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Survey of cell wall structure in some Florideophycidae Rusanowski, Paul Charles 1970

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A SURVEY OF CELL WALL STRUCTURE IN SOME FLORIDEOPHYCIDAE by PAUL CHARLES RUSANOWSKI B.A., San Fernando V a l l e y S t a t e C o l l e g e , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f Botany We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Paul Rusanowski (In absentia) Department of Botany  The University of British Columbia Vancouver 8, Canada Date June 8, 1970 ABSTRACT C e l l w a l l s t r u c t u r e was i n v e s t i g a t e d i n 20 d i f f e r e n t r e d a l g a e . R e p r e s e n t a t i v e s f r o m a l l 4- f a m i l i e s o f t h e o r d e r C e r a m i a l e s and one f a m i l y o f t h e o r d e r G i g a r t i n a l e s were i n v e s t i g a t e d . Of t h e s e , 3 g e n e r a , P o l y s i p h o n i a , P t e r o s i p h o n i a and A n t i t h a m n i o n were i n v e s t i g a t e d w i t h r e g a r d s t o b o t h t h e c e l l u l o s i c and m u c i l a g i n o u s p o r t i o n s o f t h e c e l l w a l l . A new s t a i n i n g t e c h n i q u e u t i l i z i n g a c o m b i n a t i o n o f r u t h e n i u m r e d and osmium t e t r o x i d e as a p o s t f i x a t i o n was used i n t h e l a t t e r p o r t i o n o f t h e s t u d y . The u l t r a s t r u c t u r e o f p i t c o n n e c t i o n s was examined i n a l l a l g a e . The i n n e r c e l l u l o s i c p o r t i o n o f t h e c e l l w a l l c o n s i s t s o f a r e t i c u l a t e p a t t e r n o f m i c r o f i b r i l s w h i c h appear d e n s e l y s t a i n e d , I n P t e r o s i p h o n i a t h i s c e l l u l o s i c p o r t i o n was f o u n d t o c o n s i s t o f 2 l a y e r s ; an i n n e r l a y e r o f m i c r o f i b r i l s w h i c h e n s h e a t h e d i n d i v i d u a l c e l l s and an o u t e r l a y e r o f m i c r o f i b r i l s w h i c h e n s h e a t h e d t h e e n t i r e t h a l l u s and was i n c o n t a c t w i t h t h e m u c i l a g i n o u s c o a t . The m i c r o f i b r i l s i n t h e i n n e r l a y e r a ppear n e a r l y c r o s s - s e c t i o n e d , w h i l e t h o s e i n t h e o u t e r l a y e r appear more l o n g i t u d i n a l l y o r i e n t e d t o t h e p l a n e o f s e c t i o n i n g . The o u t e r m u c i l a g i n o u s c o a t c o v e r s t h e e n t i r e t h a l l u s . I t c o n s i s t s o f 4 l a y e r s . The f i r s t o r o u t e r m o s t l a y e r c o n s i s t s o f l o o s e bunches o f m i c r o f i b r i l s e x t e n d i n g out from t h e , s e c o n d l a y e r . The wecond l a y e r c o n s i s t s o f a zone o f medium e l e c t r o n d e n s i t y a p p r o x i m a t e l y 750 A i n t h i c k n e s s . The t h i r d l a y e r i s w h o l l y c o n t a i n e d w i t h i n t h e s e cond l a y e r . I t i s composed o f a d e n s e l y s t a i n i n g band o f m i c r o f i b r i l s extending from a s i m i l a r l y s t a i n i n g membrane-like structure. The fourth layer i s a densely stained membrane-like structure i n contact with the c e l l u l o s i c portion of the c e l l w all. An a d d i t i o n a l l a y e r , the D l a y e r , i s sometimes found i n the c e l l w all. When present i t i s found i n the outermost portion of the c e l l u l o s i c wall and obscures the fourth layer of the mucilaginous coat. It consists of a densely s t a i n i n g amorphous material. Investigation of the p i t connection showed the occurrence of 2 stages of one basic p i t structure. One stage, the single d i s c stage-p i t s t r u c t u r e , has been found i n a l l algae investigated. I t consists of a s o l i d , l e n t i c u l a r , membrane-bound plug situated within an aperture i n the c e l l w a l l . The plug consists of a granular material surrounded by a zone of densely s t a i n i n g amorphous material. The other stage, the double d i s c stage p i t s t r u c t u r e , i s a modification of the s i n g l e d i s c stage. It i s not found i n young c e l l s near the apex of the t h a l l u s , but only i n c e l l s which have, or are undergoing, r a p i d elongation and vacuolation. This p i t structure has only been observed i n a x i a l c e l l s of the family Ceramiaceae i n the order Ceramiales. The double di s c stage p i t structure d i f f e r s ;from the s i n g l e disc stage i n that the granular material of the plug i s segregated into 2 regions or p l a t e s , one on e i t h e r side of the plug. The c e n t r a l region of the plug at f i r s t appears c l e a r but l a t e r appears to be p a r t i a l l y occupied by a granular to f i b r i l l a r material. The d i f f e r e n t i a t i o n of the double di s c stage p i t structure from the single d i s c stage has been described. These observations are thought to support and confirm the e a r l i e r work of Jungers (25). However, h i s observations have been extended through the use of electron microscopy i n th i s study. I t has been proposed that the terms used i n th i s study, s ingle disc stage- and double disc stage p i t s t ructures , replace the terms Polysiphonia and G r i f f i t h s i a p i t s used by Jungers. TABLE OF CONTENTS PAGE L i s t of tables . . . . . . . . . . . . . . . . i i i L i s t of plates i v L i s t of appendicies • v INTRODUCTION . . . . . . . . . . . . . . 1 LITERATURE REVIEW . 2 C e l l wall . .2 P i t connections • 3 MATERIALS AND METHODS 6 RESULTS . . . . . . . . . 9 Light microscopy . . . . . . . . 10 Electron microscopy 11 C e l l wall . . . . . . . . . . . . . . . . ..11 P i t u l t r a s t r u c t u r e • • 13 Cytoplasm • 17 DISCUSSION 20 C e l l w a l l 20 P i t structure 22 CONCLUSIONS 29 BIBLIOGRAPHY 32 APPENDICIES . . ' 36 Appendix I i 36 Appendix II . . 38 Appendix III . . . . . . . . 39 KEY TO ABBREVIATIONS . . . . 41 ( i i ) LIST OF TABLES PAGE Table I. L i s t of algae, c o l l e c t i o n and u t i l i z a t i o n data 7-8 ( i i i ) TABLE OF PLATES PAGE Plates 1-2 -Light micrographs of a p i c a l region of the t h a l l u s and p i t s . with. both, f r e s h and f i x e d and sectioned material • .42-43 Plates 3-4 Ult r a s t r u c t u r e of the a p i c a l c e l l and derivatives . .44-4-5 Plates 5-7 Ult r a s t r u c t u r e of the single d i s c stage p i t 46-48 Plate 8 Ul t r a s t r u c t u r e and development of the double d i s c stage p i t . . . . . . . . 49 Plates 9-12 Ul t r a s t r u c t u r e of cytoplasmic organelles and in c l u s i o n s . . . • • 50-53 Plates 13-14 Ultra s t r u c t u r e of the c e l l w all .54-55 (i v ) LIST OF APPENDICIES PAGE I. Culture formulae 36 I I . Light microscope techniques and stains 38 I I I . Electron microscopy technique and formulae 39 C v ) 1 INTRODUCTION One of the unique features of the Rhodophyceae i s the possession of p i t connections. These p i t connections are absent from the sub-class Bangiophycideae. They are a prominent feature of c e l l morphology i n the sub-class Florideophycidae to which most of the red algae belong. P i t connections appear oval to c i r c u l a r i n shape and are of var i a b l e s i z e , depending on t h e i r age. In some red algae p i t connections can occupy the e n t i r e wall between two c e l l s . Although p i t connections have been the subject of numerous investigations both t h e i r structure and function remain obscure. Recent inv e s t i g a t i o n s using electron microscopy have done much toward r e s o l v i n g the structure of p i t connections, however, opinions are s t i l l divided as to t h e i r actual chemical and ph y s i c a l structure. The present studies were undertaken i n an attempt to determine the structure of the c e l l wall and p i t connection i n several red algae, by means of electron microscopy. Of e s p e c i a l importance was an i n v e s t i -gation i n t o the structure of the outermost layer of the c e l l w a l l , the so-c a l l e d p e c t i c coat, using a previously untried s t a i n i n g procedure. In a d d i t i o n , a comparison of p i t u l t r a s t r u c t u r e between d i f f e r e n t groups of algae u t i l i z i n g the same preparative procedures was undertaken. Members of a l l four f a m i l i e s of the order Ceramiales and one family of the order Gigartinales were investigated. 2 LITERATURE REVIEW CELL WALL The c e l l w a l l i n t h e F l o r i d e o p h y c i d a e i s g e n e r a l l y c o n s i d e r e d t o be u n i f o r m i n s t r u c t u r e . I t c o n s i s t s o f two p a r t s : an i n n e r c e l l u l o s i c p o r t i o n and an o u t e r p e c t i c l a y e r ( 1 6 , 19, 2 8 ) . I n some ca s e s a c u t i c l e i s a l s o formed ( 2 8 ) . I n P o r p h y r a , a member o f t h e B a n g i a l e s , t h e c u t i c l e has been shown t o c o n t a i n a v e r y l a r g e amount o f p r o t e i n ( 2 0 ) . I n some a l g a e , e s p e c i a l l y members o f t h e f a m i l y C e r a m i a c e a e , c e l l w a l l s show a d i s t i n c t s t r a t i f i c a t i o n ( 7 , 1 9 ) . The i n n e r c e l l u l o s i c p o r t i o n o f t h e c e l l w a l l c o n s i s t s o f a p a t t e r n o f r e t i c u l a t e m i c r o f i b r i l s , w h i c h a r e e a s i l y seen under t h e e l e c t r o n m i c r o s c o p e ( 1 1 , 12, 4-0, 4 9 ) . The m i c r o f i b r i l s a r e embedded i n an amorphous m a t r i x ( 2 6 ) . P r e s t o n (44) has c l a s s i f i e d w a l l m a t e r i a l o f a l g a e i n t o t h r e e g r o u p s : Group I , c e l l u l o s e I b e i n g t h e main component w i t h r e g u l a r l y o r i e n t e d m i c r o f i b r i l s ; Group I I , randomly o r i e n t e d m i c r o f i b r i l s o f c e l l u l o s e I I , and Group I I I , w a l l composed o f o t h e r m i c r o f i b r i l s . The m i c r o f i b r i l s have been i d e n t i f i e d as c e l l u l o s e I I i n G r i f f i t h s i a ( 3 7 ) , and most r e d a l g a l c e l l w a l l s have been p l a c e d i n Group I I o f P r e s t o n ' s c l a s s i f i c a t i o n ( 1 2 , 1 3 ) . The c e l l u l o s i c l a y e r •surrounds i n d i v i d u a l c e l l s o f t h e t h a l l u s ( 4 9 ) . The m u c i l a g i n o u s o r p e c t i c c o a t , on t h e o t h e r hand, c o v e r s t h e o u t e r s u r f a c e o f . t h e t h a l l u s o n l y ( 1 2 , 4 9 ) . Dawes e t a l . (12) r e p o r t e d t h i s m u c i l a g i n o u s c o a t t o c o n s i s t o f r e t i c u l a t e m i c r o f i b r i l s embedded i n an amorphous m a t r i x ; w h i l e B i s a l p u t r a e t a l . (2) have been a b l e t o r e s o l v e i t initio f o u r d i s t i n c t 3 layers of complex nature. Surface a c t i v i t i e s at the plasmalemma have been described and r e l a t e d to c e l l w all deposition (2). PIT CONNECTIONS P i t connections i n the red algae have received considerable attention. One of the f i r s t workers, Schmitz, described the p i t as being closed by a membrane i n which two plates were si t u a t e d , one on e i t h e r side of the membrane (52). The plates stained densely with haematoxylin, and plasmodesmata were observed passing through the membrane separating the plat e s . Falkenberg, working with members of the Ceramiales, agreed with Schmitz, but could not f i n d plasmodesmata (17). Mangenot, using G r i f f i t h s i a (Ceramiales), claimed that the p i t had no c l o s i n g membrane and that the cytoplasm was continuous through the p i t connection,(34). Miranda, using Bornetia (Ceramiales), was of the opinion that the c l o s i n g membrane was present and that there were protoplasmic connecting strands (plasmodesmata) through the region (35). Jungers was the f i r s t to propose that the confusion i n t h i s area had probably re s u l t e d from the f a c t that there i s more than one type of p i t connection i n the red algae (25). He recognized two types of p i t structures, the Polysiphonia-type and the G r i f f i t h s i a - t y p e (25). The Polysiphonia-type has two densely stained plates separated by a membrane, with no plasmodesmata passing through the membrane; the G r i f f i t h s i a - t y p e was described as a dense, biconvex^lens-shaped body, without a c l o s i n g membrane. Muldorf suggested that the plates of the Polysiphonia-type p i t were a c t u a l l y rings which formed part of the c e l l w all (36). K y l i n , using Bonnemaissonia (Nemalionales), was i n 4 agreement with Muldorf's r e s u l t s (28). These l a t t e r two inte r p r e t a t i o n s are s i m i l a r to the e a r l i e r work of Mangenot (34) i n that cytoplasmic continuity i s maintained from c e l l to c e l l by means of the p i t connection. In 1959 the f i r s t u l t r a s t r u c t u r e studies of p i t connections were published by Myers et a l . (38). Using osmium tetroxide f i x e d material they described two types of p i t s . One from Rhodymenia (Rhodymeniales), was described as an open pore on the c e l l w a l l , through which the cytoplasm was continuous. They regarded i t as equivalent to Jungers' G r i f f i t h s i a - t y p e p i t . The second p i t str u c t u r e , from Laurencia (Ceramiales), was closed by a c e l l w all membrane and was considered equivalent to Jungers' Polysiphonia-type p i t . Later Dawes et a l . (12), using chemically cleared and macerated c e l l walls from members of a l l the orders of the Florideophycidae, also described two types of p i t connections. These were termed open and closed p i t s . Open p i t s were described as a duct or channel between two c e l l s , as i n Rhodymenia. Closed p i t s , as found i n Laurencia, consisted of a p i t c l o s i n g membrane perforated by protoplasmic strands. No attempt was made to correlate- these two p i t types with those of Jungers. In contrast to e a r l i e r electron microscopy e f f o r t s , Bouck (5), working with Lomentaria (Rhodymeniales), observed a d i s c r e t e , membrane-bound, biconvex plug blocking the p i t aperture. The plug was shown to have a dense rim with a less dense c e n t r a l region. This same type of p i t structure has been observed by Bischoff (4) i n Thorea (Nemaliona-l e s ) , B i s a l p u t r a et a l . (2) i n Laurencia (Ceramiales), Hawkins (21) and Peyriere (42) i n Ceramium (Ceramiales) and Ramus (46) i n Pseudogloiophlea (Nemalionales). 5 Minor var ia t ions i n p i t u l t ras t ructure have also been reported. Bouck (5) observed papi l lae on the plug surface. Bisalputra et a l . (2) reported a dense amorphous material extending from the edge of the plug into the middle of the c e l l w a l l . Ramus reported the deposition of material around the edge of the p i t , to produce a d i s t i n c t border or r i ng around the aperture and plug (47). Ramus has also described the development of a plug i n Pseudogloiophlea (46). Through c e l l d i v i s i o n an incomplete septum was formed between daughter c e l l s . Nichols et a_l. (39) reported the same type of septum formation i n Compsopogon. However, no plug i s formed i n Compsopogon and the aperture gradually disappears (39). Plug material next condenses along flat tened v e s i c l e s , of possible endoplasmic ret iculum o r i g i n , which become oriented long i tud ina l ly wi th in the p i t aperture. The f lat tened membranes disappear as deposi-t ion of plug material takes p lace , r e su l t i ng i n the formation of a s o l i d plug blocking the aperture. 6 MATERIALS AND METHODS I n t h e p r e s e n t s u r v e y , r e p r e s e n t a t i v e s o f a l l f o u r f a m i l i e s o f t h e o r d e r C e r a m i a l e s and one f a m i l y o f t h e o r d e r G i g a r t i n a l e s were s t u d i e d . These a l g a e , as w e l l as t h e c o l l e c t i o n s i t e s and s p e c i f i c p a r t s o f t h e t h a l l i i n v e s t i g a t e d , a r e l i s t e d i n T a b l e I . M a t e r i a l was m a i n t a i n e d e i t h e r i n a c o l d room, a t 10 C+2, o r a P e r c i v a l i n c u b a t o r w i t h a 12 C/10 C+l d a y / n i g h t t e m p e r a t u r e c y c l e . Day l e n g t h s were v a r i e d s e a s o n a l l y from 10.to 16 h o u r s . C o o l w h i t e f l o u r e s c e n t l i g h t s were used f o r i l l u m i n a t i o n , w i t h an i n t e n s i t y f r o m 10-100 f t - c . C u l t u r e s were m a i n t a i n e d e i t h e r i n P r o v a s o l i ES e n r i c h e d s e a w a t e r (45) o r C h i h a r a marine media I I (8) (app. I ) . A s o l u t i o n o f 0.1% germanium d i o x i d e was added t o a l l c u l t u r e media (31) t o e l i m i n a t e d i a t o m c o n t a m i n a t i o n o f c u l t u r e s . F o r l i g h t m i c r o s c o p y e i t h e r f r e s h m a t e r i a l o r m a t e r i a l p r e p a r e d f o r e l e c t r o n m i c r o s c o p y and s e c t i o n e d 0.25-0.50 u i n t h i c k n e s s was used. S e c t i o n e d m a t e r i a l was s t a i n e d w i t h b a s i c f u c h s i n and c r y s t a l v i o l e t ( 1 8 ) , t o l u i d i n e b l u e and b o r a x , o r m e t h y l g r e e n w i t h p y r o n i n ( 1 0 ) . The s t a i n i n g p r o c e d u r e used f o r t h e s e s t a i n s i s l i s t e d i n a p p e n d i x I I . O b s e r v a t i o n s were made w i t h a N i k o n Model S m i c r o s c o p e . P h o t o g r a p h s were t a k e n w i t h a N i k o n EFM camera on 35 mm Kodak Panatomic X o r I I f o r d FP 3 f i l m s . A v a r i e t y o f f i x a t i o n s f o r e l e c t r o n m i c r o s c o p y was used d u r i n g t h i s s t u d y . Those t h a t p r o v e d t o be t h e most s a t i s f a c t o r y were t h e TABLE I L I S T OF A L G A E , COLLECTION AND U T I L I Z A T I O N DATA Name o f a l g a e C o l l e c t i o n s i te ; ( ; s ) P a r t s i n v e s t i g a t e d C u l t u r e - X f i e l d - 0 A n t i t h a m n i o n p a c i f i c u m ( H a r v e y ) K y l i n A_. s u b u l a t u m ( H a r v e y ) J . A g a r d h . 9 Ceramium s p . R o s a r i o B e a c h , W a s h . , S o o k e , B r i t . C o l . , C o r o n a t i o n I s l a n d , A l a s k a R o s a r i o B e a c h , W a s h . , S o o k e , B r i t . C o l . C o r o n a t i o n I s l a n d , A l a s k a a p i c a l r e g i o n , a x i a l c e l l s o f v a r i o u s ages as i n A . p a c i f i c u m a p i c a l r e g i o n , c o r t i c a l c e l l s X , 0 X , 0 P l a t y t h a m n j o n r e v e r s u m ( S e t c h e l l and G a r d n e r ) K y l i n P l e o n o s p o r i u m v a n c o u v e r - ianum J . A g a r d h . D a s y o p s i s densa S m i t h D e l e s s e r i a d e c i p i e n s J . A g a r d h . Membranop te ra m u l t i r a m o s a ^ G a r d n e r P o l y n e u r a l a t i s s i m a ( H a r v e y ) K y l i n S o o k e , B r i t . C o l . R o s a r i o B e a c h , Wash. R o s a r i o B e a c h , Wash. S o o k e , B r i t . C o l . R o s a r i o B e a c h , Wash. R o s a r i o B e a c h , Wash. a p i c a l r e g i o n , young a x i a l c e l l s a p i c a l r e g i o n a p i c a l r e g i o n and b r a n c h l e t s young b l a d e young b l a d e a p i c a l r e g i o n , young b l a d e X , 0 X , 0 0 X X , 0 Laurencia s p e c t a b i l i s Postels and Ruprecht Odonthallia floccosa (Esper) Falkenberg 0_. l y a l l i i (Harvey) J. Agardh. Polysiphonia -sp. Polysiphonia p a c i f i c a Hollenberg var. p a c i f i c a P_. p a c i f i c a var. g r a c i l i s Hollenberg P_. paniculata Montagne Pterosiphonia bipinnata (Postels and Ruprecht) Falkenberg var. bipinnata Pt. g r a c i l i s K y l i n A gardhiella c o u l t e r i Rosario Beach, Wash. Rosario Beach, Wash. Rosario Beach, Wash. Goronation Island, Alaska Sooke, B r i t . Col. Rosario Beach, Wash. Rosario Beach, Wash. Rosario Beach, Wash. Rosario Beach, Wash. Rosario Beach, Wash. Rosario Beach, Wash. a p i c a l region a p i c a l region a p i c a l region a p i c a l region a l l parts ex-cept the hold-f a s t region as f o r P. p a c i f i c a var. p a c i f i c a as i n P. p a c i f i c a var. p a c i f i c a a p i c a l region, p e r i c e n t r a l c e l l s a p i c a l region p e r i c e n t r a l c e l l s a p i c a l region X, 0 X, 0 X, 0 X, 0 X, 0 -'•The algae were i d e n t i f i e d by the author with the use of several references (15,23,24,27,4-3,51,53). ^Reference i d e n t i f i c a t i o n material f o r these 2 algae was l o s t ; p o s i t i v e species i d e n t i f i c a t i o n was not possible with the amount of f i x e d and embedded material. ^Membranoptera multiramosa was co l l e c t e d from Rosario Beach, Washington, on March 18, 1968. It was te t r a s p o r i c at the time of the c o l l e c t i o n . This c o l l e c t i o n i s believed to be a new northward extension of the occurrence of t h i s alga, previous to t h i s occasion, appears to be Coos Bay, Oregon (15). 9 following: 1. 2.5% glutaraldehyde i n sea water (ph 6.8 and 8.0) or 0.1 M sodium cacodylate buffer (ph 6.8); postfixed i n 1.0% osmium tetroxide i n Dalton's buffer (ph 6.8) or 0.1 M sodium cacodylate buffer (ph 6.8). 2. 2.5% glutaraldehyde or formaldehyde (41) i n 0.1 M sodium cacodylate buffer (ph 6.8); postfixed i n 1.0% osmium tetroxide plus 0.1% ruthenium red i n 0.1 M sodium cacody-l a t e b u f f e r made up i n d i s t i l l e d water (ph 6.8). Specimens were f i x e d f o r 1 hr. e i t h e r at room temperature i n the laboratory or at ambient f i e l d temperature. Pos t f i x a t i o n s i n e i t h e r case were f o r 1 hr. i n the laboratory. An alcohol-propylene oxide dehydration ser i e s was used, and the tissues were embedded i n maraglas according to Bi s a l p u t r a and Weier (3). Sections were cut with glass knives, a GeFeRi or Dupont diamond k n i f e , using e i t h e r a Porter-Blum Mt-1 or an LKB Ultrotome I microtome. Specimens were examined,, a f t e r uranyl acetate and lead c i t r a t e (48) s t a i n i n g , with a Hitachi HS-7S or a Zeiss EM-9A electron microscope. RESULTS In t h i s study a l l species of algae were surveyed with regards to p i t u l t r a s t r u c t u r e . Only three genera, Polysiphonia, Pterosiphonia and Antithamnion were used i n the observations by l i g h t microscopy on the cytoplasm and c e l l w a l l , and c e l l w all u l t r a s t r u c t u r e . 10 LIGHT MICROSCOPY Examination of l i v i n g apices of Polysiphonia, Pterosiphonia  Antithamnion species shows the a p i c a l c e l l to be t y p i c a l l y dome-shaped ( f i g s . 1, 2). In Polysiphonia the a p i c a l c e l l i s sometimes hidden by t r i c h o b l a s t filaments ( f i g . 2). The s i z e of the a p i c a l d e r i v a t i v e s increases r a p i d l y with the distance from the apex ( f i g s . 2, 3). P i t connections and c e l l u l a r d e t a i l are not r e a d i l y apparent i n the a p i c a l region of the t h a l l u s . In sectioned material the a p i c a l c e l l , again, appears dome-shaped ( f i g . 3). Derivatives appear, at f i r s t , as very narrow t i e r s under the a p i c a l c e l l ( f i g . 3), which undergo r a p i d eleongation as the distance from the a p i c a l c e l l increases ( f i g s . 3, 4, 5). L a t e r a l d i v i s i o n of the d e r i v a t i v e s i s noticeable within the fourth derivative (arrow, f i g . 3). Vacuoles of various sizes and a nucleus with a prominent, densely s t a i n i n g nucleolus are observed more e a s i l y i n each de r i v a t i v e than i n the case of fresh material ( f i g s . 3-6). The nucleoplasm i s stained l e s s intensely than the cytoplasm, and the nucleolus appears oval to round ( f i g . 5) with occasional vacuoles or i n c l u s i o n s , which may r e s u l t , i n some cases, i n a r i n g - l i k e appearance ( f i g . 6). The p i t connection i n both young and mature c e l l s of Polysiphonia and Pterosiphonia appears as an aperture through the c e l l w a l l , blocked by a plug ( f i g . 5). This plug appears homogeneous and stains approxi-mately the same as the cytoplasm ( f i g s . 4, 5). P i t connections are also observed i n the t r i c h o b l a s t filaments, of Polysiphonia ( f i g . 4). In the genus Antithamnion the plug of the p i t connection consists of an inner l i g h t zone surrounded by a densely s t a i n i n g band of material ( f i g . 7). With higher magnification the densely s t a i n i n g band of material appears as an outer dark rim, possibly due to r e f r a c t i o n , with an inner and somewhat less dense band of material ( f i g . 8). The densely s t a i n i n g band of material almost completely disappears i n the mid-region of the c e l l w all (arrow, f i g . 8). The mucilaginous coat of the c e l l wall has been stained very heavily i n these preparations, whereas the c e l l u l o s i c wall shows v a r i a t i o n s i n i n t e n s i t y of s t a i n i n g The middle lamella region of the wall stains s i m i l a r l y to the mucilag nous coat of the c e l l w all ( f i g s . 4, 7). The lamellar nature of the wall i s seen i n Antithamnion but not i n the other algae examined ( f i g . 7). ELECTRON MICROSCOPY CELL WALL The appearance of the c e l l w all i s dependent upon the type of f i x a t i o n and s t a i n i n g procedure employed. With glutaraldehyde or formaldehyde f i x a t i o n s followed by p o s t f i x a t i o n i n osmium t e t r o x i d e , and s t a i n i n g with uranyl acetate and lead c i t r a t e , the wall can be d i f f e r e n t i a t e d into two phases; the outer mucilaginous coat and the inner c e l l u l o s i c wall ( f i g . 41). The c e l l u l o s i c wall appears as an amorphous matrix e i t h e r nearly devoid of m i c r o f i b r i l s ( f i g s . 13, 38), or with a very loose r e t i c u l a t e pattern of m i c r o f i b r i l s present ( f i g . 41). The mucilaginous coat appears to be composed of four layers ( f i g . 42). These are an outer layer of r e t i c u l a t e m i c r o f i b r i l s of 12 medium electron density, followed by a zone of densely s t a i n i n g material, a region of r e l a t i v e l y low electron density, and an innermost layer, a membrane-like structure which i s firml y appressed to the c e l l u l o s i c portion of the c e l l w a l l . When an osmium tet r o x i d e -ruthenium red p o s t f i x a t i o n i s used i n place of the previously described p o s t f i x a t i o n wall u l t r a s t r u c t u r e i s revealed i n much more d e t a i l . Four regions are again distinguished within the mucilaginous coat of the c e l l w all. These regions w i l l be r e f e r r e d to as " I , I I , I I I , and IV" ( f i g s . 42, 43). The outermost l a y e r (I) consists of loose m i c r o f i b r i l s extending out approximately 750 A from the surface of the second layer. The second layer (II) i s a zone of medium electron density, extending from the f i r s t layer down to the fourth layer, and i s approximately 750 A thick ( f i g s . 42, 43). The t h i r d layer (III) e x i s t s e n t i r e l y within the second layer. It consists of a very densely s t a i n i n g band of m i c r o f i b r i l s (arrows, f i g . 42) which extend outward from a s i m i l a r l y s t a i n i n g membrane-like layer. The t h i r d layer i s approximately 370 A thi c k . The fourth layer (IV) forms the innermost p o r t i o n of the mucilaginous coat. This layer appears to be a membrane-like structure of approximately 60 A thickness. It i s i n contact with the inner c e l l u l o s i c wall ( f i g s . 41-43). Occasionally observed i s a region of amorphous mate r i a l , which can be of e i t h e r medium or high electron density, located d i r e c t l y under the fourth l a y e r , within the outermost portion of the c e l l u l o s i c wall ( f i g s . 38, 40). This layer has been designated the D layer and, when present, completely obscures the innermost l a y e r of the mucilaginous coat. With the use of ruthenium red-osmium tetroxide p o s t f i x a t i o n the c e l l u l o s i c w a l l appears to consist of a very densely s t a i n i n g r e t i c u l a t e pattern of m i c r o f i b r i l s embedded i n an amorphous matrix ( f i g s . 42-44). The c e l l u l o s i c wall can be d i f f e r e n t i a t e d f u r t h e r , i n Pterosiphonia, into two subregions. There appears to be an inner portion which ensheaths the i n d i v i d u a l c e l l s ( f i g s . 42, 44). This portion of the w a l l . i s approximately 375 mu t h i c k . There i s also distinguishable an outer c e l l u l o s i c l a y e r , approximately 700 mu t h i c k , forming a sheath around the en t i r e t h a l l u s ( f i g s . 42, 44). The differ e n c e between these two portions of the c e l l u l o s i c wall appears to be r e l a t e d to the density and o r i e n t a t i o n of m i c r o f i b r i l s i n each region. The inner portion which ensheaths the. i n d i v i d u a l c e l l s has both fewer and le s s compacted m i c r o f i b r i l s than the outer portion which ensheaths the e n t i r e t h a l l u s . This i s possibly r e l a t e d to the stages of development of the two portions of the c e l l u l o s i c w a l l . It could also be due to the o r i e n t a t i o n of m i c r o f i b r i l s i n these two layers of the wall i n which the m i c r o f i b r i l s i n layer L l are oblique to the plane of se c t i o n i n g , whereas the m i c r o f i b r i l s i n layer L2 appear to be mostly oriented l o n g i t u d i n a l to the plane of sectioning. Extensive surface a c t i v i t y of the plasmalemma has also been observed ( f i g . 41). Membrane-bound droplets, approximately 35 to 90 mu i n diameter, can be seen at the c e l l surface, attached to or at some distance from the plasmalemma (arrows, f i g . 41). PIT ULTRASTRUCTURE Only one p i t structure has been found i n the algae investigated so f a r . However, two d i f f e r e n t stages of t h i s p i t structure have been 14 observed. The most common stage observed i s exemplified by P o l y s i - phonia and Pterosiphonia and.will be r e f e r r e d to as the s i n g l e d i s c stage p i t . The other stage, r e f e r r e d to as the double d i s c stage p i t , i s exemplified by the genus Antithamnion and i s of l i m i t e d occurrence. SINGLE DISC STAGE PIT The s i n g l e disc stage p i t structure has been observed i n a l l algae examined. This p i t structure b a s i c a l l y consists of an aperture or pore through the c e l l w all ( f i g . 13). A l e n t i c u l a r plug i s s i t u a t e d within t h i s aperture ( f i g s . 13-23). The plug exhibits a groove around i t s periphery which appears to f i r m l y p o s i t i o n i t within the aperture ( f i g s . 13-23). A membrane of approximately 100 A thickness separates the plug from the adjacent cytoplasm ( f i g s . 13, 14, 19). This membrane often appears loosely appressed to the surfaces of the plug f a c i n g the cytoplasm ( f i g s . 13-20). The membrane across the face of the plug i s i n contact with the plasmalemma at the rim of the p i t aperture (arrows, f i g s . 13, 14, 20, 26). The plug consists of a t h i n , dense, amorphous peri p h e r a l zone of material, possibly proteinaceous, enclosing a c e n t r a l region of homogeneous, f i n e l y granular material ( f i g . 13). The inner granular matrix consists of granules approximately 100 A i n diameter. The outer, dense zone varies from 240 to 620 A i n thickness i n the d i f f e r e n t algae examined ( f i g s . 13-23). MINOR VARIATIONS IN THE SINGLE DISC STAGE PIT MORPHOLOGY Minor v a r i a t i o n s i n plug morphology have been observed. In Dasyopsis the peripheral zone of the plug was found to be up to 1000 A 15 i n thickness and less dense than the inner granular matrix ( f i g . 17). A dense amorphous material i s sometimes associated with the groove region of the plug and extends i n t o the middle portion of the c e l l w a ll i n Odonthallia and Laurencia ( f i g . 16). The shape of the groove region of the plug also varies i n many of the algae examined ( f i g s . 13, 16, 19), and i s considered to be due to the plane of sectioning. However, i n the p i t structures of a x i a l c e l l s of Polysiphonia and Antithamnion a consistent v a r i a t i o n i s apparent. The same v a r i a t i o n i s observed i n p i t structures between the basal c e l l s of l a t e r a l branches and a x i a l c e l l s i n Antithamnion. What appears to be a ridge-l i k e extension of the inner granular matrix i n t o the mid-region of the c e l l w all i s present ( f i g s . 21-23). This ridge does not occupy the en t i r e groove region, but i s o f f s e t s l i g h t l y to one s i d e . DOUBLE. DISC STAGE PIT STRUCTURE A major v a r i a t i o n i n the single d i s c stage p i t structure has been observed ( f i g s . 24-29). It occurs only i n the mature elongated a x i a l c e l l s of Antithamnion and Platythamnion species, which are characterized by being extremely vacuolated ( f i g s . 7, 8). This p i t structure seems to occur only i n the Ceramiaceae of the Ceramiales. The plug of the s i n g l e d i s c stage p i t structure becomes modified r e s u l t i n g i n what I have termed the double disc stage p i t structure. This p i t structure consists of an aperture through the c e l l wall which i s blocked by a biconvex, lens-shaped plug ( f i g s . 24-29). The f i n e granular plug matrix, however, appears to be segregated into two approximately equal discs or plates ( f i g s . 24-29), d i s t i n g u i s h i n g i t 16 from the homogeneous plug matrix of the single d i s c stage p i t . The two discs or plates are separated by a region of low electron density, which extends across the e n t i r e diameter of the plug ( f i g s . 25, 28, 29). In older c e l l s a fibrous to granular material may be found scattered within t h i s c e n t r a l region ( f i g s . 25, 29). DEVELOPMENT OF THE DOUBLE DISC STAGE FROM THE SINGLE DISC STAGE PIT As the plug of the s i n g l e d i s c stage p i t increases i n age, the mid-region of the plug undergoes a ser i e s of changes which are chrono-l o g i c a l l y associated with increasing age. I n i t i a l l y a zone of d i f f e r e n t i a t i o n i s developed i n which the.inner fin e granular matrix of the plug appears to become clumped and more densely stained ( f i g . 26). This zone i s approximately 700 to 800 A wide and extends across the e n t i r e mid-region of the plug. Transparent spaces appear f i r s t near the edges of the plug ( f i g . 26).and continue to develop c e n t r i -p e t a l l y within t h i s zone of d i f f e r e n t i a t i o n . This r e s u l t s i n the plug material being segregated into two discs or plates as described above ( f i g s . 25, 27-29). The newly formed surfaces of the two discs bordering the c e n t r a l electron transparent region appear rough and uneven with protrusions of the f i n e granular matrix ( f i g s . 27, 28). Throughout these changes the outer, dense, amorphous zone remains i n t a c t around the periphery of the plug. F i n a l l y the electron transparent region becomes, p a r t i a l l y to completely, occupied by a fibrous to granular matrix of approximately the same density as the cytoplasm of the c e l l s connected by the p i t structure ( f i g s . 24, 25, 29). 17 CYTOPLASM CHLOROPLASTS Proplastids have been observed only i n the a p i c a l c e l l ( f i g s . 9-11). Also present i n the a p i c a l c e l l are d i f f e r e n t i a t i n g chloroplasts which can be distinguished from proplastids by the occurrence of inner photosynthetic lamellae ( f i g s . 10, 11). Proplastids are oval to round and range from 0.15-1.2 u. They are bounded by a double membrane envelope enclosing a heterogeneous matrix or stroma ( f i g . 30). Within each p r o p l a s t i d there i s an inner double membrane c l o s e l y p a r a l l e l i n g the envelope, which i s designated the outermost photosynthetic lamella by Brown and Weier (19). Both systems of membranes are approximately 125 A t h i c k , and are separated from one another by a distance of 300-380 A ( f i g s . 30). The outermost photosynthetic lamella occasionally shows interruptions which become e s p e c i a l l y noticeable i n developing chloroplasts ( f i g . 31). The granular stroma has a density s i m i l a r to the surrounding cytoplasm ( f i g s . 30, 31). There are, however, regions i n the stroma of low electron density which can be quite extensive ( f i g s . 30-33). These regions contain DNA f i b r i l s of 25-30 A diameter, which are p a r t i a l l y clumped together ( f i g . 33). In the process of d i f f e r e n t i a t i o n of chloroplasts the outermost photosynthetic lamella gives r i s e to the inner photosynthetic lamellae by invagination (arrows, f i g . 33). The newly formed photosynthetic lamellae vary i n length and o r i e n t a t i o n . Developing chloroplasts range from 1.8-3.3 u i n length ( f i g s . 33, 44). In the mature chloroplast a variable number of f l a t , l o n g i -t u d i n a l l y p a r a l l e l photosynthetic lamellae are separated by a f a i r l y uniform distance of 740 A (figs.35, 38). Thylakoids occasionally 18 branch or fuse with one another, e s p e c i a l l y near the t i p s of the chloroplast. The stroma i s homogeneous but f o r the presence of p l a s t o g l o b u l i (32) and occasional electron trasparent regions. Chloroplasts have been observed i n various stages of d i v i s i o n . D i v i s i o n occurs by a simple c o n s t r i c t i o n of the chloroplast envelope and the outermost photosynthetic lamella ( f i g . 35). The thylakoids appear to separate i n t o two during the process of d i v i s i o n . An anomaly to the previously described chloroplast structure has been observed i n one culture of Polysiphonia p a c i f i c a . When mature chloroplasts from a x i a l c e l l s were examined the chloroplasts were usually found to contain a variable number of appressed lamellae. From two to eight lamellae appear to associate and separate randomly at various points ( f i g . 34-). P e r i c e n t r a l c e l l s of t h i s same culture showed r e l a t i v e l y poor preservation of chloroplast u l t r a s t r u c t u r e . NUCLEUS The nucleus i s round to oval i n shape and from 3-6 u i n diameter ( f i g s . 9, 37-39). It possesses a prominent, c e n t r a l l y located, nucleolus ( f i g s . 9, 37-39). The a p i c a l c e l l i s uninucleate ( f i g s . 9,11), but the derivatives are sometimes binucleate. The nuclear matrix consists of both f i n e granular and coarse granular components ( f i g s . 37, 38). The f i n e granular portions of the nuclear matrix, may, i n part, correspond to chromatin ( f i g . 37). The nuclear envelope i s approximately 220 A t h i c k , but i t may exhibit extensive swelling between the inner and outer membranes of the envelope ( f i g . 37). Occasionally i n t e r r u p -tions i n the nuclear envelope, or nuclear pores, are observed. In face 19 view these pores appear to be approximately 400 A i n diameter ( f i g . 130. The nucleolus, a very densely s t a i n i n g structure, varies i n s i z e and shape ( f i g s . 37-39). I t i s usually oval to round and possesses a s l i g h t l y i r r e g u l a r margin ( f i g s . 9, 37-39). Present within the nucleolus are one or more regions of lower electron density with a matrix s i m i l a r to the nuclear matrix ( f i g s . 9, 37-39). These regions have been variously termed vacuoles (29), nucleolar inclusions (22) and internucleonema regions (6). In some sections such a region may give the nucleolus a d i s t i n c t r i n g - l i k e appearance ( f i g . 39). These vacuole? l i k e or low electron density regions are common i n the n u c l e o l i of plant c e l l s (29) and were frequently observed i n the n u c l e o l i of the algae investigated i n t h i s survey. INCLUSIONS C r y s t a l l i n e structures have been observed i n both Laurencia and Polysiphonia. These structures are absent from f i e l d material c o l l e c t e d and f i x e d i n winter and spring. In sectioned material c r y s t a l l i n e structures appear quite large, ranging from 0.5-1.5 u or more i n diameter, and possess three of more s t r a i g h t sides ( f i g . 36). No l i m i t i n g membrane has been found surrounding them. These structures appear to be composed of a very uniform, densely s t a i n i n g , granular material. 20 DISCUSSION CELL WALL Re l a t i v e l y l i t t l e work has been done on the u l t r a s t r u c t u r e of the c e l l w all of red algae using t h i n sectioning techniques. Thus i t i s too early to generalize on red a l g a l wall structure. However, a useless and complicated p r o l i f e r a t i o n of terminology should also be avoided. Where differences e x i s t between previously described species and those under consideration an attempt has been made to r e l a t e unique and previously described wall structures to the e x i s t i n g terminology. B i s a l p u t r a et a l . (2) have investigated the u l t r a s t r u c t u r e of the p e c t i c coat of the c e l l w a l l . They were able to d i s t i n g u i s h four layers or zones composing the p e c t i c coat, which were designated, s t a r t i n g from the outside, the A, B, C, and D layer s . These layers are s i m i l a r but not i d e n t i c a l to layers II and III of the p e c t i c coat and the D layer of the c e l l u l o s i c wall described i n t h i s paper. With a new s t a i n i n g procedure u t i l i z i n g a combination of ruthenium red and osmium tetroxide as a p o s t f i x a t i o n i t has been possible to show the p e c t i c coat of the c e l l w all i n considerably more d e t a i l . The outer-most lay e r , ( I ) , consists of loose bunches of m i c r o f i b r i l s extending from the surface of the second layer. The second l a y e r , ( I I ) , described here includes the A and C layers of Bisalputra et_ a l . (2). It consists of a zone of medium electron density, approximately 750 A i n thickness. Likewise the B layer described by Bisalputra et_ a l . (2) as a heavily s t a i n i n g layer i s s i m i l a r to the t h i r d l a y e r , ( I I I ) , presented here. 21 This B layer has been shown to consist of a densely s t a i n i n g band of m i c r o f i b r i l s extending from a s i m i l a r l y s t a i n i n g membrane-like structure. The fourth l a y e r , (IV), a densely stained membrane-like structure i n contact with the c e l l u l o s i c w a l l , i s described here f o r the f i r s t time. It forms the innermost layer of the p e c t i c portion of the c e l l wall; In addition to these four layers there i s a band of material occasionally seen associated with the p e c t i c coat of the c e l l w a l l . This layer has been termed the D band, and, when present, completely obscures the fourth layer of the p e c t i c coat. The D band consists of a densely s t a i n i n g amorphous ma t e r i a l , and i s analogous to the D layer described by B i s a l p u t r a et a l . (2). Recently Hanic and Craigie (20) have shown that the c u t i c l e i n Porphyra i s highly proteinaceous and comprised 1/50-1/100 of the c e l l w a l l . The p e c t i c coat described i n t h i s work from members of the Ceramiales constitutes a s i m i l a r amount of the t o t a l thickness of the c e l l w a l l . However, i t cannot be determined at t h i s time whether the c u t i c l e i s o l a t e d by Hanic and Craigie i s s i m i l a r to the e n t i r e p e c t i c coat, to j u s t one of the layers comprising i t , or i s not found at a l l in the algae used i n t h i s study. With the use of the ruthenium red-osmium tetroxide p o s t f i x a t i o n the r e t i c u l a t e nature of the m i c r o f i b r i l s i n the c e l l u l o s i c layer of the c e l l wall i s r e a d i l y apparent ( f i g . 43). In Pterosiphonia there appears to be an o r i e n t a t i o n of m i c r o f i b r i l s i n which the inner layer of m i c r o f i b r i l s has a s l i g h t l y d i f f e r e n t o r i e n t a t i o n than m i c r o f i b r i l s i n the r e s t of the c e l l w a l l . This can be seen i n f i g u r e 44 where the inner one-third of the c e l l u l o s i c wall appears to be almost a cross-s e c t i o n a l o r i e n t a t i o n of m i c r o f i b r i l s whereas i n the r e s t of the wall 22 the m i c r o f i b r i l s appear to be more l o n g i t u d i n a l l y oriented, The m i c r o f i b r i l s i n L2 also appear to be more compact than those i n LI. This o r i e n t a t i o n of m i c r o f i b r i l s can be explained i n one of two ways. It i s possible that there i s a l a y e r i n g of wall material r e s u l t i n g i n two layers of d i f f e r e n t l y oriented m i c r o f i b r i l s within the c e l l u l o s i c w all. Such a condition has not been reported as occurring i n any of the red algae thus f a r studied. The other p o s s i b i l i t y i s that layers LI and L2 may r e f l e c t the r e l a t i v e ages of these respective -layers of the c e l l u l o s i c w a l l . The d i f f e r e n t o r i e n t a t i o n and les s compact arrangement of m i c r o f i b r i l s i n LI merely r e f l e c t i n g the r e l a t i v e l y recent deposition of t h i s portion of the c e l l u l o s i c wall. This l a t t e r p o s s i b i l i t y seems l i k e l y since young and a c t i v e l y growing a p i c a l regions were used i n t h i s study. PIT STRUCTURE In t h i s survey two morphological stages of one basic p i t structure were found, the sing l e d i s c stage and the double d i s c stage p i t structures. The sing l e disc stage p i t structure consists of an aperture through the c e l l w all which i s blocked by a membrane-bound plug. The plug consists of an amorphous peripheral zone of material of v a r i a b l e thickness and density, enclosing a homogeneous f i n e l y granular material. This s i n g l e d i s c stage has been observed i n a l l Florideophycidae examined i n t h i s survey. The structure of the sing l e disc stage p i t structure i s i n agreement with reports on p i t u l t r a -structure by Bouck (5), Bischoff (4), Bisalputra et a l . (2) and Ramus (46). 23 Besides the sing l e d i s c stage p i t structure a second stage, the double di s c stage p i t structure, i s present i n those members of the family Ceramiaceae of the order Ceramiales examined i n t h i s survey, with the exception of Ceramium sp. i n which a x i a l c e l l s were not examined. The double disc.stage p i t structure d i f f e r e n t i a t e s from the sing l e disc stage p i t structure concurrent with r a p i d elongation and vacuolation. It d i f f e r s from the l a t t e r stage i n that material within the plug of the double d i s c stage p i t appears to be segregated into two discs separated by a region of low electron density. The distance at which the double d i s c stage p i t f i r s t occurs i n the a x i a l filament has not been determined. I t i s not present i n the a p i c a l c e l l nor i n early derivatives or c e l l s of l a t e r a l branchlets. It i s present, however, i n a x i a l c e l l s of Antithamnion which are at least twice as long as they are wide. The d i f f e r e n t i a t i o n of the double di s c stage p i t structure occurs at a l a t e r time i n the p i t s associated with the basal c e l l s of l a t e r a l branches. So f a r , the double d i s c stage p i t i s found i n only 1 c e l l type within one family of the order Ceramiales, which may also suggest the p o s s i b i l i t y of t h i s stage being a f i x a t i o n a r t e f a c t . Such an i n t e r p r e t a t i o n tends to be supported by the f a c t that i n comparison to other c e l l s , older a x i a l c e l l s of the Ceramiaceae could not be adequately f i x e d . It should be noted, however, that regardless of the f i x a t i o n s used and the degree of cytoplasmic preservation, p i t u l t r a -structure remained uniform i n a l l red algae except those i n the family Ceramiaceae. This seems to i n d i c a t e , therefore, that some sort of d i f f e r e n t i a t i o n has taken place i n the ce n t r a l region of the plug r e s u l t i n g i n the double di s c stage p i t structure. Furthermore, the 24 d i f f e r e n t i a t i o n of the double d i s c stage p i t occurs over a distance of several c e l l s , and various stages of d i f f e r e n t i a t i o n are chronologically-associated with a x i a l c e l l s of d i f f e r e n t ages. There i s also evidence f o r the occurrence of the double disc stage p i t from other researchers. Peyriere (42) has observed the u l t r a -structure of p i t s i n Ceramium. Though not discussing them i n her report, i t appears from the published micrograph ( r e f . 42, f i g . 7) that the p i t shown agrees i n appearance with an early stage i n the d i f f e r e n -t i a t i o n of the double d i s c stage p i t structure. Hawkins (21) has recently examined the p i t s i n Ceramium. She was able to discern that the plug was membrane-bound. The writer considers the p i t structures shown by her at the Phycological Society of America meetings held i n 1968 at Columbus, Ohio also correspond to early stages i n the d i f f e r e n t i a t i o n of the double di s c stage p i t . These reports and the lack of other data lend support to the r e s t r i c t i o n of the double di s c stage p i t to the family Ceramiaceae. Besides the r e s t r i c t e d occurrence of the double d i s c stage p i t there are two important differences between i t and the si n g l e d i s c stage p i t . The f i r s t i s that of the r e l a t i v e width of theppit structure i n the septum of the a x i a l c e l l . In the double di s c stage p i t , the p i t structure occupies only about one-eighth of the end w a l l , whereas i n comparable c e l l s which contain the single disc stage p i t almost the e n t i r e end wall i s composed of the p i t structure. In both instances the p i t connection between the a p i c a l c e l l and the f i r s t d e r i v a t i v e occupies less than one-eighth of the septum. The second difference may be due mainly to the composition of the plug i t s e l f . The s i n g l e d i s c stage p i t plug i s composed of an amorphous p e r i p h e r a l zone s u r r o u n d i n g a homogeneous f i n e l y g r a n u l a r m a t e r i a l . I n t h e d o u b l e d i s c s t a g e p i t t h e f i n e l y g r a n u l a r m a t e r i a l i s r e s t r i c t e d t o two a r e a s a l o n g t h e p e r i p h e r y o f t h e p l u g . The c e n t r a l r e g i o n i s o c c u p i e d by an e l e c t r o n t r a n s p a r e n t r e g i o n . T h i s r e g i o n c o u l d be t h e r e s u l t o f e i t h e r a l o s s o f m a t e r i a l d u r i n g f i x a t i o n , t h a t i s , a f i x a t i o n a r t e f a c t as d i s c u s s e d e a r l i e r , o r an a c t u a l d i f f e r e n t i a t i o n w i t h i n t h e p l u g . I n t h e l a t t e r c a s e t h i s c o u l d o c c u r i n one o f two ways. E i t h e r t h e r e i s a s p l i t t i n g o f t h e p l u g due t o an i n c r e a s e i n t h e s i z e o f t h e p l u g w i t h o u t an i n c r e a s e i n t h e p l u g m a t e r i a l , o r t h e r e i s a d e g r a d a t i o n o f m a t e r i a l w i t h i n t h e c e n t r a l r e g i o n o f t h e p l u g . S i n c e t h i s p l u g s t r u c t u r e i s l i m i t e d t o o l d e r a x i a l c e l l s and t h e r e i s o n l y a l i m i t e d i n c r e a s e i n t h e s i z e o f t h e p l u g i t seems l i k e l y t h a t t h i s d i f f e r e n t i a t i o n i s due t o a d e g r a d a t i o n o f m a t e r i a l i n t h e m i d r e g i o n o f t h e p l u g . E a r l i e r e l e c t r o n m i c r o s c o p i c s t u d i e s by Myers e t a l . (38) and Dawes e t a l . (12) d e s c r i b e d t h e o c c u r r e n c e o f two d i f f e r e n t p i t s t r u c -t u r e s i n t h e F l o r i d e o p h y c i d a e . Myers e t a l . ( 3 8 ) , u s i n g Rhodymenia and L a u r e n c i a , e q u a t e d t h e p i t s t r u c t u r e s t h e y d e s c r i b e d t o t h e G r i f f i t h s i a and P o l y s i p h o n i a t y p e s r e s p e c t i v e l y o f J u n g e r s ( 2 5 ) . However, t h e w r i t e r c o n c u r s w i t h Bouck ( 5 ) , who has p o i n t e d out t h a t t h e r e s o l u t i o n o f t h e i r m i c r o g r a p h s l e a v e s t h e p r o b l e m u n r e s o l v e d . An e x a m i n a t i o n o f t h e m i c r o g r a p h s o f Myers e t a l . (38) shows t h a t t h e p i t s t r u c t u r e d e s c r i b e d from Rhodymenia appears t o be an a l o g o u s t o t h e s i n g l e d i s c s t a g e p i t . The m i c r o g r a p h s o f t h e p i t s t r u c t u r e from L a u r e n c i a a r e o f i n s u f f i c i e n t r e s o l u t i o n t o make c o m p a r i s o n p o s s i b l e . Dawes e t a l . (12) s u r v e y e d t h e p i t s t r u c t u r e s i n s e v e r a l members o f 26 the Rhodophyta and reported two types of p i t s which they have termed "open" and "closed" p i t s . Unfortunately they used chemically cleared and macerated c e l l w all material which i s a technique i l l - s u i t e d f o r t h i s problem. Several algae studied by Dawes et a l . (12) (Polysiphonia, Ceramium, Pterosiphonia) are shown i n t h i s study to contain the single disc stage p i t structure. The writer considers, therefore, that the open pit-may be analogous to the sing l e d i s c stage p i t structure. There i s not enough evidence to substantiate the occurrence of the closed p i t reported by Dawes et_ al_. (12). It i s necessary, nevertheless, to repeat t h e i r survey before any d e f i n i t e conclusion can be made. Ramus (46, 47) showed that the plasmalemma was continuous from one c e l l to the next through the p i t aperture and that the p i t membrane joined the plasmalemma at the rim of the aperture. The amorphous perip h e r a l zone was divided i n t o two parts separated by the p i t membrane (46). In the present survey the plasmalemma was also seen to be continuous through the p i t aperture. With respect to the p i t membrane the r e s u l t s are s l i g h t l y d i f f e r e n t . The p i t membrane shows up very c l e a r l y across the face of the plug, and i s not fi r m l y attached to i t . It occupies the region immediately adjacent to the outer •surface of the perip h e r a l amorphous zone of the plug. It i s possible that both membrane associations occur, since Ramus made h i s observations on a member of the Nemalionales whereas the observations i n the present survey are based on members of the Ceramiales. In a survey of the l i t e r a t u r e on p i t u l t r a s t r u c t u r e i t can be seen that the sing l e d i s c stage p i t occurs inijthe Nemalionales (4, 46) and Rhodymeniales (5, 38) as well as the Ceramiales and Gigartinales reported i n t h i s survey. It seems l i k e l y , therefore, that the sing l e 27 d i s c stage p i t i s the most common p i t structure i n the Florideophycidae, since i t s presence has been established i n four of the s i x orders i n t h i s c l a s s . I t i s also evident i n the present study that the single d i s c stage p i t can be s t r u c t u r a l l y modified to produce another morpho-l o g i c a l l y d istinguishable stage - the double di s c stage p i t structure. At present t h i s double di s c stage p i t i s r e s t r i c t e d to one family of the order Ceramiales. Ramus (47) has found that the cross wall region surrounding the p i t can also undergo modification to produce an a d d i t i o n a l v a r i a t i o n i n p i t morphology. He has observed the deposition of material on the septum around the periphery of the aperture to produce a d i s t i n c t r i n g - l i k e swelling surrounding the p i t . This type of structure might possibly account f o r the desc r i p t i o n of the p i t as rings by Muldorf (36) and Ky l i n (28). It i s i n t e r e s t i n g to compare the r e s u l t s obtained i n t h i s survey with the e a r l i e r work of Jungers (25). He proposed that two types of p i t s e x i s t i n the red algae, the Polysiphonia and the G r i f f i t h s i a p i t s . These p i t types appear to be analogous to the sing l e d i s c stage and the double d i s c stage p i t s r e s p e c t i v e l y of t h i s survey. It appears that the Polysiphonia and G r i f f i t h s i a p i t s can be explained i n a manner s i m i l a r to that f o r the sing l e disc stage and double di s c stage p i t s . That i s , the G r i f f i t h s i a p i t (double d i s c stage) i s d i f f e r e n t i a t e d from the Polysiphonia type p i t (single disc stage). There are several s i m i l a r i t i e s between t h i s survey and the work of Jungers which seem to support such an i n t e r p r e t a t i o n . F i r s t l y , the Polysiphonia p i t can occupy almost the entire septum of the end wall 28 i n a x i a l c e l l s , whereas, i n algae with p i t structures s i m i l a r to the G r i f f i t h s i a type, p i t structures occupy only a small portion of the end wall septum. The same r e l a t i o n s h i p occurs between the s i n g l e d i s c stage and the double d i s c stage p i t s . Secondly, the Polysiphonia p i t was described by Jungers from Polysiphonia and Delesseria, both shown to contain the s i n g l e disc stage p i t i n t h i s survey. T h e ' G r i f f i t h s i a p i t was described from G r i f f i t h s i a and Ceramium, both members of the Ceramiaceae. The double disc stage p i t was also described i n t h i s survey from members of the Ceramiaceae. T h i r d l y , by comparing the drawings of the G r i f f i t h s i a p i t by Jungers with the electron micrographs of the double d i s c p i t i t can be seen that the d e s c r i p t i o n of the double disc stage p i t presented here also f i t s the d e s c r i p t i o n f o r the G r i f f i t h s i a p i t ( r e f . 25, f i g s . 15, 16). Fourthly, the s i n g l e d i s c stage p i t described i n t h i s survey and as described by others (2, 4, 5, 46) shows quite c l e a r l y that the Polysiphonia p i t consists of a single homogeneous plug blocking the p i t aperture. It i s proposed that the terms "single d i s c stage p i t " which includes Jungers' Polysiphonia p i t and most p i t structures described i n the l i t e r a t u r e , and the "double d i s c stage pit," which includes Jungers! G r i f f i t h s i a p i t , be adopted. The use of t h i s terminology seems more appropriate since these terms are based upon a morphological de s c r i p t i o n of p i t structures rather than the group of algae i n which they were f i r s t described. The terminology proposed would eliminate the use of species names f o r structures which are of general occurrence i n the Florideophycidae. These terms also eliminate the problem a r i s i n g 29 from the occurrence of more than one stage of p i t structure i n the same alga, as occurs i n Antithamnion and Platythamnion species. CONCLUSIONS 1. The c e l l w all has been described i n considerable d e t a i l following the use of ruthenium red-osmium tetroxide p o s t f i x a t i o n . The c e l l u l o s i c portion of the c e l l wall i s composed of a r e t i c u l a t e arrangement of m i c r o f i b r i l s . In Pterosiphonia t h i s r e t i c u l a t e arrangement of m i c r o f i b r i l s can be subdivided into two regions: an inner region where the m i c r o f i b r i l s appear to be mostly obliquely sectioned and an outer and much thicker region where the micro-f i b r i l s are mostly l o n g i t u d i n a l l y oriented. The mucilaginous or p e c t i c coat of the c e l l w all appears quite complex. It has been found to be composed of four d i s t i n c t i v e l y structured l a y e r s . In a d d i t i o n , a f i f t h l ayer, which i s associated with the outermost portion of the c e l l u l o s i c w a l l , i s sometimes present. 2. Two stages of one p i t structure have been described from the red algae examined i n t h i s survey, the single d i s c stage and the double disc stage p i t structures. 3. The s i n g l e d i s c .stage p i t occurs i n a l l algae investigated. Most p i t structures reported i n the l i t e r a t u r e can be equated to t h i s stage. It occurs i n four of the s i x orders of the Florideophycidae. An extensive i n v e s t i g a t i o n of a l l t i s s u e types, except the holdfast region, i n Polysiphonia showed only the s i n g l e d i s c stage p i t . The occurrence of minor v a r i a t i o n s i n the groove region of the plug have been reported. 30 4 . The double disc stage p i t has been reported and described from u l t r a s t r u c t u r e . I t has been found i n older a x i a l c e l l s of members of the family Ceramiaceae of the order Ceramiales, It d i f f e r s i n structure from the'single disc stage p i t i n that the matrix of the plug of the double d i s c stage p i t i s segregated i n t o two discs separated by a region of low electron, density, which i s i n contrast to the homogeneous matrix of the si n g l e d i s c stage p i t . The d i f f e r e n t i a t i o n of t h i s double d i s c stage from that of the si n g l e d i s c stage p i t has been described and postulated to take place by the degradation of plug material. 5. The plug i s membrane-bound. The plasmalemma passes through the aperture of the p i t . The p i t membrane i s loosely appressed to the outermost face of the plug and jo i n s with the plasmalemma at the edge of the p i t aperture. 6. The p i t structures presented here substantiate Jungers' e a r l i e r l i g h t microscope work. His o r i g i n a l d e s c r i p t i o n s , however, have been c l a r i f i e d and extended with the use of electron microscopy. The si n g l e disc stage and the double d i s c stage p i t s used here correspond to Jungers' Polysiphonia and G r i f f i t h s i a p i t s r e s p e c t i v e l y . In a d d i t i o n , i t has been proposed that the terms "s i n g l e d i s c stage p i t " and "double disc stage p i t , " based on an u l t r a s t r u c t u r a l d e s c r i p t i o n of these s t r u c t u r e s , replace the e a r l i e r terms used by Jungers. 7. Cytoplasmic d e t a i l i n Antithamnion, Polysiphonia and Pterosiphonia species .was found to not d i f f e r s i g n i f i c a n t l y from each other and other red algae. A c r y s t a l l i n e s t ructure , which d i f fe rs from most other reports s i m i l a r structures i n plants by the absence of a surrounding membrane, has been reported i n Polysiphonia and Laurencia. 32 BIBLIOGRAPHY 1. B i s a l p u t r a , T. P e r s o n a l communication. 2. , P. C. R u s a n o w s k i , and W. S..Walker. 1967. S u r f a c e a c t i v i t y , c e l l w a l l , and f i n e s t r u c t u r e o f p i t c o n n e c t i o n s i n t h e r e d a l g a L a u r e n c i a s p e c t a b i l i s . J . U l t r a s t r u c t . Res. 20: 277-289, 3. , and T. E. Weier. 1963. The c e l l w a l l o f Scenedesmus q u a d r i c a u d a . Amer. J . B o t . 50: 1011-1019. 4. B i s c h o f f , H. W. 1965. Thorea r e i k e i s p. nov. and r e l a t e d s p e c i e s . J . P h y c o l . 1: 111-117. 5. Bouck, G. B. 1962. Chromatophore development, p i t s , and o t h e r f i n e s t r u c t u r e i n t h e r e d a l g a , L o m e n t a r i a b a i l e y a n a ( H a r v . ) F a r l o w . J . C e l l B i o l . 12: 553-569. 6. B r e s n i c k , E., and A. S c h w a r t z . 1968. F u n c t i o n a l dynamics o f t h e C e l l . Academic P r e s s , New Y o r k . pp. 165-216. 7. Chadefaud, M. 1962. Sur q u e l q u e s d e t a i l s de 1 ' o r g a n i s a t i o n m o r p h o l o g i q u e des p a r o i s c e l l u l a i r e s chez l e s F l o r i d e e s f i l a m e n t u e s e s . B u l l Soc. B o t . F r a n c e . 109: 148-156. 8. C h i h a r a , M. P e r s o n a l communication. 9. Conn, H. J . , M. A. Darrow and V. M. Emmel. 1960. ' S t a i n i n g P r o c e d u r e s . B i o l o g i c a l S t a i n Commission, U n i v e r s i t y o f R o c h e s t e r M e d i c a l C e n t e r , R o c h e s t e r , N. Y. , W i l k i n s Co. , B a l t i m o r e . 10. D a l t o n , A. J . 1955. A chrome-osmium f i x a t i v e f o r e l e c t r o n m i c r o s c o p y . A n a t . Rec. 121: 281. 11. Dawes, C. J . , F. M. S c o t t and E. B o w l e r . 1960. L i g h t and e l e c t r o n m i c r o s c o p e s t u d y o f c e l l w a l l s o f brown and r e d a l g a e . S c i e n c e , 132: 1663-1664. 12. :[ ••. , and . 1961. A l i g h t and e l e c t r o n m i c r o s c o p e s u r v e y o f a l g a l c e l l w a l l s . Amer. J . B o t . 48: 925-934. 13. D e s i k a c h a r y , T. V. 1960. S u b m i c r o s c o p i c morphology o f a l g a e . I n P r o c e e d i n g s o f t h e Symposium on A l g a e , New D e l h i , 1959. P_. K a c h r o o , ed. I n d i a n C o u n c i l o f A g r i c u l t u r a l R e s e a r c h . Job P r e s s L t d . Kanpur. pp. 70-77. 14. D i x o n , P. S. P e r s o n a l c o m m u n i c a t i o n . 33 15. D o t y , N. S. 1947. The marine a l g a e o f Oregon. P t . I I Rhodophyceae. F a r l o w i a 3: 159-215. 16. Drew, K. M. 1951. Rhodophyta. I n Manual o f P h y c o l o g y . G. M. S m i t h , ed. R o n a l d P r e s s Co. New Y o r k . pp. 167-192. 17. F a l k e n b e r g , P. 1901. D i e Rhodomelaceen des G o l f e s von N e a p e l und d e r angrezenden M e e r e s a b s c h n i t t e . Fauna und F l o r a des G o l f e s von N e a p e l . Bd. 26, B e r l i n . ( n o t s e e n , c i t e d f r o m r e f . 2 8 ) . 18. F r a s e r , B. P e r s o n a l c o m munication. 19. F r i t s c h , F. E. 1945. The S t r u c t u r e and R e p r o d u c t i o n o f t h e A l g a e , volume I I . Cambridge U n i v e r s i t y P r e s s , 939 pp. 20. H a n i c , L. A., and J . S. C r a i g i e . 1969. S t u d i e s on t h e a l g a l c u t i c l e . J . P h y c o l . 5: 89-102. 21. Hawkins, E. K. 1968. C e l l c o n t a c t i n t h e r e d a l g a e : t h e f i n e s t r u c -t u r e o f t h e p i t c o n n e c t i o n s i n Ceramium diaphanum. ( A b s t r . ) J . P h y c o l . 4 ( s ) : 6. 22. Hay, E. D. 1968. S t r u c t u r e and f u n c t i o n o f t h e n u c l e o l u s i n d e v e l o p i n g c e l l s . I n U l t r a s t r u c t u r e i n B i o l o g i c a l Systems. A. J . D a l t o n and F. Haguenau, eds. Academic P r e s s . New Y o r k . 3: 474-483. 23. H o l l e n b e r g , G. J . 1942. An a c c o u n t o f t h e s p e c i e s o f P o l y s i p h o n i a on t h e P a c i f i c Coast o f N o r t h A m e r i c a . I . O l i g o s i p h o n i a . Amer. J . B o t . 29: 772-785. 24. . 1944. An a c c o u n t o f t h e s p e c i e s o f P o l y s i p h o n i a on t h e P a c i f i c Coast o f N o r t h A m e r i c a . I I . P o l y s i p h o n i a . Amer. J . Bot.' 31: 474-483. 25. J u n g e r s , V. 1933. Recherches s u r l e s plasmodesmes chez l e s v e g e t a u x . I I . Les synapses des a l g u e s r o u g e s . La C e l l u l e 17: 1-28. 26. K r e g e r , D. R. 1962. C e l l w a l l s . I n P h y s i o l o g y and B i o c h e m i s t r y o f t h e A l g a e . R. A. L e w i n , ed. Academic P r e s s . New Y o r k , pp. 315-336. 27. K y l i n , H. 1925. The marine a l g a e i n t h e v i c i n i t y o f t h e b i o l o g i c a l s t a t i o n a t F r i d a y H a r b o r , Washington. Lunds U n i v . A r s s k r . N. F. Avd. 2, 2 1 ( 9 ) : 1-87. 28. 1956. D i e G a t t u n g e n Der Rhodophyceen. CWK G l e e r u p s F o r l a g . Lund. 673 pp. 34 29. L a f o n t a i n e , J . G. 1968. S t r u c t u r a l components o f t h e n u c l e u s i n m i t o t i c p l a n t c e l l s . I n U l t r a s t r u c t u r e i n B i o l o g i c a l Systems. A. J . D a l t o n and F. Haguenau, eds. Academic P r e s s . New Y o r k . 3: 152-196. 30. L e a k , L. V. 1967. F i n e s t r u c t u r e o f t h e ' m u c i l a g i n o u s s h e a t h o f Anabaena sp. J . U l t r a s t r u c t . Res. 21; 61-74. 31. L e w i n , J.. 1966. S i l i c o n m e t a b o l i s m i n d i a t o m s . V. Germanium d i o x i d e , a s p e c i f i c i n h i b i t o r o f d i a t o m growth. P h y c o l o g i a 6: 1-12. 32. L i c h t e n t h a l e r , H. K. 1968. P l a s t o g l o b u l i and t h e f i n e s t r u c t u r e o f p l a s t i d s . Endeavor 27: 144-149. 33. L u f t , J . H. 1965. F i n e s t r u c t u r e o f c a p i l l a r i e s : t h e e n d o c a p i l l a r y l a y e r . A n a t . Rec. 151: 380. 34. Mangenot, G. 1924. Sur l e s communications p r o t o p l a s m i q u e s dans 1' a p p a r e i l sporogene de q u e l q u e s F l o r i d e e s . Rev. A l g o l . 1: 376. ( n o t s e e n , c i t e d f r o m r e f . 1 9 ) . 35. M i r a n d a , F. 1930. Las Communicaciones i n t e r p r o t o p l a s m i c a s . e n " B o r n e t i a s e c u n d i f l o r a " ( J . Agardh.) T h u r e t . Ebenda, T. 30. (not s e e n , c i t e d from r e f . 2 8 ) . 36. M u l d o r f , A. 1937. Das p l a s m a t i s c h e wesen d e r p f l a n z l i c h e n z e l l b r u c k e n . B e i h . B o t ; C e n t r a l b l a t t A . 56: 171-364. (not s e e n , c i t e d from r e f . 2 8 ) . 37. M y e r s , A., R. D. P r e s t o n , F. R. S. and G. W. R i p l e y . 1956. F i n e s t r u c t u r e i n t h e r e d a l g a e . I . x - r a y and e l e c t r o n m i c r o s c o p e i n v e s t i g a t i o n o f G r i f f i t h s i a f l o s c u l o s a . P r o c . Roy. Soc. London, B/ 144: 450-459. 38. . 1959. An e l e c t r o n m i c r o s c o p e i n v e s t i g a t i o n i n t o t h e s t r u c t u r e o f t h e f l o r i d e a n p i t . Ann. B o t . N. S. 23: 257-262. 39. N i c h o l s , H. W., J . E. Ridgeway and H . C. B o l d . 1966. A p r e l i m i n a r y u l t r a s t r u c t u r a l s t u d y o f t h e f r e s h w a t e r r e d a l g a Compsopogon. Ann. M i s s o u r i B o t . Gard. 53: 17-27. 40. N o r t h c o t e , D. H. 1963. The b i o l o g y and c h e m i s t r y o f t h e c e l l w a l l s o f h i g h e r p l a n t s , a l g a e , and f u n g i . I n I n t e r n a t i o n a l Review o f  C y t o l o g y . G. H. Bourne and J . F. D a n i e l l i , eds. Academic P r e s s . New Y o r k . 14: 223-259. 41. P e a s e , D. 1964. H i s t o l o g i c a l T e c h n i q u e s f o r E l e c t r o n M i c r o s c o p y . Academic P r e s s , New Y o r k . 42. P e y r i e r e , M. 1963. Les p l a s t e s e t l ' a m i d o n f l o r i d e e n chez q u e l q u e s Rhodophycees. C. R. Acad. S c i . P a r i s 257: 730-732. 35 43. P h i l l i p s , R. C , and R. L. Vadas. 1967. M a r i n e a l g a e o f Whidbey I s l a n d , Washington. J . I n s t . R es., S e a t t l e P a c i f i c C o l l e g e . A ( 4 ) . 82 pp. 44. P r e s t o n , R. D. 1959. W a l l s t r u c t u r e o f marine a l g a e . P r o c . IX I n t e r n . B o t . Congr. ( A b s t r . ) 2: 310. 45. P r o v a s o l i , L. 1962. H a s k i n s L a b o r a t o r y B u l l e t i n . 46. Ramus, J . 1969. P i t c o n n e c t i o n f o r m a t i o n i n t h e r e d a l g a Pseudo-g l o i o p h l e a . J . P h y c o l . 5: 57-63. 47. . 1969. P i t c o n n e c t i o n d i morphism i n t h e r e d a l g a Pseudo-g l o i o p h l e a . J . C e l l B i o l . 41: 340-345. 48. R e y n o l d s , E. S. 1963. The use o f l e a d c i t r a t e a t h i g h pH as an e l e c t r o n - o p a q u e s t a i n i n e l e c t r o n m i c r o s c o p y . J . C e l l B i o l . 17: 208-213. 49. R o e l o f s e n , P. A. 1959. The p l a n t c e l l w a l l . I n E n c y c l o p e d i a o f P l a n t Anatomy. W. Zimmerman and P. G. Ozenda, eds. B o r n t r a e g e r , B e r l i n . 3 ( 4 ) . 50. S a b a t i n i , D. D., K. B e n s c h , and R. J . B a r r n e t t . 1963. C y t o c h e m i s t r y and e l e c t r o n m i c r o s c o p y . The p r e s e r v a t i o n o f c e l l u l a r u l t r a s t r u c t u r e and e n z y m a t i c a c t i v i t y by a l d e h y d e f i x a t i o n . J . C e l l B i o l . 17: 19-58. 51. S c a g e l , R. F. 1957. An a n n o t a t e d l i s t o f t h e ma r i n e a l g a e o f B r i t i s h C o l u m b i a and N o r t h e r n Washington. Nat. Museum Canada B u l l . No. 150. 289 pp. 52. S c h m i t z , F. 1883. Untersuchungen u b e r d i e b e f r u c h t u n g d e r f l o r i d e e n . S i t z u n g s b e r . Akad. W i s s e n s c h . B e r l i n , I . ( n o t s e e n , c i t e d from r e f . 3 8 ) . 53. S m i t h , G. M. 1944. M a r i n e A l g a e o f t h e Monterey P e n i n s u l a C a l i f o r n i a . S t a n f o r d U n i v e r s i t y P r e s s , S t a n f o r d , C a l i f o r n i a , pp. 256-381. 36 APPENDIX I CULTURE FORMULAE I . P r o v a s o l i ES e n r i c h m e n t (4-5). ES e n r i c h m e n t : S t o c k Amount o f s o l u t i o n s t o c k / 1 0 0 ml H 20 NaN0 3 , . . 350 mg/100 ml 10 ml . .. 50 ti 1 ml Fe (as EDTA; 1:1 m o l a r ) 1 25 ml P I I m e t a l s 2 25 ml . . 10 ug/100 ml 1 ml . . 0.5 mg/100 ml 1 ml . . 5 ug/100 ml 5 ml mg/100 ml 5 ml The ES e n r i c h m e n t s o l u t i o n i s t h e n f i l t e r e d , u s i n g m i l l i p o r e f i l t e r s number GSWP 047 00, and s t o r e d a t 4- C. Use 2 m l . o f ES e n r i c h m e n t f o r each 100 ml o f f i l t e r e d and steamed s e a w a t e r . 1 D i s s o l v e 351 mg. Fe(NH^) 2.6H 20 and 330 mg. o f Na 2EDTA i n 500 m l . d i s t i l l e d w a t e r . One m l . o f t h i s s o l u t i o n e q u a l s 0.1 mg. Fe. 2 P I I m e t a l s : d i r e c t i o n s f o r making 100 ml o f m e t a l mix. boron - HgBOg 0;114 g. i r o n - F e C l 3 . 6 H 2 0 4.9 mg. Manganese - MnS0^.4H 20 16.4 mg. z i n c - ZnS0 1 +.7H 20 2.2 mg. c o b a l t - C o S O ^ f ^ O 0.48 mg. Na 2EDTA 100 mg. 37 I I . C h i h a r a m a r i n e media I I ( 8 ) . se a w a t e r 1000 ml. min o r e l e m e n t s 2 m l . NaNOg 0.2 g, NaH 2P0 4.12H 20 0.025 g. min o r e l e m e n t s : d i s t i l l e d w a t e r • 1000 ml. Na 2EDTA . . . • 3.0 g. F e C l 3 .0.08 g. M n C l 2 0.12 g. Z n C l 2 0.015 g. C o C l 2 0.003 g. C u C l 2 0.0012 g. Na 2Mo0 1 +.2H 20 0.05 g. H 3 B 0 3 .0.6 g. I I I . P r e p a r a t i o n o f germanium d i o x i d e a d d i t i v e ( 1 4 ) . G e 0 2 (BDH) 1.0 g. d i s t i l l e d w a t e r 1000 m l . Shake i n t e r m i t t e n t l y f o r 3 days. Use 1-3 m l . p e r l i t e r o f pre-p a r e d media. 38 APPENDIX I I LIGHT MICROSCOPE TECHNIQUE AND STAINS I . S t a i n i n g t e c h n i q u e : Use \ - 3g m i c r o n s e c t i o n s . 1. soak i n e t h y l e n e d i c h l o r i d e f o r 1 m i n u t e . 2. r i n s e i n d i s t i l l e d w a t e r and t h e n i n 50% a c e t o n e . 3. heat s l i d e u n t i l d r y . 4. s t a i n f o r 1-2 m i n u t e s - h e a t s l o w l y d u r i n g s t a i n i n g u n t i l steam r i s e s f r o m t h e l i q u i d on t h e s l i d e . 5. r i n s e i n d i s t i l l e d w a t e r 3 t i m e s . 6. h e a t s l i d e u n t i l d r y . 7. r e p e a t s t e p s 4-6 : i f 2 s t a i n s a r e employed. 8. mount i n immersion o i l o r permount. I I . S t a i n s : 1. B a s i c f u c h s i n - c r y s t a l v i o l e t c o m b i n a t i o n ( 1 8 ) . Make up 1% aqueous s o l u t i o n s o f b a s i c f u c h s i n and c r y s t a l v i o l e t , f i l t e r w e l l b e f o r e use. T r e a t s e c t i o n s w i t h b a s i c f u c h s i n and t h e n c r y s t a l v i o l e t . 2. T o l u i d i n e b l u e and b o r a x . Make up a 1% aqueous s o l u t i o n o f t o l u i d i n e b l u e i n a weak b o r a x s o l u t i o n . 3. M e t h y l g r e e n w i t h p y r o n i n ( 9 ) . me'thyl g r e e n l . O g . p y r o n i n y o r b 0.25 g. 95% a l c o h o l 5.0 m l . " g l y c e r o l 20 m l . 2% aqueous p h e n o l 100 ml. 39 APPENDIX I I I ELECTRON MICROSCOPY TECHNIQUE AND FORMULAE I . S t a n d a r d t e c h n i q u e : p r i m a r y f i x a t i o n . 1 hour wash 3-6 t i m e s i n b u f f e r 1-3 h o u r s p o s t f i x a t i o n . 1 hour wash i n a s o l u t i o n o f \ b u f f e r and Jg d i s t i l l e d w a t e r 2 t i m e s Jg h o u r wash t h r o u g h a g r a d e d s e r i e s o f s e a w a t e r ( 3 0 % , 15%) t o d i s t i l l e d w a t e r . . . . . . . Jg hour a l c o h o l d e h y d r a t i o n s e r i e s ( 3 0 % , 50%, 70%, 90%, 3 changes o f 100%) 2*g hours 50% a l c o h o l - 5 0 % p r o p y l e n e o x i d e wash \ h o u r 3 changes o f 100% p r o p y l e n e o x i d e 1 hour i n f i l t r a t e t o 50% maraglas by w/v 4 hours l e a v e o v e r n i t e i n 50% m a r a g l a s ( o p t i o n a l ) t r a n s f e r f t o 75% m a r a g l a s by w/v 1 h o u r t r a n s f e r t o 100% m a r a g l a s 2-3 hours change t o f r e s h 100% m a r a g l a s and embed i n c a p s u l e s ; c u r e a t 65-70 C f o r 24- h o u r s . I I . P r e p a r a t i o n o f p r i m a r y f i x a t i v e s : 1. n e u t r a l i z e d g l u t a r a l d e h y d e . Use 25 o r 50% g l u t a r a l d e h y d e s t o c k s o l u t i o n . Add enough b a r i u m c a r b o n a t e t o produce a 3% c o n c e n t r a t i o n i n t h e above s o l u t i o n . Shake i n t e r m i t t e n t l y f o r 1-2 h o u r s , t h e n f i l t e r u n t i l c l e a r . Check pH ( 6 - 7 ) ; i f l o w , r e p e a t above p r o c e d u r e . 40 2. f o r m a l d e h y d e ( 4 1 ) . Add p a r a f o r m a l d e h y d e t o d i s t i l l e d w a t e r by weight-volume up t o a c o n c e n t r a t i o n o f 40%. Heat t h e s o l u t i o n t o 65 C and add c o n c e n t r a t e d NaOH u n t i l t h e s o l u t i o n t u r n s c l e a r . 3. r u t h e n i u m red-osmium t e t r o x i d e f i x a t i v e ( m o d i f i e d from r e f s . 30, 3 3 ) . Add t o a 1% osmium t e t r o x i d e s o l u t i o n i n d i s t i l l e d w a t e r ( o r b u f f e r made w i t h d i s t i l l e d w a t e r ) enough r u t h e n i u m r e d (w/v) t o produce a f i n a l c o n c e n t r a t i o n o f 0.1% r u t h e n i u m r e d . L e t s t a n d 15 mi n u t e s b e f o r e u se. I I I . B u f f e r s and embedding media: 1. sodium c a c o d y l a t e I ( a f t e r r e f . 5 0 ) . sodium c a c o d y l a t e 2.14 g. d i s t i l l e d w a t e r 10 ml. f i l t e r e d s e a w a t e r 90 m l . A d j u s t t h e pH t o 6.8 w i t h H C l . 2. sodium c a c o d y l a t e I I ( 1 ) . sodium c a c o d y l a t e 1 g, d i s t i l l e d w a t e r 25 ml. f i l t e r e d s e a w a t e r 25 m l . A d j u s t t h e pH t o 6.8 w i t h H C l . 3. D a l t o n ' s b u f f e r ( a f t e r r e f . 1 0 ) . P r e p a r e a 5% w/v s o l u t i o n o f p o t a s s i u m d i c h r o m a t e . A d j u s t t h e pH t o 6.8 w i t h 2.5N KOH, and d i l u t e t o 4% w i t h d i s t i l l e d w a t e r . M i x 1 p a r t o f above s o l u t i o n w i t h 1 p a r t o f f i l t e r e d s e a w a t e r . Use 1 p a r t o f t h i s b u f f e r and 1 p a r t f i l t e r e d s e a w a t e r t o make f i x a t i o n b u f f e r . 4. ma r a g l a s ( 3 ) . Ma r a g l a s 655 18.3 g. C a r d o l i t e NC 513 7.2 g. DMP 30 0.8 g. Cure a t 65-70 C f o r 24 h o u r s . 41 KEY TO ABBREVIATIONS I , I I , I I I , IV - l a y e r s o f t h e m u c i l a g i n o u s w a l l AC - a p i c a l c e l l AM - amorphous m a t e r i a l a s s o c i a t e d w i t h groove o f p i t CE - c h l o r o p l a s t e n v e l o p e CH - c h l o r o p l a s t CR - c e n t r a l r e g i o n i n d o u b l e d i s c s t a g e p i t CS - c r y s t a l l i n e s t r u c t u r e CW - c e l l w a l l , c e l l u l o s i c CY - c y t o p l a s m D l , D2 - i n d i v i d u a l d i s c s o f d o u b l e d i s c s t a g e p i t D l - d i c t y o s o m e E - amorphous D l a y e r o f m a t e r i a l a s s o c i a t e d w i t h t h e o u t e r m o s t p o r t i o n o f t h e c e l l u l o s i c w a l l F - DNA f i b r i l s G - groove o f p i t L I - i n n e r p o r t i o n o f c e l l u l o s i c w a l l L2 - o u t e r p o r t i o n o f c e l l u l o s i c w a l l M - plasmalemma MI - m i t o c h o n d r i o n ML - m i d d l e l a m e l l a MW - m u c i l a g i n o u s c o a t N - n u c l e u s NI - n u c l e o l a r i n c l u s i o n NU - n u c l e o l u s OL - o u t e r m o s t p h o t o s y n t h e t i c l a m e l l a P - p l u g PM - p i t membrane PP - p r o p l a s t i d PZ - amorphous p e r i p h e r a l zone o f p l u g R - r i d g e i n groove r e g i o n o f p l u g S - f l o r i d e a n s t a r c h g r a i n T - t o n o p l a s t TC - t r i c h o b l a s t f i l a m e n t c e l l TS - t r a n s p a r e n t space V - v a c u o l e ZD - zone o f d i f f e r e n t i a t i o n 42 PLATE 1 f i g . 1 A n t i t h a m n i o n s p . showing g e n e r a l morphology o f t h e a p i c a l r e g i o n and dome shaped a p i c a l c e l l . P i t c o n n e c t i o n s and c y t o p l a s m i c d e t a i l a r e n o t r e a d i l y a p p a r e n t . L i v i n g m a t e r i a l . x l 8 0 . f i g . 2 P o l y s i p h o n i a s p . showing morphology o f t h e a p i c a l r e g i o n and th e o c c u r r e n c e o f t r i c h o b l a s t f i l a m e n t s . The a p i c a l c e l l i s c o l o r l e s s and dome shaped. L i v i n g m a t e r i a l . x40. f i g . 3 P_. p a n i c u l a t a showing t h e a p i c a l r e g i o n and a p i c a l c e l l i n median l o n g i t u d i n a l s e c t i o n . The amount o f v a c u o l a t i o n o b s e r v e d i n c r e a s e s w i t h t h e d i s t a n c e from t h e apex o f t h e t h a l l u s . N u c l e i , w i t h n u c l e o l i , a r e r e a d i l y v i s i b l e . G l u t a r a l d e h y d e f i x a t i o n i n sodium c a c o d y l a t e b u f f e r and s t a i n e d w i t h b a s i c f u c h s i n - c r y s t a l v i o l e t . x780. f i g . 4 P. p a n i c u l a t a showing o l d e r a p i c a l r e g i o n and arrangement o f t r i c h o b l a s t f i l a m e n t . Note p i t c o n n e c t i o n s between t r i c h o -b l a s t c e l l s . The m i d d l e l a m e l l a i s a l s o c l e a r l y v i s i b l e i n th e t r i c h o b l a s t c e l l s . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . S t a i n e d w i t h m e t h y l g r e e n - p y r o n i n . x 750. 43 PLATE 2 f i g . 5 P_. p a n i c u l a t a showing a x i a l p i t c o n n e c t i o n s ( s i n g l e d i s c s t a g e p i t ) . N u c l e i , w i t h d e n s e l y s t a i n e d n u c l e o l i , occupy t h e c e n t r a l r e g i o n s o f t h e c e l l s . G l u t a r a d e h y d e f i x a t i o n i n sodium c a c o d y l a t e b u f f e r and s t a i n e d w i t h b a s i c f u c h s i n -c r y s t a l v i o l e t . " x l 9 0 0 . f i g . 6 P_. p a n i c u l a t a showing n u c l e o l i ( a r r o w s ) w h i c h have a r i n g - l i k e appearance due t o n u c l e o l a r i n c l u s i o n s . G l u t a r a l d e h y d e f i x a t i o n i n sodium c a c o d y l a t e b u f f e r and s t a i n e d w i t h t o l u i -d i n e b l u e w i t h b o r a x . x2000. f i g . 7 A_. s u b u l a t u m showing a x i a l p i t c o n n e c t i o n ( d o u b l e d i s c s t a g e p i t ) and w a l l l a m e l l a e i n a mature a x i a l c e l l . Note t h e e x t r e m e l y l a r g e v a c u o l e r e g i o n . The p l u g o c c u p i e s a p p r o x i -m a t e l y 1/8 o f t h e c e l l w a l l . A l a t e r a l b r a n c h can be seen i n c r o s s s e c t i o n t o t h e l e f t o f t h e a x i a l f i l a m e n t . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium . c a c o d y l a t e b u f f e r . S t a i n e d w i t h b a s i c f u c h s i n - c r y s t a l v i o l e t . x900. f i g . 8 A_. s u b u l a t u m showing d e t a i l o f a x i a l p i t c o n n e c t i o n ( d o u b l e d i s c s t a g e p i t ) . The d i s c s o f t h e p i t a l m o s t c o m p l e t e l y d i s a p p e a r i n t h e groove r e g i o n o f t h e p l u g ( a r r o w ) . Note t h a t t h e p l u g o c c u p i e s o n l y a s m a l l p o r t i o n o f t h e c e l l w a l l . F i x a t i o n and s t a i n i n g as i n f i g . 7. x2600. 44 PLATE 3 f i g . 9 P t e r o s i p h o n i a b i p i n n a t a showing d e t a i l o f a p i c a l c e l l . Note t h e l a r g e n u c l e u s and n u c l e o l u s and t h e o c c u r e n c e o f numerous p r o p l a s t i d s and m i t o c h o n d r i a . Double f i x a t i o n i n g l u t a r a l d e -hyde and osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . x l 4 7 0 0 . f i g . 10 P t . g r a c i l i s s howing d e t a i l o f a p i c a l c e l l . V a c u o l a r r e g i o n i s s m a l l and l i m i t e d t o t h e a p i c a l r e g i o n o f t h e c e l l . Many p r o p l a s t i d s and m i t o c h o n d r i a a r e e v i d e n t , as w e l l as s e v e r a l d i c t y o s o m e s . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x l 0 5 0 0 . 45 PLATE 4 f i g . 11 P o l y s i p h o n i a s p. showing t h r e e - c e l l e d l a t e r a l f i l a m e n t . Note arrows s h owing e a r l y c r o s s - w a l l f o r m a t i o n i n t h e a p i c a l c e l l . P r o p l a s t i d s appear t o be l i m i t e d t o t h e a p i c a l c e l l . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . x4760. f i g . 12 P t . g r a c i l i s showing d e t a i l o f d e r i v a t i v e s o f a p i c a l c e l l . Note t h e abundance o f young c h l o r o p l a s t s . M i t o c h o n d r i a can a l s o be seen s c a t t e r e d t h r o u g h o u t t h e c y t o p l a s m . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x5500. 46 PLATE 5 f i g . 13 Pt. g r a c i l i s showing general morphology of the single d i s c stage p i t . Mitochondria with tubular c r i s t a e are c l e a r l y evident i n the cytoplasm. Note the nuclear pores (NP), shown i n surface and oblique view i n the nuclear membrane. Also shown i s the attachment of the p i t membrane to the plasmalemma•(arrow). Double f i x a t i o n i n glutaraldehyde i n sea water, osmium tetroxide i n sodium cacodylate buffer. xl7550. 47 PLATE 6 f i g . 14 L a u r e n c i a s p e c t a b i l i s showing s i n g l e d i s c s t a g e p i t morphology and p i t membrane. Arrow i n d i c a t e s where c o n t a c t between t h e p i t membrane and t h e plasmalemma o c c u r s . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x48000. f i g . 15 P_. p a n i c u l a t a showing s i n g l e d i s c s t a g e p i t between a p i c a l c e l l and t h e f i r s t d e r i v a t i v e . F i x a t i o n as i n f i g . 14. X800000. f i g . 16 O d o n t h a l l i a l y a l i i showing s i n g l e d i s c s t a g e p i t and a s s o c i a t e d amorphous m a t e r i a l (AM) e x t e n d i n g i n t o t h e m i d d l e o f t h e c e l l w a l l . M i t o c h o n d r i a and t h e t o n o p l a s t can a l s o be s een. Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x24000. f i g . 17 D a s y o p s i s densa showing s i n g l e d i s c s t a g e p i t . Note t h a t t h e p e r i p h e r a l zone (PZ) o f t h e p l u g i s l e s s dense t h a n t h e i n n e r m a t r i x o f t h e p l u g . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . x68400. f i g . 18 P_. p a n i c u l a t a showing s i n g l e d i s c s t a g e p i t f r o m a t r i c h o b l a s t c e l l . The c y t o p l a s m i s r e s t r i c t e d t o a narrow band around t h e edge o f t h e c e l l w a l l due t h e e x t r e m e l y l a r g e v a c u o l e . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n s e a w a t e r . x54100. f i g . 19 L. s p e c t a b i l i s showing a t a n g e n t i a l s e c t i o n t h r o u g h a s i n g l e d i s c s t a g e p i t . Note t h e mature c h l o r o p l a s t and c r o s s s e c t i o n o f a m i t o c h o n d r i o n . The p i t membrane can be s e e n c l e a r l y a l o n g one s i d e o f t h e p l u g . Double f i x a t i o n as i n f i g . 14. x22000. 48 PLATE 7 f i g . 20 P o l y s i p h o n i a sp. showing s i n g l e d i s c s t a g e p i t and p i t membrane. P i t membrane and plasmalemma c o n t a c t can be seen a t t h e a r r ow. N u c l e u s , p l a s t i d s and m i t o c h o n d r i a can be seen i n t h e c y t o p l a s m . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . x l 8 0 0 0 . f i g . 21 P_. p a c i f i c a showing r i d g e - l i k e e x t e n s i o n o f p l u g i n p i t o f mature a x i a l c e l l . The p l u g o c c u p i e s a l m o s t t h e e n t i r e end w a l l o f t h e c e l l i n c o n t r a s t t o t h e mature p l u g i n A n t i t h a m n i o n . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x33500. f i g . 22 P_. p a c i f i c a v a r . g r a c i l i s showing r i d g e - l i k e e x t e n s i o n o f p l u g i n mature a x i a l c e l l . O n ly one edge o f t h e p l u g i s shown, as t h e p l u g o c c u p i e s a l m o s t t h e e n t i r e c e l l w a l l . Double f i x a t i o n i n f o r m a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x75700. f i g . 23 A_. s u b u l a t u m showing r i d g e - l i k e e x t e n s i o n o f p l u g i n young p i t c o n n e c t i o n between a x i a l c e l l and l a t e r a l b r a n c h . Compare t h i s m i c r o g r a p h w i t h f i g . 29 showing a mature p l u g from an a x i a l c e l l . F i x a t i o n as i n f i g . 22. x27000. 49 PLATE 8 f i g . 24 P l a t y t h a m n i o n reversum,showing d o u b l e d i s c s t a g e p i t i n a x i a l ' c e l l . C e n t r a l r e g i o n appears t o c o n t a i n a f i b r i l l a r m a t e r i a l . P i t s t r u c t u r e o c c u p i e s o n l y about 1/8 o f t h e c e l l w a l l . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x36000. f i g . 25 P I . r e v e r s u m showing an o l d e r a x i a l c e l l t h a n i n f i g . 24 w i t h a mature d o u b l e d i s c s t a g e p i t . F i b r i l l a r m a t e r i a l i s p r e s e n t w i t h i n t h i s p i t as w e l l , b u t i s n o t r e a d i l y a p p a r e n t i n t h e m i c r o g r a p h . Note t h a t t h e g r a n u l a r m a t e r i a l i s l i m i t e d t o 2 d i s c s on e i t h e r s i d e o f t h e p l u g ( D l , D2). The p i t o c c u p i e s a p p r o x i m a t e l y 1/8 o f t h e c e l l w a l l . F i x a t i o n as i n f i g . 24. x53700. f i g . 26 A_. s u b u l a t u m showing e a r l y s t a g e o f d i f f e r e n t i a t i o n o f d o u b l e d i s c s t a g e p i t from t h e s i n g l e d i s c s t a g e i n an a x i a l c e l l . Note t h e o c c u r r e n c e o f o n l y a t h i n l a y e r o f c y t o p l a s m a c r o s s th e f a c e o f t h e p l u g . T r a n s p a r e n t spaces (TS) can be seen a t t h e edge o f t h e p l u g and d e v e l o p i n g w i t h i n t h e zone o f d i f f e r e n t i a t i o n (ZD). Double f i x a t i o n i n fo r m a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x24000. f i g . 27 A. s u b u l a t u m showing l a t e r d i f f e r e n t i a t i o n s t a g e o f dou b l e d i s c ' s t a g e p i t . T r a n s p a r e n t spaces (TS) e x t e n d n e a r l y a c r o s s t h e e n t i r e p l u g . F i x a t i o n as i n f i g . 26. x31300. f i g . 28 A. s u b u l a t u m showing a l a t e r s t a g e i n t h e d i f f e r e n t i a t i o n o f . t h e dou b l e d i s c s t a g e p i t t h a n i n f i g . 27.- A t r a n s p a r e n t r e g i o n e x t e n d s a c r o s s t h e e n t i r e m i d r e g i o n o f t h e p l u g . F i x a t i o n as i n f i g . 26. x32400. f i g . 29 A. s u b u l a t u m showing mature d o u b l e d i s c s t a g e p i t . Note t h e s i m i l a r i t y o f t h i s p i t t o t h a t shown i n f i g . 8. The c e n t r a l c l e a r r e g i o n i s w e l l d i f f e r e n t i a t e d from t h e p e r i p h e r a l g r a n u l a r d i s c s ( D l , D2). A g r a n u l a r t o f i b r i l l a r m a t e r i a l p a r t i a l l y f i l l s t h e c e n t r a l r e g i o n . The p i t o c c u p i e s a p p r o x i m a t e l y 1/8 o f t h e c e l l w a l l . F i x a t i o n as i n f i g . 26. x l 3 5 0 0 . 50 PLATE 9 f i g . 30 P o l y s i p h o n i a s p . showing d e t a i l e d morphology o f a p r o p l a s t i d . A d i c t y o s o m e can be seen t o t h e r i g h t o f t h e p r o p l a s t i d . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a . w a t e r , osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . . x51400. f i g . 31. P t . g r a c i l i s showing young c h l o r o p l a s t s . P l a s t o g l o b u l i can be seen i n t h e m a t r i x o f one o f t h e c h l o r o p l a s t s . Note a l s o t h e t o n o p l a s t a d j a c e n t t o t h e p l a s t i d s . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . xM-6000. f i g . 32 P t . g r a c i l i s showing young c h l o r o p l a s t , m i t o c h o n d r i o n , and d i c t y o s o m e . The t o n o p l a s t i s shown i n t h e upper r i g h t p o r t i o n o f t h e m i c r o g r a p h . F i x a t i o n as i n f i g . 31. x53700. 51 PLATE 10 f i g . 33 P t . g r a c i l i s showing c h l o r o p l a s t d e t a i l and e x t e n s i v e e l e c t r o n t r a n s p a r e n t r e g i o n s c o n t a i n i n g DNA f i b r i l s . - Note t h e a t t a c h -ment o f t h y l a k o i d s t o t h e outermost p h o t o s y n t h e t i c l a m e l l a ( a r r o w s ) . A l s o v i s i b l e i s t h e plasmalemma (upper l e f t ) , m i t o c h o n d r i a , and t o n o p l a s t (upper and l o w e r r i g h t ) . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x42000. 52 PLATE 11 f i g . 34 P_. p a c i f i c a showing abnormal c h l o r o p l a s t u l t r a s t r u c t u r e . Note t h e a s s o c i a t i o n and . s e p a r a t i o n o f l a m e l l a e ( t h y l a k o i d s ) w i t h i n t h e c h l o r o p l a s t . Double f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x49900. f i g . 35 P_. p a n i c u l a t a showing a d i v i d i n g c h l o r o p l a s t . The t h y l a k o i d s appear t o have b r o k e n i n two a t t h e p o i n t o f c o n s t r i c t i o n . D aughter c h l o r o p l a s t s e v e n t u a l l y p i n c h o f f f r o m one a n o t h e r . D o u b l e . f i x a t i o n i n g l u t a r a l d e h y d e and osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . x20300. f i g . 36 P_. p a n i c u l a t a showing a c r y s t a l l i n e s t r u c t u r e i n t h e c y t o p l a s m . I t a p p e ars t o be n o t membrane bound. Numerous s t a r c h g r a i n s (S) a r e e v i d e n t i n t h e c y t o p l a s m . F i x a t i o n as i n f i g . 34. x80000. 53 PLATE 12 f i g . 37 P. p a n i c u l a t a showing d e t a i l e d s t r u c t u r e o f t h e n u c l e u s . Arrow i n d i c a t e s a r e g i o n o f f i n e g r a n u l a r i t y w i t h i n t h e n u c l e a r m a t r i x ( p o s s i b l y c h r o m a t i n ) . The d e n s e l y s t a i n i n g n u c l e o l u s o c c u p i e s a n e a r l y c e n t r a l p o s i t i o n . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , osmium t e t r o x i d e i n D a l t o n ' s b u f f e r . x l 7 5 0 0 . f i g . 38 A_. s u b u l a t u m showing t h e s t r u c t u r e o f t h e n u c l e u s and c e l l w a l l . The n u c l e u s , n u c l e o l u s w i t h n u c l e o l a r i n c l u s i o n s , c h l o r o p l a s t s and m i t o c h o n d r i a a r e v i s i b l e i n t h e c y t o p l a s m . Note t h e l a y e r o f amorphous m a t e r i a l (E) i n t h e o u t e r m o s t p o r t i o n o f t h e c e l l u l o s i c w a l l . Double f i x a t i o n i n g l u t a r a l -dehyde and osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x l 5 0 0 0 . f i g . 39 P_. p a c i f i c a showing, n u c l e o l u s w i t h n u c l e o l a r i n c l u s i o n . Note t h e r i n g - l i k e appearance o f t h e n u c l e o l u s due t o t h e n u c l e o l a r i n c l u s i o n . F i x a t i o n as i n f i g . 38. x l 8 0 0 0 . 54 PLATE 13 f i g . 40 P_. p a c i f i c a showing d e t a i l s of the c e l l w a l l . The mucila-ginous portion of the c e l l w all i s very densely stained and shows no d e t a i l . A l e s s densely stained amorphous region (E) can be seen beneath the mucilaginous coat.. The c e l l u l s o i c portion of the c e l l w all appears as a r e t i c u l a t e arrangement of m i c r o f i b r i l s . Double f i x a t i o n i n glutaraldehyde and ruthenium red-osmium tetroxide i n sodium cacodylate b u f f e r . xl5000. f i g . 41 P_. paniculata showing d e t a i l s of the c e l l w a l l . Four layers can be distinguished within the mucilaginous w a l l . Compare the c e l l u l o s i c portion of the c e l l w a l l , which shows few m i c r o f i b r i l s , with the same portion of the c e l l wall i n f i g s . 40, 42-44 i n which ruthenium red-osmium tetroxide p o s t f i x a t i o n was employed. Note also the surface a c t i v i t y of the plasma-lemma (arrows). Double f i x a t i o n i n glutaraldehyde and osmium tetroxide i n sodium cacodylate buffer. x24000. f i g . 42 • Pt. bipinnata showing d e t a i l of c e l l wall and mucilaginous coat. Note the complexity of the mucilaginous coat as compared to that i n f i g . 41. Four layers are again v i s i b l e , but i n much greater d e t a i l . Two layers (LI, L2) can also be distinguished within the c e l l u l o s i c portion of the c e l l w a ll. Double f i x a t i o n i n glutaraldehyde i n sea water, osmium tetroxide-ruthenium red i n sodium cacodylate buffer. x40000. 55 PLATE 14 f i g . 43 P t . b i p i n n a t a showing t r a n s v e r s e s e c t i o n o f c e l l w a l l . Note t h e r e t i c u l a t e arrangement o f m i c r o f i b r i l s i n t h e c e l l u l o s i c p o r t i o n o f t h e c e l l w a l l . The 4 l a y e r s o f t h e m u c i l a g i n o u s c o a t a r e a l s o v i s i b l e . Double f i x a t i o n i n g l u t a r a l d e h y d e i n s e a w a t e r , r u t h e n i u m red-osmium t e t r o x i d e i n sodium c a c o d y l a t e b u f f e r . x240Q0. f i g . 44 P t . b i p i n n a t a showing c e l l w a l l l a y e r s ( L l , L2) o f t h e c e l l u l o s i c p o r t i o n o f t h e c e l l w a l l i n r e l a t i o n t o t h e p e r i c e n t r a l c e l l s . L a y e r L l can be seen t o be c o n t i n u o u s around each c e l l ; w h i l e l a y e r L2 i s n o t p r e s e n t between c e l l s and o n l y c o v e r s t h e e n t i r e t h a l l u s . F i x a t i o n as i n f i g . 43. x l 3 2 0 0 . 

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