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Fine structural studies on some marine algae from the Pacific Coast of British Columbia and Washington Bourne, Victor Laurence 1971

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FINE STRUCTURAL STUDIES ON SOME MARINE ALGAE FROM THE PACIFIC COAST OF BRITISH COLUMBIA AND WASHINGTON by VICTOR LAURENCE BOURNE B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1964-A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FCR THE DEGREE OF DOCTOR OF PHILOSOPHY I n t h e Department o f BOTANY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1971 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Botany  The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e A p r i l 2 1 , 1971 i i ABSTRACT Fine structural studies on some marine algae from the Pacific Coast of British Columbia and Washington In a fine-structure study of Phaeostrophion irregulare (Dictyosiphonales) most characteristics of the organelles were found to be similar to those previously reported for other brown algae. However, the pyrenoid which i s present i n the c e l l s of the sporeling i s absent from the thallus which grows direc t l y from i t . This i s significant because i t draws attention to the possible implications of observing a limited number of heterogeneous tissue types, such as spores, sporeling, and mature thallus, i n comparative studies. Phaeostrophion irregulare retains i t s intermediate position i n the phylogenetic cla s s i f i c a t i o n of the brown algae. It possesses some characteristics of the more advanced brown algae, such as the absence of a pyrenoid i n the adult thallus and the absence of a physical association of the chloroplast and nucleus. Also i t possesses more primitive characteristics such as a s t r i c t l y perinuclear Golgi apparatus and a diffuse distribution of plasmodesmata. The red algae Porphyra perforata and Bangia f uscopurpurea. both of the order Bangiales, subclass Bangiophycidae, were also studied. In the former, the thallus, bipolar sporeling, and conchocelis phase were considered, and i n the lat t e r , the thallus only. Cells of a l l i i i the tissues that were studied have very similar fine structural characteristics. I t was noted also, that the fine structural features of these tissues were similar to other Bangiophycidae and the Florideophycidae. A f a i r l y constant association was noted between the mitochondria and the forming face of the Golgi bodies. Most c e l l s contained numerous lomasome-like bodies throughout the cytoplasm. Two types of c e l l d ivision were observed i n the Porphyra thallus c e l l . The possible function of several organelles i s discussed. In addition to these features seen i n the thallus, c e l l s of the young conchocelis have phycobilisomes i n the chloroplasts and a l l c e l l s have a typical floridean p i t connection, confirming earlier l i g h t microscope observations. This characteristic had previously been considered f a i r l y definitive for the Florideophycidae, I t i s suggested that with these data and other published results the status of subclass for the Bangiophycidae and Florideophycidae may have to be reviewed. . i v TABLE OF CONTENTS PREFACE 1 PART ONE Phaeophyceae Some observations on the fine structure of the marine brown alga Phaeostrophion irregulare.... 3 Introduction 3 Materials and Methods 4-Observations 6 Chloroplasts 6 Pyrenoid 8 Nucleus. • • • • 8 Golgi complex. 9 O i l Bodies 10 C e l l wall and associated structures 11 Other cytoplasmic bodies 13 Discussion 14-Literature Cited 19 PART TWO Rhodophyceae A comparison of cellular fine structure of the vegetative t h a l l i of Porphyra perforata and Bangia fuscopurpurea and certain phases i n the l i f e history of Porphyra perforata 28 V TABLE OF CONTENTS (CON'T) Introduction , 28 Materials and Methods 30 Observations 3^ Vegetative c e l l s of the thallus and bipolar sporelings of Porphyra perforata and the thallus of Bangia fuscopurpurea J6 Conchocelis phase c e l l s of Porphyra perforata.. 4-6 Discussion 53 Literature cited 62 GENERAL CONCLUSIONS 91 APPENDICES I.On the ultrastructure of p i t connections i n the conchocelis phase of the red alga Porphyra perforata J. Ag 93 II.A preliminary experiment using radiotracer techniques i n an attempt to follow the path of polymerized glucose, mannose and xylose i n the macroscopic thallus of Porphyra perforata 97 Introduction. 97 Materials and Methods 98 Observations and Discussion. 99 Literature cited 100 III.A simplified technique for the application of nuclear emulsion i n electron microscopic autoradiography.... 102 v i LIST OF FIGURES FIGURES 1 Chart showing possible interrelationships of the orders within the Phaeophyceae and Rhodophyceae. Phaeostrophion irregulare: 2 Photograph of mature plant of P. irregulare . 3 - Cross section of a young maturing blade as seen by light microscopy. k Electron micrograph of a surface cell. 5 View of a portion of a cortical cell of the blade. 6 Pyrenoid and part of a chloroplast in a sporeling c e l l . 7 A chloroplast in a cortical cell with a lateral out-pocketing or non-lamellar area. 8 Part of a medullary vacuolated cell containing numerous o i l bodies. 9 Chloroplast from a plant kept under ar t i f i c a l illumination for an extended period. 10 Oil body adjacent to a chloroplast. 11 Chloroplast of vacuolated c e l l seen in Figure 8 . 12 Nucleus with nucleolus and nuclear envelope with pores. 13 Centriolar bodies . 14- Spheroid body with a limiting membrane between the plasmalemma and ce l l wall, 15 Portion of a pore area in a cortical cell wall. 16 Longitudinal section of plasmodesmata showing continuity through the wall. v i i Two typical mitochondria. Portion of a vacuolated c e l l showing an accumulation of small vesicles between the plasmalemma and c e l l wall. Lamellar body located i n a vacuole showing the paired lamellae and inner vesicles. Porphyra perforata; vegetative thallus. Map of the collection sites of algae used i n this study. Diagram of the tentative l i f e cycle of Porphyra perforata . Blades collected from Ucluelet, Vancouver Island. Blades collected from Point Grey, Vancouver. A l i g h t micrograph of a portion of the blade showing the o< spores, ,3 spores and vegetative c e l l s . An electron micrograph of a single vegetative c e l l . A cross section of the blade showing similar organelles to those of Figure 25. Section through the c e l l surface showing convoluted plasmalemma, and vesicles between cytoplasm and c e l l w a ll. Section showing the vegetative chloroplast with single lamellae. A portion of a vegetative c e l l . Two mitochondria with associated Golgi bodies Section showing the Golgi apparatus and mitochondrion. Two nuclei i n a vegetative c e l l . Single vegetative nucleus . Highly lobate vegetative nucleus, which may be the i n i t i a l stage of nuclear division. I n i t i a l stage of cytokinesis . A later stage of c e l l division . v i i i FIGURE 37 Two nuclei i n the c e l l separated by invaginated plasmalemma. 38 A later stage of this type of c e l l d ivision. 39 Relationship of thick layered c e l l wall to rhizoidal process. 40 Cross section of rhizoidal process with highly convoluted plasmalemma. 41 Longitudinal section of rhizoidal process. 42 Scanning electron micrograph of the surface of the vegetative thallus. 43 Section through ©< spore producing portion of the thallus . 44 Numerous small f i b e r - f i l l e d vesicles near the c e l l wall of differentiating c* spores . 45 Concentric lamellar body with tubules . 46 Portion of a vegetative c e l l with c e l l wall fibers attached to the plasmalemma. 47 Concentric lamellar body i n the c e l l w a ll. 48 A c e l l with concentric lamellar bodies i n the cytoplasm, and i n the chloroplast. 49 A concentric lamellar body i n close proximity to a polyvesicular body. 50 Finger-like projections of cytoplasm into the c e l l wall. 51 Membranes interconnecting the concentric lamellar bodies and polyvesicular bodies i n the vegetative c e l l . 52 Polyvesicular body with tubular inner vesicles . 53 Polyvesicular body located near the c e l l periphery. ix FIGURE. -5k Polyvesicular body i n contact with the c e l l wall. 55 Complex elaboration of the plasmalemma. 56 Endoplasmic reticulum i n the central portion of a c e l l . Porphyra perforata: bipolar sporeling. 57 Light micrograph of released aplanospores -58 Young bipolar sporeling with rhizoidal process at basal end. 59 Older bipolar sporeling . 60 Electron micrograph of c e l l from older bipolar sporeling showing the thick c e l l wall and convoluted plasmalemma-61 C e l l showing chloroplasts and pyrenoid, and single lamellae. 62 Chloroplast with pyrenoid and osmiophilic droplets. 63 C e l l with thick c e l l wall. 64- Ce l l with chl&roplasts, starch granules, and a Golgi apparatus which i s near a mitochondrion. Bangia fuseopurpurea: vegetative thallus. 65 Light micrograph of a filament. 66 Electron micrograph of a c e l l i n the filament. 67 Cell surface with highly convoluted plasmalemma. 68 Lobate nucleus with prominent nucleolus , 69 Section through a vegetative c e l l showing endoplasmic reticulum, connected to a concentric lamellar body, a mitochondrion and Golgi apparatus, 70 A concentric lamellar body i n a chloroplast. 71 Round concentric lamellar body i n the cytoplasm, 72 Concentric lamellar body i n a c e l l wall. X FIGURE 73 Irregularly shaped concentric lamellar body i n cytoplasm. 74 Cell d i v i s i o n . 75 Thick multilayered c e l l wall between two cells(aplanospores). Bangia fuscopurpurea; development of aplanospores. 76 Endoplasmic reticulum i n the peripheral cytoplasm and c e l l wall material surrounding the developing spore. 77 Developing aplanospore _ showing separation of spore wall. 78 Developing aplanospore. 79 Aplanospore released from the spore mother c e l l wall but s t i l l i n the lumen. Porphyra perforata:conchocelis phase. 80 Light micrograph of spores released from mature thallus. 81 Light micrograph of a young conchocelis filament• 82 Electron micrograph of a cross section of a conchocelis filament. 83 Cross section of conchocelis filament showing chloroplast, phycobilisomes attached to thylakoids • 84 Conchocelis c e l l s showing chloroplast, starch and pyrenoid. 85 Light micrograph of conchocelis filament. 86 Electron micrograph of f i r s t conchocelis c e l l and the empty spore case. 87 Empty spore case and f i r s t c e l l of conchocelis-88. Plugged p i t connection between two vacuolated c e l l s . 89 Plugged p i t connection between two conchocelis c e l l s • 90 Cell wall adjacent to a plugged pi t connection. 91 Plugged p i t connection between two older conchocelis c e l l s • x i FIGURE 92 Two pyrenoids i n one chloroplast, and accumulation of starch granules. 93 Chloroplast and pyrenoid of older conchocelis c e l l . 94- Longitudinal section of conchocelis c e l l . 95 Light micrograph of conchospore branches. 96 Conchospore branch c e l l showing projection of the c e l l wall. 97 Conchospore branch cells showing convoluted c e l l wall, chloroplast and pyrenoid. 98 Conchospore branch c e l l with nucleus,nucleolus, and chloroplast. 99 Plugged p i t connection between two conchospore branch c e l l s . 100 Scanning electron micrograph of the surface of conchocelis filaments . 101 Scanning electron micrograph of the surface of conchospore branch c e l l s . 102 Light micrograph of the connection between vegetative conchocelis c e l l s and the conchospore branch c e l l s . 103 Scanning electron micrograph of the connection between vegetative conchocelis c e l l s and conchospore branch ce l l s . 104- Light micrograph of conchocelis filaments. 105 Low magnification electron micrograph of p i t connection . 106 High magnification electron micrograph of the p i t connection i n Figure 105. R a d i o t r a c e r experiment. Diagram of bubble chamber used f o r the r a d i o t r a c e r experiment. A p p l i c a t i o n o f nuclear emulsion -Diagram showing r e l a t i o n s h i p o f emulsion, g r i d and c e n t r i f u g e s p i n d l e . E l e c t r o n micrograph showing the undeveloped s i l v e r h a l i d e granules a f t e r a p p l i c a t i o n o f e l e c t r o n beam . E l e c t r o n micrograph showing s i l v e r p a r t i c l e s a f t e r exposure t o l i g h t and development i n D-19. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. Kathleen Cole, whose patient perseverance and guidance during the course of these studies made i t possible to complete them. Thanks are also extended to the other committee members: Drs. R.F. Scagel, K.I. Beamish, E.B. Tregunna, F.J.R. Taylor and J.R. Maze for helpful suggestions and cr i t i c i s m and to Mrs. S-C, Lin and Mr. D.L. McBride for many interesting discussions on the subject matter. The author expresses his indebtedness to Mr. L.L. Veto of the University of B r i t i s h Columbia biological sciences electron microscopy laboratories for his excellent technical assistance, to Miss E. Packham of Point No Point resort on whose property soma of the specimens were collected, and to Mr. J. Thorpe for various forms of assistance and the use of the f a c i l i t i e s of the Department of Botany, University of British Columbia, Thanks are also extended to Dr. E, Conway for help with the culturing of Porphyra perforata while she was a v i s i t i n g professor i n this department. Thanks are also extended to Dr. L, M, Srivastava of the Department of Biological Sciences at Simon Eraser University for the use of the RCA, EMU-3H and Zeiss, EM9-A electron microscopes and to Dr. A.E, Curzon of the Department of Physics, Simon Eraser University for the use of the Philips EM 300 electron microscope. Financial assistance from the National Research Council of Canada i s recognized for a scholarship 1967-70 and grant A-64-5 to Dr. K. Cole which supplied equipment and materials. 1 PREFACE Studies on the cytology of marine algae are of potential importance. Such studies w i l l aid i n our understanding the relationships between various algal groups. It i s a fundamental axiom of taxonomy that the more information upon which a c l a s s i f i c a t i o n i s based the more natural i t w i l l be. The taxonomy of algae i s of some considerable interest. I t i s i n the large and diverse group of algae that biochemical characteristics were f i r s t put to significant use. There i s also some indication that studies on the fine structure of the algal c e l l may be of taxonomic significance. A second valuable aspect of cytological studies of marine algae i s that whenever a new organism i s studied a considerable amount of information i s added to our knowledge of organelle structure and function. This assumes considerable significance i n a f i e l d such as electron microscopy. Too often, sweeping generalizations on organelle structure and function are made,, based on observations from a very limited number of organisms. This study was i n i t i a t e d to provide ultrastructural information on some l o c a l species of marine algae. These and previously published findings were used to examine hypotheses of taxonomic relationships i n the Phaeophyceae and Rhodophyceae (Fig.l) and hypotheses relating to organelle structure and function i n Porphyra perforata. F i g u r e 1 Chart showing p o s s i b l e i n t e r r e l a t i o n s h i p s o f the orders w i t h i n the Phaeophyceae and Rhodophyceae A Phenetic r e l a t i o n s h i p s w i t h i n the Phaeophyceae. B Phenetic r e l a t i o n s h i p s w i t h i n the Rhodophyceae. Laminariales Desraarestiale Fucales Sporochnales Ectocarpales Phaeophycidae Cyclosporidae A Ceramiales Bangiales Goniotrichales B Bangiophycidae Rhodymeniale s \ Gigartinales \ Cryptonemiale s Florideophycidae Figure 1 Chart showing possible interrelationships of the orders within the Phaeophyceae and Rhodophyceae, 3 PART ONE Phaeophyceae Some observations on the fine structure of the marine brown alga Phaeostrophion irregulare^ Introduction The fine structure of a small number of Phaeophyceae and other related groups has been investigated during the past few years and an interesting feature has been reported. According to several authors (Gibbs,1962a; Greenwood, 1964; Bouck,1965; Manton,1966; Cole, Bourne, and Lin,1968; Cole and Lin ,1968; and Cole,1969,1970) there i s a physical association of the nucleus, chloroplast, pyrenoid, and other organelles i n various brown algae and. other related species. Evans (1966,1968) has presented evidence that the presence or absence of the pyrenoid i s of phylogenetic importance. Previous publications have covered only a limited number of species and i t i s obvious that more information on the ultrastructure of an increased number of Phaeophyceae i s required to determine the true phylogenetic significance of these findings. Only one member of the Dictyosiphonales, Dictyosiphon foeniculaceus (Evans,I966), has been reported i n the literature. Consequently a study of the fine structure of another species of the same order, Phaeostrophion irregulare, was i n i t i a t e d , 1 This part of the thesis i s based on an a r t i c l e by V.L. Bourne and K, Cole which appeared i n the Canadian Journal of Botany 46; 1369-1375 (1968). The text of the original a r t i c l e has been brought up to date by including subsequent findings where appropriate. M a t e r i a l s and Methods The brown a l g a Phaeostrophion i r r e g u l a r e S.et G., a member of the f a m i l y Punctariaceae i n the order D i c t y o s i p h o n a l e s , was o r i g i n a l l y d e s c r i b e d i n 1924 ( S e t c h e l l and Gardner,1924), but d e t a i l s o f i t s l i f e h i s t o r y and ecology have only r e c e n t l y been e l u c i d a t e d (Mathieson, 1967). I t i s s i m i l a r t o s e v e r a l other s p e c i e s i n t h i s order i n having a d i r e c t type of l i f e c y c l e . M e i o s i s i s probably suppressed i n the u n i l o c u l a r sporangium although t h i s has not been determined c y t o l o g i c a l l y . Phaeostrophion i r r e g u l a r e was c o l l e c t e d d u r i n g low t i d e i n the w i n t e r months from 1965 t o 1967 a t P o i n t No P o i n t ( G l a c i e r P o i n t ) , Vancouver I s l a n d , B.C. The mature t h a l l u s c o n s i s t s o f a few s t r a p -l i k e blades attached t o a b a s a l h o l d f a s t ( F i g . 2 ) . The blades are o f t e n i r r e g u l a r l y t o r n and v a r y i n l e n g t h from 1.0 t o 25.0 cm and i n w i d t h from 0.5 t o 4 ,5 cm. A cross s e c t i o n o f the blade r e v e a l s b o t h c o r t i c a l and medullary l a y e r s ( F i g . 3 ) . C u l t u r e s o f the fila m e n t o u s stage were i n i t i a t e d by washing the blades c o n t a i n i n g mature u n i l o c u l a r and p l u r i l o c u l a r sporangia i n s t e r i l e seawater, then p l a c i n g them i n f r e s h s t e r i l e seawater t o r e l e a s e the spores. The spores were p i p e t t e d i n t o another d i s h c o n t a i n i n g E r d s c h r e i b e r s o l u t i o n ( S t a r r , 1964) and maintained i n a c u l t u r e room a t 10 "C, under S y l v a n i a c o o l white f l u o r e s c e n t tubes (F48 T12-CW) f o r 12 hours per day a t an i n t e n s i t y o f approximately 100 f . c . Some a d d i t i o n a l blades were c o l l e c t e d i n the f i e l d and maintained i n the l i v i n g c o n d i t i o n i n seawater a t 5 °C under the same 5 l i g h t conditions for several months before fixation. For l i g h t microscopy the blades were fixed directly i n 3si ethanol-acetic acid for Zk hr. They were then washed i n 70$ ethanol and embedded i n paraffin wax through a graded ethanol-tertiary butyl alcohol series. Sections were cut on a Spencer rotary microtome and stained with bismarck brown. For study by electron microscopy, blades approximately 5 cm long were fixed on the beach i n either Dalton's solution (Dalton, 1955) or i n 6$ glutaraldehyde i n 1/15 M phosphate buffer at pH 7.2 for 2-48 nr., washed thoroughly i n buffer, and then postfixed i n 1$ or 2$ osmium tetroxide i n the same buffer for 2-24- hr. Cultured sporelings (developing plethysmothalli) and stored t h a l l i were fixed i n the same manner. The fixed samples were washed in.buffer, dehydrated i n a graded ethanol series, and then i n f i l t r a t e d with propylene oxide followed by an epoxy resin mixture which was polymerized i n an oven at 60°C for 4-8 hr. The plastic resin mixture consisted of Maraglas 655, &5p, Cardolite NC 513, 20$} dibutyl phthalate,15$s and benzyl-dimethylamine catalyst, an extra 2 ml per 100 ml resin (Freeman and Spurlock,1962), Both tangential and cross sections of the blade were cut on an LKB Dltrotome I by use of a glass knife. They were stained i n lead citrate (Reynolds, 1963) and subsequently examined with Hitachi HU-11A and HS-7S electron microscopes. 6 Observations Chloroplasts Several small chloroplasts, measuring approximately 6 u x 2 u i n longitudinal section, are located i n each c e l l of the blade of Phaeostrophion irregulare (Figs, k,7). The chloroplast fine structure characteristics of this alga are very similar to those previously reported for other brown algae. Bands of three thylakoids or discs traverse the whole length of each chloroplast and a peripheral band usually completely encircles i t underneath the chloroplast double membrane. Each band i s approximately 58 mu wide and 4-.5 u long. The thylakoids are about 11 mu thick and are not closely appressed. An exchange of discs between adjacent bands often occurs i n a regular pattern so that the number of thylakoids per band tends to remain at three (Fig.7 ) , rather than to vary from two to four as reported for Chorda filum (Bouck,1965), Fucus serratus, and Pelvetia  canaliculata (Evans,1968), This phenomenon has been observed previously i n P y l a i e l l a l i t t o r a l i s (Evans,1966). A constant three-thylakoid handing with no exchange of discs has been recorded i n some culture sporelings of Leathesia difformis (Cole, Bourne,and Lin, 1968). Membrane-free areas containing fibers presently believed to be deoxyribonucleic acid (DNA) (Bouck,1965; Bisalputra and Bisalputra, 1967) ar© formed at each end of the chloroplast between the peripheral band and the termination of the longitudinal bands. Osmiophilic droplets are occasionally located between the bands (Figs, k,7). An out-pocketing of stroma appeared on one side of a few chloroplasts, leaving a large non-lamellar area between the thylakoids and the chloroplast-limiting membrane (Fig, 7 ) . Outpockets have recently been seen on the chloroplasts of other brown algae, Leathesia difformis (Cole and Lin, 1968), Budesme virescens (Cole,1969)> and Scytosiphon lomentaria (Cole, 1970). Something similar was shown by Dodge (1968) i n the dinoflagellate Aureodinium pigmentosum, although the non-lamellar area of this organism was subterminal l a t e r a l rather than median l a t e r a l as i n Phaeostrophion. This feature i n Phaeostrophion does not quite resemble the rudimentary pyrenoid of some fucoids (Evans,1968) i n which there i s a folding back of the chloroplast envelope to delimit the pyrenoid. There was an interesting alteration i n the internal morphology of a number of chloroplasts i n material which had been stored for a long period at 5°C under a r t i f i c i a l illumination. In some, the bands tended to pack together i n several areas forming larger bands consisting of 12 to 18 t i g h t l y associated discs (Fig. 9 ) . In other parts of the same chloroplast the thylakoids separated and formed irregular patterns (Fig,9 ) . Chloroplasts have also been observed without the wide bands but with the separation of thylakoids i n an intermediate region (Fig. 11) . This type of packing and separation of discs has not been reported previously i n the brown algae. Intermediate stackes of 40 to 60 long discs have been noted i n the chloroplasts of some c e l l s of cultured Leathesia difformis (Cole, Bourne, and Lin, 1968), and have been shown to be a stage i n chloroplast division (Cole and Lin, 1968). However, these differed from the wide bands i n Phaeostrophion since they represented 8 the formation of extra bands between the regular ones. Also, a l l the thylakoids of L. difformis remained i n a loosely bound arrangement whereas those of P. irregulare became tightly associated i n some areas. Because of these basic differences, i t i s unlikely that the cytological changes reported i n P. Irregulare are a manifestation of c e l l division. Pyrenoid Pyrenoids were observed i n ce l l s of Phaeostrophion sporelings (Fig.6 ) , but not i n the thallus (Figs. 4-,5). They have a granular, uniformly dense internal matrix lacking thylakoids, typical of those i n several other species of brown algae (Gibbs,1962b; Bouck, 1965; Evans,1966; Cole and Lin,1968j Cole,1969,1970). In presenting electron microscopic evidence that pyrenoids occur i n some orders of brown algae but not i n others, Evans (1966) recorded the occurrence of pyrenoids i n thallus c e l l s of one of the Dictyosiphonales, Dictyosiphon foeniculaceus. although he did not include an electron micrograph to i l l u s t r a t e i t . In the present study no typical pyrenoids were noted i n the thallus c e l l s of Phaeostrophion. However, a pyrenoid has been noted i n the mature thallus c e l l s of a thir d genus of the same order, Punctaria sp. (Cole, unpublished data). Pyrenoids have also been observed i n some members of the related order Scytosiphonales (Cole,1970). Nucleus Some nuclear ultrastructural characteristics of Phaeostrophion are very similar to those reported for Chorda filum and Giffordia sp. (Bouck,1965). The uniformly granular nucleus i s approximately 3-5 P-i n diameter (Fig. 12), and i s encompassed by a double membrane which i s permeated by pores. It contains one or more nucleoli, about 1.0 u i n diameter, which are more densely granular than the surrounding nucleoplasm. There i s no evidence that the outer nuclear membrane extends and envelops the chloroplast i n the c e l l s of the mature blade. However, this feature has been reported i n the thallus of Chorda  filum (Bouck,1965), motile zoospores of Pylaiella l i t t o r a l i s and Ectocarpus confervoides (Evans,1966), cultured sporelings of Leathesia  difformis (Cole, Bourne and Lin, 1968), Eudesme (Cole, 1969) and some Scytosiphonales (Cole, 1970). Although a l l stages of development have not been f u l l y investigated as yet, there also does not appear to be any connection between the nuclear envelope and the chloroplasts i n the unilocular sporangium of Phaeostrophion. Golgi complex The Golgi complex of Phaeostrophion i s similar i n most respects to that described for Giffordia sp. and Chorda filum (Bouck,1965), Leathesia difformis (Cole and Lin, 1968), Eudesme virescens (Cole,1969) and Petalonia d e b i l i s (Cole, 1970). The "forming" face i s closely associated with the nuclear membrane and the "maturing" face i s directed toward the cytoplasm or vacuole (Fig. 12). It consists of many flattened cisternae, some with a slight swelling at either end when viewed i n cross section. The Golgi body seems to be produced by the coalescence of smaller vesicles which have formed from blebs on the 10 outer nuclear membrane. This i s also much the same process of formation reported previously (Bouck,1965). As the Golgi cisternae of Phaeostrophion increase i n size towards the maturing face, fibrous contents of l i g h t electron density, similar i n appearance to those observed i n Fucus vesiculosus (McGully,1968) can be clearly seen within them (Fig. 12) , As the Golgi complex was seldom seen near the wall i t i s not known i f the fibrous material observed i n i t was direc t l y involved i n c e l l wall formation. The Golgi complex has been shown, using autoradiographic and other techniques, to be involved i n c e l l wall production i n other species (Barton, 1968; Wooding,1968} Bailey and Bisalputra,1969} Brown,1969). O i l Bodies Large dense vesicles with very l i t t l e internal structure, which are probably o i l bodies, are found i n most vacuolated and nonvacuolated c e l l s of Phaeostrophion irregulare. They range i n size from 0.5 u to 5 M and the number per c e l l varies. They can become so numerous that they exclude much of the cytoplasm or vacuolar material (Figs.5, 8 ) . Most o i l bodies are free i n the cytoplasm or vacuole and when appressed to a chloroplast or nucleus there seems to be no communication between the contents of these vesicles and the other organelles (Fig.10) . Since they are so extremely dense i t i s d i f f i c u l t to determine whether they have a limiting membrane. In general, they are very similar to what are referred to as the physodes in other species observed with the electron microscope: Egregia menziesii (Bisalputra ,1966) , Fucus vesiculosus (McCully,1968), Asperococcus fistulosus. Leathesia 11 difformis, and Nereocystis luetkeana (Cole, unpublished results). Using centrifugation and electron microscopy Neushul and Liddle (1968) and Liddle and Neushul (I969) separated the o i l droplets and physodes i n Zonaria eggs and noted that the physodes were not as electron dense as the o i l droplets. The o i l i s probably a storage product. Ce l l Wall and associated structures The c e l l wall of Phaeostrophion thallus i s composed of two structurally different layers, an outer primary alginic layer (Siegel ,1962) containing randomly oriented microfibrils with many spaces and inclusions, and an inner secondary cellulose-like layer (Siegel ,1962; Cole 1964) made up of microfibrils of paral l e l orientation (Fig. 4 ) . This type of c e l l wall construction has been observed previously i n the brown algae, Macrocystis pyrifera (Ziegler, 1963)» Fucus vesiculosus (McCully,1965) and Ectocarpus acutus (Bailey and Bisalputra ,1969) . The c e l l wall of the Phaeostrophion sporeling has not yet been f u l l y studied due to technical d i f f i c u l t i e s encountered with this phase of the l i f e cycle, A few structures with a spherical to ovoid shape and ranging i n size from 1 u to 2 u were attached to the inner part of the c e l l wall (Fig, 14) . They extended into a portion of the lumen that had previously been occupied by the cytoplasm, and were separated from the c e l l contents by the plasmalemma. Some appeared to be firmly attached to the c e l l wall material by thin fibers which were very similar to 12 the cellulosic fibers of the wall. These attached bodies had an external limiting membrane. Some showed l i t t l e internal structure except for a fine granularity, while others appeared extremely dense. Similar objects have been observed attached to the inner wall of orange epicarp (Thomson, 1967) a n c* Fucus vesiculosus (McCully ,1968). A large accumulation of small circular vesicles was occasionally observed between the plasmalemma and the c e l l wall i n the thallus (Fig. 18) . Each was bounded by a single membrane and measured approximately 0.07 to 0,30 u. The whole aggregation f i t t e d into a depression i n the c e l l wall. This •resembled a buildup of vesicles seen next to the inner wall i n orange epicarp(Thomson,1967) and pine resin canal c e l l s (Wooding and Northcote,1965). Further studies are required before definite statements can be made about the origin and significance of any of these structures seen between the c e l l wall and the plasmalemma, Plasmodesmata occur i n Phaeostrophion. connecting adjacent c o r t i c a l and medullary c e l l s of the blade through pores i n the c e l l wall (Figs. 15,16), They are not confined to a well-defined primary p i t f i e l d as i n Egregia menziesii (Bisalputra ,1966) and Dictyota flabellata (Dawes, Scott and Bowler,196l), but are spread over a more diffuse area. The plasmodesmata of Leathesia (Cole and Liri ,1968) , and some Scytosiphonales (Cole,1970) also are spread over a diffuse area. An electron-transparent area separates the plasmodesma from the pore opening (Fig.15) and i n some cases f i b r i l l a r projections extend from the unit membrane to the surrounding c e l l wall 13 material, Bisalputra (1966) mentioned that similar spaces i n Egregia resulted from plasmolytic shrinkage but did not comment on the connecting f i b r i l s which were also evident i n his micrographs. It i s more l i k e l y that the electron-transparent area represents a deposit of callose similar to that which surrounds the sieve pores of Macrocystis pyrifera (Ziegler,1963) and Laminaria (Ziegler and Ruck,1967). Other Cytoplasmic Bodies Centriolar bodies resembling those described i n Fucus  vesiculosus (Bouck,1965), Colpomenia peregrina (Evans,1966) and Petalonia debilis (Cole,1970) were observed near the central part of one c e l l ( F i g i l 3 ) . Since the c e l l was i n a mature part of the thallus and was not a sporangium i t i s concluded that these are centrioles and not the basal bodies of f l a g e l l a . The overall diameter of each i s approximately 300 mu. It i s composed of nine longitudinally oriented fibers, each fiber consisting of three subfibers. This i s a common centriolar pattern i n the brown algae previously studied (Bouck,1965; Evans 1966), Mitochondria are not p l e n t i f u l i n the thallus c e l l s of P. irregulare. In section, they vary i n shape from round to e l l i p t i c and measure from 0,25 u to 0,75 u i n width and 0,80 to 1.5 u i n length (Fig,17), These mitochondria have characteristic brown algal tubular cristae which are continuous with the inner mitochondrial membrane (Bouck,1965). Lamellar structures measuring approximately 1.2 u by 1.7 u i n cross section were observed i n the cytoplasm and vacuoles of several c e l l s of the thallus (Fig. 19) . Each consisted of many-concentric rings or layers of paired lamellae 30 mu apart. A space of 10 mu separated the two lamellae each of which was 6 mu thick. Bodies closely resembling these lamellar structures have been observed i n Fucus vesiculosus (McCully,1968) and i n Leathesia  difformis, Nereocystis luetkeana. Petalonia debilis (Cole and Lin, 1970) and Punctaria sp. (Cole, unpublished data). These bodies also bear some morphological similarity to the "lamellasome" described i n Anacystis nidulans (Echlin, 196*0 except that there was no separate vesicles i n the middle of these structures i n the blue-green alga. Although the lamellasome i n Anacystis nidulans was shown to be derived from the photosynthetic lamellae, nothing conclusive can be stated about i t s function or derivation i n Phaeostrophion at the present time. Discussion On the basis of pigmentation, storage products, flagellation, and c e l l wall components, the brown algae are now established as constituting a di s t i n c t class, the Phaeophyceae (Seagel ,1966). Certain characteristics including flagellation, clearly distinguish the Fucales from other orders i n this class and fine-structure studies seem to be corroborating this distinction. However, some controversy has arisen about the status of several other orders within the group. The Dictyosiphonales, for example, i s not well delimited from the Ectocarpales on general morphological grounds (Russell, 196*0. Consequently i t would be useful to know i f there are some differences i n the fine-structure of species within these orders which could be 15 of taxonomic significance. From results of the current electron microscopic investigation on Phaeostrophion irregulare (Dictyosiphohales) i t i s obvious that this species has many features typi c a l of other brown algae studied to date. Certain variations which were observed might be of importance phylogenetically. However i t i s clear that before any definite statements can be made, the entire l i f e cycle of many more species i n the Dictyosiphonales and Ectocarpales must be studied. The taxonomic significance of pyrenoids i n certain orders and families of Phaeophyceae has been hypothesized by Evans (1966,1968). From his observations he has suggested that pyrenoids are present i n orders such as the Ectocarpales, which are regarded as primitive on other grounds, and absent or rudimentary i n the more advanced ones, such as the Laminariales and Fucales. However, his studies were almost exclusively restricted to certain l i f e cycle stages of only one or two species i n each order, Pyrenoids have since been recorded i n the f i e l d material of Leathesia difformis (Chordariales) (Cole and Lin, 1968), Eudesme virescens (Chordariales)(Cole,1969) and Colpomenia  sinuosa and Petalonia d e b i l i s (Scytosiphonales) (Cole,1970), These are a l l considered as "lower orders" within the Phaeophyceae. It i s obvious from information obtained i n the present ultrastructural study of Phaeostrophion that caution must be taken i n drawing conclusions from limited data. For example, within the Dictyosiphonales the thallus c e l l s of Pietyosiphon foeniculaceus (Evans, 1966) and Punctaria sp. (Cole, unpublished data) have pyrenoids while those of Phaeostrophion do not. On the other hand, the sporeling c e l l s of Phaeostrophion do have well-defined pyrenoids characteristic of most brown algae. The p o s s i b i l i t y exists that the out-pocketing on the side of a few chloroplasts i n Phaeostrophion (Fig. 7) could represent a v e s t i g i a l pyrenoid i n the adult tissues, thus placing this alga i n an intermediate evolutionary position regarding this structure. An outpocket was also noted i n f i e l d material of Leathesia difformis (Cole and Lin, 1968), Eudesme virescens (Cole, 1969) and Soytosiphon  lomentaria (Cole, 1970). However, the rudimentary pyrenoid reported by Evans (1968) i n fucalean eggs i s more obviously delimited than the one i n Phaeostrophion (Fig. 7 ) . Dodge (1968) has suggested that a. similar out-pocketing i n the chloroplast of the dinoflagellate Aureodinium may be either a developing pyrenoid or just an area where the lamellae are s t i l l forming. It i s impossible to make any firm statement regarding the nature of this characteristic i n Phaeostrophion at present. Since the sporelings of Phaeostrophion produce the thallus directly, at least two points should be investigated: the stage during thallus development when the pyrenoid i s no longer produced, and the reason for i t s elimination from the differentiated tissue. I t i s possible that there i s some change i n the type of carbohydrate storage products during maturation which may be correlated with the disappearance of the pyrenoid. Simple analytical techniques could be used to demonstrate whether there i s a change from insoluble polysaccharide storage i n the germling to a soluble form i n the blade but this type of study i s made more d i f f i c u l t by the numerous problems of culturing Phaeostrophion. It has been stated that the chloroplast endoplasmic reticulum may be a typical fine-structural feature of the Phaeophyta and Chrysophyta and direct connections between i t and the nuclear membrane have been noted i n several species of brown algae (Bouck,1965; Evans, 1966; Kirk and Tilney-Bassett,1967; Cole, Bourne and Lin,1968; Cole, 1969,1970). However, no interrelationship between the chloroplast envelope and the nuclear membrane has been observed i n Fucus and Giffordia (Bouck, 1965) or the Phaeostrophion thallus material. Current studies i n this laboratory indicate that there i s an association of nucleus and chloroplast which i s limited to particular phases of the l i f e cycle of some Laminariales (Cole, unpublished data). Consequently, i f this characteristic i s variable within a species during i t s l i f e cycle one must be very certain to observe a l l stages before using i t taxonomically. Another ultrastructural characteristic which may be significant i n phylogenetic studies of the brown algae has been proposed. From observations on one species within each of the orders Ectocarpales, Laminariales, and Fucales, Bouck (1965) suggested that the association of the Golgi apparatus with a portion of the endoplasmic reticulum as well as the nuclear membrane i n the phylogenetically more advanced fucalean species could be of evolutionary significance, since i t i s consistently perinuclear i n the more primitive ones. The Golgi bodies have been observed to be s t r i c t l y perinuclear i n members of two other "lower orders", the Chordariales, Leathesia (Cole and Lin, 1968) and Eudesme (Cole, 1969) and the Scytosiphonales, Colpomenia, Petalonia and Scytosiphon (Cole, 1970). This would also seem to be the case for Phaeostrophion. which has been placed lower than the Fucales i n the evolutionary scale and i n which the Golgi complex i s associated with only the nuclear envelope. Centrioles have been noted i n light-microscopic studies of brown algae, but only infrequently or uncertainly, e.g. Nereocystis  luetkeana (Kemp and Cole, 196 l ) , Fucus sp. (Evans,1962), Halidrys  silquosa (Roberts, 1966) and Eudesme virescens (Cole, 1967). As a result of recent ultrastructural studies on Himanthalia lorea (Berkaloff, 1963), Fucus vesiculosus (Bouck,1965), Colpomenia peregrina (Evans, 1966), and Petalonia debilis (Cole,1970), as well as the current one on Phaeostrophion, i t i s becoming more evident that centrioles are of general occurrence i n c e l l s of the Phaeophyceae. Deviations from the usual three-thylakoid banding i n the Phaeophyceae, which were observed i n c e l l s of stored Phaeostrophion blades, most l i k e l y resulted from the a r t i f i c i a l culture conditions rather than from imperfect fixation since typical chloroplasts were also observed i n the same tissue (Fig.9 ) . The deviation of Phaeostrophion might indicate early stages i n degeneration. This could offer potential material for the study of senescence. Cole, Bourne and Lin (1968) have suggested that the illumination could possibly have been responsible for the production of extensive thylakoid stacking i n cultured sporeling c e l l s of Leathesia difformis. another brown alga. This thylakoid stacking i n Leathesia has since been shown to be part 19 of chloroplast division (Cole and Lin, 1968). While the abnormal stacking phenomenon i n Leathesia differed from that observed i n Phaeostrophion, i t i s interesting to note that both species were maintained with the same medium and l i g h t arid very similar temperature .conditions. The fact that the Leathesia chloroplasts were i n actively growing sporeling c e l l s while those of Phaeostrophion were i n mature c e l l s of thallus tissue may explain the difference i n type of thylakoid stacking between the two species. In conclusion, Phaeostrophion irregulare retains i t s intermediate position i n the phylogenetic cl a s s i f i c a t i o n of the brown algae. I t possesses some characteristics of the more advanced brown algae, such as the absence of a pyrenoid i n the adult thallus and the absence of a physical association of the chloroplast and nucleus. Also i t possesses more primitive characteristics, a s t r i c t l y perinuclear Golgi apparatus and a diffuse distribution of plasmodesmata. Literature Cited Bailey, A, and Bisalputra, T. 1969. Some structural aspects of the c e l l wall of Ectocarpus acutus Setchell and Gardner and Elachista fucicola (Valley) Areschoug. Phycologia 8: 57-63 Barton, R. 1968. Autoradiographic studies on wall formation: i n Chara. Planta (Berl.) 82i 302-306 Berkaloff, C. 1963. Les cellules meristematiques d 1Himanthalia lorea (L.) S,F. Gray, Etude au microscope electronique, J. Microscop. 2: 213-228 Bisalputra, T. 1966. Electron microscopic study of the protoplasmic continuity i n certain brown algae. Can. J. Bot. 44: 89-93 Bisalputra, T. and Bisalputra, A.A, 1967. Chloroplast and mitochondrial DNA i n a brown alga Egregia menziesii. J_. C e l l B i o l . 33: 511-520 Bouck, G.B. 1965. Fine structure and organelle associations i n brown algae. J. Cell B i o l . 26s 523-537 Brown, R.M. 1969. Observations on the relationship of the Golgi apparatus to wall formation i n the marine chrysophycean alga, Pleurochrysis s c h e r f f e l i i Pringsheim. J. Cell Biol. 4-1:109-123 Cole, K. 1964-. Induced fluorescence i n gametophytes of some -Laminariales. Can. J. Bot. 4-2: 1173-1181 Cole, K. 1967. The cytology of Eudesme virescens (Carm.)J. Ag. I. Meiosis and chromosome number. Can. J. Bot. 4-5: 665-673 Cole, K. 1969. The cytology of Eudesme virescens (Carm.) J.Ag. II. Dltrastructure of corti c a l c e l l s . Phycologia 8: 101-108 Cole, K. 1970. Ultrastructural characteristics i n some species i n the order Scytosiphonales. Phycologia 9» 275-283 Cole, K., Bourne, V., and Lin, S-C. 1968. Electron microscope observations on chloroplasts of cultured Leathesia difformis (L.) Aresch. Can. J. Genet. Cytol. 10: 63-67 Cole, K., and Lin, S-C. 1968. The cytology of Leathesia difformis. I. Fine structure of vegetative c e l l s i n f i e l d and cultured material. Syesis 1 : 103-119 Cole, K. and Lin, S-C. 1970. Plasmalemmasomes i n sporelings of the brown alga Petalonia deb i l i s . Can. J. Bot. 4-8:265-268 Dalton, A.J, 1955. A chrome-osmium fixative for electron microscopy. Anat. Record 121: 281 Dawes, C.J., Scott, F.M., and Bowler, E. 196l. A l i g h t and electron-microscopic survey of algal c e l l walls. I. Phaeophyta and Rhodophyta. Am. J. Bot. 48: 925-934 Dodge, J.D. 1968. The fine structure of chloroplasts and pyrenoids i n some marine Dinoflagellates. J. C e l l Sci. 3: 41-48 Echlin, P. 1964. Intra-cytoplasmic membranous inclusions i n the blue-green alga, Anacystis nidulans. Arch. Mikrobiol. 49: 267-274 Evans, L.V. 1962. Cytological studies i n the genus Fucus. Ann. Bot. (N.S.) London 26: 345-360 Evans, L.V. 1966. Distribution of pyrenoids among some brown algae. J. C e l l Sci. 1: 449-454 Evans, L.V. I968. Chloroplast morphology and fine structure i n B r i t i s h fucoids. New Phytologist 67: 173-178 Freeman, J.A. and Spurlock, B.D. 1962. New epoxy embedment for electron microscopy. J_. C e l l B i o l . 13: 437-443 Gibbs, S.P. 1962a, Nuclear envelope-chloroplast relationships i n algae. J. Cell B i o l . l 4 i 433-444 Gibbs, S.P. 1962b. The ultrastructure of the pyrenoids of algae, exclusive of the green algae. J_. Ultrastruct. Res. 7s247-261 Greenwood, A.D. 1964. The structure of chloroplasts i n lower plants. Abstr. 10th Intern. Botan. Congr. Edinburgh1212-213 Kemp, L. and Cole, K. 1961. Chromosomal alternation of generations i n Nereocystis luetkeana (Mertens) Postels and Ruprecht. Can. J. Bot. 39s 1711-1724 Kirk, J.T.O. and Tileny-Bassett, R.A.E. 1967. The plastids. W.H. Freeman and Co., San Francisco. Liddle. L.B. and Neushul, M. 1969. Reproduction in Zonaria farlowii. I I . Cytology and ultrastructure. J . Fhycology 5* 4-12 McCully, M.E. 1965. A note on the structure of the ce l l walls of the brown alga Fucus. Can. J . Bot. 43: 1001-10C4 McCully, M.E. 1968. Histological studies on the genus Fucus. I H . Fine structure and possible functions of the epidermal cells of the vegetative thallus. J . Cell Sci . 3: 1-16 Manton, I . 1966. Further observations on the fine structure of Chrysochromulina chiton, with special reference to the pyrenoid. J . Cell Sci . 1 : 187-192 Mathieson, A.C. 1967. Morphology and l i f e history of Phaeostrophion irregulare S.et G. Nova Hedwigia 13t 293-318 Neushul, M. and Liddle, L. 1968. A light-and electron-microscopic study of primary heterogeneity in the eggs of two brown algae. Am. J . Bot. 55: 1068-1073 Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J_. Cell Biol . 17: 208-212 Roberts, M. 1966. The Phaeophyceae. Part H . in The chromosomes of the algae. Edited by M.B.E. Godward, Edward Arnold Ltd . , London, pp. 149-167 Russell, G. 1964. Systematic position of Pilayella l i t tora l i s and status of the order Dictyosiphonales. Brit . Phycol. Bull . 2 : 322-326 Scagel, R.F. 1966. The Phaeophyceae in perspective. Oceanogra. Mar. Biol . Ann. Rev. 4 : 123-194 Setchell, W.A. and Gardner, N.L. 1924. Phycol. Contrib; V I I . Univ. Calif . Publ. Bot. 13$ 1-13 Siegel, S.M. 1962. The plant c e l l wall. Pergamon Press Ltd., Oxford Starr, R.C. 1964. The culture collection of algae at Indiana University. Am. J. Bot. 51» 1013-1044 Thomson, W.W. 1967. Electron microscope studies on some modifications of the plasmalemma i n oranges. J. Ultrastruct. Res. 17; 475-486 Wooding, F.B.P. I968. Radioautographic and chemical studies of incorporation into sycamore vascular tissue walls. J. C e l l Sci. 3: 71-81 Wooding, F.B.P. and Northcote, D.H. 1965. The fine structure of the mature resin canal c e l l s of Pinus pinea. J. Ultrastruct. Res. 131 233-244 Ziegler, H. I963. Untersuchungen uber die Feinstruktur des Phloems. II. Mitteilung. Die Siebplafcten bei der Braunalge Macrocystis pyrifera (L.) Ag. Protoplasma 57: 786-799 Ziegler, H. and Ruck, I. 1967. Untersuchungen uber die Feinstruktur des Phloems. III. Mitteilung. Die "Trompetenzellen" von Laminaria-arten. Planta (Berl.) 73: 62-73 24 Figure 2 Photograph of mature plants of P. irregulare showing the narrow, short stipes» the small disc-like holdfast (H), and the irregularly torn blades, X 1/3 Figure 3 Cross section of a young maturing blade as seen by l i g h t microscopy, showing the developing medullary c e l l s (MC), and unilocular sporangia (US). X 1,000 Figure 4 Electron micrograph of a surface c e l l which may be a unilocular i n i t i a l . Note the numerous small chloroplasts (Ch), o i l bodies (Ph), c e l l wall (CW), and the small vacuole (V). X 4,000 Figure 5 View of a portion of a c o r t i c a l c e l l of the blade showing the numerous o i l bodies, vacuole, and c e l l wall consisting of an outer primary layer (l'CW) and inclusions (arrows) and an inner secondary layer (2°CW). X 7,000 Figure 6 The pyrenoid (Py) and part of a chloroplast i n a sporeling c e l l . X 34,000 Figure 7 A chloroplast i n a c o r t i c a l c e l l with a l a t e r a l out-pocketing (OP) or non-lamellar area. Note the location of the correlated thylakoid interchange (arrows) and the osmiophilic droplet (0). X 22,000 Figure 8 Part of a medullary vacuolated c e l l containing numerous o i l bodies (Ph). X 5,000 Figure 9 Chloroplast from plant kept under a r t i f i c i a l illumination for extended period. Note the clustering of lamellar bands into larger groups and separation of bands into single thylakoids (arrows). X 10,000 Figure 10 O i l body adjacent to a chloroplast. The chloroplast envelope (CE) separates the two (arrows). X 36,000 26 Figure 11 Chloroplast of vacuolated c e l l seen i n Figure 8. Note especially the lamellar separations (arrows). X 32,000 Figure 12 Nucleus (N) with nucleolus (Nl) and nuclear envelope (NE) with pores (NP). Blebs (arrows) on the outer nuclear membrane are forming small circular vesicles. These coalesce to form the Golgi apparatus (G), which consists of the forming face ( f f ) and maturing face (mf). X 31,000 Figure 13 Centriolar bodies. Note the three subunits per subfiber (arrows). X 25,000 Figure 14 Spheroid body with a limiting membrane (LM) between the piasmalemma and c e l l wall. Note the connecting f i b r i l s (arrows) between the body and the c e l l wall material X 40,000 Figure 15 Portion of a pore area i n a c o r t i c a l c e l l wall. Note each plasmodesma (PI), unit membrane, and the eleotron-transparent area, which may be callose (double arrow) between i t and the c e l l wall material. F i b r i l s (arrows) extend from the plasmodesma to the wall material. X 67,000 Figure 16 Longitudinal section of plasmodesma ta showing continuity through the wall. X 70,000 Figure 17 Two typical mitochondria (M). The inner membrane i s continuous with the tubular cristae (arrow). X 38,000 Figure 18 Portion of a vacuolated c e l l showing an accumulation of small vesicles (arrows) between the plasmalemma and the c e l l wall. X 15,000 Figure 19 Lamellar body located i n a vacuole showing the paired lamellae (arrows) and inner vesicles (IV). X 40,000 PART TWO Rhodophyceae A comparison of cellular fine structure of the vegetative t h a l l i of Porphyra perforata and Bangia fuscopurpurea and certain phases i n the l i f e history of Porphyra perforata Introduction It has been generally accepted that the Bangiophycidae (Bangioideae) are simple rhodophycean forms which exhibit diffuse growth, re l a t i v e l y unspecialized sex organs, carposporangia formed by direct division of the zygote and have no p i t connections or aggregation of filaments. On the other hand most of the Florideophycidae (Florideae) are more complex Rhodophyceae and usually consist of aggregated filaments with p i t connections, highly differentiated sex organs, and carposporangia formed on filamentous gonimoblasts derived from the zygote (Fritsch, 194-5). These c r i t e r i a were used to separate the two subclasses macroscopically and by l i g h t microscopy (Fritsch,194-5). However, with the application of electron microscopic techniques we may be approaching the time when the separation of the two subclasses, Bangiophycidae and Florideophycidae, w i l l have to be re-evaluated, There i s an increasing interest i n the ultrastructure of the red algae. For example, within the Bangiophycidae there have been reports on Bangia (Honsell,1963), Porphyridium (Brody and Vatter*1959; Speer, Dougherty, and Jones,1964; Gantt and Conti,1965,1966a and b; Gantt, Edwards, and Gonti 1968); Compsopogon (Nichols, Ridgway,and Bold,1966), Smithora (McBride and Cole, 1969,1971), Rhodella (Evans, 1970), and Porphyra (Yokomura,1967; Kito and Akiyama,1968; Lee and Fultz,1970; Bourne, Conway, and Cole,1970;, and Kazama and Fuller, 1970). However many authors considered only one or two organelles i n most species, or i n the case of Porphyra only one stage i n the l i f e cycle. The basic l i f e cycle of the Bangiales consists of a macroscopic form alternating with a filamentous microscopic form, both being capable of asexual reduplication by neutral or monospores. Numerous studies have been made on the l i f e history of various Porphyra species. Some workers have completed the l i f e cycle i n culture (Kurogi,1953; Tseng and Chang,1955; Graves,1955; Hollenberg,1958; Kornmann,196l; Conway,1964b; and Chen, Edelstein, Ogata,and McLachlan, 1970), A specific example of the basic l i f e cycle of P. perforata i s given by Hollenberg (1958) where the mature thallus produces spores which develop into the filamentous conchocelis phase. This latter phase i n turn produces spores which develop into the mature thallus. Although Hollenberg (1958) did not observe the production of neutral spores i n summer material, he did not exclude the p o s s i b i l i t y that they existed at another time. Several researchers have shown that a few species produce aplanospores that develop into bipolar sporelings (Conway 1964b,1966), and neutral spores that reduplicate the bipolar sporelings (Conway 1964b,1966), whereas others have demonstrated the role of monospores i n the conchocelis phase (Conway, 1964b; Krishnamurthy,1969; Chen, Edelstein, Ogata, and McLachlan,1970). 30 The purpose of this study was to extend the ultrastructural knowledge of the Bangiophycidae by examining and comparing the thallus c e l l s of two l o c a l species, Porphyra perforata and Bangia  fuscopurpurea, and comparing the c e l l s of several phases of the l i f e history of P. perforata. This information would be used i n comparison with published data on other red algae, to determine any phylogenetical significance of the ultrastructure. Materials and Methods Porphyra and Bangia are included i n the family Bangiaceae, order Bangiales, subclass Bangiophycidae. Porphyra perforata J.G. Agardh grows on rocks i n the i n t e r t i d a l region of the Pacific coast of North America. The mature blade i s monostromatic, has one chloroplast per c e l l and i s highly variable i n size and shape ranging from 2 x 10 cm to 15 x 30 cm (Figs 22,23). The blade i s held to the substratum by a holdfast consisting of rhizoidal processes extending from the basal c e l l s . The color varies from a deep purple-red to dark brown. Bangia fuscopurpurea (Dillwyn) Lyngbye grows i n much the same area and has similar coloring to P. perforata. However the external morphology i s somewhat different. The mature thallus of B, fuscopurpurea i s a multiseriate filament, with the basal c e l l s extending rhizoidal processes downward to form a holdfast. Mature blades of P.perforata were collected from Amphitrite Point, Uclueletj Whiffin Spit and Point No Point (Glacier Point), Vancouver Island; Brockton Point, Stanley Park and Point Grey, Vancouver, General collections were made for fixation during afternoon low tides i n the springs of 1968,1969, and 1970. Filaments of B, fuscopurpurea were collected from a pi l i n g at Rosario Beach, Washington, U.S.A. i n July I968. The collection sites of P. perforata and B, fuscopurpurea are indicated i n Figure 20, Collections for culturing P. perforata were made during November 1969 and January to A p r i l 1970. They were kept cool and brought to the University of B r i t i s h Columbia laboratories where they were placed i n p e t r i dishes containing a sea water culture medium. The culture medium consisted of a l s l mixture of enriched sea water (ster i l e sea water to make 1 l i t r e plus: NaN03 - 200 mg., Na2HP04-7H20 - 20 mg., KN03 -50 mg., EDTA - 1 mg., Tris buffer - 500 mg., s o i l water - 50 ml., Vitamin B 12 - 1 mg.) and ste r i l e sea water. After spore release, the t h a l l i were removed and the media changed frequently during the subsequent growth period to reduce contamination. Cultures were maintained i n a Sherer culture chamber under GE F.20.T.12 cool white fluorescent tubes at a le v e l of illumination of about 1000 lux or 93 f.c. The l i g h t regime was 8 hours of l i g h t and 16 hours of darkness at a constant temperature of 10°C, The growth of the germlings was observed for a period of several months. The culturing for this study was done i n collaboration with Dr. Elsie Conway. A l l materials were fixed for 2-4 hours i n 4$ glutaraldehyde i n 1/15 M phosphate buffer,pH 7.4 at 5°C and brought to room temperature. They were then washed i n buffer, post-fixed i n 1$ 0s04/phosphate for one hour, dehydrated i n an alcohol-propylene oxide series, and 3 2 Figure 2 0 Map of the collection sites of algae used i n this study 1 Ucluelet, Vancouver Island 2 Point No Point (Glacier Point), Vancouver Island 3 Whiffin Spit, Vancouver Island 4 Point Grey, Vancouver 5 Brockton Point, Stanley Park, Vancouver 6 Rosario Beach, Washington subsequently embedded i n an epoxy resin. The Maraglas epoxy (Spurlock, Kattine and Freeman, 1963) was polymerized at 60°C for 48 hours. Sections were cut with glass knives on an LKB Ultrotome I and a Reichert 0M-U2, stained with lead citrate (Reynolds, 1963) and uranyl acetate (Watson,1958) and subsequently examined with Hitachi HS-7S, HU-11A, Zeiss EM-9A, RCA EMU-3H and Philips EM300 electron microscopes. Specimens for surface study were fixed i n 1$ aqueous 0sC4, dehydrated and coated with Au/Pd 60:40 under vacuum. They were then observed i n a Cambridge Stereoscan scanning electron microscope at 20 kv.. Some confusion might arise regarding the terms used to designate the spores produced by Porphyra during different stages of i t s l i f e history. Consequently the terminology used i n this study i s defined: <* spores - are sli g h t l y smaller than the vegetative c e l l s and are deeply pigmented. They are usually produced i n packets of 8 - 32 by repeated division of a vegetative c e l l i n the mature macroscopic thallus. They produce the conchocelis phase (Conway,1964a), and are equivalent to the type III spores of Drew (1956), (3 spores - are very much smaller than the vegetative c e l l s and are almost colorless. They are produced on the macroscopic thallus much l i k e the o t spores, but with more divisions (Conway,1964a), The /3 spores are produced 32-128 per packet and are also equivalent to the type III spores of Drew (1956). They are commonly referred to as spermatia. 34 are approximately the same size as the vegetative ce l l s and are produced on the leafy thallus by differentiation of a whole c e l l . These are equivalent to the type II spores of Drew (1956) and develop into bipolar sporelings. are produced by differentiation of whole c e l l s i n the bipolar sporelings and develop into another generation of bipolar sporelings. are produced terminally on the conchocelis filaments by differentiation of whole c e l l s and grw into another generation of conchocelis filaments, are produced on specialized branches of the conchocelis phase and develop into bipolar sporelings. Observations The l i f e history of Porphyra perforata observed i n this laboratory (Fig.21) was basically similar to that described by Hollenberg(1958) for the same species although the development of the conchospore into the thallus was not obtained. The production and development of neutral and monospores, which Hollenberg (1958) did not see i n his material, were observed and found to be similar to that of other Porphyra species (Drew,1949,1954; Kurogi,1953; Tseng and Chang,1955; Graves,1955; Kornmann,196l; Conway,1964a and b; Krishnamurthy,1959,1969; Chen, Edelstein, Ogata, and McLachlan,1970) and Bangia (Sommerfeld and Nichols,1970). T h a l l i of P. perforata collected during October,1969, aplanospores -neutral spores -monospores -conchospores -35 Figure 21 Diagram of the ten ta t i ve l i f e cycle of Porphyra per forata ». por t ion of the l i f e cycle observed i n t h i s study ~ por t ion of the l i f e cycle not observed i n t h i s study but i n fe r red from previous publ icat ions (see p.33 , t e x t ) f e r t i l i z a t i o n ? f e r t i l i z a t i o n and the r o l e of spores i n t h i s process have not ye t been conclusively demonstrated Figure 21 Diagram of the t e n t a t i v e l i f e c y c l e of Porphyra p e r f o r a t a 36 released aplanospores from small patches scattered over most of the blade. These spores underwent bipolar germination (Figs. 57»58). The young bipolar sporelings quickly developed rhizoidal c e l l s (Figs. 58 f59) and grew into small blades about 2 cm across. Many cel l s i n the sporelings formed neutral spores, reduplicating the bipolar sporeling. T h a l l i collected during January 1970 released both large <x spores and smaller ft spores or spermatia (Fig. 24), from small patches scattered throughout the blade. The fi spores (Fig. 80) when released and germinated alone, degenerated within two days. These results are similar to those obtained with P. umbilicalis (Krishnamurthy 1959, Conway 1964b). The released o< spores (Fig. 80) germinated into the conchocelis phase (Fig. 81). After about 2-3 weeks, branched filaments appeared i n culture. Many of the branches terminated i n monospores. Upon release the monospores produced a new generation of conchocelis filaments. This process repeated i t s e l f for several months yielding a very profuse growth of filaments, as i n P. umbilicalis (Conway,1964b), After approximately five weeks the conchocelis filaments developed some side branches composed of almost isodiametric c e l l s (Figs, 95,102), which were probably the conchospore branches. These c e l l s had a stellate chloroplast with a central pyrenoid similar to that i n the vegetative c e l l s of the macroscopic thallus, Vegetative c e l l s of the thallus and bipolar sporelings of Porphyra perforata, and the thallus c e l l s of Bangia fuscopurpurea The vegetative ce l l s of P. perforata thallus and bipolar 37 sporelings and of B. fuscopurpurea thallusare basically similar with a large stellate chloroplast and central pyrenoid, a small nucleus, mitochondria, Golgi bodies, starch-like storage products and a loose gelatinous c e l l wall. The general ultrastructure of these c e l l s i n most respects resembles that already reported for both subclasses of the Rhodophyceae, Porphyridium (Brody and Vatter,1959» Speer, Dougherty,and Jones,1964;and Gantt and Conti ,1965), Lomentaria (Bouck, 1962), Polysiphonia, Nemalion and Kylinia (Gibbs,1962a and b), Bangia (Honsell ,1963), Laurencia (Bisalputra, Rusanowski,and Walker, 1967), Batrachospermum (Brown and Weier, 1968,1970), Porphyra yezoensis (Kito and Akiyama,1968), Pseudogloiophloea (Ramus,1969a and b) and Smithora (McBride and Cole, 1969). The most conspicuous organelle i s the highly lobate central chloroplast (Figs. 25 , 2 6 , 6 1 , 6 7 , 76 ) . It i s enclosed by a continuous double membrane (Figs. 2 6 , 6 2 , 6 7 , 7 6 ) . The thylakoids, 20 mu thick are arranged p a r a l l e l to one another, separated by about 100 mu (Figs. 28 ,30 , 63 ,67 ) . The overall shape of the chloroplasts of Porphyra and Bangia resembles that of Porphyridium (Brody and Vatter, 1959; Gantt and Conti ,1965; and Gantt, Edwards,and Conti ,1968) and Smithora (McBride and Cole, 1 9 6 9 ) . Finger-like projections of the chloroplast extend through the cytoplasm. There i s no peripheral thylakoid, and i n some lobes a group of thylakoids terminates before reaching the chloroplast limiting membrane (Figs. 3 0 , 3 4 , 6 4 , 7 6 ) . A similar phenomenon has been seen i n Porphyridium (Gantt and Conti ,1965), and Smithora (McBride and Cole, 1969). However, many Florideophycidae such as Batrachospermum (Brown and Weier, 1968,1970) do possess a 38 peripheral thylakoid paralleling the limiting envelope. No loose thylakoid stacking such as that reported for Smithora (McBride and Cole, 1969) was observed i n Porphyra. Some interconnections between thylakoids (Figs. 28,76) were noted as i n Porphyridium (Gantt and Conti, 1965) and Smithora (McBride and Cole, 1969). Phycobilisomes were not obvious on the thylakoids of Porphyra. This resembles Smithora (McBride and Cole, 1969), and contrasts sharply with the chloroplast lamellae of Porphyridium where the phycobilisomes are very d i s t i n c t (Gantt and Conti, 1965,1966aj and Gantt, Edwards, and Conti, 1968). Small electron transparent areas containing clumped f i b r i l s 1 to 10 mu thick are located between the photosynthetic lamellae throughout the chloroplast (Figs. 28,63). These are similar to the DNA localizations i n Laurencia (Bisalputra and Bisalputra,1967) and Smithora (McBride and Cole, 1969). Numerous osmiophilic droplets occur throughout the chlaroplast. The pyrenoid of Porphyra and Bangia i s quite similar to that reported for other Bangiophycidae, Porphyridium (Brody and Vatter,1959; Gantt and Conti,1965) and Smithora (McBride and Cole, 1969). I t i s also quite similar to that reported for some Florideophycidae, Nemalion and Kylinia (Gibbs 1962a and b). It i s located i n the central portion of the chloroplast and i s traversed by several thylakoids which are continuous with those i n the chloroplast (Figs. 25,26,34,63, 66,77). Small thylakoid cisternae i n the pyrenoid are sli g h t l y distended. Many of the pyrenoid lamellae are p a r a l l e l (Fig, 25), a feature which corroborates Krishnamurthy1s (1959) l i g h t microscope observations on the internal pyrenoid structure. In the Porphyra bipolar sporelings a pyrenoid was noted within several of the chloroplast arras i n some vegetative c e l l s . This suggests that the pyrenoid was very lobate, extending through the chloroplast or that there was more than one pyrenoid present i n the chloroplast. Since this i s unlike the central pyrenoid i n the thallus c e l l s , i t may have been a cultural aberration. On the other hand, there may have been more than one chloroplast present i n these c e l l s . This was d i f f i c u l t to ver i f y by li g h t microscopy. The large numbers of osmiophilic droplets which occur i n the chloroplast of the bipolar sporeling c e l l s (Figs, 62,63) may also be due to culture conditions. Floridean starch granules occur singly or i n groups outside the chloroplast membrane (Figs. 30*32,34,63,64). These bodies are sausage-shaped and approximately 0.1 x 0.1 x 0,4 u i n size. They were never observed inside the chloroplast and i t i s assumed that they are formed outside the chloroplast limiting membrane. This feature has also been recorded i n other red algae, Porphyridium (Brody and Vatter, 1959{ Speer, Dougherty, and Jones,19.64; and Gantt and Conti, 1965), Ldmentaria (Bouck, 1962), Kylinia. Polysiphonia and Nemalion (Gibbs, 1962a and b), Pseudogloiophloea (Ramus,1969a) and Smithora (McBride and Cole, 1969). Elements of the endoplasmic reticulum (ER) are located throughout the cytoplasm and are especially prevalent i n some areas (Figs. 34,46, 47,50,56,60,69,?1,76). The E R often occurs i n the form of a single cisterna near and paral l e l to the plasmalemma (Figs. 46,47), as i n Porphyridium (Gantt and Conti,1965). It may also form several cisternae which extend throughout the cytoplasm (Fig. 56) very much 40 l i k e Lomentaria (Bouck, 1962), Since the E R i s continuous for some distance through a section i t i s considered to be lamellar o rather than tubular. The membranes are about 80 A thick and the o cisternae are 200 A wide. Some cisternae become sl i g h t l y distended (Fig. 56). The E R i s much more extensive i n Porphyra than i n either Porphyridium (Gantt and Conti, 1965; Gantt, Edwards, and Conti,1968) or Smithora (McBride and Cole, 1969). The ribosomes do not appear to be attached to the E R membranes, A more densely staining membrane believed to be E R extends throughout some c e l l s (Fig, 50). This has also, been noted i n Smithora (McBride and Cole, 1969). The mitochondria (Figs. 28-31,69,76) resemble those reported for Smithora (McBride and Cole, 1969) and Pseudogloiophloea (Ramus, 1969a), They are approximately 0,5 to 1 u i n section are round to oval shaped, and the cristae are continuous with the inner membrane. No long branched mitochondria l i k e those of Porphyridium (Gantt and Conti,I965) nor any ring shaped mitochondria as recorded occasionally i n Smithora (McBride and Cole, 1969) were seen i n Porphyra or Bangia, Very few lamellar cristae were noted, i n contrast to Lomentaria (Bouck, 1962), The mitochondria were often seen near the nucleus (Figs. 29,31). The Golgi apparatus i s generally small (Figs. 28-31,64,69), consisting of several flattened cisternae. Towards the maturing face the extremities of each cisterna are swollen and contain fibrous material. Several free vesicles containing the same material were noted beyond the maturing face. The forming face of the Golgi apparatus i s very often close to a mitochondrion (Figs. 28-31,64,69) as in 41 Corallina (Bailey and Bisalputra,1970) and Batrachospermum (Brown and Weier,1970) . The Golgi bodies and associated vesicles are morphologically similar to those reported for Lomentaria (Bouck ,1962), Porphyridium (Gantt and Conti,1965 ; Gantt, Edwards, and Conti,1968) and Smithora (McBride and Cole, 1 9 6 9 ) . The plasmalemma i s a typical single membrane and portions of i t i n each c e l l are highly convoluted (Figs. 2 7 , 3 7 , 4 0 , 5 5 , 6 0 , 6 7 ) . Many wall fibers extend i n between the various infoldings and a few are continuous with the plasma membrane (Figs. 4 6 , 4 9 , 5 0 , 5 5 ) . Convolutions i n the plasmalemma have also been observed i n Lomentaria (Bouck ,1962), and Porphyra yezoensis (Kito and Akiyama,1968) and surface a c t i v i t i e s have been demonstrated i n the red alga Laurencia (Bisalputra, Rusanowski, and Walker 1 9 6 7 ) . Nuclear shape and position appear to be influenced by the large chloroplast. The chloroplast tends to compress the nucleus into a peripheral location. The nucleus i s at times highly lobate (Figs. 3 4 , 6 8 , 7 8 ) , a condition previously observed by l i g h t microscopy, and discussed by Dixon ( 1 9 6 6 , p , 1 7 7 ) . This suggests the p o s s i b i l i t y of a very active nucleus. No close association was noted between the nucleus and the pyrenoid and chloroplast as i n Rhodella (Evans,1970). The nucleoplasm i s granular and surrounded by a very porous nuclear envelope (Figs. 3 4 , 6 8 ) , The single large nucleolus with areas of lower electron density appeared to have no constant position within the nucleus (Figs. 2 5 , 3 3 , 3 4 , 3 6 , 3 8 , 6 8 , 7 8 ) . It was not necessarily oriented towards the chloroplast as i n Porphyridium (Gantt and Conti, 42 1 9 6 5 ) . The nucleolar shape and position resembled more closely that of Smithora (McBride and Cole, 19&9). Nuclear structure i s very similar to that of other members of the Bangiophycidae, Porphyridium (Gantt and Conti 1 9 6 5 , Gantt, Edwards, and Conti 1 9 6 8 ) and Smithora (McBride and Cole, I 9 6 9 ) . Nuclear division precedes cytokinesis and the daughter nuclei separate and are usually positioned at opposite ends of the c e l l well i n advance of protoplasmic division i n Porphyra perforata (Figs.35t36). This characteristic has also been noted i n l i g h t microscope observations on other Porphyra species (Krishnamurthy, 1 9 5 9 , Pringle and Austin,1970 ) , as well as Porphyridium (Gantt and Conti, 1 9 6 5 ) . Centriolar bodies were not observed i n P. perforata thallus and bipolar sporeling or B, fuscopurpurea thallus. Cytokinesis i n the thallus nf Porphyra  perforata collected i n the spring i s generally priented longitudinally, i . e . p a r a l l e l to the long dimension of the c e l l , an observation also recorded by Krishnamurthy ( 1959 ) and Conway ( 1 9 6 4 b ) i n vegetative and sporing areas of P. umbilicalis. Drew ( 1 9 5 4 ) , however, suggested that i n some Porphyra species the i n i t i a l plane of division i n vegetative c e l l and monospore production i s longitudinal and that for carpospore ( o< spore) production i s transverse. Cytokinesis i n the blade of P. perforata appears to be of two types. The f i r s t i s very much li k e that seen i n Porphyridium (Gantt and Conti, 1 9 6 5 ) . It commences with the i n i t i a t i o n of a cross wall at the c e l l periphery. This wall i s about the same thickness as the surrounding walls (Fig. 35). The wall growth continues towards the center of the c e l l , the thickness remaining the same (Fig. 36). The 43 chloroplast and pyrenoid are the l a s t major organelles to divide, actually being caught i n the opening l e f t by the forming cross wall. The second, more common type of division observed i n this material, commences with the division of the chloroplast and pyrenoid followed by the invagination of the plasmalemma deep into the c e l l (Fig, 37). This invagination process continues u n t i l the plasmalemma crosses the c e l l completely. Unlike the previous method of c e l l division no wall material i s l a i d down during this a c t i v i t y . The two c e l l s are separated by only the two plasma membranes. There i s then an increased Golgi and/or plasmalemma ac t i v i t y on both sides with the formation of small fiber-containing vesicles. This i s followed by a deposit of wall fibers at various locations between the two plasma membranes u n t i l a wall i s completed between the two cells (Fig. 38). It i s not yet possible to t e l l i f the wall formed at this point remains thin or thickens later. Similar c e l l division was also seen i n Bangia (Figs. 66,74). It i s d i f f i c u l t to provide an explanation for the two types of cytokinesis. The second type seems to be more general i n the Porphyra species which have previously been studied cytologically. Krishnamurthy (1959) noted that a thicker wall formed between daughter c e l l s i n the vegetative area than between those i n the spore producing regions. Relatively thin c e l l walls characterize «x spore development i n the current study (Fig. 43). The production of the o< spores i n Porphyra i s by repeated division of the spore mother c e l l , and c e l l size decreases with each division. The «=< spores contain c e l l organelles similar to those i n 44 the vegetative c e l l s of the thallus (Figs. 43,44) . The c e l l walls between the developing spores are usually much thinner than the surrounding mother c e l l wall. Near the c e l l surface, there are a large number of small vesicles which are packed with a fibrous material similar to the wall (Fig, 44) , The function of these vesicles i s not known, but the fibrous contents suggest that they may contribute towards the production of the spore wall. Some stages of aplanospore development and release have been observed i n Bangia (Figs. 76-79). These spores are about the same size as the vegetative c e l l . Their ultrastructural details are much l i k e those of the vegetative c e l l . The spore wall i s much thinner than the surrounding mother c e l l wall (Figs. 78,79) and becomes separated from i t as development nears completion (Fig, 79). Previous reports (reviewed by Drew, 1956: Sommerfeld and Nichols,1970) indicate that the Bangia spore i s released naked. The presence of a c e l l wall observed i n the present study i s possibly due to the fact that the spore i s starting to germinate i n sit u . This has also been reported previously (reviewed by Drew, 1956; Cole, 1971). Paramural bodies and associated structures such as concentric lamellar bodies and polyvesicular bodies are seen i n the vegetative c e l l s . The most conspicuous of these are the concentric lamellar bodies which are 0.5 to 1.5 u i n diameter and have concentrically arranged membranes (Figs. 45-51» 69-73). Some consist of many single or paired thick lamellar layers (Figs. 49,73) whereas others have much thinner, tightly packed membranes (Figs. 48,71) . They may also contain small tubules (Fig. 45) . The position of these bodies i s somewhat diverse; some are at the edge of the c e l l (Figs, 46,4-9), others through the cytoplasm (Figs, 45,48,71,73), and a few are located i n the chloroplast (Figs, 48,70) . Lamellar bodies similar to those i n the cytoplasm occasionally occur i n the wall (Figs. 47,72) near the c e l l surface and can be adjacent to cytoplasmic lamellar bodies i n the cytoplasm. Wall fibers attached to the plasmalemma near a lamellar body are shown i n Figure 46, Similar configurations were seen i n Porphyridium (Gantt and Conti, 1965,1966b) and Batrachospermum (Brown and Weier,1970). Polyvesicular or lomasome-like structures about 0,7 )x i n diameter are observed i n the cytoplasm. These consist of one or two enveloping membranes, containing several smaller vesicles, the outermost of which may contact the c e l l wall (Fig. 49, 51-54). They resemble the lomasomes of Moore and McAlear (196 l ) . They are often i n close association with concentric lamellar bodies (Fig. 49 ) , therefore their function may be related to that of the latte r structures, Bouck (1962) noted a similar polyvesicular body i n Lomentaria between the c e l l membrane and the c e l l wall, but the ontogeny of this was not known. Also, Ramus (1969a) demonstrated structures that resembled these i n the red alga Pseudogloiophloea, both i n the cytoplasm and between the plasmalemma and wall. He concluded that these lomasomes were involved i n c e l l wall production. Lee and Fultz (1970) showed similar structures i n the conchocelis phase of Porphyra  leucosticta. The Porphyra c e l l wall i s composed of xylan microfibrils and a mannan-xylan embedding matrix (Frei and Preston, 1964), It i s a 46 loose network of f i b r i l s embedded i n an amorphous matrix and resembles the sheath of Porphyridium (Gantt and Conti, 1965)and walls of Laurencia (Bisalputra, Rusanowski, and Walker,1967) and Smithora (McBride and Cole, 1969). It consists of several layers. Although the fibers of both the inner and outer layers are reticulate, the more conspicuous fibers i n the outer layers (Figs. 25,33) are p a r a l l e l to the c e l l surface whereas those i n the inner layers tend to be perpendicular to the c e l l surface (Figs. 34,36). Sections through c e l l s i n the holdfast region (Figs. 39-41) of Porphyra show the very thin rhizoidal processes of the c e l l s surrounded by extremely thick walls (Fig. 39). The plasmalemma of these processes i s highly convoluted (Figs, 40,41), Scanning electron microscopy of the Porphyra f i e l d material reveals a highly folded surface of the whole thallus (Fig, 42), The material has been fixed and dried so thi s may not r e f l e c t the true picture i n nature. Cross sections of Porphyra fixed and embedded i n plastic also show an undulating surface, although i t i s less pronounced. Conchocelis phase ce l l s of Porphyra perforata On germination, the o< spore (Fig. 80) forms a tube into which the entire cytoplasmic mass eventually migrates. A cross wall- i s then formed sealing off the empty spore case (Fig,85) (Drew, 1949,1954} Krishnamurthy, 1959,1969). Fine structural studies confirm t h i s . The spore cases (Figs, 86,87) were devoid of any cytoplasm, and a plugged p i t connection had formed between the spore case and the conchocelis c e l l (Fig, 86). The P. perforata conchocelis phase c e l l s are long and narrow (Fig. 85) about 20 u by 7 u. As i n the c e l l s of the macroscopic thallus, the chloroplast of the conchocelis phase i s the most prominent organelle (Figs. 83,84,94-), It i s not stellate and i t s shape seems to be determined by the long tubular c e l l shape. Projections of the chloroplast extend throughout the individual c e l l , f i l l i n g i t i n certain areas (Figs. 83,84) as i n P. leucosticta (Lee and Fultz, 1970).The thylakoids are single, about 15 mu thick and separated by 100 mu. Interconnections of individual lamellae occur (Fig. 83) , as i n the chloroplasts of the macroscopic thallus as well as i n Smithora (McBride and Cole, 1969). No association of thylakoids, such as those seen i n Smithora (McBride and Cole, 1969) were noted. Although not always the case (Fig. 82) , the chloroplasts often have a peripheral thylakoid (Figs. 84 ,92 ,94), This characteristic was also noted by Lee and Fultz (1970) i n P. leucosticta conchocelis. However i t should be mentioned that a peripheral thylakoid i s not shown i n a l l of their figures. Osmiophilic droplets occur between the photosynthetic lamellae and often at the periphery of the pyrenoid (Figs. 84,93). In the young pink (2r3 week) conchocelis filaments there were numerous round phycobilisomes attached to the chloroplast lamellae (Fig. 83), whereas i n the old brown (6-8 week) conchocelis and the brown colored thallus none were observed (Figs. 92-94). Round phycobilisomes were also noted i n Porphyridium (Gantt and Conti, 1965,1966a} and Gantt, Edwards, and Conti,1968). It i s quite unlike Batrachospermum (Lichtle and Giraud,1970) which has rod shaped phycobilisomes, and unlike the macroscopic thallus of Porphyra with 48 i t s apparent absence of phycobilisomes. Phycobilisomes were observed only i n the young pink conchocelis of Porphyra perforata. It i s probable that the phycobilisomes contained more phycoerythrin and were therefore more round and easily observed than the other coin-shaped form, seen i n Porphyridium (Gantt and Conti, 1 9 6 6 b ; Gantt, Edwards, and Conti,1968 ) . As the conchocelis grew older i t was subjected to a l i g h t intensity that may have been f a i r l y high for i t ( 100 f . c . ) . This possibly helped to s h i f t the phycoerythrin/phycocyanin ratio and changed the shape of the phycobilisomes so that they resembled those i n the adult thallus. The thallus grows near the high water mark, where the l i g h t intensity i s much greater than i n deep water. Since the phycobilisomes are sensitive to fixation at the best of times (Gantt and Conti,1966a) the less easily seen forms probably became almost unnoticeable. I t would be interesting to know the relative amounts of the accessory pigments i n Batrachospermum virgatum which has rod shaped phycobilisomes (Lichtle and Giraud ,1970) Small areas of low electron density containing a f i b r i l l a r material resembling the DNA-like patches i n the f i e l d material are clumped between the photosynthetic lamellae (Figs. 83,84). The pyrenoid (Figs. 84,92 ,93,94) i n the chloroplast i s traversed by several thylakoids, continuous with those i n the chloroplast, and i s very similar to the pyrenoid i n the macroscopic plant. The vegetative c e l l s of the conchocelis sometimes have more than one pyrenoid (Figs. 9 2 , 9 4 ) , a phenomenon which may have been induced by culture conditions. 49 The nucleus, about 1-2 u diameter (Figs. 82,94), i s usually appressed to one side of the c e l l , and can be p a r t i a l l y surrounded by the chloroplast or. other organelles. It i s similar to the nucleus of the f i e l d material, possessing a double nuclear membrane, which i s permeated by numerous prominent pores. Condensed chromatin was seen i n most sections of nuclei (Fig. 82) , which could correspond to heterochromatic bodies seen by l i g h t microscopy i n the nuclei of germinating conchocelis i n Porphyra umbilicalis (Krishnamurthy,1959). No centriolar bodies were observed i n the conchocelis phase of Porphyra perforata. The vacuole (Fig. 86) i s quite large, i n some sections occupying most of the c e l l . It i s enclosed by a single membrane. The starch bodies (Figs, 84,86,92,94) are very similar i n size and shape to those i n the vegetative c e l l s of the macroscopic phase and are also found-only outside the chloroplast membrane. In contrast to the c e l l s of the macroscopic phase which have a considerable amount, the endoplasmic reticulum i s not very extensive i n most of the c e l l s of the conchocelis phase (Fig. 82) . The mitochondria (Figs. 83,89,91) have tubular cristae continuous with the inner membrane and are very similar to those of the macroscopic phase. The wall of the conchocelis vegetative c e l l (Figs. 82,83,86) i s much thinner than the wall of the macroscopic thallus. The f i b r i l s are so densely packed that i t i s d i f f i c u l t to distinguish them. In contrast, i t i s interesting that the walls of both the monospore and o< spore resemble much more closely the wall of the macroscopic phase • (Figs. 86,8?). The conchocelis c e l l s possess typical florideophycidean plugged p i t connections (Figs. 86,88-91). They are similar to those reported i n the conchocelis of P. leucosticta (Lee and Fultz ,1970) and have already been reported i n P. perforata (Bourne,Conway, and Cole,1970, Appendix I ) . The plug i s bounded by a membrane, and the plasmalemma of adjacent c e l l s i s firmly attached to i t , as can be seen i n s l i g h t l y plasmolyzed c e l l s (Fig. 88) . The plasmalemma takes on a d i s t i n c t l y different appearance at the point of attachment to the plug. I t becomes very diffuse and loses i t s three-layered organization (Fig.88) . Brown and Weier (1970) showed that the plasmalemma i n Batrachospermum was continuous from c e l l to c e l l and did not cross the face of the plug. In the wall, at the l e v e l of the. middle lamella, adjacent to and surrounding the plug are a series of small vesicles (Figs . 88, 90) . The origin and function of these i s not known. However, since they did not appear to penetrate the wall i t i s unlikely that they are plasmodesmata. In older c e l l s there was a build up of a second layer of wall material on the inside of the original wall (Fig. 91) very much li k e that which occurred i n Pseudogloiophloea (Ramus, 1969b). Several differences were noted between the conchocelis of Porphyra perforata described here and the conchocelis of P. leucosticta (lee and Fultz, 1970). P. perforata had phycobilisomes, and a very narrow, dense wall. No prominent nucleolus was observed i n t h i s study, P. leucosticta had a sl i g h t l y wider and less dense c e l l wall, a prominent nucleolus, and no phycobilisomes. These may be innate species differences. On the other hand, they are more easily explained on the basis of different culture conditions and developmental stages. •51. P. leucosticta conchocelis was obtained from the algal culture collection at Indiana University, which has been maintained for several years, whereas P. perforata conchocelis was grown immediately from spores released by f i e l d material. The conchospore branch c e l l s of the conchocelis are isodiametric measuring about 12 u across (Figs. 95,97). The stellate chloroplast, pyrenoid, nucleus, nucleolus, vacuoles and starch grains (Figs. 96-99) are very similar to those found i n the macroscopic phase. The wall separating the conchospores within a branch i s permeated by a typica l floridean plugged p i t connection (Fig. 99). These c e i l s are surrounded by a rather unique thick c e l l wall, which has many projections from the surface (Figs. 96,97). It i s very wrinkled i n surface view, as revealed by the scanning electron microscope (Fig. 101). The appearance i s most striking when i t i s compared with the much smoother surface of the conchocelis vegetative filament (Fig,100). Drew and Richards (1953) and Drew (1954) have shown that the outer c e l l walls of P. umbilicalis conchospore branches remain intact while the cross walls break down to form a tube. The conchospores are then released from the end of the tube. The reason given for the tube formation was that the branch was buried i n the surface of a shel l . Chen, Edelstein, Ogata and McLachlan (1970) noted that the conchospores of P. miniata were released through a pore located between the second and terminal conchosporangium. The plugged p i t connections between the conchospore branch c e l l s may be the site of i n i t i a l wall breakdown and f a c i l i t a t e tube formation. This process could be similar to the dissolution of pit-containing walls during formation of the 52 carposporophyte i n G r i f f i t h s i a pacifica (Ramus, 1971). There are three possible functions of the thick surface walls of the conchospore branch c e l l s . These are: 1) substrate attachment after release, i f the spores are released with these walls intact; 2) a dispersal mechanism with the protuberances attaching to passing objects, resulting i n shaking of the branch; this function would apply i f the spores are released through a tube formed by the outer conchospore branch c e l l walls; 3) protection during overwintering of the spores. I t i s d i f f i c u l t to make a definite choice. However, the fact that the conchospores of Porphyra can be released naked through a tube formed by the outer conchospore branch c e l l walls (Drew,1954; Chen, Edelstein, Ogata,and McLachlan, 1970) indicates a slight preference for the second interpretation. Using scanning electron microscopy, a collar was seen between adjacent c e l l s of the vegetative filaments and also adjacent conchospore branch c e l l s i n surface view (Fig. 103). No collar was noted i n the region of attachment between the conchocelis filament and the conchospore branch. The collar could be an a r t i f a c t , the result of the preparation techniques used for scanning electron microscopy. The severe drying under vacuum possibly caused the c e l l s to shrink. Therefore, the support of the internal cross wall resulted i n a raised collar between ce l l s of the filaments. No collar was seen i n the transmission electron micrographs (Figs. 91,97), or i n the l i g h t micrographs (Fig. 102). 53 Discussion The internal morphology of vegetative c e l l s of Porphyra perforata and Bangia fuscopurpurea have been observed i n d e t a i l i n the present study and i t has been found that these two genera i n the order Bangiales are quite similar. The fine structure of B. fuscopurpurea found l o c a l l y appears very similar to that of B. fuscopurpurea found i n Naples, Italy (Honsell ,1963). The chloroplasts of the t h a l l i are stellate, possess a large central pyrenoid, are bounded by a double membrane, and lack a peripheral thylakoid. The single thylakoids occasionally penetrate the pyrenoid. Although the thylakoids appear to be free i n the stroma, i t i s possible that some association may exist since a functional stacking has been demonstrated i n Porphyridium by freeze etching (Neushul,1970). There i s an alternate layering of thylakoids and phycobilisomes which results i n a resonance contact between layers. Close stacking, when the phycobilisomes are not present, has been shown i n Smithora (McBride and Cole, 1969,1971). The endoplasmic reticulum, Golgi apparatus, nuclei, mitochondria, plasmalemma and c e l l wall are also very similar i n both genera. In addition, both genera contain paramural bodies, such as concentric lamellar bodies and polyvesicular bodies. The major difference between the two genera i s the external morphology of the macroscopic thallus. Cells of the thallus and conchocelis phase of Porphyra perforata have some si m i l a r i t i e s ! the pyrenoid located within the chloroplast, separate thylakoids some of which penetrate the pyrenoid, nuclear and mitochondrial morphology. However, there i s some variation between 54 these d i s t i n c t l y different growth forms of the same species, the thallus and conchocelis phases, relating to c e l l shape, chloroplast morphology and presence of a vacuole. In addition, the thallus c e l l s possess more endoplasmic reticulum and thicker, less dense c e l l walls; and lack the plugged p i t connection which occurs between conchocelis c e l l s . Several general morphological : characteristics can be used to separate the two rhodophycean subclasses Bangiophycidae and Florideophycidae. These are the number of carpospores per carposporangium, number of spermatia per spermatium, number of secondary p i t connections, •and number of nuclei per c e l l . Although diffuse or parenchymatous growth i s considered to be a characteristic of the Bangiophycidae, i t can be found i n some members of the Delesseriaceae, order Ceramiales and Corallinaceae, order Cryptonemiales (Florideophycidae) (Fritsch ,1945; Taylor,1957) and likewise, growth by an axial c e l l , a characteristic of the Florideophycidae, i s seen i n Compsopogon (Bangiophycidae) (Fritsch , 1945) . The specialization of the female sexual apparatus has been used as a major criterion for separating the two subclasses. However, this characteristic forms an almost continuous spectrum of increasing complexity from the most primitive members of the Bangiophycidae to the most advanced members of the Florideophycidae. It i s obvious that comparative ultrastructure i s as va l i d as comparative morphology for cl a s s i f i c a t i o n purposes. Therefore as more observations are made i t becomes possible to correlate certain taxonomic groups and various ultrastructural characteristics. Perhaps as 55 t h i s i s done some re-evaluation of the c l a s s i f i c a t i o n could be profitable. A characteristic which seems limited to the Rhodophyceae i s the association between the Golgi apparatus and a mitochondrion. This has been shown i n the Bangiophycidae, Porphyra i n this study, and Porphyridium (Gantt and Conti, 1965), and i n the Florideophycidae, G r i f f i t h s i a (Peyriere ,1969) and Corallina (Bailey and Bisalputra,1970). Although not spe c i f i c a l l y mentioned i t can be seen i n micrographs of other genera, Lomentaria (Bouck,1962), Pseudogloiophloea (Ramus,1969b) and Batrachospermum (Brown and Weier,1970). Another feature distinctive to the red algae i s the plugged p i t connection. Although i t i s mostly restricted to the Florideophycidae i t has also been shown to occur i n the conchocelis phase of the l i f e cycle of the Bangiophycidae i n an essentially identical form (Lee and Fultz,1970} Bourne, Conway, and Cole, 1970). The thylakoids are generally similar i n the Bangiophycidae and Florideophycidae. They are single, interconnect and often have attached phycobilisomes. A major difference i s the single large chloroplast with pyrenoid and no peripheral thylakoid i n the Bangiophycidae vs. the several small chloroplasts with peripheral thylakoids,' and lack of pyrenoid i n the Florideophycidae. However, these features are not completely diagnostic for either group. Some Nemalionales (Florideophycidae) such as Nemalion have a single chloroplast with a pyrenoid (Fritsch,194-5), whereas Goniotrichopsis (Bangiophycidae) has many small chloroplasts and no pyrenoid (Drew,1951). I t i s not known i f Nemalion lacks a peripheral thylakoid or i f Goniotrichopsis has one. A peripheral thylakoid commonly absent i n the 56 Bangiophycidae has been recorded as a regular feature of the chloroplasts of Smithora (McBride and Cole, 1969). It i s interesting not only to compare the ultrastructural features of members of the Bangiophycidae with those of the Florideophycidae, but also to note that there can be as much or more variation of characteristics between alternate phases of the l i f e cycle of one species (Porphyra perforata^ as there i s between the members of two subclasses. As more characteristics are found that are common to both subgroups the distinction between the subclasses w i l l tend to become less pronounced. If i t i s eventually found that the taxa Bangiophycidae and Flonideophycidae are no longer natural, there i s no doubt that these terms would remain i n common use for some time and that the orders presently included i n both subclasses would remain i n the class Rhodophyceae, It i s possible that several c e l l structures are involved i n the process of wall production i n P. perforata. These include the Golgi apparatus, polyvesicular bodies, concentric lamellar bodies, endoplasmic reticulum and plasmalemma. The Golgi apparatus i n Porphyra consists of a number of small flattened cisternae, forming vesicles which contain fibers similar to those of the wall. These may be involved i n wall material deposition. The Golgi apparatus i s most often cited i n connection with c e l l wall production i n plants (Mollenhauer, Whaley, and Leech,196ls Wooding and Northcote,1964j. Northcote and Pickett-Heaps, 1966; Pickett-Heaps, 1967a and bj Barton, 1968; Gantt, Edwards, and Conti, 1968: Wooding,19685 and review i n Beams and Kessel,1968). The frequent association of the forming face of the 57 Golgi apparatus and a mitochondrion i n Porphyra (Figs. 28-31) and other red algae (Gantt and Conti, 1965} Peyriere ,1969; Bailey and Bisalputra,1970; and Brown and Weier,1970) suggests that the Golgi body may originate from the mitochondrial outer membrane. A direct connection between the two has been demonstrated i n Corallina (Bailey and Bisalputra,1970). An alternative explanation of the association i s that the mitochondrion i s supplying energy to the Golgi apparatus for the synthesis of some product.. Both p o s s i b i l i t i e s are equally l i k e l y . There seems to be an increasing number of reports of polyvesicular bodies, lamellar bodies, and other lomasome-like structures similar to those seen i n Porphyra, i n a wide range of plant species (Marchant and Robards, 1968), They were f i r s t described i n fungi (Girbardt 1958,1961; Moore and McAlear ,196l) . Following this there was a number of observations on both higher plants and algae, and studies on the Rhodophyta i n particular have demonstrated their presence i n Lomentaria (Bouck,1962), Porphyridium (Gantt and Conti,1965), Laurencia (Bisalputra, Rusanowski, and Walker,1967), Pseudogloiophloea (Ramus, 1969b), Smithora (McBride and Cole, 1969) and Porphyra  leucosticta (Lee and Fultz , 1970) . Polyvesicular bodies similar to these observed i n Porphyra have also been observed i n the higher plants, Helianthus (Walker and Bisalputra, 1967), Helleborus (Echlin and Godwin,1968) and Beta vulgaris (Esau, Cheadle, and Gill , 1 9 6 6 ) . Marchant and Robards (1968) have considered the origin and function of these bodies which apparently are formed within the cytoplasm, move to the c e l l surface, and are released into the c e l l wall. Walker and Bisalputra (196?) also showed a sequence in Helianthus involving vesicular migration to the c e l l surface, with a subsequent release of materials into the wall, Tt i s possible that material i s carried to the c e l l surface by the polyvesicular bodies i n Porphyra. The green algae Chara and Nitel l a have been shown to possess complex elaborations of the plasmalemma or "charasomes" on the c e l l wall side (Crawley,1965; Barton, 1965a and b). However, their function i s s t i l l open to question. Barton (1968) demonstrated that radioactive glucose reaches the c e l l wall i n Chara by way of the Golgi bodies and not these iomasome-liket structures. Also using autoradiography (radioactive sugars), Pickett-Heaps (1967a and b) found that the Golgi apparatus was involved i n the deposition of c e l l wall precursors i n Triticum while at the same time he found no evidence of involvment of lomasomes etcetera i n this a c t i v i t y (personal communication to Fowke and Setterfield , 1 9 6 9 ) . This may be taken as evidence that the Golgi bodies are involved i n c e l l wall formation while the paramural bodies are not. However, i t must be kept i n mind that c e l l wall metabolism involves more than just deposition of simple sugars and other precursors. These paramural structures may also be involved i n wall breakdown, which occurs i n Porphyra, Bangia and other plants during such times as spore release. However, there i s no evidence to confirm this, such as build up prior to spore release. Although i t has been speculated that the various paramural structures are involved i n carbohydrate deposit i n the c e l l wall, attempts to trace the path of carbohydrate into the wall of Porphyra 59 perforata f a i l e d to show any deposit of tracer material (Appendix II). The true function of the paramural bodies i n P. perforata must remain unknown u n t i l further studies have been made. They are probably not art i f a c t s as claimed by Fowke and Setterfield (1969) for the following reasons: their structure i s so complex that there must have been a "phenomenally rapid growth of this membrane during fixation" (Cole and Lin, 1970); there i s no large vacuole present i n t h i s c e l l to supply the membrane material to a fixation a r t i f a c t as i n Helianthus (Fowke and Setterfield , 1 9 6 9 ) ; and similar structures have been shown by freeze-etching i n the red alga Corallina (Bailey and Bisalputra,1970) and i n the fungus Vertic i l l i u m (Griffiths,1 9 7 0 ) . Heath and Greenwood (1970) concluded that plasmalemmasome formation i n fungi was the result of plasmalemma production exceeding wall expansion. The E R i s quite discernable close to the plasmalemma i n several areas of Porphyra and Bangia and i t may be engaged i n c e l l wall synthesis or modification. The E R has been implicated i n c e l l wall production i n a number of other species. Porter and Machado ( I960) showed that the E R could develop into the elements of the phragmoplast i n Allium root t i p s . Hepler and Newcomb (1964) gave some evidence i n favor of the possible role of the E R i n wall production. Some of their figures show clusters of f i b r i l l a r elements within cisternae of the E R of Cpleus. These were believed to have been produced for eventual deposition i n the wall.In Haemanthus katherinae (Hepler and Jackson,1968) few Golgi bodies were noted but many E R lamellae were associated with the phragmoplast during plate formation. Pickett-Heaps (1967a and b) used radiotracers to demonstrate rather conclusively that both the Golgi bodies and E R i n wheat seedlings (Triticum vulgare) took up the wall precursors during production of c e l l plate and secondary wall thickenings. A formation i n Porphyra which must be considered along with those previously mentioned i s a system of densely staining membranes which extend throughout the cytoplasm linking various concentric lamellar bodies and polyvesicular bodies (Figs. 50,51). The membranes of these conspicuous features appear thicker than the endoplasmic reticulum (E R) membranes. Since a few connections have been observed between these membranes and the E R i t i s quite possible that these are modified elements of the E R. In some cases the cisternae expand forming an enlarged lumen which contain concentric lamellar bodies and polyvesicular bodies (Fig. 51), while i n other cases i t simply extends into finger-like cytoplasmic extensions i n the wall (Fig.50). The convoluted portions of the plasmalemma i n Porphyra (Figs. 40,50) with attached wall fibers are possibly areas of fiber elaboration. Convolutions i n the plasmalemma have also been observed i n the red algae Lomentaria (Bouck, 1962) and Porphyra  yezoensis (Kito and Akiyama, 1968) as well as i n other plants, and are generally associated with a c t i v i t i e s such as transport of wall materials out of the c e l l (Freywyssling,1962), Surface a c t i v i t i e s were also demonstrated i n the red alga Laurencia spectabilis (Bisalputra, Rusanowski, and Walker,196?). Small membrane-bounded droplets at the cytoplasm-cell wall interface of this species were interpreted as material released from the c e l l into the wall. Cronshaw and Bouck (19&5) implicated the plasmalemma i n metabolic a c t i v i t i e s such as c e l l wall elaboration i n Avena coleoptiles by suggesting that some thick portions of the membrane might contain enzymes on the outer surface associated with the f i n a l synthesis and orientation of wall materials. In conclusion, i t has been shown that the thallus c e l l s of Porphyra perforata and Bangia fuscopurpurea are ultrastructurally similar. This i s not unexpected since they have been c l a s s i f i e d i n the same order. However, the ultrastructural differences between the two phases of the l i f e cycle of P. perforata are interesting and could be of some phylogenetic significance. These are: the plugged p i t connection between adjacent conchocelis c e l l s which i s absent from the thallus, phycobilisomes i n the chloroplasts of the young conchocelis c e l l not present i n the thallus c e l l s , the ribbon-like chloroplast i n the conchocelis c e l l s and stellate chloroplast i n the thallus. There i s much similarity between the ultrastructure of the two rhodophycean subclassesBangiophycidae and Florideophycidae, with no ultrastructural feature restricted to either one. A number of organelles including the Golgi apparatus, endoplasmic reticulum paramural bodies and plasmalemma may be involved i n c e l l wall production and/or modification i n Porphyra perforata. Liter attire Cited Bailey, A., and Bisalputra, T. 1 9 7 0 . A preliminary account of the application of thin-sectioning, freeze-etching, and scanning electron microscopy to the study of corralline algae. Phycologia 9 : 8 3 - 1 0 1 . 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An electron microscopic study of DNA-like f i b r i l s i n chloroplasts. Cytologia 3 2 : 3 6 1 - 3 7 7 . Figures 22 - 56 Vegetative thallus of Porphyra perforata Figure 22 Blades collected from Ueluelet, Vancouver Island X 1/3 Figure 23 Blades collected from Point Grey, Vancouver x 1/3 Figure 24 A l i g h t micrograph of a portion of the blade showing the packets of large, densely pigmented <* spores and adjacent packets of smaller, lighter colored (i spores. The single c e l l s are vegetative c e l l s . X 100 Figure 25 An electron micrograph of a single vegetative c e l l showing the large pyrenoid (Py) surrounded by the chloroplast, the nucleus (Nu), nucleolus (Nl) and reticulate c e l l wall (CW). This section i s p a r a l l e l to the blade surface. X 7,000 Figures 26 - 29 Electron micrographs of the vegetative thallus oells of Porphyra perforata Figure 26 A cross section of the blade showing similar organelles to those of Figure 25. Note the nucleus (Nu) and pyrenoid (Py) are located approximately midway between the two blade surfaces. X 2,500 Figure 27 Section through the c e l l surface showing the convoluted plasmalemma (Pi) and numerous vesicles (arrows) between cytoplasm and c e l l wall (CW), X 150,000 Figure 28 Section showing the vegetative chloroplast (Ch) with single lamellae. Note the interconnections (arrows) between numerous lamellae. Also note the DNA f i b r i l s (double arrows) and the close association between mitochondrion (M) and Golgi apparatus (G). X 30,000 Figure 29 A portion of a vegetative c e l l . Note the forming face of the Golgi apparatus i s directed towards the mitochondrion (M) and the maturing face oriented towards the c e l l wall (CW). X 30,000 Figures 30 - 32 Electron micrographs of c e l l s i n the vegetative thallus of P i perforata Figure 30 Two mitochondria (M) with associated Golgi bodies (G). Note the floridean starch granules (arrows). X 36,000 Figure 31 Section showing the Golgi apparatus (G) and mitochondrion (M), Note the small vesicles between the mitochondrion and the forming face of the Golgi body. Observe the fibrous material i n Golgi vesicles (GV). X 36,000 Figure 32 Two nuclei (or possibly two lobes of one nucleus) i n a vegetative c e l l . A portion of one nucleus i s directed towards the opposite end of the c e l l . X 16,000 Figures 33 - 36 Stages of one type of c e l l division i n P. perforata Figure 33 Single vegetative nucleus (Nu), appressed to one side of the c e l l . Note convoluted portions of the plasmalemma (PI). X 5,000 Figure Highly lobate vegetative nucleus, which may be the i n i t i a l stage of nuclear division. Note the nuclear pores (NP), floridean starch (St) and the elements of the endoplasmic reticulum (ER) near the c e l l surface. X 26,000 Figure 35 I n i t i a l stage of cytokinesis. The two daughter nuclei (Nu) are at opposite ends of the c e l l . The dividing wall i s starting to form from the c e l l periphery. X 7,000 Figure 36 A later stage of c e l l division. The c e l l wall (CW) extends most of the way across the c e l l . Note the pyrenoid and chloroplast have not yet divided and are caught i n the annular opening, X 10,000 75 Figures 37 and 38 Stages in another type of cel l division in P. perforata Figure 37 The two nuclei (Nu) in this ce l l are separated by the invaginated plasmalemma. In this section the plasmalemma extends most of the way across the ce l l (arrows). No ce l l wall material has been laid down yet. X 16,500 Figure 38 A later stage of this type of ce l l division. The plasmalemma (PI) is convoluted in this area. A number of ce l l wall fibers have been laid down between the two cells . X 18,000 Figures 39 - 4 l Holdfast region of P. perforata Figure 39 Note the very thick layered c e l l wall i n relation to the small rhizoidal process i n the center. Wrinkles are due to section stretching. X 6 , 5 0 0 Figure 40 Cross section of the rhizoidal process with highly convoluted plasmalemma (PI). X 31,000 Figure 41 Longitudinal section of rhizoidal process showing thick c e l l wall (CW) and convoluted plasmalemma, X 15,000 Figure 42 Scanning electron micrograph of the surface of the vegetative thallus. X 1,200 Figures 4 3 - 4 4 <=< spore producing portion of thallus of P. perforata Figure 43 Section through c< spore producing portion of the thallus. Note the thin c e l l wall between developing <x spores. X 5i700 Figure 44 Numerous small f i b e r - f i l l e d vesicles near the c e l l wall (CW) of differentiating <* spores. X 32,000 Figures 45 - 47 Paramural bodies i n the vegetative thallus of P. perforata Figure 45 Concentric lamellar body (CLB) with tubules located inside the outer membrane. X 52,000 Figure 46 Portion of a vegetative c e l l . Note the c e l l wall fibers attached to the plasmalemma (PI) part i c u l a r i l y i n the v i c i n i t y of the concentric lamellar body. X 68,000 Figure 47 Concentric lamellar body i n the c e l l wall (CW). Note the endoplasmic reticulum (ER) closely p a r a l l e l l i n g the plasmalemma (PI). X 52,000 Figures 4 8 - 51 Paramural bodies in Porphyra perforata Figure 4 8 A cell with concentric lamellar bodies (CLB)in the cytoplasm, and in the chloroplast (arrow). X 13,000 Figure 4 9 A concentric lamellar body (CLB) in close proximity to a polyvesicular body (PVB). Both are located near the ce l l surface. X 40 ,000 Figure 50 Finger-like projections of cytoplasm into the cell wall (CW). Note the membranes that extend into these projections (arrows). X 21,000 fp Figure 51 Note the membranes (arrows) interconnecting the concentric lamellar bodies (CLB) and polyvesicular bodies (PVB), in the vegetative c e l l . X 32,000 Figures 5 2 - 5 5 Paramural bodies i n P. perforata Figure 52 Polyvesicular body with tubular inner vesicles. X 97,000 Figure 53 Polyvesicular body (PVB) located near the c e l l periphery. X 35,000 Figure 5^ Polyvesicular body i n contact with the c e l l wall (CW). X 56,000 Figure 55 A complex elaboration of the plasmalemma, located between the plasmalemma and c e l l wall (CW). X 29,000 Figure 56 Endoplasmic reticulum (ER) i n the central portion a c e l l . Note the numerous membranes. X 90,000 80 Figures 57 - 60 Bipolar sporelings of Porphyra perforata Figure 57 Light micrograph of released aplanospores. X 2,000 Figure 58 Young bipolar sporeling with rhizoidal process at basal end. X 1,600 Figure 59 Older bipolar sporeling. Note the rhizoidal processes at the basal end. X 4,700 Figure 60 Electron micrograph of c e l l from older bipolar sporeling showing the thick c e l l wall and convoluted plasmalemma. X 32,000 81 Figures 6 1 - 6 4 Finestructural details of bipolar sporeling c e l l s of P. perforata Figure 61 Portion of a c e l l showing chloroplasts with pyrenoid and single lamellae. X 33,000 Figure 62 Chloroplast with pyrenoid and osmiophilic droplets. X 33,000 Figure 63 Cell with thick c e l l wall (CW), Note the numerous osmiophilic droplets between the chloroplast lamellae and the chloroplast DNA. f i b r i l s (arrows). X 18,000 Figure 64 Portion of a c e l l with chloroplasts (Ch), starch granules (St), and a Golgi apparatus (G) which i s situated near a mitochondrion. X 12,000 82 Figures 65 - 79 Micrographs of Bangia fuscopurpurea Figure 65 Light micrograph of a filament. X 2,500 Figure 66 Electron micrograph of a c e l l i n the filament. Note that one c e l l contains two chloroplasts (Ch) and two pyrenoids (Py), I t i s probably undergoing division. X 7,600 Figure 67 Cell surface with highly convoluted plasmalemma (PI). X 44,500 Figure 68 Lobate nucleus (Nu) with prominent nucleolus (Nl) similar to the vegetative nucleus of Porphyra  perforata (Figs. 33,34). X 13,000 Figure 69 Section through a vegetative c e l l showing endoplasmic reticulum (ER) connected to a concentric lamellar body, and a mitochondrion (M) with a closely associated Golgi apparatus (G). X 30,000 Figures 70 - 75 Electron micrographs of Bangia fuscopurpurea thallus Figure 70 A concentric lamellar body (CLB) i n a chloroplast (Ch). X 40,000 Figure 71 A round concentric lamellar body i n the cytoplasm (aplanospore). X 31,000 Figure 72 Concentric lamellar body i n a c e l l wall.X 48,000 Figure 73 An irregularly shaped concentric lamellar body i n the cytoplasm. X 31,000 Figure 74 C e l l division. Note the membrane (arrows) separating the two chloroplasts and pyrenoids (Py). X:ll6,000 Figure 75 Thick multilayered c e l l wall between two c e l l s (aplanospores). X 16,000 Figures 76 - 79 Development of aplanospores i n Bangia  fuscopurpurea Figure ?6 Note the extensive endoplasmic reticulum (ER) i n the peripheral cytoplasm and layer of c e l l wall material surrounding the developing spore, and the interconnections of the thylakoids (arrows). X 18,000 Figure 77 Developing aplanospore showing p a r t i a l separation of the spore wall from the mother c e l l wall. Note the highly lobate chloroplast with i t s central pyrenoid, X 4,000 Figure 78 Developing aplanospore. Note that the spore c e l l wall consists of several layers. X 5,000 Figure 79 Aplanospore released from the spore mother c e l l wall but s t i l l i n the lumen. Again note that the spore c e l l wall consists of several layers. X 5,000 85 Figures 80 - 103 Micrographs of the conchocelis phase of Porphyra perforata Figure 80 Light micrograph of spores released from the mature thallus. The larger spore i s the «x spore and the smaller, the /3 spore. X 2,000 Figure 81 Light micrograph of a young conchocelis filament. X 3,000 Figure 82 Electron micrograph of a cross section of a conchocelis filament showing a nucleus (Nu), with some chromatin attached to the nuclear membrane, chloroplast (Ch), and endoplasmic reticulum (ER). X 27,000 Figure 83 Cross section of conchocelis filament showing a chloroplast (Ch) with phycobilisomes (Ph) attached to the thylakoids. Note that some of the thylakoids interconnect. X 27,000 Figures 84-87 Electron micrographs of the conchocelis phase of P. perforata Figure 84 Conchocelis c e l l s showing chloroplast (Ch), starch (St), pyrenoid (Py). X 6,600 Figure 85 Light micrograph of conchocelis filament showing the empty spore case (Sp). X 1,500 Figure 86 Electron micrograph of f i r s t conchocelis c e l l and the empty spore case (Sp). Note the vacuole (V), plugged p i t connection between the f i r s t c e l l and the spore case and the difference between c e l l walls of the conchocelis filament and sporev X 23,000 Figure 87 Empty spore case and f i r s t c e l l of conchocelis. Note the thick, less dense c e l l wall of the spore case and the thin,very dense wall of the conchocelis. X 6,000 87 Figures 88 - 91 Electron micrographs of the wall between adjacent conchocelis c e l l s of P. perforata Figure 88 Plugged p i t connection (Pg) between two vacuolated conchocelis c e l l s . Note the attachment of the plasmalemma to the plug, the small vesicle i n the wall beside the plug (arrow/)) and the floridean starch granules (St). X 56,000 Figure 89 Plugged p i t connection (Pg) between two conchocelis c e l l s . X 42,000 Figure 90 Ce l l wall adjacent to a plugged p i t connection. Note the small vesicle i n the wall next to the plug (arrow). X 35,000 Figure 91 Plugged p i t connection (Pg) between two older conchocelis c e l l s . Note the paramural body next to the plug and secondary thickening of the c e l l wall (arrows). X 15,000 88 Figures 9 2 - 9 4 Older conchocelis vegetative c e l l s of P. perforata Figure 92 Note the two pyrenoids (Py) i n one chloroplast, and the accumulation of starch granules. X 7,200 Figure 93 Chloroplast (Ch) and pyrenoid (Py) of older conchocelis c e l l . Note the absence of phycobilisomes. X 9,000 Figure 94 Longitudinal section of conchocelis c e l l . Note the two pyrenoids and the nucleus (Nu). X 7,200 Figure 95 Light micrograph of conchospore branches. X 500 89 Figures 96 - 99 Electron micrographs of conchospore branch ce l l s of P. perforata Figure 96 Conchospore branch c e l l showing projection of the c e l l wall (CW). X 9,800 Figure 97 Conchospore branch c e l l s showing convoluted c e l l wall, chloroplast and pyrenoid (Py). X 4,300 Figure 98 Conchospore branch c e l l with nucleus (Nu), nucleolus (Nl), and chloroplast (Ch). X 15,000 Figure 99 Plugged p i t connection (Pg) between two aonchospore branch c e l l s . Note the large number of starch, granules (St). X 15,000 Figures 100 - 103 Conchocelis phase of P. perforata Figure 100 Scanning electron micrograph of the surface of conchocelis filaments. Note the re l a t i v e l y smooth c e l l wall. X 7,000 Figure 101 Scanning electron micrograph of the surface of conchospore branch c e l l s . Note the highly convoluted c e l l wall. X 7,000 Figure 102 Light micrograph of the connection between the vegetative conchocelis c e l l s and the conchospore branch c e l l s . X 2,000 Figure 103 Scanning electron micrograph of the connection between the vegetative conchocelis c e l l s and the conchospore branch c e l l s (arrow). Note the collar between adjacent conchocelis c e l l s and between adjacent conchospore branch c e l l s . Also note that there appears to be no collar between the conchocelis c e l l and the conchospore branch c e l l . X 5,000 GENERAL CONCLUSIONS ( PART I AND II) On the basis of ultrastructure, Phaeostrophion irregulare possesses some characteristics common to the higher orders of the Phaeophyceae: a, absence of a pyrenoid i n the adult thallus b, absence of a physical association of the chloroplast and nucleus. It also has some characteristics common to the lower orders of the Phaeophyceae: a. s t r i c t l y perinuclear Golgi apparatus b. diffuse distribution of plasmodesmata Consequently, this species i n the order Dictyosiphonales retains i t s intermediate phyletic position which had previously been determined using more gross characteristics. The ultrastructure of Porphyra perforata and Bangia fuscopurpurea both i n the order Bangiales (Rhodophyceae), i s very similar. The ultrastructure of the conchocelis phase of P. perforata i s basically similar to that of the vegetative thallus. However the following differences were noted: a. the plugged p i t connection between adjacent conchocelis c e l l s i s absent from the thallus b. phycobilisomes i n the chloroplasts of the young conchocelis are not present i n the thallus c e l l s c. chloroplast shape i s ribbon-like i n the conchocelis and stellate i n the thallus. 9 2 4 There i s much similarity between the ultrastructure of the two rhodophycean subclasses Bangiophycidae and Florideophycidae. 5 A number of organelles including the Golgi apparatus, endoplasmic reticulum, and paramural bodies may be involved i n c e l l wall production and/or modification i n Porphyra  perforata. APPENDIX I. On the ultrastructure of p i t connections i n the conchocelis 2 ^ phase of the red alga Porphvra perforata J.Ag. * J As Dixon (1963) has pointed out, there i s s t i l l doubt about the presence and structure of p i t connections i n the Bangiophycidae. Fan (I960) l i s t e d early reports of connections seen by l i g h t microscopy, and these were followed by Magne (i960), Belcher (i960), and Krishnamurthy (1969). However, Nichols, Ridgway, and Bold (1966) were unable to confirm with the electron microscope Fan's observations on Compsopogon. In our study of Porphyra perforata, p i t connections i n the transverse walls of conchocelis filaments have been observed both by l i g h t and electron microscopy. Filaments of conchocelis grown i n culture for 3 to 4 weeks were fixed i n a glutaraldehyde-osmium f i x and embedded i n Maraglas. The sections were stained with uranyl acetate and lead citrate (McBride and Cole, 1969). In the transverse walls of most ce l l s a p i t connection of about 0.5 u diameter was obvious with the l i g h t microscope (Fig.104), but no details could be seen. Ultrastructurally, the longitudinal and outer edges of the transverse walls of the filaments were 0.25 u thick and composed of closely packed, electron dense microfibrils. There were This a r t i c l e by V.L. Bourne, E. Conway and K. Cole appeared i n Phycologia 9: 79-81 (1970). The study was supervised by E, Conway and K. Cole. An a r t i c l e by Lee and Fultz (1970) appeared while this was i n press describing a similar p i t connection i n the conchocelis phase of Porphyra leucosticta. It has been discussed earlier (p. 5 0 ) . l o c a l increases i n thickness i n wall areas adjacent to the p i t and at the meeting points of longitudinal and transverse walls (Fig. 105). The p i t , centrally located within the transverse wall, was blocked by a membrane-bounded plug. The plasmalemma of adjacent c e l l s appeared to be closely associated with the plug membrane (Fig. 106), The core of the plug consisted of material less electron dense than that of the surrounding wall. No. cytoplasmic continuity was observed through the plug. These general ultrastructural details of the p i t connections appear to be similar to those already known i n the Florideophycidae (Myers, Preston, and Ripley,1959$ Bouck, 1962} Bischoff, 1 9 6 5 ; Bisalputra, Rusanowski, and Walker, 1 9 6 7 ! and Ramus, 1 9 6 9 a and b). Literature Cited Belcher, J.H. I 9 6 0 . Culture studies of Bangia atropurpurea (Roth) Ag. New Phytol. 59t 367-373 Bisalputra, T., Rusanowski, P.C., and Walker, W.S. 1 9 6 7 . Surface a c t i v i t y , c e l l wall, and fine structure of p i t connection i n the red alga Laurencia spectabilis. J. Ultrastruct. Res. 2 0 : 2 7 7 - 2 8 9 Bischoff, H.W, 1 9 6 5 . Thorea r i c k e i sp, nov, and related species, J. Phycol. 1 : 1 1 1 - 1 1 7 Bouck, G.B, 1 9 6 2 . Chromatophore development, p i t s , and other fine structure i n the red alga, Lomentaria baileyana (Harv.) Farlow, J. C e l l Biol. 1 2 : 553-569 Dixon, P.S, 1 9 6 3 . The taxonomic implications of the " p i t connexions" reported i n the Bangiophycidae, Taxon 1 2 : 108-110 Fan, K-C. i960. On pit-connections i n the Bangiophycidae. Nova Hedwigia 1 : 305-307 Krishnamurthy, V. 1969. -The conchocelis phase of three species of Porphyra i n culture, J_. Phycol . 5' 42-47 McBride, D.L., and Cole, K, 1969. Ultrastructural characteristics of the vegetative c e l l of Smithora naiadum (Rhodophyta), Phycologia 8: 177-186 Magne, F. I960. Le Rhodochaete parvula Thuret (Bangioidee) et sa reproduction sexuee, Cahiers B i o l . Mar. Is 407-420 Myers, A,, Preston, R.D, and Ripley, G.W. 1959. An electron microscope investigation into the structure of the Floridean p i t . Ann. Bot. Lond. 23: 257-260 Nichols, H.W., Ridgway, J.E. and Bold, H.C. 1966. A preliminary ultrastructural study of the freshwater red alga Compsopogon. Ann. Missouri Bot. Gard. 53: 17 - 27 Ramus, J. 1969a. Dimorphic p i t connections i n the red alga Pseudogloiophloea. J. Ce l l B i o l . 4 1 : 340-345 Ramus, J. 1969b. P i t connection formation i n the red alga Pseudogloiophloea. J_. Phycol. 5: 73-83 Literature Cited i n Footnotes Lee, R.E., and Fultz, S.A', 1970. Ultra structure of the conchocelis stage of the marine red alga Porphyra leucosticta. J_. Phycol. 6: 22-28 Figure 104 Light micrograph of conchocelis filaments showing the p i t connection (arrows) between adjacent c e l l s . X 2,000 Figure 105 Low magnification electron micrograph of a p i t connection. X 13,000 Figure 106 High magnification electron micrograph of the p i t connection i n Figure 105,Note the c e l l wall (CW), plug (Pg), chloroplast (Ch), plasma membrane (PM), and plug membrane (arrows). X 120,000 97 APPENDIX II A preliminary experiment using radiotracer techniques i n an attempt to follow the path of polymerized glucose, mannose, and xylose i n the macroscopic thallus of Porphyra perforata. Introduction Since Frei and Preston (19&0 demonstrated that the wall of Porphyra consists of mannose and xylose i n a particular configuration l i t t l e has been done to trace the path followed by the wall precursors. This seemed unfortunate i n the l i g h t of successful attempts to show the path of glucose polymers through the Golgi apparatus into the cellulosic fraction of the wall of Chara (Barton,1968) and Triticum (Pickett-Heaps, 1967). A number of organelles have been observed i n Porphyra c e l l s ( page 4-h) that are morphologically similar to organelles i n other species which are suspected, of being involved i n c e l l wall deposition and/or organization. These observations indicated that some hypothesis concerning the synthesis, and deposition of wall material i n Porphyra could be formed and tested. The hypothesis formulated was that some or a l l of the organelles seen i n Porphyra are involved i n the direct synthesis and/or deposition of wall material. It was tested by using electron microscopic autoradiographic techniques to localize the deposition of polymerized mannose and xylose i n and around the c e l l . This would of course test only for synthesis and deposition of carbohydrate materials. Therefore, a negative result would not exclude the p o s s i b i l i t y of any of the organelles being involved i n deposition of protein or i n the reorganization of wall material. Materials and Methods Porphyra blades for this experiment were collected during evening low tides and kept at 5°C overnight. The material for the glucose test was collected on January 20th,1969» f ° r raannose, March 3rd,1969: and for xylose on A p r i l 5th,1969. The blades were placed i n a culture solution composed of 25 ml autoclaved sea water with 200 mg NaN03 and 20 mg Na2HP04«?H20 added plus 1 mc of glucose-6-tritium: 1 mc of mannose-l-tritium: and 0.05 mc of D-xylose C 1^ a l l at approximately 10°C. The culture vessel was a glass bubble chamber (Fig. 107) which allowed the easy removal and reimmersion of the material i n the culture solution. Samples were collected by clipping a small piece from the blade at t o t a l times of approximately 0,3,8,15,20,30,40,70,100,190 and 350 minutes. As each piece was removed i t was fixed and embedded as previously described (page 31 ). The embedded material was cut and stained (see page 3 3). The grids were coated with Gevaert Scientia NUC 3.07 emulsion (Bourne, and Cole, 19&9) a " d stored at 5-8°C for 1 to 10 months. Upon removal from storage the emulsion coated grids were developed for 3 minutes i n Kodak D 19-b developer, fixed, washed and examined i n an Hitachi HU - 11A electron microscope. Test grids were exposed to white l i g h t and developed to check the emulsion coating. 99 Observations and Discussion Cross wall formation and Golgi bodies containing fibrous material were observed i n the sectioned c e l l s and i t might be expected that sugars would be incorporated at these areas of metabolic a c t i v i t y . However, very few silver grains were observed and there was no dis t i n c t correlation of silver grains with a specific organelle. There were as many grains off the sections as on them. Test grids exposed to l i g h t and developed at the same time showed that the emulsion was coated onto the grids. Since very l i t t l e s i l v e r was reduced i n the emulsion i t can be questioned whether or not there was any incorportion of the labelled sugars at a l l . This could be tested by macerating and dissolving the whole material and then measuring the radioactivity with a liquid s c i n t i l l a t i o n counter. An absence of evident incorporation could be explained by a number of factors; the sugars did not penetrate the walls, the alga was not metabolizing these sugars at those times, perhaps the test should have been run i n the dark, the sugars were not polymerized enough and were therefore s t i l l soluble, or possibly the reactions that were being studied were i n a closed chain that the labelled material could not enter. On the other hand, i f there was incorporation i t i s possible that i t was not detected because the storage temperature was too low or the exposure not long enough. 100 Literature Cited Barton, R. 1968. Autoradiographic studies on wall formation i n Chara. Planta (Berl.) 82: 302-306 Bourne, V.L., and Cole, K. 1969. A simplified technique for the application of nuclear emulsion i n electron microscopic autoradiography. Can. J. Bot. 4?: 1821-1822 Frei, E.. and Preston, R.D. 1964. Non-cellulosic structural polysaccharides i n algal c e l l walls, II. Association of xylan and mannan i n Porphyra umbilicalis. Proc. Roy. Soc. 160: (Ser. B), 314-327 Pickett-Heaps, J.D. 1967. The use of radioautography for investigating wall secretion i n plant c e l l s . Protoplasma 64: 49-66 F i g u r e 107 Diagram o f bubble chamber used f o r the r a d i o t r a c e r experiment Diagram o f bubble chamber showing immersion o f Porphyra  p e r f o r a t a b lades i n the r a d i o a c t i v e sugar s o l u t i o n . The blades are attached t o the g l a s s support r o d . The r o d and b l a d e s can be removed e a s i l y f o r sampling purposes. F i g u r e 10? Diagram of bubble chamber used f o r the r a d i o t r a c e r experiment. 102 APPENDIX III A simplified technique for the application of nuclear emulsion h i n electron microscopic autoradiography Several techniques for the application of nuclear emulsions i n electron microscopic autoradiography have been developed i n the l a s t 10 years. However, most of these present inherent d i f f i c u l t i e s related either to the preparation and application of the emulsion, or to the interpretation of results. The silver evaporation technique of Silk, Hawtrey, Spence and Gear ( 1 9 6 1 ) has been c r i t i c i z e d because i t has low radio sensitivity and high chemical sensitivity (Caro and van Tubergen, 1962} Koehler, Muhlethaler, and Freywyssling, 1 9 6 3 ; Pele, 1 9 6 3 ; Caro, 1 9 6 6 ) . The dipping and dropping methods devised and published by a number of workers including O'Brien and George ( 1 9 5 9 ) ; Pelc, Coombes, and Budd ( 1 9 6 1 ) ; Przybylski, ( 1 9 6 l ) ; Kopriwa and LeBlond,(1962); Hay and Revel, ( 1 9 6 3 ) ; and Salpeter and Bachmann,(1964) lead to uneven or excessively thick layers of emulsion (Caro and van Tubergen,1962; Koehler, Muhlethaler, and Freywyssling ,1963). The semiautomatic instrument of Kopriwa ( 1 9 6 6 ) i s a modification of the dipping procedure and requires the use of a piece of equipment which may not be readily available i n some laboratories. The use of a wire loop (George and Vogt, 1 9 5 9 ; Revel and Hay, 1 9 6 1 ; Kayes, Maunsbach, and Ullberg , 1 9 6 2), centrifugation (Dohlman, Maunsbach, 4 This a r t i c l e by V.L, Bourne and K. Cole appeared i n the Canadian Journal of Botany 47: 1821-1822 (1969). The study was supervised by K. Cole, 103 Hammarstro'm,and Appelgren, 1964), and the agar block method (Caro and van Tubergen,1962), require complex observations and manipulations under safe-light conditions. The rotating disc method of Koehler, Mtihlethaler and Freywyssling (1963) seems the most promising of a l l the techniques reported to date since i t requires a minimum of equipment while maintaining easy-reproduction of thin, even emulsion coats. However, i n addition to the carbon coat applied to the sections to prevent chemical reduction of the emulsion by the tissues (Salpeter and Bachmann, 1964), i t introduces an extra layer of forravar between the carbon-coated sections and the emulsion. This layer could not only reduce microscopic resolution by electron scatter (Sjostrand,1956) but also reduce autoradiographic resolution by moving the emulsion layer beyond the ideal distance from the radioactive source (Pelc, Coombes and Budd 1961, Caro and van Tubergen. 1962). Consequently a simple modification of this technique was designed to improve and simplify i t for use i n a moderately well-equipped E M laboratory. The modified rotating disc technique (Fig. 108) was tested using grids coated f i r s t with collodion and then with a layer of carbon. I f necessary the collodion base may be removed leaving only the carbon support before attaching sections. Under indirect yellow-green safelight illumination (Wratten series OA) a short strip of •§• i n . masking tape was attached to the center of the head of a small centrifuge. The centrifuge was then spun at low speed to observe the center of rotation. The rotor was stopped and the grid placed at the center. Only extremely l i g h t pressure was needed for the grid to remain on the tape even at high speeds. I t was d i f f i c u l t to remove when fastened with too much pressure. The grid was then spun at about 10,000 r.p.m. Undiluted Gevaert Scientia NUC 3.07 emulsion was melted i n a water bath which had been preheated to 40°C. The emulsion was picked up i n a pipette and a single drop about 3 mm . i n diameter was released onto the center of the spinning grid. The size of the drop may not be too c r i t i c a l since most of the emulsion i s thrown off immediately after application. The grid was spun for about •§• to 1 minute to allow the emulsion to dry, then removed with a pair of fine forceps and placed i n a capsule. To demonstrate the evenness of the coating, some undeveloped grids as well as others which had been developed i n Kodak D-19 after exposure to white l i g h t for several minutes were observed and photographed i n the electron microscope. D-19 developer was used to obtain the characteristic developed silver pattern of a tangled skein, seen i n Fig. 110. I f a higher resolution with a much finer grain i s needed then a solvent developer such as Microdol-X (Caro and van Tubergen, 1962) or a phenidone base developer (Paweletz,1967) may be used. Since a r e l a t i v e l y high illumination l e v e l i s required for successful completion of the operation i t was necessary to test the effect of the safelight on the emulsion. Therefore a number of prepared grids were placed about 3 f t . d i r e c t l y i n front of the yellow-green l i g h t for 20 minutes and subsequently developed and examined. A f a i r l y even distribution of silve r halide granules was produced i n a l l groups i n the test (Figs. 1 0 9 t H 0 ) . Similar results were obtained by Koehler, Miihlethaler and Freywyssling (1963). However, this modification of their technique i s an improvement, since i t eliminates the extra layer of formvar over the carbon-coated sections before emulsion application. Also, i t i s simpler, reducing the number of operating steps and making i t possible to ooat a large number of grids i n quick succession. The gelatin.was not removed i n these tests, btrt may be i f desired. Although Przybylski (1961), Salpeter and Bachmann (1964), and Caro (1966) indicated that the emulsions can be used safely under a yellow-green l i g h t , some workers seem to use the dark red lamp (Kopriwa and LeBlond 1962 * Northcote and Pickett-Heaps 1966), Obviously, the efficiency and speed of the rotating disc method are improved i f the operation i s carried out under the brighter illumination. It was evident from the present tests that this i s possible, since exposure to direct yellow-green l i g h t for periods of 20 minutes had no effect on the emulsion. Literature Cited Caro, L.G. 1966, Progress i n high-resolution autoradiography. Progr. Biophys. Mol. Biol. 16: 171-190 Caro, L.G. and van Tubergen, R.P, 1962, High-resolution autoradiography. I, Methods. J. Cell B i o l . 15s 173-188 Dohlman, G.F,, Maunsbach, A.B., Hammarstrom, L., and Appelgren, L-E. 1964, Electron microscopic autoradiography, A method for producing uniform monolayers of silver halide crystals using centrifuge sedimentation, J, Ultrastruct. Res. 10$ 293-303 106 George, L.A. and Vogt, G.S, 1959. Electron microscopy of autoradiographed radioactive particles. Nature 184s1474-1475 Hay, E.D. and Revel, J.P. 1963. The fine structure of the DNP component of the nucleus. An electron microscopic study u t i l i z i n g autoradiography to localize DNA synthesis. J. Cell B i o l . I 6 s 2 9 - 5 1 Kayes, J., Maunsbach, A.B. and Ullberg, S. 1 9 6 2 , Electron microscopic autoradiography of radioiodine i n the thyroid using the extra-nuclear electrons of I : . J. Ultrastruct. Res. 7s 339-345 Koehler, J.K., Muhlethaler, K, and Freywyssling, A. 1963. Electron microscopic autoradiography. An improved technique for producing thin films and i t s application to H3-thymidine-labelled Maize nuclei. J. C e l l B i o l . l6s 73-80 Kopriwa, B.M. 1 9 6 6 . A semiautomatic instrument for the radioautographic coating technique, J_, Histochem. Cytochem. 14: 923-928 Kopriwa, B.M, and Leblond, C.P. 1 9 6 2 , Improvements i n the coating technique of radioautography. J_. Histochem. Cytochem. 10 :269-284 Northcote, D.H, and Pickett-Heaps, J.D. 1966. A function of the Golgi apparatus i n polysaccharide synthesis and transport i n the root-cap c e l l s of wheat. Biochem. J. 98: 159-167 O'Brien, R.T. and George, L.A, 1959 , . Preparation of autoradiograms for electron microscopy. Nature 183: 1461-1462 Paweletz, N, 1 9 6 7 . Specimen preparation methods for electron microscopic autoradiography. Siemens Review 34: 32-37 Pelc, S.R. 1 9 6 3 . Theory of electron autoradiography, J, Roy. Microsc. Soc. 8 1 : 131-139 Pelc, S.R., Coombes, J.D, and Budd, G,C. 196l , On the adaptation of autoradiographic techniques for use with the electron microscope. Exp. Cell Res. 24: 192-195 Przybylski, R.J. 1961. Electron microscope autoradiography, Exp. C e l l Res. 24: 181-184 Revel, J.P. and Hay, E.D. I96I. Autoradiographic localization of DNA synthesis i n a specific ultrastructural component of the interphase nucleus. Exp. Cell Res. 25: 474-480 Salpeter, M.M. and Bachmann, L, 1964. Autoradiography with the electron microscope. A procedure for improving resolution, sensitivity, and contrast. J . Cell Biol. 22: 469-477 Silk, M.H., Hawtrey, A.O., Spence, I.M., and Gear, J.H.S. 1961. A method for intracellular autoradiography i n the electron microscope, J. Biophys. Biochem. Cytol. 10: 577-587 Sjostrand, F.S. 1956. A method to improve contrast i n high resolution electron microscopy of ultrathin tissue sections. Exp. C e l l Res. 10: 657-664 Figure 108 Diagram showing the relationship of the emulsion, grid, and centrifuge spindle. Figure 109 Electron micrograph showing the undeveloped silver halide granules just after application of the electron beam, X 18,000 Figure 110 Electron micrograph showing the silver particles after exposure to l i g h t and development i n D-19. X 10,000 

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