t i o n s , even though f r e e mastigonemes can be seen.., T h i s c o u l d be because the whiplash i s so l a b i l e t h a t i t d i s i n t e g r a t e s completely when i t i s removed. a f t e r adhesion o f the a n t e r i o r f l a g e l l u m , the zoospore i s drawn to the s u b s t r a t e b y withdrawal of the axoneme i n t o the spore. In some s e c t i o n s f l a g e l l a are appressed t o ( F i g . 65), or f u s e d with the plasmalemma ( F i g s . 107, 108). The axoneme may be drawn i n e i t h e r s t r a i g h t or by f u s i o n of the f l a g e l l a r membrane with the plasmalemma ( F i g . 109). T h i s probably depends on the o r i e n t a t i o n of the spore body with r e s p e c t to the f l a g e l l u m , which would vary under circumstances of water motion. The axonemes preserve t h e i r o r g a n i z a t i o n l o n g a f t e r withdrawal. For a time the two b a s a l bodies may r e t a i n t h e i r a s s o c i a t i o n f o l l o w i n g withdrawal of a t l e a s t part of one axoneme ( F i g . 110), but e v e n t u a l l y they move away from the s i t e 31 o f i n s e r t i o n ( F i g . A15). S i m i l a r l y , the axonemes w i l l be found l a t e r removed from the plasmalemma and deeper i n s i d e the c e l l (Figs..111-120). . Once the spore has rounded up a t about the time of axoneme r e s o r p t i o n , the c y t o s k e l e t o n i s not d e t e c t a b l e and has probably been disassembled.. Presumably a t l e a s t p a r t i a l disassembly would be r e g u i r e d f o r movement of the b a s a l bodies during r e s o r p t i o n of the axonemes. The adhesion v e s i c l e s are not extruded ( F i g . 112) u n t i l a f t e r axonemes are i n s i d e the spore., The v e s i c l e s remain stuck t o the e x t e r i o r of the plasmalemma a f t e r e x o c y t o s i s ( F i g s . 112, 119), and o f t e n open ( F i g s . , 113, 115, 116, 118), r e l e a s i n g t h e i r contents which glue the spore t o the s u b s t r a t e . As the spore begins to form a w a l l , v e s i c l e s d e p o s i t m a t e r i a l o u t s i d e the plasmalemma, i n t e r c a l a t i n g between the plasmalemma and any attached remnants of adhesion v e s i c l e s ( Fig..117). The axonemes are s t i l l evident as more wa l l m a t e r i a l i s l a i d down ( F i g s . 118, 119), but they are s c a t t e r e d through the cytoplasm., They can s t i l l be seen a f t e r f o r m a t i o n of the germination tube commences ( F i g . 120). In whole mount p r e p a r a t i o n s , the diameter of the s e t t l e d spores i s l a r g e r than that of spores s t i l l i n the swimming s t a t e (121). T h i s i n c r e a s e i n diameter, and the appearance o f other s e t t l e d spores as "double b l o b " f i g u r e s ( F i g s . 122, 123) can be a t t r i b u t e d t o the presence of the withdrawn axonemes i n s i d e the c e l l s . The mastigonemes may be c a s t o f f the f l a g e l l a d u r i n g withdrawal, because they are seen s c a t t e r e d about ( F i g . 124), or forming a t r a i l t h a t l e a d s to the s e t t l e d spore ( F i g . 125) . 32 I I . , Sperm A. A n t h e r i d i a A n t h e r i d i a develop on male gametophytes, u s u a l l y i n a t e r m i n a l or l a t e r a l p o s i t i o n ( F i g . 127) ; i f i n t e r c a l a r y , at l e a s t one of the n e i g h b o r i n g c e l l s i s another an t h e r i d i u m ( F i g . , 1 2 9 ) . They may be formed s i n g l y or i n c l u s t e r s t h a t are e s s e n t i a l l y rudimentary p l u r i l o c u l a r s t r u c t u r e s ( p l u r i l o c s ) ( F i g s . 130-133), f o r a c o n f l u e n t o u t e r w a l l i s o f t e n d e t e c t a b l e around adjacent a n t h e r i d i a ( F i g s . 131, 132).. They are more or l e s s c o n i c a l i n shape, measuring 5 t o 9 urn i n l o n g e s t dimension. The apex of the cone i s the s i t e of dehiscence, and i t i s l i n e d with a "cap" o f d i f f e r e n t i a l l y s t a i n i n g m a t e r i a l t h a t i s g r a n u l a r i n the e l e c t r o n microscope ( F i g s . , 133-136). , The w a l l s are s u r p r i s i n g l y e f f e c t i v e b a r r i e r s to p e n e t r a t i o n of f i x a t i v e s , and o f t e n nearby v e g e t a t i v e c e l l s show s u p e r i o r p r e s e r v a t i o n . , B. / Sperm S t r u c t u r e One sperm i s formed per antheridium. H i t h i a the antheridium, sperm measure from 1.7 x 7.2 um to 2.5 x 5 um. . They appear t o be f o l d e d before r e l e a s e (Figs.,137-139), l i k e Fucus sperm. E a r l y i n the course of d i f f e r e n t i a t i o n of the a n t h e r i d i a l cytoplasm the... f u t u r e b a s a l bodies can be seen (Figs./140-141) ; t h e m i c r o t u b u l a r assemblages are not p o s i t i o n e d p e r p e n d i c u l a r to each other as c e n t r i o l e s would be. 33 The sperm possesses a band of microtubules i n the a n t e r i o r m o s t p o r t i o n o f the c e l l which runs p a r a l l e l t o the b a s a l bodies i n the f o l d of the c e l l t h a t e n c l o s e s the f l a g e l l a ( F i g s . 137, 143-147). At t h i s time a p a r t of the a n t e r i o r m i c r o t u b u l a r band i s a s s o c i a t e d with the b a s a l bodies, which are p a r a l l e l t o each other, s i m i l a r to the s i t u a t i o n i n the zoospores ( F i g s . 137, 142B-D, 145). A f t e r r e l e a s e , the sperm u n f o l d s so t h a t the angle between the b a s a l bodies i n c r e a s e s , p o s s i b l y t o as much as 180 degrees, as i n the Fucus sperm, and the a n t e r i o r band o f the c y t o s k e l e t o n l o o p s around the a n t e r i o r p e r i p h e r y of the c e l l , as i n the zoospore (Figs.,148, 149). The a n t e r i o r f l a g e l l u m of the sperm i s e s s e n t i a l l y t h e same as t h a t of the zoospore, f o r i t has a b a s a l p o r t i o n 16-18 um long t h a t b ears mastigonemes ( F i g s . 152, 155, 157, 158), and a d i s t a l p o r t i o n at l e a s t 10 um long that i s a c o n t i n u a t i o n of the two c e n t r a l microtubules ( F i g s . 150, 153, 154)., Fewer sperm were o b t a i n a b l e than zoospores; consequently these measurements probably do not r e p r e s e n t the dimensions of the f u l l y i n t a c t a n t e r i o r f l a g e l l u m , because here again the d i s t a l whiplash i s a p p a r e n t l y l a b i l e and d i f f i c u l t t o preserve. The most remarkable f e a t u r e of the sperm i s the p o s t e r i o r f l a g e l l u m , which i s up t o 38 um long and t a p e r s d i s t a l l y ( F i g s . 150-152, 154-160). Observations of unshadowed and unstained whole mounts ( F i g s . 151 # 155, 157, 158) and c r o s s -s e c t i o n s of f l a g e l l a w i t h i n a n t h e r i d i a (Figs./161-163) both show t h a t t h i s t a p e r i s the r e s u l t of a decrease i n the number of m i c r o t u b u l e s i n the axoneme, with a change o f the d o u b l e t s i n t o s i n g l e t s . The t e r m i n a t i o n p o i n t of microtubules can be 34 detected when a f r a y i n g of the p o s t e r i o r f l a g e l l u m occurs (Figs.,164, 165); a microtubule not anchored i n place a t i t s d i s t a l end may t e a r loose from the axoneme s h o r t of the end of the f l a g e l l u m , a phenomenon t h a t v i r t u a l l y never occurs i n a f l a g e l l u m i n which a l l the axonemal microtubules end at a common terminus. P l a s t i d s The p l a s t i d s of the sperm a r e s m a l l e r than those o f the zoospores or v e g e t a t i v e c e l l s o f the gametophytes, measuring from 0.7 x 1.5 um to 0.33 x 2.0 um i n s e c t i o n . They are o v a l i n v i r t u a l l y a l l s e c t i o n planes, r a r e l y bent o r f o l d e d . , The usual 3 - t h y l a k o i d s t a c k s are present, but only 3-4 per p l a s t i d . In c o n t r a s t t o the spores, t h e r e are 2-3 p l a s t i d s i n each sperm, u s u a l l y next to the nucleus ( F i g s . 166-168, 173, 175). As i n the zoospores, there i s no eyespot./ Mitochondria As many as seven m i t o c h o n d r i a l s e c t i o n s , measuring up to 0.54 x 1.65 um, can be seen i n one s e c t i o n of a sperm. They are u s u a l l y p o s i t i o n e d a t the a n t e r i o r and v e n t r a l s u r f a c e s (Figs..137, 142A). The t u b u l a r c r i s t a e possess the same t u b u l a r i n c l u s i o n s found i n the mitochondria of zoospores ( F i g . 169). 35 Nucleus The nucleus i s g l o b u l a r t o i r r e g u l a r , r e f l e c t i n g a c l o s e r packing of the membrane-bound o r g a n e l l e s than i n zoospores (Figs. 137-139, 172, 177). I t i s u s u a l l y elongated along the a n t e r i o r - p o s t e r i o r a x i s , from 2.2 x 2.8 um to 1.6 x 3.5 um i n dimensions. I t i s p o s i t i o n e d between the d o r s a l , p o s t e r i o r p l a s t i d s and the v e n t r a l , a n t e r i o r mitochondria. Heterochrony t i n i s r a r e l y e v i d e n t , i n c o n t r a s t to the zoospore n u c l e u s . No n u c l e o l i were seen., Endomemforane System The g o l g i apparatus i s a c t i v e i n e a r l y stages o f d i f f e r e n t i a t i o n o f a n t h e r i d i a ( F i g . 170), c i s t e r n a e forming v e s i c l e s t h a t empty i n t o the channel w i t h i n which the f l a g e l l a appear ( F i g s . 143, 171).. In more mature sperm the g o l g i apparatus i s much l e s s apparent than i n the zoospores. Stacks of ES sometimes occur on the d o r s a l s i d e of the n u c l e u s ( F i g . 172) , and e l e c t r o n - t r a n s p a r e n t v e s i c l e s , s i m i l a r to those i n the d e v e l o p i n g zoospores, produce the m a t e r i a l t h a t f i l l s the e x t r a c y t o p l a s m i c space which surrounds the mature sperm ( F i g s . 142B-D, 175). V e s i c l e s producing membranous m a t e r i a l are a p p a r e n t l y i n v o l v e d i n formation of the f l a g e l l a ( F i g . 143) , and f r e e membrane aggregations ( F i g s . 173, 177), o f t e n a s s o c i a t e d with the f l a g e l l a , may occur o u t s i d e the cytoplasm.. Four t o f i v e g r a n u l a r v e s i c l e s up t o 0.55 um i n diameter, s i m i l a r t o the "type B vacuoles" of the zoospores, are p o s i t i o n e d c h a r a c t e r i s t i c a l l y i n the a n t e r i o r of the sperm 36 (Figs..137, 142, 171, 176, 178). Electron-dense g l o b u l e s may be the c o u n t e r p a r t s of s i m i l a r bodies seen i n zoospores (Figs.,137, 142, 146). However, because a bounding membrane was never v i s i b l e , and they were found i n anomalous p o s i t i o n s at times, such as the f l a g e l l a and o u t s i d e the cytoplasm, these g l o b u l e s may r e p r e s e n t an a r t i f a c t o f osmium p r e c i p i t a t i o n . / F i g . 183 i s a g e n e r a l i z e d sperm model based on my o b s e r v a t i o n s . , £• Sperm Function Upon r e l e a s e , sperm are a c t i v e swimmers, p r o p e l l e d by b e a t i n g of the a n t e r i o r f l a g e i l u m . , According to l i g h t microscope o b s e r v a t i o n s , the p o s t e r i o r f l a g e i l u m does not beat, but i t may show a bend when the sperm i s not moving r a p i d l y ( F i g s . 180-182)•, A sinuous waveform demonstrates the g r e a t e r a c t i v i t y and f l e x i b i l i t y of the a n t e r i o r f l a g e i l u m . S h o r t l y a f t e r r e l e a s e , masses o f sperm gather around eggs, demonstrating t h a t the sperm a r e chemotactic. 37 CHAPTER 4., DISCOSSIGN I w i l l d i s c u s s my r e s u l t s with the L a m i n a r i a l e s i n l i g h t of what i s known o f other brown a l g a l r e p r o d u c t i v e s t r u c t u r e s so t h a t the s i g n i f i c a n c e o f my f i n d i n g s i s made c l e a r . , I. Sporangia A. U n i l o c u l a r Sporangia O n i l o c u l a r s p o r a n g i a may be d e f i n e d as spora n g i a t h a t at some stage i n t h e i r development c o n t a i n more than two n u c l e i ( t h i s e x c l u d e s both c o n v e n t i o n a l p l u r i l o c u l a r s t r u c t u r e s and one-chambered, a i t o t i c a l l y - d e r i v e d s t r u c t u r e s ) . , T h i s d e f i n i t i o n emphasizes t h e i r l o n g - r e c o g n i z e d r o l e as the onl y known s i t e o f meiosis i n brown alga e ( F r i t s c h 1945), although i n many l i f e h i s t o r i e s i t has been shown t h a t meiosis f a i l s to occur d u r i n g unispore f o r m a t i o n (Bold and Wynne 1978). O n i l o c s may be s p h e r i c a l or o v o i d t o c l a v a t e , p e d i c e l l a t e , s e s s i l e or immersed i n the t h a l l u s . , The most p r i m i t i v e u n i l o c u l a r s p o r a n g i a may be considered t o be those t h a t a r e s o l i t a r y and g l o b u l a r i n form. O n i l o c s t h a t a re t i g h t l y packed i n a sorus may be c l a v a t e (e.g., L a m i n a r i a l e s ) . O n i l o c s may be s o l i t a r y or c l u s t e r e d , o c c a s i o n a l l y i n catenate s e r i e s (e.g., A r t h r o c l a d i a , A s c p s e i r a , P i l a y e l l a l or may occur i n d i s t i n c t i v e a g g r e g a t i o n s termed s o r i , which may i n c l u d e s p e c i a l i z e d n o n - s p o r a n g i a l c e l l s . . When s o r i a re 38 immersed i n the t h a l l u s the r e s u l t i n g s t r u c t u r e i s c a l l e d a c o n c e p t a c l e (e. g.,•• Splachnidium, Notheia, A s c o s e i r a . " F u c a l e s " sensu l a t o ) . S o r i vary from i r r e g u l a r , s c a t t e r e d groups of sporangia too s m a l l t o be seen by the unaided eye (e.g., P u n c t a r i a ) , t o l a r g e r aggregations with a d i s t i n c t o r g a n i z a t i o n (e.g., Soranthera. with s p e c i a l i z e d m u l t i c e l l u l a r "paraphyses" and a c e n t r a l t u f t of t r i c h o t h a l l i c h a i r s ) , t o very l a r g e continuous expanses [ e . g . , Himantothallus (Boe and S i l v a 1977), a l l L a m i n a r i a l e s j t h a t may be c o n f i n e d t o s p e c i a l i z e d l a t e r a l s (e.g., M a r i a . Lessoniopsis) . The elongated sporangia of L a m i n a r i a l e s , r e s u l t i n g from the c l o s e packing of s p o r a n g i a and paraphyses i n the s o r u s , c l e a r l y demonstrate the high degree of o r g a n i z a t i o n which must e x i s t w i t h i n any such s t r u c t u r e . T h i s o r g a n i z a t i o n i s e v i d e n t i n s e c t i o n s taken through the sporangium; the cytoplasm i s d i s t r i b u t e d along an a x i s i n a way t h a t cannot be r e a d i l y seen i n globose sporangia of other groups such as E c t o c a r p a l e s ( c f . Loiseaux 1973, Baker and Evans 1973a, Harkey and S i l c e 1976a). Each nucleus i s l o c a t e d c l o s e t o the periphery o f the cytoplasm and the f l a g e l l a a l l protrude r a d i a l l y , n e i t h e r o f which i s p o s s i b l e i n a s p h e r i c a l sporangium. T h i s accounts f o r the l a c k of f l a g e l l a r v e s i c l e s w i t h i n the cytoplasm and suggests t h a t there a r e no fundamental d i f f e r e n c e s i n the way f l a g e l l a are formed among brown algae (cf. Markey and w i l c e 1976a p. 168)• The most h i g h l y modified u n i l o c u l a r sporangia are found i n Hotheia with i t s micro- and macrosporangia (Nizamuddin and Homersley 1960) or Syrinqoderma a b y s s i c o l a , which produces c r o s s - w a l l s w i t h i n the sporangium (Walker and Henry 1978). 39 fi*- P l u r i l o c u l a r Sporangia In p l u r i l o c u l a r sporangia c r o s s - w a l l s are always formed a f t e r each m i t o t i c d i v i s i o n ( t h e l a c k o f c r o s s - w a l l s r e p o r t e d by Knight (19 23) i n P i l a y e l l a has been disproved by Harkey and a i l c e (1976a) J. ,• P l u r i l o c u l a r s p o r a n g i a occur on d i p l o i d sporophytes o f only the v e g e t a t i v e l y l e s s advanced brown alga e (they a re not known i n L a m i n a r i a l e s ^ D e s m a r e s t i a l e s , Sporochnales, C u t l e r i a l e s , D i c t y o t a l e s , o r " F u c a l e s " sen.su l a t o ( F r i t s c h 1945)], where they serve to r e c y c l e t h e sporophyte. S i m i l a r p l u r i l o c u l a r s t r u c t u r e s t h a t occur on gametophytes are co n s i d e r e d gametangia, even i f the "gametes" may develop p a r t h e n o g e n e t i c a l l y ( F r i t s c h 1945, Bold and Wynne 1978). These sporangia may a l s o be s o l i t a r y , c l u s t e r e d , or grouped i n t o s o r i . I I . Spores 4* V a r i a b i l i t y Among Spores Few previous s t u d i e s have examined r e l e a s e d spores or gametes except as whole mounts, f o r once free of the p r o t e c t i o n o f the sporangium or gametangium, the swarmer appears t o be very f r a g i l e . For example, mastigonemes of a c t i v e l y swimming c e l l s a r e u s u a l l y not preserved u n l e s s an i n i t i a l osmium f i x a t i o n i s used, yet t h i s r e s u l t s i n very poor p r e s e r v a t i o n of the c e l l c o ntents ( c f . , Cheignon 1964)*, Swarmers are very l i k e y to be f i x e d b e f o r e or a f t e r t h e i r p e r i o d o f optimum 40 v i t a l i t y i f a t t e n t i o n i s not paid t o c o n d i t i o n s o f r e l e a s e {Chi 1973) or t i m i n g of f i x a t i o n (Toth 1976a). The p r i m i t i v e zoospore has a l r e a d y been d e s c r i b e d i n the I n t r o d u c t i o n ; very l i t t l e v a r i a t i o n among brown a l g a l zoospores has been reported to date. The l a r g e {to 12 urn i n l e n g t h ) spores from the u n i l o c u l a r s p o r a n g i a of Ectocarpus were found to c o n t a i n up t o s i x dictyosomes r a t h e r than the one of most other (smaller) s p o r e s , and the p l a s t i d was not appressed to the nucleus (Baker and Evans 1973b) ; both these m o d i f i c a t i o n s can be a t t r i b u t e d to the l a r g e s i z e of the c e l l . !• The M i s s i n g Eyespot In most r e s p e c t s l a m i n a r i a l e a n zoospores resemble those of other brown algae. The most s i g n i f i c a n t d i f f e r e n c e from other orders i s the l a c k of an eyespot. This f e a t u r e had been reported by some pr e v i o u s workers {Sauvageau 1918, Kanda 1936, 1938, Papenfuss 1942, Bold and Bynne 1978 (no a u t h o r i t y c i t e d ) ], but not by others (Meyers 1928, angst 1929, H o l l e n b e r g 1939); a l l (Sauvageau 1918, K y l i n 1918, Kanda 193 8, Toth 1974, Bold and Mynne1978) agree, however, t h a t Chorda possesses an eyespot [and a l s o perhaps, S a c e o r h i z a (Sauvageau 1918, Kain 1969, Norton and Burrows 1969)]. The s u p p o s i t i o n of C h i (1973) and S a l k e r (1980) t h a t o s m i o p h i l i c g l o b u l e s i n the cytoplasm of the zoospore of H a c r o c y s t i s and N e r e o c y s t i s are e x t r a p l a s t i d i c eyespot g l o b u l e s can be d i s c o u n t e d f o r s e v e r a l reasons: 1) although the g l o b u l e s may be aggregated a t the p o s t e r i o r of the c e l l i n a s s o c i a t i o n with a band of m i c r o t u b u l e s , i n other p a r t s of the c e l l a p p a r e n t l y i d e n t i c a l g l o b u l e s can be found. 41 2) The mass of globules i s not bounded by a membrane; although eyespots of some a l g a l groups may lack a memirane when located outside a p l a s t i d (Dodge 1973), t h i s i s never the case i n the f l a g e l l a t e groups thought to be most closely related to brown algae (Chrysophyceae, Xanthophyceae). 3) Although the globules are osmiophilic, they are not necessarily l i p i d ; i n f a c t , they are much more osmiophilic than known phaeophycean eyespots and could be composed of some other osmiophilic substance. They resemble vesic l e s i n the paraphyses of the sorus s i m i l a r to those in Fucus epidermal c e l l s thought to contain phenolic material (HcCully 1968b)., 4) Light microscopy f a i l s to reveal the presence of any pigmented bodies i n the l i v e zoospore other than the p l a s t i d ; a l l other a l g a l eyespots are v i s i b l e i n the l i g h t microscope. 5) There i s no association of the globules with the f l a g e l l a r apparatus; such an association i s known in a l l brown algae and related groups i n which any eyespot i s found., 6) Numerous observers (Hollenberg 1939, Papenfuss 1942, Suto 1950, Saito 1975, my own observations) have noted the lack of phototaxis i n the behavior of zoospores of many Laminariales; by contrast, i n Chorda, shown by electron microscopy (Toth 1974) to possess an eyespot, reports of phototaxis appear to be universal (Kylin 1918, Sauvageau 1918, Toth 1976 a) . I propose that t h i s loss of the eyespot can he accounted for by a consideration of the the reproductive ecology of Laminariales. A l l Laminariales except Chorda produce sporangia i n s o r i that cover an expanse of blade much larger than the sporangial surface of most other algae, and most species occur 42 i n dense stands, even forming " f o r e s t " - l i k e k e l p beds (e.g., H a c r o c y s t i s ) . Spore r e l e a s e by such c l o s e a g g r e g a t i o n s of p l a n t s can r e s u l t i n dense s e t t l e m e n t s of spores near the parent sporophytes./ Such dense s e t t l e m e n t s may r e s u l t i n poor growth because of shading by the parents (Anderson and North 1965). under these c o n d i t i o n s t h e r e may be s e l e c t i o n f o r a mechanism t o prevent s e t t l i n g of the spores too soon. A l o s s of the eyespot and a s s o c i a t e d phototaxis would ensure t h a t spores would f i n d a s u b s t r a t e at random, r a t h e r than s e e k i n g the sea bottom by negative p h o t o t a x i s ( c f . K y l i n 1918). By s t a y i n g m o t i l e l o n g e r , spores would be more l i k e l y to be c a r r i e d f u r t h e r by water c u r r e n t s . £• How flany P l a s t i d s ? I n v a r i o u s other orders of brown algae there may be from one to s e v e r a l p l a s t i d s per swarmer. , S e v e r a l authors (Chi and Neushul 1972, C o l l i n s and Kugrens 1975) have rep o r t e d the occurrence of more than one p l a s t i d i n zoospores of L a m i n a r i a l e s . T h i s c o n c l u s i o n has most l i k e l y been based on s e c t i o n s which show two l o b e s of the p l a s t i d as i t l i e s f o l d e d around the nucleus ( c f . Sauvageau 1918) . Hy o b s e r v a t i o n s agree with those of Toth (1976a) t h a t there i s only one p l a s t i d per s p o r e , which begins to d i v i d e a f t e r the spore i s r e l e a s e d from the sporangium. Because I observed no pyrenoids, I c o n s i d e r r e p o r t s of pyrenoids (Chi 1973, Walker 198Q) i n pre-r e l e a s e spores q u e s t i o n a b l e ; the matrix of these " p y r e n o i d s " does not appear d i f f e r e n t i a t e d from the stroma., These appendages of the p l a s t i d may be l o b e s formed at p l a s t i d 43 d i v i s i o n . p_. M i t o c h o n d r i a The t u b u l a r i n t r a c r i s t a l i n c l u s i o n s found i n mitochondria i n l»oth zoospores and sperm of L a m i n a r i a l e s appear i d e n t i c a l t o those i n f i v e other orders of brown algae (Markey and H i l c e 1976a # C a s s e l l and P o l l o c k 1979, Henry 1979)., Because they are not e v i d e n t i n v e g e t a t i v e c e l l s , or the sporangia b e f o r e f l a g e l l a d i f f e r e n t i a t e , i t seems l i k e y t h a t t h e i r presence i s r e l a t e d t o the r e s p i r a t o r y demands of m o t i l i t y . E. The C y t o s k e l e t o n The c y t o s k e l e t o n of swarmers has r e c e i v e d l i t t l e a t t e n t i o n s i n c e Manton (1964) compared the fucacean " p r o b o s c i s " to the c y t o s k e l e t a l m i c r o t u b u l a r bands i n C y s t o s e i r a sperm and S c y t o s i p h o n spores. Toth (1976a) f a i l e d to observe the c y t o s k e l e t o n i n zoospores of Chorda, but he a p p a r e n t l y was l o o k i n g a t spores t h a t were rounded up, and i n such spores the c y t o s k e l e t o n of n e c e s s i t y has been disassembled. Although Manton (1964) discounted the s k e l e t a l f u n c t i o n o f c y t o p l a s m i c m i c r o t u b u l e s , t h e i r appearance as the zoospore becomes elongated and disappearance as i t rounds up i n s e t t l i n g c o n s t i t u t e s t r o n g c i r c u m s t a n t i a l evidence of t h e i r s k e l e t a l r o l e . A c l o s e a s s o c i a t i o n with the b a s a l bodies of the f l a g e l l a i s a l s o c o n s i s t e n t with mechanical support, because th e f o r c e s generated by beating of the f l a g e l l a must be t r a n s m i t t e d to the body of the spore through a s t r u c t u r e 44 capable o f withstanding such s t r e s s e s without p e r m i t t i n g damage or deformation of the c e l l . The microtubules extending from the r e g i o n of the b a s a l bodies c o u l d d i s t r i b u t e these f o r c e s w i t h i n t h e cytoplasm so that no one component would be o v e r l y s t r e s s e d . T h i s r a t i o n a l i z a t i o n accounts f o r the wide, i f not u n i v e r s a l , occurrence of f l a g e l l a r r o o t s of v a r i o u s forms among f l a g e l l a t e s and c i l i a t e s ( P i t e l k a 1963, Hibberd 1976). F.. The F l a g e l l a Host of the c h a r a c t e r i s t i c s of l a m i n a r i a l e a n zoospore f l a g e l l a demonstrated by t h i s study have been p r e v i o u s l y encountered by other workers, but some asp e c t s a r e obscure and o t h e r s are s u b j e c t t o c o n t r o v e r s y . , Several authors (Bouck 1969^ Loiseaux and Best 1970, Markey ana S i l c e 1976a, 1976b, L a C l a i r e and Best 1978, Toth 1974) have noted t h a t brown a l g a l mastigonemes are found i n p r e - r e l e a s e swarmers o n l y on one s i d e of the f l a g e l l u m . , Some (Loiseaux and ifest 1970, Toth 1976a, L a C l a i r e and West 1978) b e l i e v e t h a t mastigonemes i n the swimming spore are arranged u n i l a t e r a l l y or h e l i c a l l y , and t h a t the b i l a t e r a l appearance i n whole-mount p r e p a r a t i o n s i s an artifact.„ However, s e c t i o n s of free-swimming Fugus sperm (Cheignon 1964), and whole mounts of "exploded" f l a g e l l a show that the attachment of the mastigonemes i s b i l a t e r a l i n the a c t i v e swarmer. Indeed, a h e l i c a l arrangement can be r u l e d out because i n t h a t case, the mastigonemes should show a p e r i o d i c "wavy" p a t t e r n i n whole mounts. S i m i l a r l y , a u n i l a t e r a l d i s t r i b u t i o n of mastigonemes should y i e l d many f l a g e l l a with asymmetric d i s t r i b u t i o n s of mastigonemes t o one s i d e or the 45 other i n many specimens, , k uniform d i s t r i b u t i o n around the f l a g e l l a r c i r c u m f e r e n c e might show a symmetrical a r r a y , but t h i s i s incompatible with the rows of mastigonemes a s s o c i a t e d with axqnemal microtubules t h a t can be seen i n "exploded" f l a g e l l a . Although only l i m i t e d attempts have been made to analyse the hydromechanical f u n c t i o n of f l a g e l l a , some ( H o l w i l l 1.974) suggest t h a t a b i l a t e r a l arrangement o f mastigonemes i s necessary f o r the r e v e r s a l of t h r u s t e v i d e n t i n a l l f l a g e l l a with s t i f f , r i g i d l y anchored mastigonemes. A l l a n t e r i o r l y d i r e c t e d f l a g e l l a t h a t beat s i n u s o i d a l l y seem t o share t h i s c o n s t r u c t i o n . T h e r e f o r e , i t i s p o s s i b l e to r e j e c t on hydromechanical grounds Caram's (1975) c o n t e n t i o n that i n the sperm of C u t l e r i a the a n t e r i o r f l a g e l l u m i s s h o r t and simple and the p o s t e r i o r i s long and adorned with mastigonemes. The whiplash was found on the a n t e r i o r f l a g e l l u m of both zoospores and sperm i n a l l s p e c i e s of L a m i n a r i a l e s s t u d i e d . Numerous r e f e r e n c e s t o the adhesive f u n c t i o n of the a n t e r i o r f l a g e l l u m a r e c i t e d by F r i t s c h (1945) and Friedmann (1961). T h i s whiplash has been shown to be as l o n g as the mastigoneme b e a r i n g p o r t i o n i n many specimens (Petersen e t a l . 1958), Mu l l e r and F a l k 1973, Toth 1976a)• I t s presence has been repor t e d i n C h o r d a r i a (Petersen e t a l , 1958), Ectocarpus and §2F9£^£R§§ ( H o l l e r 1965), Chorda (Toth 1976a), Desmarestia, Soranthera. Coilodesmev A n a l i p u s , Syringoderma. and Fucus (unpub. o b s . ) . I t i s v i s i b l e under ph a s e - c o n t r a s t i n the l i g h t microscope (Muller 1965), appearing as a loop or l o o s e l y wound h e l i x . / But i t i s not preserved i n most e l e c t r o n microscope p r e p a r a t i o n s a f t e r s t a n d a r d aldehyde or osmium f i x a t i o n ( M u l l e r 46 and Falk 1973). Because the t i p o f the a n t e r i o r f l a g e i l u m i s the s i t e of f i r s t s u b s t r a t e c o n t a c t by s e t t l i n g swarmers ( F r i t s c h 1945), the e x i s t e n c e of a s p e c i a l i z e d s t r u c t u r e f a c i l i t a t i n g attachment should not come as a s u r p r i s e . The c o i l i n g o f the whiplash observed i n the l i g h t microscope i s sometimes preserved i n e l e c t r o n microscope p r e p a r a t i o n s ( M u l l e r and F a l k 1973, Toth 1976a), but s i n c e other c o n f i g u r a t i o n s have been found (Petersen e t a l . 1958), i t may i e t h a t i t s form i s a l t e r e d by the pr e p a r a t o r y methods, or j u s t t h a t i t i s not r e a d i l y v i s i b l e by l i g h t microscopy i f i t i s not c o i l e d . The r a r i t y o f p r e s e r v a t i o n o f the whiplash i n p r e p a r a t i o n s f o r the e l e c t r o n microscope has been a t t r i b u t e d to f a i l u r e of f i x a t i o n ( H o l i e r and F a l k 1973), but a second p o s s i b i l i t y remains, t h a t the whiplash i s not formed u n t i l the svarmer i s ready to s e t t l e . T h i s would account f o r so few whiplashes appearing i n s e c t i o n s of swarmers before r e l e a s e ; those t h a t are seen are attached t o the p o s t e r i o r f l a g e i l u m . N e v e r t h e l e s s , the h y p o t h e s i s o f extreme l a b i l i t y i s c o n s i s t e n t with the f a c t t h a t the a n t e r i o r f l a g e i l u m i s d i s r u p t e d much more f r e q u e n t l y than the p o s t e r i o r , and always s t a r t i n g from the d i s t a l end. adhesive f u n c t i o n migist be t i e d t o an i n t r i n s i c f l u i d i t y of the whiplash membrane, making f i x a t i o n d i f f i c u l t . . The l o s s of mastigonemes a f t e r aldehyde f i x a t i o n demonstrates t h a t even the "b e s t " f i x a t i v e s may f a i l t o preserve c e r t a i n components. I t would be i n t e r e s t i n g to look f o r a s i m i l a r s t r u c u t u r e i n f u n g a l zoospores of s i m i l a r design (e.g., Oomycetes) and Chrysophyta known to have s e t t l e d stages i n t h e i r l i f e c y c l e s . The p o s t e r i o r f l a g e i l u m of zoospores of L a m i n a r i a l e s 47 d i f f e r s fom those of most brown a l g a l swarmers i n l a c k i n g a b a s a l s w e l l i n g , s i n c e t h e r e i s no eyespot. I t should be noted that the s h o r t whiplash of t h i s f l a g e i l u m i s u s u a l l y w e l l p r e s e r v e d . Although the c o n s t r u c t i o n seems t o be the same as the l o n g a n t e r i o r whiplash (2 c e n t r a l s i n g l e m i c r o t u b u l e s covered by membrane), i t s ease o f p r e s e r v a t i o n may be a s s o c i a t e d with i t s l a c k of involvement i n s e t t l i n g , and conseguent l a c k of f l u i d i t y . T h i s f l a g e i l u m i s r i g i d (Friedmann 1.961, Toth 1976a) but may execute b e a t i n g motions (Couch 1941, M u l l e r and F a l k 1973) . That i t i s not necessary f o r m o t i l i t y i s suggested by the f a c t t h a t i n the sperm of D i c t y o t a i t i s missing., No mastigonemes have been seen on t h i s f l a g e i l u m [ I have concluded t h a t the r e p o r t by Loiseaux and West (1970) of s h o r t t u f t e d mastigonemes on the p o s t e r i o r f l a g e i l u m of G i f f o r d i a p y i g e r can be dismissed as contamination of t h e i r c u l t u r e s by a c o l o r l e s s f l a g e l l a t e , probably a bodonid ]• Adhesion v e s i c l e s A l l c y t o p l a s m i c v e s i c l e s seen i n t h i s study of l a m i n a r i a l e a n zoospores are comparable to those seen i n other brown a l g a l zoospores by p r e v i o u s a u t h o r s . However, the great v a r i a t i o n i n the appearance o f the contents of the adhesion v e s i c l e s ( " l a m e l l a t e v e s i c l e s " of C h i (1973), "plagues" of Walker (1980) ] among the s p e c i e s of L a m i n a r i a l e s examined was unexpected. T h e i r d e r i v a t i o n from the g o l g i apparatus has been e s t a b l i s h e d (Baker and Evans 1973a, 1973b, C h i 1973, Walker 1980). Some of the v a r i a t i o n may be a t t r i b u t a b l e t o d i f f e r e n t 48 stages of f o r m a t i o n , and t h i s makes establishment of s t r u c t u r a l types dubious a t present., However, i t i s probably s a f e to make a few g e n e r a l i z a t i o n s . Most of the adhesion v e s i c l e s i n L a m i n a r i a l e s show more s t r u c t u r a l complexity than those of "lower" brown alga e (cf• Baker and Evans 1973a, 1973b, Loiseaux 1973, my unpub. obs.). I n t e r n a l membranous components have been shown i n f i x a t i o n s with added ruthenium red (Chi 1973) or chroma te ( F i g . , 85). The s t r i a t i o n s formed by the g r a n u l a r contents i n Cymathere were observed c o n s i s t e n t l y i n s e v e r a l d i f f e r e n t c o l l e c t i o n s and f i x a t i o n s . although v e s i c l e types encountered i n t h i s study c o u l d not be c o r r e l a t e d with f a m i l y concepts ( F r i t s c h 1945) i n the L a m i n a r i a l e s , i t i s noteworthy t h a t the v e s i c l e s d e s c r i b e d by C h i (1973) i n .Macrocystis p y r i f e r a appear i d e n t i c a l t o those found i n i n t e q r i f o l i a . M« Other M o d i f i e d Spores although l a c k of an eyespot s e t s most Laminarialean zoospores apart from those of more p r i m i t i v e brown a l g a e , spores a r e modified even more d r a m a t i c a l l y i n some other o r d e r s . , Only one i n s t a n c e o f heterospory has been r e l i a b l y recorded i n the brown algae, i n the r e p r o d u c t i o n of Nothexa, where two s i z e s of s p o r a n g i a produce macrospores and microspores (Nizamuddin and Womersley 1960). In D i c t y o t a l e s and T i l o p t e r i d a l e s , l a r g e , non-motile spores are produced i n the u n i l o c u l a r sporangium. The t e t r a -or o c t o s p o r e s of D i c t y o t a l e s are u n i n u c l e a t e products of meiosis. I t i s s i g n i f i c a n t t h a t i n the production of the 49 t e t r a s p o r e s o n l y f o u r n u c l e i are produced ( F r i t s c h 1,945), whereas i n oogonia of Fuc a l e s e i g h t n u c l e i r e s u l t , no matter how many eggs are e v e n t u a l l y produced (from one t o e i g h t ) . , In T i l o p t e r i d a l e s only one l a r g e , non-motile mono spore i s produced which i s g u a d r i n u c l e a t e , at l e a s t i n i t i a l l y { F r i t s c h 1945). In both D i c t y o t a l e s and T i l o p t e r i d a l e s the l a c k of f l a g e l l a i n these spores can be a t t r i b u t e d to t h e i r l a r g e s i z e ; a f l a g e l l u m would be u s e l e s s t o propel such l a r g e c e l l s (the same r e a s o n i n g can be a p p l i e d to the eggs o f oogamous forms) . I.II. Garnetanqia &» / P l u r i l o c u l a r Gametangia The most p r i m i t i v e gametangia are ob v i o u s l y p l u r i l o c u l a r . The chambers of the gametangia may form a simple f i l a m e n t (e.g., i n Scy to siphon), or a hollow tube (e.g.. T i l opt er i s ) ( F r i t s c h 1945). In many cases a parenchyma i s formed, even i n s p e c i e s showing no other parenchymatous s t r u c t u r e s (e.g., i n E c t o c a r p a l e s , C h o r d a r i a l e s ) . F r e q u e n t l y gametangia are grouped i n s o r i . S p e c i a l i z e d p l u r i l o c u l a r gametangia occur i n anisogamous and oogamous s p e c i e s . Anisogamy i n v o l v e s f u s i o n of gametes produced i n d i s t i n c t l y larger-chambered (female) and s m a l l e r -chambered (male) gametangia (e.g., C u t l e r i a ) , although r a r e l y the two s i z e s of chamber may occur i n the same gametangium I e.g., G i f f o r d i a m i t c h e l l i a e ( M u l l e r 1969) J . Even when a gametangium produces a very s p e c i a l i z e d gamete ( i . e . , i n 50 oogamy), the gametangium may r e t a i n an obvious p l u r i l o c u l a r character [e.g., antheridia of Dictyotales (Fritsch 1945) ]. In many groups, however, the gametangia have been reduced to s o l i t a r y or clustered single l o c a l e s , so that t h e i r homology with p l u r i l o c s i s obscured, as with oogonia of Dictyotales, and both types of gametangia of Laminariales, Desmarestiales and Sporochnales., Note, however, that antheridia of Dictyotales are unquestionably p l u r i l o c u l a r and that the antheridium of Nereia (Sporochnales) i s reported to be two-chambered (Fritsch 1 9 4 5 ) . Therefore, i t i s inappropriate to refer to such one-chambered gametangia as " u n i l o c u l a r " (cf. / Setche11 and Gardner 1925, p. 5 9 0 ; Kanda 1936, p.256 ), as such terminology obscures the unmistakable homologies among d i f f e r e n t orders., !• Antheridia Of Laminariales Although i n Laminariales most antheridia are borne sin g l y on the male gametophytes, occasional c l u s t e r s of gametangia show that these structures may be regarded as reduced p l u r i l o c s . The confluence of the outer walls indicates that the adjacent antheridia are formed by c e l l d i v i s i o n within a common wall, as in the formation of more conventional p l u r i l o c u l a r structures (cf.„ Markey and Wilce 1 9 7 6 b ) . The "cap" of material l i n i n g the apex of the antheridium i s undoubtedly involved i n the t r i g g e r i n g of sperm release. I t has been shown (Luning and Muller 1978, Muller et a l . 1979) c that release occurs within 12 seconds of contact with a substance produced by f e r t i l e female gametophytes i n several Laminariales. , Such a rapid response implies a s p e c i a l i z e d 51 mechanism f o r tri g g e r i n g dehiscence of the antheridium; the d i f f e r e n t i a l staining of the an t h e r i d i a l cap shows that i t i s chemically d i f f e r e n t i a t e d from the other a n t h e r i d i a l contents and wall. I t s s u p e r f i c i a l resemblance to the cap found i n the apex of Laminariales zoosporangia may indicate that sporangial release in t h i s order i s induced by a more sp e c i a l i z e d mechanism (e.g., an enzymatic breakdown of the t i p of the sporangium) than i s the case i n "lower" brown algae i n which no predetermined s i t e of dehiscence i s discernible, release being triggered by osmotic forces. IV. Gametes A. , Variation Among Gametes Three d i f f e r e n t patterns of sexual reproduction i n brown algae have been recognized t r a d i t i o n a l l y : isogamy, anisogamy and oogamy (Fritsch 1945)., "Isogametes" are most commonly produced among the Ectocarpales (sensu F r i t s c h 1945).. The few that have been studied u l t r a s t r u c t u r a l l y show l i t t l e to dis t i n g u i s h them from zoospores, or the males from the females (Harkey and S i l c e 1976a, 1976b, Muller and Falk 1973). Ectocarpus bas been the most c a r e f u l l y studied (Papenfuss 1935, F r i t s c h 1945, Huller 1967, 1972a, 1972b, 1975, HuHer and Falk 1973), and as i n a l l other "isogaaous" species that have been examined rigorously, the male and female swarmers are d i f f e r en t i a te d behayiorally, and so should not be termed isogamous i n the s t r i c t sense of the word. The female swims 52 oaly f o r a s h o r t time, perhaps 15 minutes, while the male may remain m o t i l e f o r e i g h t hours or more, Opon s e t t l i n g , the female begins t o s e c r e t e a pheromone t h a t a t t r a c t s the males. The males make c o n t a c t with the s e t t l e d female with the t i p of the a n t e r i o r f l a g e i l u m , and when one succeeds i n f u s i n g with the female, pheromone production e v i d e n t l y s t o p s , because t h e other male gametes swim away. Gametes may a l s o s e t t l e without f u s i n g and regenerate the h a p l o i d gametophyte. Although t h e r e are r e p o r t s of f u s i o n s of swimming gametes i n isogamous s p e c i e s (Papenfuss 1934, Loiseaux 1967), these o b s e r v a t i o n s have never been confirmed by c y t o l o g i c a l evidence and should be regarded with s u s p i c i o n . , There are few well-documented examples of what has been c l a s s i c a l l y r e c o g n i z e d as anisogamy, i n which both gametes are m o t i l e but the female i s l a r g e r than the male. The best known i s C u t l e r i a , i n which the female gametes have about 30 times the volume of the male gametes (Jaenicke 1977) and possess s e v e r a l p l a s t i d s ( L a c l a i r e and Best 1978), while the male has but one p l a s t i d (Caram 1975), although both have eyespots. Here again the female gamete s e t t l e s promptly, s e c r e t e s a sex pheromone ( d i f f e r e n t from the one produced by Ectocarpus) and males are a t t r a c t e d as i n Ectocarpus ( H n l l e r 1974)., In Cplpomenia p e r e q r i n a (Clayton 1979) and G i f i; or d i a m i t c h e l l i a e ( H u l l e r 1969) the s i z e d i f f e r e n c e between the two gametes i s not as great as i n C u t l e r i a . but the m a l e g a m e t e s are n o t i c e a b l y p a l e r . E s s e n t i a l l y the same mating r e a c t i o n occurs as was observed i n C u t l e r i a , but i n Colpomenia both gamete types could develop p a r t h e n o g e n e t i c a l l y . 53 In oogamy, a motile sperm f u s e s with a l a r g e , u n f l a g e l l a t e d egg,/ In the sperm the complement of o r g a n e l l e s i s o f t e n reduced or modified from t h a t found i n zoospores or isogametes. In D i c t y o t a only the a n t e r i o r f l a g e l l u m p r o t r u d e s from the sperm, decorated with a row o f s m a l l s p i n e s along .with the u s u a l mastigonemes, and the p l a s t i d i s v e s t i g i a l , 'Sperm of Hi m a n t h a l i a , Xiphophora (Manton 1956), and Hormosira (Forbes and Hallam 1978) i n the F u c a l e s a l l possess a l a r g e r s p i n e near the terminus of the mastigoneme-bearing flagellum,.. In the Fucaceae of the Northern Hemisphere the sperm bear a " p r o b o s c i s " of membrane-bound micr o t u b u l e s , and the p o s t e r i o r f l a g e l l u m i s l o n g e r than the a n t e r i o r (Manton 1964, my unpub. obs,) . P e l v e t i a (Oltmanns 1922, my unpub. o b s . ) , P e l v e t i o p s i s , and Hesperophycus (my unpub. obs.) sperm a l l l a c k eyespots i n c o n t r a s t to JFucus and ftscophyllum. .. Some s p e c i e s of Saraassum ( F l e t c h e r and F l e t c h e r 1975) and C y s t o s e i r a (Sauvageau 1911, Roberts 1978) l a c k e y e s p o t s , even though t h e i r sperm are otherwise s i m i l a r t o more p r i m i t i v e •gametes of isogamous s p e c i e s , s i n c e t h e i r f l a g e l l a are unmodified (Manton 1964, my unpub. obs.). Less i s known of the sperm of oogamous S p h a c e l a r i a l e s (Moore 1951) , Desmarestiales ( F r i t s c h 1945) or Sporochnales (Caram 1965), as no e l e c t r o n microscopy and l i t t l e l i g h t microscopy has been done with them.. I t does appear t h a t eyespots may be absent i n a l l . L a m i n a r i a l e s , D e s m a r e s t i a l e s and Sporochnales a l l share a tendency t o r e t a i n t h e i r eggs on the oogonia of t h e i r d i m i n u t i v e gametophytes; t h i s common aspect of t h e i r r e p r o d u c t i o n might be expected to have some 54 bearing on t h e i r f e r t i l i z a t i o n b i o l o g y and thus the s t r u c t u r e of t h e i r sperm. The eggs of Fucus have been shown (Muller and J a e n i c k e 1973) t o produce a s p e r m - a t t r a c t i n g pheromone, and as a l r e a d y mentioned, f e r t i l e female gametophytes of s e v e r a l L a m i n a r i a l e s produce a substance t h a t not only a t t r a c t s sperm but i s a l s o r e s p o n s i b l e f o r t h e i r r e l e a s e from the antheridium (Luning and d u l l e r 1978, M u l l e r e t a l . 1979). B.,/ Sperm Of L a m i n a r i a l e s D e s c r i p t i o n s of sperm of L a m i n a r i a l e s have shown c o n s i d e r a b l e disagreement ( F r i t s c h 1945).; They have r e p o r t e d presence (McKay 1933, Hollenberg 1939, Kemp and Cole 1961) and absence ( G h e r a r d i n i and North 1972) of p l a s t i d s , presence (Sauvageau 1918, McKay 1933, Ho l l e n b e r g 1939) and absence (Kanda 1936, 1938, Kemp and Cole 1961) of eyespots; a l o n g e r a n t e r i o r and s h o r t e r p o s t e r i o r f l a g e i l u m (McKay 1933), "the usual two l a t e r a l l y a t t a c h e d f l a g e l l a " ( F r i t s c h 1945), " f l a g e l l a o f d i f f e r e n t l e n g t h s . . . s i z e d i f f e r e n c e . . . not as s h a r p l y pronounced as i n the zoospore" (Kemp and Cole 1961) or a p o s t e r i o r f l a g e i l u m as l o n g as the a n t e r i o r (Kanda 1936, 1938). I have found t h a t the g e n e r a l a r c h i t e c t u r e of the sperm c e l l of L a m i n a r i a l e s shows resemblance to both L a m i n a r i a l e s zoospores and fucacean sperm.„ The mi c r o t u b u l a r bands of the c y t o s k e l e t o n i n the a n t e r i o r o f the L a m i n a r i a l e s sperm are undoubtedly homologous with those of the zoospore, but w i t h i n the antheridium the sperm i s f o l d e d , s i m i l a r t o the p r e - r e l e a s e 55 Fucus sperm {Manton and C l a r k e 1956, B e r k a l o f f and Eousseau 1979)• , The f l a g e l l a of the sperm are l i k e w i s e formed w i t h i n a channel or pocket that i n the l a t e r stages of spermatogenesis p e r s i s t s as the a n t e r i o r f o l d . T h i s f o l d , with i t s c y t o s k e l e t a l m i c t r o t u b u l a r band, i s r e o r g a n i z e d upon r e l e a s e so that i t p o i n t s forward. T h i s change i n the o r i e n t a t i o n of the band i s accompanied by a change i n the angle between the b a s a l bodies from z e r o to 180 degrees, as i n t h e Fucus sperm, i n c o n t r a s t to the 90 degree angle found i n the zoospores., The very long and t a p e r i n g p o s t e r i o r f l a g e i l u m of the sperm of L a m i n a r i a l e s has no known c o u n t e r p a r t i n other brown algae. The c o n s t r u c t i o n of t h i s f l a g e i l u m resembles t h a t r e p o r t e d i n the h y p e r m a s t i g i n i d f l a g e l l a t e Trichonympha (Gibbons and Grimstone 1960)., In both the l a m i n a r i a l e a n sperm and Trichonympha. t a p e r i n g o f the f l a g e i l u m i s the r e s u l t of a r e d u c t i o n i n the number of m i c r o t u b u l a r doublets to s i n g l e t s , then r e d u c t i o n i n the number of s i n g l e t s . The o u t l i n e of these s i n g l e t m icrotubules becomes somewhat r e c t i l i n e a r towards the t i p , i n d i c a t i n g an unusual s u b s t r u c t u r e . , although more than one p l a s t i d i s present i n the sperm, they are reduced i n s i z e from those of the zoospore., T h y l a k o i d morphology i s normal, so they may be assumed to be f u n c t i o n a l . Having s e v e r a l s m a l l p l a s t i d s may f a c i l i t a t e f o rmation of the elongated shape of the sperm, s i n c e a l a r g e p l a s t i d would tend t o dominate the volume a v a i l a b l e , r e s u l t i n g i n a more globose, l e s s hydrodynamically e f f i c i e n t shape. The l a c k of eyespots i n the sperm i s c o n s i s t e n t with the importance o f chemotaxis i n sperm f u n t i o n . Lack of adhesion v e s i c l e s i s c o n s i s t e n t with 56 the g o a l of f u s i o n with the egg r a t h e r than adhesion to an i n e r t s u b s t r a t e . C. comparison With Other Brown A l g a l Sperm There are s e v e r a l p o i n t s of s i m i l a r i t y between l a m i n a r i a l e a n - sperm and those of o t h e r oogamous orde r s , as i s i l l u s t r a t e d diagrammatically i n F i g . v 184. , The v a r i o u s s p e c i a l i z e d sperm types can be d e r i v e d from the p r i m i t i v e , z o o s p o r e - l i k e sperm found i n isogamous s p e c i e s and (apparently) even some F u c a l e s . , The r e p o r t e d l a c k of eyespots i n some Fu c a l e s , Sporochnales, D i c t y o t a l e s and S p h a c e l a r i a l e s has a l r e a d y been mentioned. Desmarestia v i r i d i s sperm a l s o l a c k eyespots and have a prominent a n t e r i o r c y t o s k e l e t a l l o o p and a l o n g e r hind f l a g e l l u m , but t h i s f l a g e l l u m i s c o n s t r u c t e d d i f f e r e n t l y (see Appendix). A long p o s t e r i o r f l a g e l l u m (which r e t a i n s the "9 + 2" c o n s t r u c t i o n throughout i t s length) i s found i n s p e c i a l i z e d fucacean sperm t h a t l a c k s p i n e s on the a n t e r i o r f l a g e l l u m (Fucaceae o f Northern Hemisphere). Elongated sperm bodies a l s o occur i n Fucaceae and D u r v i l l a e a , as w e l l as Desmarestia v i r i d i s . , T h i s presumably r e s u l t s i n g r e a t e r hydrodynamic e f f i c i e n c y . The longer p o s t e r i o r f l a g e l l u m may serve as a s t a b i l i z i n g rudder, necessary to ensure s t a b i l i t y i n the presence of a very r a p i d l y b e a t i n g a n t e r i o r f l a g e l l u m . The l o n g e r f l a g e l l u m , as w e l l as the elongated c e l l shape and the " p r o b o s c i s " of some Fucaceae, a l l c o n t r i b u t e to a l a r g e r s u r f a c e area, important i n the r e c e p t i o n of chemotactic s i g n a l s . I t has been shown i n Ectocarpus (Jaenicke 1977) t h a t the sex pheromone i s f i r s t taken up by the 57 a n t e r i o r f l a g e i l u m of the male gamete. An elongated r e c e p t o r mechanism should allow more s e n s i t i v e d e t e c t i o n of c o n c e n t r a t i o n g r a d i e n t s of pheromones, f a c i l i t a t i n g l o c a t i o n of the s o u r c e o f emission (the female gamete). while D i c t y p t a sperm have no p o s t e r i o r f l a g e i l u m , they have s p i n e s on the a n t e r i o r f l a g e i l u m , r e c a l l i n g the s e v e r a l F u c a l e s (Himanthalia, £±£hophora, Hormosira) with s h o r t p o s t e r i o r f l a g e l l a and an a n t e r i o r f l a g e l l a r spine, I have i n d i c a t e d with q u e s t i o n marks i n F i g . 184 the p o s s i b i l i t y t h a t these spiny f l a g e l l a might l a c k whiplashes (they have not been r e p o r t e d ) ; i t i s c o n c e i v a b l e t h a t i n these i n s t a n c e s the f a i l u r e t o observe t h i s s t r u c t u r e r e f l e c t s i t s absence, the s p i n e s s u b s t i t u t i n g i n some way f o r the whiplashes i n sperm-egg c o n t a c t . The s i g n i f i c a n c e of these v a r i o u s sperm morphologies f o r swimming behavior o r gamete i n t e r a c t i o n has not been e s t a b l i s h e d . Some of these q u e s t i o n s might be answered by motion s t u d i e s of swimming, or u l t r a s t r u c t u r a l examination o f gamete c o n t a c t . An understanding of the f u n c t i o n a l s i g n i f i c a n c e of sperm morphologies w i l l be r e q u i r e d to make the best use of these c h a r a c t e r i s t i c s i n taxonomy., F o r t u n a t e l y , oogamy i s widespread among the o r d e r s of the brown a l g a e , and the e v o l u t i o n of c e r t a i n m o d i f i c a t i o n s (e.g., longer p o s t e r i o r f l a g e i l u m , spines) i n d i f f e r e n t p h y l e t i c l i n e s appears t o have been accomplished by d i f f e r e n t mechanisms, so merely analogous s t r u c t u r e s are r e a d i l y d i s t i n g u i s h e d from true homologies., I have t h e r e f o r e i n d i c a t e d i a F i g . 184 t h a t sperm m o d i f i c a t i o n s found among oogamous orders of brown a l g a e have occurred as a r a d i a t i o n from a p r i m i t i v e a n c e s t r a l form, r a t h e r than as a 58 h i e r a r c h i c a l s e r i e s of changes; the l a m i n a r i a l e a n , Desmarestia, fucacean, spiny f u c a l e a n and D i c t y p t a sperm types can be c o n s i d e r e d t o be a t comparable l e v e l s of advancement. Where some m o d i f i c a t i o n s have occurred l e s s widely (e.g., l o s s o f eyespots i n Fucales) p h y l e t i c s e r i e s w i t h i n f a m i l i e s may be made e v i d e n t . Seemingly anomalous f e a t u r e s may point t o taxa that have been ne g l e c t e d or m i s i n t e r p r e t e d i n morphological s t u d i e s (e.g., D u r v i l l a e a ) . The "isogamous" and anisogamous brown algae may yet show i n t e r e s t i n g v a r i a t i o n s i n swarmer s t r u c t u r e . . One might expect to f i n d adhesion v e s i c l e s i n gametes capable of parthenogenesis, or the t i m i n g of adhesion v e s i c l e f o r m a t i o n might be delayed u n t i l a f t e r r e l e a s e . B e t t e r understanding of the c y t o l o g i c a l mechanisms i n v o l v e d i n gamete f u s i o n might prevent the m i s i n t e r p r e t a t i o n of l i f e c y c l e s t h a t has plagued s t u d i e s of lower (and some higher) brown algae. E s p e c i a l l y i n the Southern Hemisphere, where the Sporochnales, D e s m a r e s t i a l e s , D i c t y o t a l e s , S p h a c e l a r i a l e s and " F u c a l e s " a l l a t t a i n t h e i r g r e a t e s t d i v e r s i t y , u l t r a s t r u c t u r a l i n v e s t i g a t i o n of swarmers can be expected t o provide important i n f o r m a t i o n f o r a b e t t e r understanding of the brown algae. 7. I m p l i c a t i o n s For P h v l e t i c s Of L a m i n a r i a l e s The unique zoospores and sperm of L a m i n a r i a l e s s e t t h i s order d i s t i n c t l y a p a r t from other brown algae, c o r r o b o r a t i n g taxonomy based on v e g e t a t i v e f e a t u r e s . Chorda i s marked as p r i m i t i v e , i n agreement with p r e v i o u s judgements ( F r i t s c h 1945). Kanda *s (1938) r e p o r t s of a short hind f l a g e i l u m and an 59 eyespot i n Chorda sperm a r e probably c o r r e c t , judging from the f a c t t h a t he noted the longer p o s t e r i o r f l a g e l l u m and l a c k of eyespots i n other s p e c i e s . i f S a c c o r h i z a does indeed have an eyespot i n i t s zoospore, i t i s l i k e l y the most p r i m i t i v e member of the L a m i n a r i a l e s , e x c l u d i n g Chorda. I t would be most i n t e r e s t i n g then t o know what the sperm of S a c c o r h i z a look l i k e . k p r i m i t i v e zoospore i s c o n s i s t e n t with Sauvageau's (1918) r e p o r t of a few s p o r o p h y t i c embryos i n h i s S a c c o r h i z a c u l t u r e s showing an a p i c a l h a i r e a r l y i n development ( c f . Chorda)., The r e t e n t i o n of h a i r - p i t s on the a d u l t t h a l l u s (Norton and Burrows 196 9) r e c a l l s C. tormentosa. and t h e l a c k of s i e v e c e l l s and occurrence of sorus on the s t i p e and b a s a l M b u l b M , as w e l l as the blade (Norton and Burrows 1969), c o n t r a s t s with a l l other L a m i n a r i a l e s except Chorda, i n which t h e r e i s no d i f f e r e n t i a t e d s t i p e and a very simple i n t e r n a l anatomy. VI. I m p l i c a t i o n s f o r Swarmer F u n c t i o n And L i f e H i s t o r i e s Some of my f i n d i n g s , combined with c o n s i d e r a t i o n of i n f o r m a t i o n a v a i l a b l e from c u r r e n t l i t e r a t u r e , suggest s e v e r a l g e n e r a l i z a t i o n s t hat could be made concerning r e p r o d u c t i o n i n brown algae. The s t r u c t u r e o f f l a g e l l a must be s u b j e c t to hydromechanical c o n s t r a i n t s . S i n c e mastigonemes are a p p a r e n t l y c r u c i a l f o r gen e r a t i o n of a p u l l i n g f o r c e i n a n t e r i o r l y d i r e c t e d , s i n u s o i d a l l y b e a t i n g f l a g e l l a , we may s a f e l y p r e d i c t that a l l brown a l g a l swarmers w i l l e v e n t u a l l y be found to possess a n t e r i o r f l a g e l l a of the ki n d I have d e s c r i b e d . I f the 60 p o s t e r i o r f l a g e l l u m i s not e s s e n t i a l f o r p r o p u l s i o n , ( c f . D i c t y o t a sperm), then i t s form may be f r e e l y a l t e r e d t o perform other f u n c t i o n s , such as chemotaxis ( c f . L a m i n a r i a l e s and Desmarestia sperm). The s i d e (and probably u n i v e r s a l ) occurrence o f the a n t e r i o r whiplash throughout the brown algae has not been g e n e r a l l y r e c o g n i z e d . , Since i t appears t o p l a y a key r o l e i n zoospore s e t t l i n g and gamete i n t e r a c t i o n , we may be very s k e p t i c a l of r e p o r t s of gametes f u s i n g while swimming (Papenfuss 1934, Caram 1964), and doubly s u s p i c i o u s of r e p o r t s of f u s i o n s of spores from u n i l o c u l a r sporangia.. The r e l e a s e of uncleaved c y t o p l a s m i c masses from s p o r a n g i a i n d i c a t e s t h a t o b s e r v a t i o n of " f u s i o n p r o d u c t s " cannot be assumed t o provide evidence o f s e x u a l i t y . , "Fused" zoospores were r e p o r t e d i n L a m i n a r i a l e s s e v e r a l times (Kain 1979) before the l i f e h i s t o r y was f i r s t d e s c r i b e d by Sauvageau (1915). There are numerous r e p o r t s of f u s i o n s of spores from u n i l o c u l a r s p o r a n g i a ( F r i t s c h 1945, Caram 1972)+ supposedly r e s u l t i n g i n the f o r m a t i o n of d i p l o i d zygotes. But more r e c e n t l y exhaustive experimental evidence has been presented (Muller 1975) which c a s t s doubt on the l i k e l i h o o d of t h i s o c c u r r i n g . Therefore there i s good reason t o b e l i e v e t h a t fundamental d i f f e r e n c e s e x i s t between spores and gametes, though both are h a p l o i d . B e t t e r understanding of sporangium dehiscence (Toth 1976b) and s e x u a l i n t e r a c t i o n s (Luning and M u l l e r 1978) i n taxa such as the L a m i n a r i a l e s , which are c o n s i d e r e d to be w e l l understood, might improve the standards used by o b s e r v e r s d e s c r i b i n g l i f e h i s t o r i e s of more enigmatic 61 groups {cf, Caram 1965), The d i s t i n c t i o n between spores and gametes may a r i s e from the fundamental p r o p e r t i e s o f the u n i l o c u l a r sporangium, with i t s unique nu c l e a r behavior ( c o n s i d e r t h a t s e x u a l i t y i s b a s i c a l l y a n u c l e a r phenomenon)., I t appears t h a t the occurrence of f r e e nuclear d i v i s i o n s (during meiosis) i s s u f f i c i e n t t o unmistakably i d e n t i f y a sporangium as homologous with such w e l l known u n i l o c u l a r s p o r a n g i a , as those i n L a m i n a r i a l e s . By t h i s r e a s o n i n g , the f o u r - n u c l e a t e "monosporangia" of T i l o p t e r i d a l e s must be u n i l o c u l a r s p o r a n g i a , while (at l e a s t some of) the u n i n u c l e a t e "moaospores" are eggs that can develop p a r t h e n o g e n e t i c a l l y , but which have n e v e r t h e l e s s shown evidence o f s e x u a l f u n c t i o n (Sundene 1966). T h i s approach a l s o enables us t o i n t e r p r e t the l i f e h i s t o r y of the " F u c a l e s " (sensu l a t o ) as homologous with other brown alga e (a vexed question indeed) (Smith 1938, K y l i n 1940, F r i t s c h 1945, C a p l i n 1968, Jensen 1974). In " F u c a l e s " , the u n i l o c u l a r r e p r o d u c t i v e s t r u c t u r e s which produce c e l l s t h a t fuse s e x u a l l y upon r e l e a s e have been c o n s i d e r e d u n i l o c u l a r gametangia. As with u n i l o c u l a r s p o r a n g i a , these gametangia are the s i t e of m e i o s i s and thus occur on d i p l o i d p l a n t s . T r a d i t i o n a l l y a l l brown algae t h a t possess t h i s " a n i m a l s l i k e " l i f e h i s t o r y have been placed i n the F u c a l e s (they share a second c h a r a c t e r i s t i c f e a t u r e i n b e a r i n g these gametangia i n c o n c e p t a c l e s ) . , T h i s i s t r u e among a l l the c o n v e n t i o n a l l y recognized F u c a l e s , but t h e r e i s n e v e r t h e l e s s s i g n i f i c a n t v a r i a t i o n i n the c o n s t r u c t i o n of the gametangia i n t h i s group. In some, the oogonia (e.g., i n Fucns) produce 62 s e v e r a l eggs, or the a n t h e r i d i a (e.g., i n C y s t o s e i r a ) produce many sperm i n a s e r i e s o f events t h a t shows no e s s e n t i a l d i f f e r e n c e from what i s found i n a u n i l o c u l a r sporangium; m e i o s i s occurs, m i t o s i s f o l l o w s t o form 8-64 n u c l e i , and the cytoplasm c l e a v e s , the products being r e l e a s e d without f u r t h e r c o m p l i c a t i o n . In other cases, a f t e r meiosis i n the oogonium, wa l l s may be formed around the f o u r d i v i s i o n products e i t h e r b e f o r e f B i f t t r c a r i o p s i s (Jensen 1974) J or a f t e r f Xiphophora (Naylor 1954) ] subsequent m i t o s i s . S i m i l a r l y , i n a n t h e r i d i a a wal l ( c a l l e d an "endochiton") may be formed i n s i d e the o r i g i n a l g a a e t a n g i a l w a l l f Himanthalia (Manton 1964), Fucus ( B e r k a l o f f and Rousseau 1979), Xiphophora. Hormosira. D u r v i l l a e a (Moestrup 1977) 3; the w a l l develops some time between meiosis and cleavage of the cytoplasm. Although complete knowledge o f the l i f e h i s t o r y i s not y e t a v a i l a b l e , analogous i n t e r n a l w a l l s i n the u n i l o c u l a r s p o r a n g i a of A s c o s e i r a ( S k o t t s b e r g 1921, Moe and Henry unpub. obs.) and Syringoderma (Walker and Henry 1978, and unpub. obs.) l i k e l y s i g n i f y t h a t these s p e c i e s produce gametes from t h e i r u n i l o c u l a r r e p r o d u c t i v e s t r u c t u r e s , i n exact analogy with the Fucales., T h i s widespread evidence.of w a l l s or w a l l remnants w i t h i n " u n i l o c u l a r gametangia" suggests t h a t i n these i n s t a n c e s , i f not i n a l l " F u c a l e s " , the "gametes" are e x a c t l y t h a t , not spores a c t i n g as gametes as proposed by Smith (1938) and C a p l i n (1968). The e v i d e n t homologies with p l u r i l o c u l a r gametangia a l s o argue a g a i n s t K y l i n * s (1940) c o n t e n t i o n that the f u c a l e a n l i f e h i s t o r y has no c o u n t e r p a r t i n t h e other brown algae, the primary j u s t i f i c a t i o n f o r h i s s e g r e g a t i o n of the 63 Fucales i n the Class Cyclosporeae. . The variation i n form and appearance of these i n t e r n a l walls, as well as p a r a l l e l developments i n the obviously unrelated genera ftscoseira and Syringpderma. suggest that the Fucales as conceived by Kylin are polyphyletic, t h i s "animal-like" l i f e history having evolved more than once i n the brown algae. ; Further u l t r a s t r u c t u r a l studies are required to confirm these homologies., The more obvious correspondence between laminarialean gametangia and p l u r i l o c u l a r gametangia of "lower" brown algae might be more widely recognized i f the terms "uniloc" and " p l u r i l o c " were redefined or abandoned i n favor of terms which e x p l i c i t l y denote the differences i n cytology of development. The terms "mitosporangium", "meiosporangium", "mitogametangium", and "meiogametangium {von Denfer 1972) are admirably expressive, but the i r adoption f a i l s to solve c e r t a i n terminological problems. Unfortunately, the term "meiosporangium" has been used to ref e r to c e r t a i n p l u r i l o c u l a r structures {Fritsch 1945). I f t h i s archaic usage be overlooked, there s t i l l might be argument that "meiosporangium" i s not a v a l i d term i n taxa shown to lack meiosis i n the l i f e history, even though they possess structures obviously homologous with normal, meiotic " u n i l o c s " . On the other hand, although i t may be known that a taxon has a sexual l i f e h i s t ory, i n a given specimen the ploidy l e v e l may not be known, so the ro l e of the products of a m i t o t i c a l l y produced reproductive structure i s unknown ( i s i t a mitosporangium or a mitogametangium?). Perhaps a term such as "zoidangium" would be u s e f u l , because i t does not denote the r o l e of the " z o i d " . But "zoidangium" cannot be used t o r e f e r to r e p r o d u c t i v e s t r u c t u r e s with non-motile products (e.g., "monosporangia" of T i l o p t e r i d a l e s ) . Since we have no s i n g l e word f o r " r e p r o d u c t i v e s t r u c t u r e " ( i . e . , "sporangium-or-gametangium") , we cannot e a s i l y c o i n a non-committal term t h a t i s both e t y m o l o g i c a l l y e l e g a n t and e x p r e s s i v e . Perhaps "coenangium" or "meiotangium" might be used f o r meiosporangium/meiogametangium, and "mitotangium" f o r mitosporangium/mitogametangium. S i n c e d i f f e r e n c e s i n c y t o l o g y of development are i n t i m a t e l y r e l a t e d t o the r o l e s of the products of r e p r o d u c t i v e s t r u c t u r e s i n l i f e h i s t o r i e s , a more i l l u m i n a t i n g terminology would a s s i s t those attempting to analyse enigmatic l i f e h i s t o r i e s , such as T i l o p t e r i d a l e s , F u c a l e s , Syringoderma and A s c o s e i r a . We must not be s u r p r i s e d t o encounter taxa t h a t defy our e f f o r t s at t y p i f i c a t i o n . When the fundamental p r o p e r t i e s of " t y p i c a l " r e p r o d u c t i o n are understood, a grasp of the o r i g i n s and f u n c t i o n i n g of e x c e p t i o n s to the r u l e w i l l be w i t h i n reach. 65 PLATE 1 F i g . , 1. H a c r o c y s t i s p y r i f e r a . Blade c r o s s - s e c t i o n i n sorus r e g i o n . The blade s u r f a c e c u r l s inward where i t has been cut (arrow), c a u s i n g the " c u t i c l e " (C) to separate from the paraphyses. 1 um t h i c k s e c t i o n , s t a i n e d osmium t e t r o x i d e , methylene blue-azure I I . F i g . 2. L a m i n a r i a s a c c h a r i n a . C r o s s - s e c t i o n through blade showing mucilage ducts (arrow) and s o r u s . 1 um t h i c k , osmium, methylene blue-azure I I . F i g . 3. L. s a c c h a r i n a . S e c t i o n through s o r u s . Bote a b u t t i n g t i p s of paraphyses (Pa) forming p r o t e c t i v e l a y e r over s p o r a n g i a (Sp) , o s m i o p h i l i c g l o b u l e s (0) i n p a r a p h y s i s t i p s , " c u t i c l e " l i f t e d up. F i g . 4. Dictyoneurum c a l i f o r n i c u m . L o n g i t u d i n a l s e c t i o n of paraphyses and sporangia showing s w o l l e n p a r a p h y s i s t i p s , o s m i o p h i l i c m a t e r i a l i n paraphyses (arrow), g r a n u l a r m a t e r i a l (gr) i n apex of s p o r a n g i a . F i g . 5. A l a r i a t e n u i f o l i a . Postcleavage zoosporangium i n l o n g i s e c t i o n showing p a r t l y l e a c h e d "type b" v e s i c l e s (B) and g r a n u l a r m a t e r i a l at sporangium apex. , F i g . 6., L e s s o n o i p s i s l i t t o r a l i s . , C r o s s - s e c t i o n o f blade with s p o r a n g i a l a r g e l y r e l e a s e d from s o r u s . Note swollen t i p s of paraphyses (arrow)., 5 um-thick s e c t i o n i s t a i n e d with t o l u i d i n e blue. 67 PLATS 2 F i g . 7. Dictyonaurum. , Cross-section of paraphyses and sporangia. V a i l s of paraphyses are thicker than those of sporangia; top sporangium i s cleaved, the bottom i s ; not. Note osm i o i p h i l i c globules i n paraphyses. F i g . 8. Piety on eur.uffl. oblique section through sporangium showing several emergent f l a g e l l a (F), each associated at basal body with a p l a s t i d . F i g . , 9. Pietyoneurum. Longitudinal section through sporangium showing close-packing of spores, conspicuous "type b" v e s i c l e s . F i g . 10. Cymathere t r i p l i c a t a . Cross-section of sporangium showing packing of spores, prominent "type b" v e s i c l e s . F i g . 11. Pterygophora c a l i f o r n i c a . Longisection of sporangium showing close-packing of spores. 69 EMM 3 F i g . 12. EisenJla arborea. L o n q i s e c t i o n of sporangium; one spore shows c o n s i d e r a b l e e l o n g a t i o n (arrow). P i g . 13. Higher m a g n i f i c a t i o n view of spore i n d i c a t e d i n F i g . 12. Bote c y t o s k e l e t a l microtubule (arrow) . P i g . 14. L e s s o n i p p s i s . Spore wi t h i n sporangium showing f o u r p a r a l l e l c y t o s k e l e t a l microtubules (arrow) O=ostaiophilic v e s i c l e , AV=adhesion v e s i c l e . F i g . , 15. Pterygophora. Whole-mount o f spores showing w e l l -preserved "nose" (arrow)., AF=anterior f l a g e l l u m . PF=posterior flagellum..-•. F i g . , 16. A l a r i a t e n u i f o l i a . Whole-mount, shadowcastl zoospores and u n d i v i d e d s p o r a n g i a l mass (U). F i g . 17. Cymatheye. Spore w i t h i n sporangium, showing f l a g e l l u m i n c r o s s - s e c t i o n a t emergence from s p o r e , with a s s o c i a t e d band of 9 microtubules (arrow).. 71 PLATE 4 F i g . 18. .,- a a c r q c y s t i s i n t e g r i f o l i a . , S e c t i o n through f l a g e i l u m of p r e - r e l e a s e spore shoving dense m a t e r i a l (arrow) arrayed p a r a l l e l t o b a s a l body. Note a s s o c i a t i o n v i t h p l a s t i d ( P ). P i g . 19., P i e t y one urum. (Same sporangium as F i g . 9) S e c t i o n through a n t e r i o r f l a g e i l u m shoving u n i l a t e r a l attachment of mastigonemes (9a), dense m a t e r i a l (arrow) p a r a l l e l to basal body, a s s o c i a t i o n of b a s a l body v i t h p l a s t i d . F i g . 20. ., Hedqphyllum s e s s i l e . S e c t i o n through r e l e a s e d zoospore i n plane of f l a g e l l a r bases. Note m i c r o t u b u l a r band (arrow) near a n t e r i o r basal body. F i g . 21. A. t e n u i f o l i a . Beleased zoospore s e c t i o n e d through attachment of one f l a g e i l u m , shoving p a r a l l e l , p o s s i b l y t u b u l a r m a t e r i a l (arrows) next to b a s a l body., F i g . 22. A. t ^ e n u i f o l i a . Beleased zoospore shoving p o s t e r i o r f l a g e i l u m attachment, a n t e r i o r f l a g e l l u m ' s b a s a l body p e r p e n d i c u l a r ( s t r a i g h t arrow), curved band of microtubules (curved arrow). Note d i v i d i n g p l a s t i d and l a c k o f eyespot, a l s o leached "type b" v e s i c l e . ( 73 PLATE 5 P i g s . 23A-23E. A. t e n u i f olj.a. Beleased zoospore. . S e r i a l s e c t i o n s through {probably p o s t e r i o r ) f l a g e i l u m , showing p a r a l l e l , t u b u l a r band of microtubules (arrow) , and band (curved arrow) a s s o c i a t e d with other, p e r p e n d i c u l a r b a s a l body (b'b)-.,.,-P i g . 24. A. t e n u i f o i l a . S e c t i o n through r e l e a s e d zoospore showing f l a g e l l a r attachment, a s s o c i a t e d m i c r o t u b u l a r bands (arrows) and other c y t o s k e l e t a l microtubules (mt). PLATE 6 25A-25G., j l . t e n u i f o l i a . S e r i a l s e c t i o n s through r e l e a s e d zoospore showing c y t o s k e l e t a l microtubules (arrows) a t "nose" of spore. F i g . 25B; d e t a i l of m i c r o t u b u les (arrow) i n F i g . 25A. , 26. A. t e n u i f o l i a . Beleased zoospore showing convergence of c y t o s k e l e t a l m i c r o t u b u l e s i n "nose? (arrow).. 77 'LATE 7 P i g . 27. &. t e n u i f o l i a . , "Exploded" zoospore, showing m i c r o t u b u l a r s k e l e t o n s of a n t e r i o r f l a g e i l u m ^ p o s t e r i o r f l a g e i l u m , and c y t o s k e l e t o n (Cy). Note p e r s i s t e n c e o f t i p of p o s t e r i o r f l a g e i l u m , c o n n e c t i o n between b a s a l bodies {large arrow), connecting m a t e r i a l between adj a c e n t d o u b l e t s {thin arrow), Two c e n t r a l s i n g l e t m i c r o t u b u l e s have been t o r n away from r e s t of axoneme {curved arrow) , but 2 microtubules are d e t e c t a b l e d i s t a l l y ( s h o r t arrow). 79 ELATE 8 P i g . 28. A. t e j o u i f o l i a . "Exploded" zoospore showing p a r t i a l l y d i s r u p t e d a n t e r i o r c y t o s k e l e t a l hand of m icrotubules (Cy) , and p o s t e r i o r band. Note p r e s e r v a t i o n of t i p of p o s t e r i o r f l a g e l l u m . s P i g . 29. A. t e n u i f o l i a . "Exploded" spore showing p a r t i a l l y d i s r u p t e d a n t e r i o r c y t o s k e l e t a l band. F i g . 30. A. t e n u i f o l i a . "Exploded" spore showing i n t a c t a n t e r i o r c y t o s k e l e t a l band and c o nnecting m i c r o t u b u les (arrow) between b a s a l bodies. 31 PLATE 2 P i g , 31. A. t e n u i f o l i a . Beleased zoospore showing l a t e r a l i n s e r t i o n of the two f l a g e l l a . , Bote l a c k of eyespot i n p l a s t i d near p o s t e r i o r f l a g e i l u m . P i g . 32.. D e t a i l of basal bodies ( F i g . 31) and p o s t e r i o r band of c y t o s k e l e t a l microtubules (arrow) • F i g . 33. Cyaathere.„ P r e - r e l e a s e zoospore showing p a r a l l e l b a s a l bodies. P i g . , 34., Dictyoneurum. . P r e - r e l e a s e zoospore with p a r a l l e l b a s a l bodies. Note a s s o c i a t i o n with p l a s t i d , u n i l a t e r a l attachment of mastigonemes (Ha). F i g s . 35-36., Dictyoneurum. „ P r e - r e l e a s e f l a g e l l a showing u n i l a t e r a l mastigoneme attachment. 33 PLATE JO P i g . , 37. , A l a r i a nana. Sfhole mount, unshadowed, unstained zoospore showing l o n g a n t e r i o r whiplash (W), b i l a t e r a l arrangement of mastigonemes. . P i g . 38. A. t e n u i f o l i a . D e t a i l of a n t e r i o r whiplash ( l a r g e l y removed) Membrane has c o l l a p s e d around axoneme showing presence of two s i n g l e t m i c r o t u b u l e s . . P i g . , 39. A. t e n u i f o l i a . "Exploded" zoospore showing attachment of mastigonemes i n two rows on opposite s i d e s of f l a g e l l u m (arrows)., 85 PLATE 11 F i g . 40. „ A. nana., H e g a t i v e - s t a i n e d mastigonemes showing dense core (arrow) and t e r m i n a l f i l a m e n t s (TP). P i g . 41. A. t e n u i f o l i a . Shadowcast, detached mastigonemes, some with s p h e r i c a l b a s a l p i e c e (arrow)., F i g . 42. / Shadowcast, p a r t l y d i s r u p t e d f l a g e i l u m showing p e r s i s t e n c e of d i s t a l whiplash base (arrow), b i l a t e r a l mastigoneme arrangement. F i g . 43., A. t e n u i f o l i a . "Exploded" spore showing c o n n e c t i o n s between a n t e r i o r and p o s t e r i o r b a s a l bodies (arrow), b i l a t e r a l arrangement of mastigonemes.. 87 PLATE 12 F i g . 44. A. nana. N e g a t i v e - s t a i n e d mastigonemes snowing d i s t a l t e r m i n a l f i l a m e n t s . F i g . 45. A. t e n u i f o l i a . / Shadowcast spore showing p a r t l y d i s r u p t e d a n t e r i o r f l a g e i l u m with b i l a t e r a l mastigoneme attachment, i n t a c t p o s t e r i o r f l a g e i l u m . F i g . 46. A. t e n u i f o l i a . D e t a i l o f p o s t e r i o r f l a g e i l u m t i p with t e r m i n a l s w e l l i n g . F i g . 47. A. t e n u i f o l i a . 2 Shadowcast zoospores, with prominent s w e l l i n g s of the t i p s of p o s t e r i o r f l a g e l l a . 89 PLATE 13 P i g . 48. Pteryqophora. Zoosporangium showing v a r i o u s s e c t i o n s through p l a s t i d s as they e n f o l d nucleus. Note l a r g e "type b" v e s i c l e s . Fig... 49. Cost a r i a c o s t a t a . Zoospore w i t h i n sporangium. P l a s t i d showing p l a s t o g l o b u l i (pg) and membrane-bound i n c l u s i o n s ( s t r a i g h t arrow) and r i n g ge nop ho re (curved arrow) . F i g s . , 50-51., P o s t e l s i a palmeformis. Zoospores w i t h i n sporangium, showing p l a s t o g l o b u l i and membrane-bound i n c l u s i o n (arrow) • F i g . , 52. Cymathere., Beleased zoospore showing d i v i d i n g p l a s t i d , adhesion v e s i c l e s (arrow) . F i g . „ 53. „ Cymathere. S e t t l e d zoospore showing d i v i d e d p l a s t i d , r e l e a s e d adhesion v e s i c l e s (&V) , c e l l w a l l (arrowy. 91 PLATE 14 P i g . 54. M a c r o c y s t i s . Mitochondria (M) i n pre-cleavage sporangium showing l o b i n g . P i g s . 55-56. Pteryqophora. M i t o c h o n d r i a showing i n t r a c r i s t a l t u b u l e s (T, arrows). P i g . 57. c o s t a r i a . , Zoospore w i t h i n sporangium, n u c l e o l i e v i d e n t (arrows) . P i g . 58., Cymathere. Pre-cleavage sporangium n u c l e i with heterochro matin. F i g . 59. Dictyoneurum Pre-cleavage sporangium n u c l e i with heterochromatin. 93 PLATE 15 F i g . 60. P o s t e l s i a . Pre-release zoospore, nucleus with pores (arrow) associated p l a s t i d and golgi (G). Figs. 61-63. Cymathere. Pre-release zoospores, nucleus with enfolding p l a s t i d , nuclear pores (short arrows),.; Note extensions of nuclear envelope around p l a s t i d (curved arrows) and into cytoplasm (long arrows).. 95 PLATE J 6 F i g . , 6 4 . ; Hedophyllum.. P r e - r e l e a s e zoospore showing nucleus enfol d e d by p l a s t i d , a s s o c i a t e d g o l g i and endoplasmic r e t i c u l u m (er) s t a c k s . F i g . . 6 5 . Cymathere. Beleased zoospore., Note nucleus with c y t o p l a s m i c e x t e n s i o n (arrow), g o l g i opposite endoplasmic r e t i c u l u m s t a c k s (curved arrow), appressed f l a g e i l u m ( F). F i g . , 6 6 . Pteryqophora. P r e - r e l e a s e zoospore, nucleus with g o l g i i n i n v a g i n a t i o n . . F i g . , 67.,, L e s s o n i o p s i s . Pre-cleavage sporangium with endoplasmic r e t i c u l u m s t a c k s surrounding n u c l e i . F i g . , 6 8 . Hedophyllum. Pre-cieavage sporangium nucleus showing endoplasmic r e t i c u l u m s t a c k s with ribosome a r r a y s . F i g . , 6 9 . , Hedophyllum. D e t a i l of spore from F i g . 6 4 . Endoplasmic r e t i c u l u m s t a c k s with ribosome a r r a y s , adjacent g o l g i . 97 PLATE J7 F i g . , 70. Pteryqophora. Beleased zoospore showing osmiophilic ves i c l e s with d i s t i n c t bounding membrane (arrows). Fig . 7 V. &. t e n u i f o l i a . Beleased zoospore with aggregation of osmiophilic globules s u p e r f i c i a l l y resembling an eyespot (arrow).. F i g . , 72. P o s t e l s i a . Pre-release zoospore nucleus with rough endoplasmic reticulum stacks forming contents of "type b" v e s i c l e . F i g s . , 73-74. Cymathere. Pre-release zoospores treated with thiocarbohydrazide-osmium. Note retention of "type b" ves i c l e contents. 99 PLATE .18 Pigs. 75-76. Dictyonearma. Pre-cleavage sporangium with mastigonemes i n close association with precursors i n cytoplasm (curved arrow).,. Electron-dense core of mastigonemes i s evident (thin arrow). „ Pigs., 77-78. Dictyoneurum. Pre-cleavage sporangia with extracytoplasmic membrane stacks (arrow).. 100 101 PLATE 19 F i g s . 79-80. Pietyoneururn. Pre-cleavage sporangia with e x t r a c y t o p l a s m i c s t a c k s (E) e n c l o s i n g f l a g e l l a . , , F i g . 81. Pictyoneurum.. Pre-cleavage sporangium with e x t r a c y t o p l a s m i c v e s i c l e s (V) among f l a g e l l a . Note c r o s s - s e c t i o n of f l a g e i l u m with only 2 s i n g l e t m i crotubules (arrow)., F i g . 82. Hedophyllum. P r e - r e l e a s e zoospore with adhesion v e s i c l e s {AV). , F i g . , 83. H a c r o c y s t i s . P r e - r e l e a s e spore with adhesion v e s i c l e s (arrow). F i g . 84. La m i n a r i a q r o e n l a n d i c a . P r e - r e l e a s e spore with adhesion v e s i c l e s (arrow). F i g . 85. L. s a c c h a r i n a . P r e - r e l e a s e spore with adhesion v e s i c l e s . . Note apparent membrane i n negative image (arrow) (chromate f i x ) . 103 PLATE JO F i g . f 86. Agar um cribosum. P r e - r e l e a s e zoospore with adhesion v e s i c l e s (arrow) • P i g . 87. P o s t e l s i a . P r e - r e l e a s e spore with adhesion v e s i c l e s (arrow). P i g . , 88. Cymathere. P r e - r e l e a s e zoospore with adhesion v e s i c l e s . One v e s i c l e shows e l e c t r o n - t r a n s p a r e n t ; c e n t r a l core (arrow). F i g . , 89. Cymathere. Adhesion v e s i c l e i n l o n g i s e c t i o n . F i g . , 90. A. nana. Zoospore with mastigoneme clump a t t i p of a n t e r i o r whiplash., F i g . , 91., D e t a i l o f ( F i g . 90) mastigoneme clump. 105 PLATE 21 F i g . 92., Hedophyllum. Zoospore with mastigonemes adhering to a n t e r i o r whiplash (arrow). F i g . , 93., Agarum. , Zoospore with a n t e r i o r whiplash. F i g . 94. A. t e n u i f o l i a . Zoospore with a n t e r i o r whiplash. F i g . 95. Cymathere. Zoospores, a n t e r i o r whiplash not preserved. F i g . 96. ? E i s e n i a . Zoospore with a n t e r i o r whiplash.,. F i g . 97. C o s t a r i a . Zoospore with a n t e r i o r whiplash.. 107 PLATE 22 F i g . 98. L e s s o n i o p s i s . Zoospore, a n t e r i o r whiplash not pr e s e r v e d . F i g . 99. M a c r o c y s t i s . Zoospore, o n l y b a s a l remnant of a n t e r i o r whiplash preserved. whiplash. F i g . 101. Pleurophycus g a r d n e r i . Zoospore with a n t e r i o r whiplash. F i g . 10 2., P o s t e l s i a . Zoospore, a n t e r i o r whiplash and mastigonemes not preserved. F i g . 103. Pteryqpphora. Zoospores, o n l y b a s a l remnant of a n t e r i o r f l a g e l l u m preserved; f u l l extent of p o s t e r i o r f l a g e l l u m v i s i b l e . y Zoospore with a n t e r i o r 109 PLATE 23 F i g . , 104. Cymathere. S e c t i o n through p r e - r e l e a s e f l a g e l l a , one through t i p with only 2 s i n g l e t m i c r o t u b u l e s (arrow).; F i g . 105. Cymathere. S e c t i o n through p r e - r e l e a s e f l a g e l l a , one showing r e g i o n near t i p ; 9*2 p a t t e r n l o s t , t hree d o u b l e t s and two s i n g l e t s (arrow) e v i d e n t . F i g . 1 0 6 . D i c t y o n e u r u m . P r e - r e l e a s e f l a g e i l u m i n l o n g i s e c t i o n , with whiplash. , F i g s . 107-109. A. t e n u i f o l i a . S e t t l i n g zoospores showing r e s o r p t i o n of axonemes (arrows) F i g . , 110. 4. t e n u i f o l i a . Grazing s e c t i o n through r e l e a s e d zoospore near b a s a l bodies; one axoneme i s withdrawn (note b a s a l p l a t e s , [ a r r o w s ] ) , one i s s t i l l emergent. 110 111 PLATE 24 P i g . / 111., A. t e n u i f o l i a . Beleased zoospore showing p e r i p h e r a l , withdrawn axoneme (arrow). P i g . 112. A. t e n u i f o l i a . S e t t l e d zoospore e x t r u d i n g adhesion v e s i c l e s (arrow). Note withdrawn axonemes (curved arrow) . ; F i g . , 113.,; Cymathere. S e t t l e d zoospore showing r e l e a s e d , opened adhesion v e s i c l e s (arrow)., F i g . 114., Cymathere. S e t t l e d zoospore with extruded adhesion v e s i c l e s , withdrawn axonemes (arrow). F i g . 115. M a c r o c y s t i s . .. S e t t l e d zoospore showing withdrawn bas a l body (arrow), extruded adhesion v e s i c l e s . . F i g . 116. M a c r o c y s t i s . , S e t t l e d zoospore showing extruded, opened adhesion v e s i c l e s . . PLATE 25 117-120. A l a r i a t e n u i f o l i a . S e t t l e d zoospores showing extruded adhesion v e s i c l e s and wall development (arrows), F i g . 120 shows formation o f germination tube. 1 15 PLATE 26 F i g . , 121., A. t e n u i f o l i a . Shadowcast zoospores, one s e t t l e d (Se} , one s t i l l a c t i v e (Ac). Note s c a t t e r e d mastigonemes. F i g s . 122-123. , A. t e n u i f o l i a . S e t t l e d zoospore forming "double-blob 1 1 f i g u r e s . F i g . , 124., A. t e n u i f o l i a . S e t t l e d zoospores with s c a t t e r e d mastigonemes. F i g . , 125. , A. t e n u i f o l i a . / S e t t l e d zoospore showing t r a i l of mastigonemes, p o s s i b l y i n d i c a t i n g path of f l a g e l l u m withdrawal. 117 1MI1 21 F i g . 126A. Diagrammatic r e p r e s e n t a t i o n of a g e n e r a l i z e d L a m i n a r i a l e a n zoospore, shoving probable arrangement of c y t o s k e l e t a l microtubules and major o r g a n e l l e s . AI?= a n t e r i o r f l a g e l l u m , B= "type b" v e s i c l e , H= mitochondrion, Ma= mastigonemes, N= nucleus, P= p l a s t i d . F i g . 126B. Diagrammatic r e p r e s e n t a t i o n of a " d o r s a l view" of a zoospore, d e p i c t i n g c o n f i g u r a t i o n c f o r g a n e l l e s and v e s i c l e s . Av= adhesion v e s i c l e , e c - endoplasmic r e t i c u l u m , G= g o l g i apparatus, 0= o s m i o p h i l i c v e s i c l e . 1.1.9 PLATE 28 F i g . 127. M a c r o c y s t i s . Hale gametophyte. , 1 um-thick s e c t i o n s t a i n e d with methylene blue-azure I I . A n t h e r i d i a are commonest at p e r i p h e r y of sphere of f i l a m e n t s . F i g . , 128., A l a r i a t a e n i a t a . , Gametophyte f i l a m e n t showing l a t e r a l p o s i t i o n of a n t h e r i d i a (A). F i g . , 129.,, H a c r o c y s t i s . Gametophyte with i n t e r c a l a r y antheridium. Note g r a n u l a r m a t e r i a l (gr) a t apex of t e r m i n a l antheridium. F i g . 130. / A. nana. , Gametophyte.,., Note s i z e d i f f e r e n c e between p l a s t i d s of developing sperm (arrows) and v e g e t a t i v e c e l l s . , F i g s . , 131-132. H a c r o c y s t i s . , A n t h e r i d i a l c l u s t e r s showing p l u r i l o c u l a r nature of c l u s t e r s , c o n f l u e n t outer w a l l s (arrows). 121 PLATE 29 B i g . , 133. H a c r o c y s t i s . A n t h e r i d i a . -Mote confluence of outer w a l l s cf c l u s t e r (arrow). P i g . 134., H a c r o c y s t i s . 1 um-thick s e c t i o n showing d i f f e r e n t i a l s t a i n i n g of m a t e r i a l a t apex of antheridium (arrow).. Methylene blue-azure I I . F i g . , 135. A. .nana. Same antheridium as F i g . , 130.,, Bote g r a n u l a r m a t e r i a l at apex of antheridium. F i g . 136. A . , t a e n i a t a . Bote g r a n u l a r m a t e r i a l at apex of antheridium. 122 123 P1ATE 30 F i g . / 137., A. t a e n i a t a . L o n g i s e c t i o n through developing sperm showing f o l d e n c l o s i n g f l a g e l l a , a s s o c i a t i o n of p a r a l l e l b a s a l bodies with c y t o s k e l e t a l m i c r o t u b u l e s i n f o l d (arrow). Note "type b" v e s i c l e s (B). F i g . 138. A. t e n u i f o l i a . Developing sperm showing f o l d e n c l o s i n g f l a g e l l u m , e x t r a c y toplasmic membranes (£). F i g . , 139./ H a c r o c y s t i s . Developing sperm showing p a r a l l e l b a s a l bodies enclosed by f o l d o f cytoplasm.. F i g . 140. Pterygophora. E a r l y antheridium with e v i d e n t f u t u r e b a s a l bodies (arrows).. Note they are not p e r p e n d i u l a r t o each other. F i g . 141.,/ D e t a i l of b a s a l bodies (Fig 140). 125 P1AT E 31 F i g s . 142A-D. A. t a e n i a t a . S e r i a l s e c t i o n s through d e v e l p i n g sperm showing i n s e r t i o n o f b a s a l bodies i n f o l d of cytoplasm, band of microtubules i n f o l d and a s s o c i a t e d with b a s a l bodies (between arrows). Note "type ,b*! v e s i c l e s . , 126 127 PLATE 32 P i g . , 143., A l a r i a taenj.ata. E a r l y antheridium with a c t i v e g o l g i (G) , membraneous v e s i c l e s a s s o c i a t e d with f l a g e l l a (curved arrow). Note band of microtubules ( s t r a i g h t arrow). F i g . , 144. H a c r o c y s t i s . , Developing sperm showing a s s o c i a t i o n of m i c r o t u b u l a r bands (short arrow) and s i m i l a r e l e c t r o n " dense m a t e r i a l with b a s a l bodies (long arrow).,. F i g . 145. A. t a e n i a t a . Developing sperm showing c y t o s k e l e t a l m i c r o t u b u l es i n f o l d e n c l o s i n g f l a g e l l a (arrows)., Note v e s i c l e s (V) forming e x t r a c y t o p l a s m i c matrix. F i g . , 146. A. t a e n i a t a . Developing sperm. Note m i c r o t u b u l a r band p a r a l l e l t o f l a g e l l a (arrow). 128 129 PLATE 33 P i g . 147. M a c r o c y s t i s . Developing sperm., Note e l e c t r o n -dense (microtubular?) m a t e r i a l surrounding b a s a l body of f l a g e i l u m (arrow) . P i g s . . 148-149. Pteryqophora. Beleased sperm, whole mount, shadowcast., Bote 180 degree o r i e n t a t i o n o f a n t e r i o r and p o s t e r i o r f l a g e l l a , and l o o p of c y t o s k e l e t a l microtubules i n "nose" (arrow). , F i g . 150. A. t a e n i a t a . whole mount of sperm, unshadowed, unstained, showing t a p e r i n g p o s t e r i o r f l a g e i l u m , whiplash (B) on a n t e r i o r f l a g e i l u m . . F i g . , 151./ C o s t a r i a . Whole mount sperm, unstained, unshadowed, showing taper of axoneme, of p o s t e r i o r f l a g e i l u m , b a s a l remnant of a n t e r i o r whiplash. F i g . , 152. Dictyoneurum. Whole mount sperm, u n s t a i n e d , unshadowed,with w e l l - p r e s e r v e d a n t e r i o r whiplash. Many b a c t e r i a a l s o present. 131 PLATE 34 F i g . 153. D e t a i l of whiplash ( F i g . 152). F i g . , 154., L e s s o n i o p s i s . N e g a t i v e - s t a i n e d sperm showing a n t e r i o r whiplash, mastigonemes. F i g . , 155. Hedophyllum..„•• Unstained, unshadowed sperm with "exploded" a n t e r i o r f l a g e i l u m . Note a s s o c i a t i o n of mastigonemes with axonemal m i c r o t u b u l e s . F i g . , 156. Pleurophvcus. . Shadowcast sperm, showing remnant of a n t e r i o r whiplash. 1 3 3 PLATE 35 P i g s . 157-158. H a c r o c y s t i s . Unshadowed, unstained sperm c l e a r l y showing t a p e r i n g of axoneme of p o s t e r i o r f l a g e l l u m , mastigonemes, and b a s a l remnant of a n t e r i o r whiplash. P i g s . 159-160. H a c r o c y s t i s . Sperm on EM g r i d f i l m , photographed with 40x phase-contrast o b j e c t i v e . Bote taper o f p o s t e r i o r f l a g e l l u m , a n t e r i o r whiplash (arrow). , P i g . , 161. M a c r o c y s t i s . C r o s s - s e c t i o n o f t a p e r i n g p o s t e r i o r f l a g e l l u m w i t h i n antheridium., Bote d o u b l e t s have become s i n g l e t s . F i g . , 162. Pteryqophora. Sections of t a p e r i n g p o s t e r i o r f l a g e l l u m . P i g . , 163., A. t a e n i a t a . S e c t i o n s of t a p e r i n g p o s t e r i o r f l a g e l l u m . 135 PLATE 36 P i g . 164., Pterygophora. Shadowcast sperm with microtubules t o r n f r e e of ax one me (arrow) . P i g . 165. D e t a i l of f r e e microtubules ( F i g . 164).,, They appear to be d o u b l e t s c r c l o s e l y adherent s i n g l e t s . F i g . , 166., M a c r o c y s t i s . Developing sperm showing a t l e a s t three p l a s t i d s i n s e c t i o n . F i g . 167., A. t a e n i a t a . Developing sperm showing two elongate p l a s t i d s . F i g . 168. A. nana. Developing sperm with a t l e a s t 2 p l a s t i d s ; note few t h y l a k o i d s . F i g . , 169. A. t a e n i a t a . Mitochondria of developing sperm. Note i n t r a c r i s t a l t u b u l e s (arrow). 137 PLATE 37 F i g , , 170. A. nana. E a r l y antheridium shotting well-developed g o l g i . , P i g . 171., M a c r o c y s t i s . Developing sperm showing endoplasmic r e t i c u l u m a r r a y s a s s o c i a t e d with f l a g e l l a r c a v i t y (between arrows) Note "type b M v e s i c l e s . . P i g . 172., A. nana. Developing sperm showing endoplasmic r e t i c u l u m a r r a y s p a r a l l e l t o n u c l e a r membrane (arrow). F i g . , 173. A. t a e n i a t a . Developing sperm. Note e x t r a c y t o p l a s m i c membranes and v e s i c l e s producing e x t r a c y t o p l a s m i c matrix. 139 PLATE 38 P i g , 174., A. t a e n i a t a . Developing sperm with a c t i v e g o l g i producing v e s i c l e s emptying i n t o c a v i t y . F i g s . 175-177., A. t a e n i a t a . Developing sperm showing e x t r a c y t o p l a s f f l i c membranes, many a s s o c i a t e d with ... f l a g e l l a . Note "type b" v e s i c l e s . 1.4.1. PLATE 39 P i g s . 178-179.„ A. t a e n i a t a . Developing sperm with e x t r a c y t o p l a s m i c membranes. P i g s . 180-182., Pleurophvcus. L i v e sperm photographed with p h a s e - c o n t r a s t . Note s i n u s o i d a l form o f a n t e r i o r f l a g e l l u m and bend i n p o s t e r i o r f l a g e l l u m . 143 PLATE 40 F i g . 183. , Diagrammatic r e p r e s e n t a t i o n of g e n e r a l i z e d L a m i n a r i a l e a n sperm, showing c o n f i g u r a t i o n of major o r g a n e l l e s and t a p e r i n g of p o s t e r i o r f l a g e i l u m . AF= a n t e r i o r f l a g e i l u m , B= "type b" v e s i c l e , H= mitochondrion, Ha= mastigonemes, H= nucleus, P= p l a s t i d , PF= p o s t e r i o r f l a g e i l u m . PLATE jH 184. Diagrammatic r e p r e s e n t a t i o n of the better-known brown-algal sperm types., The advanced forms may be d e r i v e d by r a d i a t i o n from a p r i m i t i v e form (such as t h a t °f Ectocarpus) which resembles a zoospore. The most s i g n i f i c a n t m o d i f i c a t i o n s are the l o s s of the eyespot, e l o n g a t i o n of the body of the sperm, p r o l o n g a t i o n of the a n t e r i o r o f the sperm, and e l o n g a t i o n o f the p o s t e r i o r f l a g e i l u m or presence of s p i n e s on the a n t e r i o r f l a g e i l u m . . 146 a REFERENCES Angst, L. , 1929. Observations on the development of zoospores and gametes i n Pleurophycus g a r d n e r i . . Pub. Puget Sound B i o l . S ta. 7: 39-45. Anderson, E.K. and North, H.J. 1965.. 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B., S t e i n , J.B. and T a y l o r , T.H.C. 1965., An E v o l u t i o n a r y Survey of the P l a n t Kingdom. Wadsworth, Belmont, C a l i f . , 658 pp. S e t c h e l l , W.A. and Gardner, N.L. 1925. The marine algae of the P a c i f i c Coast of North America. / Part I I I . Helanophyceae. 0. C a l . , Pub., Bot. 8: 383-398. S k o t t s b e r g , C. 1921., Botanische Ergebnisse der schwedischen E x p e d i t i o n s nach Patagonia und dem Feuerland 1907-9. V I I I . Marine algae. 1. Phaeophyceae. Vet. Akad. H a d l i n g e r , Bd. , 61, No., 11, Stockholm., Smith, G.fl. 1938. Cryptogamic Botany. V o l . v I.„• Algae and Fungi. McGraw-Hill, N.Y.7 545~pp. Sundene, 0. 1966. Haplospora globosa K j e l l m . and Scafihospora s p e c i o s a K j e l l m . i n c u l t u r e ! Nature 209: 937-938. Suto, S.. 1950. S t u d i e s on shedding, swimming and f i x i n g of the spores of seaweeds.. B u l l . Jap. Soc. . S c i . F i s h e r i e s . 16: 1-9. „ 155 T a y l o r , F.J.E. 1978. Problems i n the development of an e x p l i c i t h y p o t h e t i c a l phylogeny o f the Lower Eukaryotes., Biosysteras 10: 67-89. Toth, B. 1974. Sporangia s t r u c t u r e and zoosporogenesis i n Chorda tormentosa ( L a m i n a r i a l e s ) . J . Phyc. 10: 170-185. •-. 1976a. The r e l e a s e , s e t t l e m e n t , and germination of zoospores i n Chorda tormentosa (Phaeophyceae, L a m i n a r i a l e s ) . J . Phycol. 12: 222-233. 1976b. A mechanism of propagule r e l e a s e from u n i l o c u l a r r e p r o d u c t i v e s t r u c t u r e s i n brown algae., Protoplasma 89: 263-278. Ba l k e r , D.C. 1980., Sorus a b s c i s s i o n from laminae of H e r e o c y s t i s leutkeana (Mert.) P. & B. Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia. 460 pp. <—.and Henry, E.C. 1978. Unusual r e p r o d u c t i v e s t r u c t u r e s i n Syrinqoderma a b y s s i c o l a (S. & G.) L e v r i n g . New Phy t o l . , 80s 1 9 3 - 1 9 7 . " 156 APPENDIX 0 I t r a s t r u c t u r e Of Sperm Of Desmarestia v i r i d i s Desmarestia v i r i d i s was s t u d i e d by the same technigues used with L a m i n a r i a l e s , The zoospores (not i l l u s t r a t e d ) are s i m i l a r t o those of most brown a l g a e ; they possess an eyespot, and the l o n g e r , a n t e r i o r , mastigoneme-bearing f l a g e i l u m . bears a d i s t a l whiplash. The sperm are elongate ( F i g s . 1,2) and l a c k an eyespot, but they c o n t a i n a nucleus, s e v e r a l mitochondria, "type b" and o s m i o p h i l i c v e s i c l e s ( F i g s . 6-8). The a n t e r i o r f l a g e i l u m i s s i m i l a r to t h a t of other brown a l g a l swarmers. The mastigoneme-bearing p o r t i o n i s about 20 um long; o c c a s i o n a l l y a d i s t a l whiplash i s preserved (not i l l u s t a t e d ) * . The p o s t e r i o r f l a g e i l u m bears a d i s t a l whiplash ( F i g s . 1-5) t h a t may extend t h i s f l a g e i l u m t o as much as 35 um i n l e n g t h . In w e l l -p r e served specimens ( F i g s . , 1,2) an a n t e r i o r rostrum, s i m i l a r to the " p r o b o s c i s " of the sperm of Vaucheria (Hoestrup 1970) i s e v i d e n t . , F i g . 2 i n d i c a t e s t h a t a c y t o s k e l e t a l loop s u p p o r t s the rostrum. , The p o s t e r i o r whiplash, l i k e the a n t e r i o r whiplash, i s an e x t e n s i o n of the two c e n t r a l m i c r o t u b u l e s of the f l a g e l l a r axoneme. In c o n t r a s t t o the a n t e r i o r whiplash, i t i s o f t e n w e l l p r e s e r v e d by osmium vapor f i x a t i o n , and i t f r e q u e n t l y i s apparent i n s e c t i o n s of p r e - r e l e a s e sperm. The elongated shape, l a c k of eyespot, and long p o s t e r i o r 157 f l a g e i l u m of the Desmarestia v i r i d i s sperm r e c a l l the sperm of L a m i n a r i a l e s , but i t i s s i g n i f i c a n t t h a t i n P. v i r i d i s f l a g e l l a r e x t e n s i o n has occurred by a d i f f e r e n t s t r u c t u r a l m o d i f i c a t i o n ( p r o l o n g a t i o n of the two c e n t r a l anonemal m i c r o t u b u l e s only) from those found i n other brown a l g a l sperm: i n the L a m i n a r i a l e s the p o s t e r i o r f l a g e i l u m t a p e r s d i s t a l l y , while i n the Fucaceae i t r e t a i n s the "9 > 2n c o n s t r u c t i o n throughout i t s e n t i r e l e n g t h . T h i s d i f f e r e n c e i n d i c a t e s t h a t d e s p i t e the s i m i l a r i t e s o f the desmarestialean and l a m i n a r i a l e a n l i f e h i s t o r i e s and gametophyte morphologies, these two or d e r s are not c l o s e l y r e l a t e d . . 1 5 8 PLATE 1 F i g . 1. Desmarestia v i r i a i s sperm. Whole mount, unstained, unshadowed. F i g . 2. Sperm showing some d i s r u p t i o n o f the a n t e r i o r part of the c e l l ; the c y t o s k e l e t o n of the rostrum has r e s i s t e d d i s r u p t i o n . , P o s t e r i o r whiplash o n l y p a r t i a l l y preserved. „ F i g . 3. Sperm with mastigonemes r e t a i n e d on a n t e r i o r f l a g e l l u m . C e l l shape i s d i s t o r t e d . F i g . 4. C e l l f i x e d with IS t a n n i c a c i d added be f o r e osmium vapor. t Hastigonemes have been preserved, but a r e s h r i v e l l e d , as i s p o s t e r i o r whiplash., C e l l shape i s d i s t o r t e d . 160 PLATE 2 F i g . , 5. ,. Sperm with w e l l preserved, mastigonemes and p o s t e r i o r whiplash. C e l l shape i s d i s t o r t e d . P i g . 6. ; S e c t i o n through sperm w i t h i n antheridium. Note c r o s s - s e c t i o n through p o s t e r i o r whiplash (arrows), nucleus (N) , mitochondria (M) . P i g . 7. Sperm w i t h i n antheridium.. Mote the f l a g e l l u m (F) l y i n g w i t h i n membrane-enclosed r e g i o n . . P i g . 8 . Sperm w i t h i n antheridium.. Note l o n g i s e c t i o n of p o s t e r i o r whiplash (arrow) , "type b w v e s i c l e s ( B ) , o s m i o p h i l i c v e s i c l e s (O) . 

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