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UBC Theses and Dissertations

Studies on some British Columbian representatives of the Erythropeltidaceae (Rhodophyceae, Bangiophycidae) McBride, Douglas Leonard 1972

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STUDIES ON SOmE BRITISH COLUMBIAN REPRESENTATIVES OF THE ERYTHROPELTIDACEAE (RHODOPHYCEAE, BANGIOPHYCIDAE) by DOUGLAS LEONARD M CBRIDE B . S c , U n i v e r s i t y of B r i t i s h Columbia* 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of Botany We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Botany The University of B r i t i s h Columbia Vancouver 8, Canada D a t e June S, 1972. i i . Supervisor: Dr. Kathleen Cole ABSTRACT Four species of the Erythropeltidaceae |smithora naiadurn (Anderson) Hollenberg, Erythrotrichia carnea (Dillwyn) J, Agardh, Erythrotrichia boryana (Montange) Berthold and Erythrotrichia pulvinata Gardner^ were observed in freshly collected and cultured conditions using light and electron microscopic techniques. The North American Pacific coast distribution of these algae was revised in view of recent collections in British Columbia and Alaska by various workers. A study of their morphologies and l i f e histories revealed new information concerning production of asexual reproductive units (rnonospores) from the basal attachment organs of Z. pulvinata and S_. naiadurn, At an ultrasfcructural level, many organelles in the vegetative cells of the Erythropeltidaceae examined were found to be similar to those reported in other members of the Rhodophyceae. However, several interesting fine structural characteristics were noted. The cellular shape was remarkably irregular, exhibiting many cytoplasmic protrusions into the c e l l wall. The single lobed chloroplast possessed a uniform lamellar arrangement and primitive thylakoid stacks or bands. In addition, multivesicular bodies occurred within the cytoplasm and in the c e l l wall near the plasmalemma. There was no evidence of any type of intercellular connection. The vegetative c e l l ultrastructurs of E. boryana and E_. pulvinata was virtually identical to S_. naiadurn but E_, carnea exhibited fewer pyrenoid-traversing lamellae and a somewhat different c e l l wall morphology. Monospore differentiation and release i n the Erythropeltidaceae was found to involve a number of specialized subcellular a c t i v i t i e s . Concomitant with a rounding of the protoplast and reduction in vacuolar area in the vegetative c a l l , was the accumulation of two products originating from dictyosornos0 The possible functions of these products ara discussed in relation to spore i i i release and attachment. Additional fine structural features of the developing monospore included an increased number of mitochondria and nuclear pores, a large amount of endoplasmic reticulum and an association between the chloroplast and the nuclear envelope. Upon release the monospore lacked a cell wall and was typified by an extensive accumulation of dictyosome product. In addition, the chloroplast exhibited a "pseudogranum-like" arrangement of thylakoids. The ultrastructural aspects of monospore dsgsneration in culture are also described. Monospore germination in S_. naiadum involved several cellular changes including formation of a call wall and a number of vacuoles. A large amount of peripheral endoplasmic reticulum and certain dictyosome populations appBared to play an important role in wall construction while other populations of dictyosomes appeared to be involved in vacuole formation. Of special interest, since i t has not been reported in the Rhodophyceae, was the occurrence of a crystalline matrix in some pyrenoids. In addition, the presence of microtubular spindle fibres was demonstrated. The alternate methods of holdfast formation in this alga are also discussed. Sexual reproduction in the Erythropeltidacsae is poorly known. In this study, an ultrastructural description of "spermatial" production in 5. naiadum is presented. The dictyosome appeared to play an important role in the maturation of these pale cells. Evidence of the process of gametogenesis and fertilization in f_. boryana is also shown. In a concluding discussion, certain ultrastructural and gross morphological information is employed in the construction of a bilateral scheme on the evolution of growth types i n the Bangiophycidae. i v . TABLE OF CONTENTS I. PREFACE. 1. I I . MATERIALS AND METHODS 4 . I I I . DISTRIBUTION, MORPHOLOGY AND LIFE HISTORY fo ) E C V "tjhy O C3? X C h X 3 e o a o o o o o v o o c e o e c o o o e e e o e * o « e » o c « « » © « « o o o * o * e • » o IV. ULTRASTRUCTURE OF THE VEGETATIVE CELL b) E r y t h g o t r i c h i a . . . . . . . . . . . . . . . . . . 34„ V. ULTRASTRUCTURAL ASPECTS OF MONOSPOROGENESIS a) D i f f e r e n t i a t i o n , Re lease and Degeneration.... 0.....o........ .44. b) G e r m i n a t i o n . . . . . . • • • . « . . . . . . « e . « . > » o . . . . « . o . < . . » . s o « o a o » . . o . < 69 • V I . ULTRASTRUCTURAL EVIDENCE OF SEXUAL REPRODUCTION 8 3 . V I I . GENERAL DISCUSSION AND CONCLUSIONS 9 4 . V I I I . LITERATURE CITED . . . . . 1 0 1 . V . LIST OF FIGURES Drawings of plants used in this study.. Map of collecting sites in British Columbia. Plate III. Light micrographs (Smithora).. „...«.„... »«......,..•• 12, Fig. 1. Basal holdfast in culture. Fig. 2„ Differentiating monospore in culture. Fig, 3. Cultured second generation monospore. Fig. 4. Cultured 2-celled stage. Fig. 5. Cultured young basal holdfast. Fig. 6. Mature blade showing monospores. Pi 8t Q I \l « (SmlJ^hOrQ ) o 4 « o 0 e 0 0 O 0 O 0 o a > o « * 0 0 « o o # 9 « o « 0 O 9 « o o c 0 0 * 0 t o o 0 0 o c 0 0 « 0 0 ^ ^ * Life cycle diagram. Plate V, Light micrographs (Erythrotrichia) ........ 19. Fig, 7. E_. carnea. Mature filaments. Fig. 8, £. carnea. Bipolar germination. Fig. 9. E. carnea. Juvenile filaments. Fig. 10, E_. carnea. In situ monospore germination. Fig, 11. E_. boryana. Mature filaments. Fig, 12. E_. boryana. Cultured basal pad. Fig. 13. E_. boryana. Association with Smithora. Plate VI. Light micrographs (Erythrotrichia pulvinata). 20« Fig. 14. Mature filament. Fig. 15. Monospore production. Fig. 16* 4-celled stage in culture. Fig. 17. Juvenile pad. v i Fig. 18. Mature pad in culture. Fig. 19, Edge of cultured pad. Plate VII. (Smithora).. 30. Fig. 2. Cross-section of vegetative c a l l . Plate VIII. (Smithora) 31. Fig, 3, Chloroplast lobe. Fig. 4, Nucleus. Fig. 5. Older c e l l . Plate IX. (Smithora) .....32. Fig. 6. Thylakoid stack. Fig. 7. Thylakoid stack. Fig. 8. Ring-shaped mitochondrion. Fig. 9. Zone of attachment. Plate X. (Smithora) .....33. Fig. 10. Multivesicular bodies. Fig, 11. Multivesicular bodies. Fig. 12. Multivesicular bodies. Fig. 13. Lamellar bodies, . Fig. 14, Vesicles near plasmalemma. Plate XI. (E. carnea) 38. Fig. 1. Light micrograph. Portion of filament. Fig, 2. Section through filament. Plate XII. (E, carnea)... ....39. Fig. 3. Portion of cel l . Fig. 4. Portion of pyrenoid. Fig, 5, Cell wall. Plate XIII. (E. bory Fig. 6. Light micrograph. Portion of filomant. v i i . Fig. 7. Section through filament. Plate XIV. (jE. boryana) 41. Fig. 8. Zone of attachment Fig. 9. ER-nuclear envelope association. Fig. 10. Portion of chloroplast lobe. Plate XV. (E_. pulvinata) 42. Fig. 11. Light micrograph. Basal cushion. Fig. 12. Section through basal cushion. Plate XVI. (E_. pulvinata) • e * f t e o « * o « « « « « o e * « o » o Q « « o e « o a a o o o o * o c o o * o o 0 0 o 43. Fig. 13. Attachment zone. Fig. 14. Portion of pyrenoid. Fig. 15. Attachment zone. Fig. 16. Peripheral region of protoplast. Plate XVII, Differentiating monospore. (Smithora) ...57. Fig. 1. Light micrograph. Surface view of sorus. Fig. 2. Section through sorus. Plate XVIII. Differentiating monospore, (Smithora) .58. Fig. 3. Nuclear area. Fig. 4. Chloroplast-nucleus association. Fig. 5. Nuclear area. Fig. 6. Nuclear envelope. Fig. 7. Nuclear pores, Plate XIX. Differentiating monospore. (Smithora) «59a Fig. 8. Dictyosome. Fig. 9. Floridean starch grain. Fig. 10. Vacuole-liko structurs-ER association. Fig. 11. Dictyosome products. v i i i . Fig. 12. Dictyo9ome products. Fig, 13, Release of dictyosome products. Plate XX, Differentiating monospore. (Smithor Fig. 14, Dictyosome. Fig. 15. Compact area of c e l l mall. Fig. 16. Liberation of monospore. Fig. 17. Thallus after spore release. Fig. 18, Remaining cytoplasm. Fig. 19. Light micrograph. Deciduous sorus, Plate XXI, Released monospore. (Smithora).... Fig. 20. Light micrograph. Released monospore Fig. 21. Section through released spore. Plate XXII. Released monospore, (Smithora) Fig. 22. Section through deciduous sorus, Plate XXIII. Released monospore. (Smithora).. Fig. 23. .Appressed chloroplast lamellae. Fig, 24. Appressed chloroplast lamellae. Fig. 25. Portion of pyrenoid. Plate XXIV. Released monospore. (Smithora) Fig. 26. Dictyosome. Fig. 27. Closely packed mitochondria. Fig. 28. Nucleus. Fig. 29. Plasmalemma. Fig. 30. Release of dictyosome product. Plate XXV. (£. boryana). Fig. 31. Unreleased monospore. Plate XXVI. (Erythrotrichia) ix. Fig. 32. F_. boryana. Portion of differentiating monospore. Fig. 33. f_. carnea. Portion of differentiating monospore. Fig. 34. F.. pulvinata. Portion of differentiating monospor Plate XVII. Degenerating monospore. (Smithora)....,....... Fig. 1, Early stage of degeneration, Plate XVIII. Degenerating monospore. (Smithora) Fig, 2. Mitochondria, * Fig. 3, Nucleus. Fig. 4. Chloroplast. Fig. 5. Portion of degenerating spore. Plate XXIX. Germinating monospore, (Smithora) Fig. 1. Light micrograph. Germinating spore. Fig. 2. Section through germinating spore. Plate XXX. Germinating monospore. (Smithora) ,. Fig. 3. Light rnicrographo Adhering germinating monospores. Fig. 4. Section through germinating sorus. Plate XXXI. Germinating monospore. (Smithora) Fig. 5. Light micrograph. Basal holdfast. Fig. 6. Dictyosome. Fig. 7. Area of c e l l wall i n i t i a t i o n . Fig. 8. Portion of developing pad. Plate XXXII. Germinating monospore, (Smithora) Fig, 9, Peripheral ER. Fig. 10. Peripheral ER. Fig. 11. Crystalline pyrenoid. Plate XXXIII. Germinating monospore. (Smithora) Fig, 12, Crystalline pyrenoid. Plate XXXIV. Germinating monospore. (Smithora) 8 2 . Fig. 1 3 . Vesicular units. Fig. 1 4 . Nuclear area. Fig. 1 5 . Nuclear area. Fig, 1 6 , Paramural body. Fig. 17. Spindle fibres. Plate XXXV. Spermatangia. (Smithora) 8 9 c Fig. 1 . Light micrograph. Cross-section of sorus. Fig. 2 . Cross-section of sorus, Plate. XXXVI„ Spermatangia. ( S m i t h o r a ) . . . . . . . . . . o . . . . . . . . . . . . . . . . . . . . . 9 0 . Fig. 3 . Immature spermatangium. Fig. 4 , Immature spermatangia. Fig. 5 . Maturing spermatangium. Vesicle production. Plate. XXXVII. Spermatangia. (Smithora) o . . . . . . . 9 1 . Fig. 6 , Release of vesicles. Fig. 7. Cell wall protrusion. Fig, 8 . Chloroplast material. Plate XXXVIII. Spermatia. (Smithora)... 9 2 . Fig. 9 . Liberated spermatium. Fig. 1 0 . Liberated spermatia. Fig. 1 1 . Light micrograph. Liberated spermatia. Plate XXXIX. (E. boryana) 9 3 . Fig. 1 . Light micrograph. Spermatium attached to filament. Fig. 2 . Spermatium attached to filament. Fig. 3 . Carpospore-like c e l l . Fig, 4. Attached spermatium. Plate XL 1 0 0 , Diagram of the evolution of growth types in the Bangiophycidae, ACKNOWLEDGEMENTS I would like to express my most sincere gratitude to Dr. Kathleen Cole for her unfailing confidence and continual academic assistance during the course of this study. Thanks are also extended to the other members of my committee: Dr. To Bisalputra, Dr. R.E. Foreman, Dr. R.F. Scagel, Dr. I.E.P. Taylor and Dr. G.H.N. Towers for their helpful criticism of this manuscript. Special thanks go to Miss E. Packham for access permission to one of the collection sites, Mr. L.L. Veto for his excellent technical assistance, Dr. J. Whyte for his companionship and aid during several collecting trips, and to the Department of Botany, University of British Columbia for the use of various facilities including the Hitachi HU 11A and AEI S01 A electron microscopes. Dr. Elsie Conway, Miss Julie Celestino, Dr. V.L. Bourne and Dr. J.R. Maze participated in many interesting discussions on the subject matter. I would also like to express my indebtedness t D my wife Joanne and to Mrs. Eileen Bennett whose constant devotion and support provided a special incentive. This study was supported by the National Research Council of Canada (Grant A645 to Dr. Cole) and by a U.B.C. Postgraduate Fellowship to the author. 1 . I . PREFACE The Rhodophyceae a r e t a x o n o m i c a l l y s e p a r a t e d I n t o tu/o s u b c l a s s e s ; t h e F l o r i d e o p h y c i d a e and t h e l e s s advanced B a n g i o p h y c i d a e . T h i s l a t t e r group i s s u b d i v i d e d i n t o s e v e r a l o r d e r s , one o f w h i c h , t h e B a n g i a l e s , c o n t a i n s two f a m i l i e s ! t h e B a n g i a c e a e and t h e E r y t h r o p e l t i d a c e a e . The E r y t h r o p e l t i d a c e a e S k u j a a r e d i s t i n g u i s h e d by t h e f o l l o w i n g c h a r a c t e r i s t i c s ; m a r i n e ; f i l a m e n t o u s o r n o n f i l a m o n t o u s t h a l l i a t t a c h e d by a r h i z o i d a l o r c u s h i o n - l i k e h o l d f a s t ; c e l l s c o n t a i n i n g a s i n g l e s t e l l a t e o r p a r i e t a l c h l o r o p l a s t w i t h a c e n t r a l l y l o c a t e d p y r e n o i d ; monospores and/or d i s t i n c t i v e , p a l e , c r e s c e n t - s h a p e d c e l l s may be formed from a s y m m e t r i c a l d i v i s i o n s o f v e g e t a t i v e c e l l s . T h i s f a m i l y has a c h i e v e d w o r l d wide d i s t r i b u t i o n and i n c e r t a i n r e s t r i c t e d h a b i t a t s a p a r t i c u l a r s p e c i e s may become t h e dominant a l g a l f o r m . There a r e f o u r g e n e r a r e p r e s e n t e d on t h e P a c i f i c c o a s t o f N o r t h A m e r i c a ; E r y t h r o c l a d i a , E r y t h r o t r i e h i a , P o r p h y r o p s i s and S m i t h o r a , The s p e c i e s c h o s e n f o r t h i s s t u d y were E r y t h r o t r i e h i a c a r n e a ( D i l l w y n ) J , Agardh, E r y t h r o t r i e h i a b o r y a n a (Montange) B e r t h o l d , E r y t h r o t r i e h i a p u l v i n a t a Gardner and S m i t h o r a naiadum (Anderson) H o l l e n b e r g . C o l l e c t i v e l y , t h e c h o s e n p l a n t s i l l u s t r a t e a maximum m o r p h o l o g i c a l v a r i a b i l i t y w i t h i n t h e group and p r o v i d e an i n t e r e s t i n g sequence o f i n c r e a s i n g s t r u c t u r a l c o m p l e x i t y . f_. c a r n e a , t h e s i m p l e s t , p o s s e s s e s a u n i s e r i a t e t h a l l u s w i t h a r h i z o i d a l a t t a c h m e n t , E_. b o r y a n a f e a t u r e s m u l t i s e r i a t e f l a t t h a l l i a r i s i n g f r o m a m o n o s t r o m a t i c d i s c - l i k e h o l d f a s t , E. p u l v i n a t a i s t y p i f i e d by m u l t i s e r i a t e f l a t t h a l l i o r i g i n a t i n g f r o m a m u l t i s t r o m a t i c b a s a l h o l d f a s t and S m i t h o r a f e a t u r e s l a r g e r , m o n o s t r o m a t i c t o d i s t r o m a t i c b l a d e s a t t a c h e d by a m u l t i s t r o m a t i c b a s a l c u s h i o n (see P I , J ) . The taxonomy o f t h e E r y t h r o p e l t i d a c e a e i s i n a somewhat c o n f u s e d s t a t e . HeBrabout (1968) has attempted to correct this situation but his efforts have been severely c r i t i c i z e d by certain authors (e.g. Dangeard, 1969). In addition, as Drew (1956) has pointed outs "... our knowledge of the reproductive processes and the l i f e history of many of these algae i s very scanty; consequently the need for precise and well documented investigations is stressed." This situation has undoubtedly been precipitated by such factors as the scarcity of many species, the epiphytic nature of others and the relatively small size of the majority of the representatives. Refined laboratory culture techniques and the use of the electron microscope have proved helpful in surmounting such barriers in recent years. These tools were applied in the present study in an attempt to add to existing information on this family. 3. Plate I. Scale drawings of the species of Erythropeltidaceae used in this study: Erythrotrichia camea, Erythrotrichia boryana, Erythrotrichia pulvinata and Smithora naiadurn. I 4. I I . MATERIALS AND METHODS Smithora naiadurn was collected intertidally at Stanley Park, Vancouver, British Columbia (Lat. 49°19» N., Long. 123°9e W.)» Sooke, Vancouver Island, British Columbia (Lat. 48°21• N ., Long. 123°43° W.) and Point No Point (Glacier Paint), Vancouver Island, British Columbia (Lat. 48°23° N., Long. 123°59* Ul.). All species of Erythrotrichia were collected at the latter site. At these locations material is most plentiful during the months of May to October in the lower and middle intertidal zones. Smithora is a specific epiphyte on the sea grasses Phyllospadix scouleri and Zostera marina. In the collection area E_. boryana is also epiphytic on these marina seed plants and on Smithora. 8oth E_. carnea and E_. pulvinata were found attached to the green alga Codium fragile. Monosporic plants were available during periods of low tides from June to October while "spermatangial" Smithora was collected from August to October. Freshly collected plants were stored on ice while being transported to the laboratory, then were isolated from host materials washed carefully and placed in plastic Petri dishes containing a modified Erdschreiber medium (sea water- 1.0 1., NaN03- 200 mg., Na2HP04'7H20- 20 mg., KNO3-50 mg,, Fe(EDTA)- 1 mg., TRIS- 500 mg., soil H20- 50 ml., vitamin 812-2 mg., Ge02- 10 mg.). Cultures were kept in a constant temperature incubator at 120C under a 12 hr,/l2 hr. light regime at 700-800 lux (fluorescent light). When culturing germinating monospores, agitation of the culture dishes was kept at a minimum resulting in many of these structures adhering to parts of the parent blade. The use of the thalli as a substrate in this manner facilitated preparation of the material for electron microscopy. Freshly collected plants were used for electron microscopy where possible. Primary fixation was carried out in the field or immediately 5. upon a r r i v a l at the laboratory, material was fix e d i n a solution of 25% glutaraldehyde (v/v)»'ISM phosphate buffer at pH 7.2 and s t e r i l i z e d sea water (1:2:2) for 1 hr., washed and followed by postfixation i n a mixture of 2% osmium tetroxide (v/v) and phosphate buffer at pH 7.2 (1:1) for 1£ hr. Fixation and postfixation were carried out at 4°C or at room temperature. After a thorough washing i n buffer, the material was then subjected to dehydration i n a graded ethanol s e r i e s , i n f i l t r a t i o n i n propylene oxide and subsequent embedding i n Epon 812 (Luft, 1961) or Maraglas 655 (Spurlock, Kattine and Freeman, 1963). Al t e r n a t i v e l y , the material was embedded i n Spurrfs medium (Spurr, 1969) d i r e c t l y after the ethanol s e r i e s . Thin sections were cut using glass knives or a Ou Pont diamond knife on a L.K.8. Ultratome I. Poststaining was carried out for 20-30 min. i n uranyl acetate (2% s o l . (w/v) i n 50% methanol (v/v)) and for 5-10 min. i n lead c i t r a t e (Reynolds, 1963). The prepared material was examined using a Hitachi HU 11A electron microscope or an AEI 801 A electron microscope operating with an accelerating voltage of 50 KV. Light microscopy was done with l i v i n g material using a Wild 0120 l i g h t microscope. When transverse sections were required, specimens were prepared with a freezing microtome. 6. III. DISTRIBUTION, LIGHT MICROSCOPIC MORPHOLOGY AND LIFE HISTORY a) Smithora Introduction, Smithora naiadum was f i r s t dsscribed as a member of the genus Porphyra (Anderson, in Blankinship and Keeler, 1892). The i n i t i a l definitive account of the morphology of this alga was given by Hus (1903): "Fronds 2-10 cm. long, obovate when young, oblanceolate when older; base cushion-shaped; fronds wine-red to blue-purple; monostromatic vegetative part 25-30 microns thick, cells square or slightly higher than broad, 15-20 microns high; surface j e l l y measuring about 5 microns, l i t t l e j e l l y between the c e l l s ; fronds dioecious ?; sporocarps with 8 carpospores." The author describes these latter structures as arising in the terminal parts of the blade. The "sporocarp" (carposporangium) gives rise to eight carpospores (two layers of four) which are released. Since this report there have been no other convincing accounts of these structures, Knox (1926) carried out more extensive investigations on "Porphyra"  naiadum and correctly interpreted the pattern of asexual reproduction in describing the development of mature plants from monospores. She made numerous unsuccessful attempts to culture these reproductive units. Her lack of success appeared to be due to poor culture f a c i l i t i e s . In addition, Knox claimed to have observed external sexual fusion, antheridial areas and c e l l division but her figures and descriptions are inconclusive. At a later date, a morphological re-evaluation carried out by Hollenberg (1959) resulted in the placement of this alga in a new genus, Smithora , and a new family, Erythropeltidaceae, Subsequently, this action has been given biochemical support by Rees and Conway (1962), Hollenberg's description of the plant is as followsi "Plants epiphytic, with numerous obovate to cuneate and monostromatic blades arising from a prostrate cushion-like perennial multistratose 7. base; cells with a single stellate chromatophore; plants with no rhizoidal processes arising from the lower cells of the blades; carpospores . formed in irregular, mostly terminal so r i , in packets of eight; spermatangia arising in irregular sori toward the middle portions of the blades as small cells cut off externally from colored cells of the locally distx-omatic portions of the blades; plants reproducing asexually by means of irregular, terminal, monostromatic gelatinous sori which are released as a unit." The account of carpospore formation is essentially based on Hus' description. Hollenberg states: " . . . i t i s practically impossible to distinguish such reproductive areas from spermatangia! areas...". Hollenberg also reported the presence of additional asexual reproductive units termed "neutral spores" which are formed at the margins of blades and a correlation of monospore release with periods of low tides. Like Knox (1926), he was largely unsuccessful in culturing the spores. Most recently, Richardson and Dixon (1969) have detailed the presence of a filamentous "conchocelis" stage in the l i f e cycle of Smithora. However, the authors observed none of the reproductive structures described above and included no light micrograph of their findings. Observations and Discussion. Distribution: The previously recorded distribution of Smithora naiadurn is from northern British Columbia to Isla Magdalena, Baja California, Mexico (Dawson, 1961). However, from collections by various workers, the following Alaskan specimens are recorded in the phycological herbarium at the University of British Columbia (WS indicates wet stack)? Barrier Is.: UBC 22153, 23467-23468, 1.VII.1965. Cape Bartolome: UBC 23466, 2.VII.1965. Cape Chiniak, Kodiak Is.: UBC 9912-9913, 23.VI. 1960. Klokachef Is.: UBC 22627-22628, 30.VI.1965. Pasagashak Pt., Kodiak Is.: UBC 8B46, 25.VI.1960. Pt. Alexander, Wrangel Narrows: UBC 23469, 1.VII.1965. Thus, the revised North American Pacific coast distribution far Smithora naiadurn is from Kodiak Island, Alaska to Isla Magdalena, 8aja California, Mexico. 8 . BRITISH COLUMBIAN COLLECTION RECORDS. WEST COAST VANCOUVER ISLAND: A m p h i t r i t e P t . , U c l u l e t : UBC 39967, 8 . V . 1969; UBC 40146, 1 8 . V . 1 9 6 9 ; UBC 40248-40249, 1 . V I 1 , 1 9 6 9 . B a m f i e l d : UBC 40794, 3 . V I . 1 9 6 9 ; UBC 41012, 1 7 . V I . 1 9 6 9 ; UBC 41154, 9 . V I 1 . 1 9 6 9 ; UBC 41588, 1 4 . V I I . 1 9 6 9 ; UBC 42064, 1 2 . V I I I . 1 9 6 9 ; UBC 42445, 2 5 . V I I . 1 9 6 9 . B lack R . : UBC 13200, 8.VI 1.1959^ Breaks P e n . : UBC 36269, 1 3 . V I I I . 1 9 6 8 . Bunsby I s . : UBC 19668, 6 . I X . 1 9 6 4 . Capo S c o t t : UBC 35769,36581,36977, 1 1 . V I I I . 1 9 6 9 . Cape S u t i l : UBC 15741,15879-15882, 2 . V I I . 1 9 6 2 . Experiment B i g h t : UBC  19353, 7 . V I I I . 1 9 6 4 . Fisherman Bay: UBC 38295,38302-38303, 3 0 . V I 1 1 . 1 9 6 8 . Garden I s . : UBC 10925-10927,11006, 2 7 . V . 1 9 5 9 , Guise Bays UBC 19770, 7 . V I I I . 1 9 6 4 . Macquinna P t . t UBC 1125B, 2 4 , V . 1 9 5 9 . M i l l s P e n . : UBC 10920- 10921,13314-13315, 7 . V I I . 1 9 5 9 . Nootka I s . : UBC 37512, 2 2 . V I 1 1 . 1 9 6 8 ; UBC 37168,37679,37687, 2 4 . VI11.1 968. Perez Rock": UBC 10922-10923,13524, 2 4 . V I . 1 9 5 9 . P o i n t No P o i n t ( G l a c i e r P t . ) : UBC 1B759, 2 , I X . 1 9 6 8 ; UBC 18760, 4 , V I I I . 1 9 6 8 ; UBC 18761, 7 . V I . 1 9 6 8 ; UBC 36795,36802, 2 8 . X I . 1 9 6 8 ; UBC 4*2359, 1 6 , V . 1 9 6 9 ; UBC 39011,39022, 2 5 . X . 1 9 6 8 ; UBC 39177, 2 2 , X I , 1 9 6 8 ; UBC 837 WS, 2 2 . X I . 1968. S_an Josef Bay: UBC 38614-38615,39023, 3 1 , V I I I . 1968. S p r i n g I s . : UBC 13525, 2 5 , V I I . 1 9 5 9 ; UBC 18985, 6 . V I . 1 9 6 4 . T o f i n o : UBC 39334, 3 . V . 1 9 6 9 . Topknot P t . : UBC 42817, 2 . I X . 1 9 6 8 . Whi f fen s p i t : UBC 38796, 2 8 . I X . 1 9 6 8 ; UBC 39010, 2 5 . X . 1 9 6 8 ; UBC 39185,39329, 2 2 . X I . 1 9 6 8 ; UBC 46065, 8 . X I I . 1 9 7 1 . Winter Harbour : UBC 11021, 3 1 . V . 1 9 5 9 , Woutuer I s . : UBC 10924,11256, 2 1 . V . 1 9 5 9 . Yel low B l u f f : UBC 37282,37505,37511, 2 1 . V I I I . 1968 ; UBC 915 WS, 2 1 . V I I I . 1 9 6 8 . EAST COAST VANCOUVER ISLAND: Cluxewe R . i UBC 17075, 5 . V I I . 1 9 6 2 . Deer I s 0 : UBC 35576-35577, 9 . V I I I . 1 9 6 8 . D iscovery I s . : UBC 38797, 3 . I X , 1 9 6 8 . F a l s e Head: UBC 38617-38616, 4 . I X . 1 9 6 8 . G a b r i o l a I s . : UBC 28375, 3 0 . I I I . 1 9 6 7 ; UBC 28826, 2 8 . I V . 1 9 6 7 ; UBC 29574,29609, 2 4 . V . 1 9 6 7 . Grassy I s . : UBC 36056, 37276,36613, 20 .V I I I . .1968 . Quadra I s . ( s o u t h ) : UBC 43793, 1 0 . I X . 1 9 7 0 . SCOTT ISLAND GROUP: Cox I s . : UBC 36055,36592,36752, 1 2 . V I I I . 1 9 6 8 . T r i a n g l e I s . t UBC 19578, 7 . I X . 1 9 6 4 . MAINLAND (JOHNSON STRAIT AREA): N e v i l l e P t . : UBC 15963, 2 9 . V I . 1 9 6 2 . PRINCE RUPERT AREA: Chatham Channe l : UBC 15110 -15111 , 8 . V I I . 1 9 6 2 . Digby I s . : UBC 16450, 2 6 . V I . 1 9 6 3 . QUEEN CHARLOTTE ISLANDS? Anthony I s . : UBC 16551,16554, 1 9 . V I . 1 9 6 3 . Marchant Reef , Graham I s „ : UBC 21343,22301, 4 . V I I . 1 9 6 5 , S t r i a e I s . : UBC 16240,16282, 2 4 . V I . 1 9 6 3 . F i e l d M a t e r i a l : F o l l o w i n g examinat ion of many of the above herbarium specimens and numerous c o l l e c t i o n s d u r i n g the p e r i o d from September, 1968 to November, 1971, I am i n genera l agreement w i t h H o l l e n b e r g ' s (1959) 9. results. However, no convincing evidence of carpospores or neutral spores was obtained. There was a marked difference among winter populations of Smithora i n different areas. At the collecting sites on Vancouver Island (Sooke and Point No Point) the basal cushions remained throughout the winter, but in Stanley Park a l l traces of the plant disappeared in mid-November only to become established again the fallowing spring. The latter collecting area has a lower salin i t y (near a freshwater outflow) and i s much more protected than the other sites . If Richardson and Dixon's conchocelis phase exists, perhaps i t serves as an overwintering mechanism in certain populations. "Spermatangia" as described by Hollenberg (1959) were observed regularly in the f a l l months of each year. However, there was no convincing indication of f e r t i l i z a t i o n taking place or having taken place. Indeed, there is. no good evidence implicating "spermatia" i n sexual fusion in any of the Bangiophycidae. Although they w i l l be referred to as "spermatangia/spermatia" in this report, perhaps they may be likened to the/3-spores of Porphyra (Conway, 1964) which are formed in a different manner. These structures w i l l be discussed further in Section VI. Cultured Material: Limited success was obtained in culturing Smithora. Sterile blades could be cultured for periods up to four months. Monosporic blades would not survive beyond one month, although during this period they would continue to differentiate and release monDspores. These structures are usually released terminally but, in culture, isolated patches of precociously released spores could be observed toward the center of the sorus (PI. I l l , Fig. 6). It appears that a r t i f i c i a l conditions somewhat disrupt the synchrony of spore production. Monospores would germinate readily, either singly or in masses, to form basal cushions (PI, III, Fig. 1). These cushions 10. would then produce single monospores (PI. I l l , Fig. 1,2,3) which were identical to those produced by the blade. After release, the spore would divide (PI. I l l , Fig. 4) to produce a new basal cushion (PI. I l l , Fig. 5). Such cushions showed no evidence of blade formation although several generations could be maintained in culture. Another characterisic of these structures to which Knox (1926) briefly alluded is their ability to adhere to one another (PI. I l l , Fig. 5). Culture of spermatangial blades proved unfruitful, although a good release of these cells could be obtained. The presently known l i f e history of Smithora is diagrammatically illustrated in PI. IV. Recently, Harlin (1971) has shown that Smithora will grow in the field on polyethylene strips approximating the dimension of Phyllospadix leaves. Present results on cultured material in this laboratory also appear to indicate that this alga is not dependent on its host for any nutritive material as was thought by some authors (e.g. Knox, 1926). However, i t suggests thata delicate set of environmental conditions is needed for the plant to produce the leafy thallus. Adjustments in culture parameters such as ingredients of the media, temperature, agitation, light intensity and light duration had no effect. 11. PLATE II. (flap of British Columbia showing collection sites of Smithora naiadurn, Erythrotrichia carnea, Erythrotrichia boryana and Erythrotrichia pulvinata as recorded in the phycological herbarium of the University of British Columbia by various workers. Legend. Acous Peninsula................2 3 . Lawton P t . o . . . . . . . . . . . . . . . . . . . . . . 3 7 . Am phi tr X t e P t o . « . . « o . o . o . o . . o . e 3 1 . Li PPy Pt . O 9 o o o . e . , o . o , a o o o » o . o » « . o o l 9 . Anthony I s . o o . . o o o . 0 . 0 . 0 0 0 0 . . • . 5 « Lookout l S e o . o e o . . o o o . » o . . « . o . e o o o o 3 2 o B a m f i e l d . . . . . . . . . . . . . . . . . . . . . . . 3 5 . Macquinna P t . . » . . . . . . . . . . . . . . . . . . . . 2 8 . Black River... 0 . . o . . . . . . . . . . . . . 3 8 . Warchant Reef...... 0 . . . . . . . . . . . . o . . 3 . Brooks Peninsula.... 2 2 . Mills P e n i n s u l a . . 0 . . . . . . . . . . . . . . . . . 3 6 . B U n Sby I S . , « o » . o o o . o . o e o o . . . o o o 7 o Neville P t o o e o o o . . o a o . * o . o o o o o . o e o » 4 6 . Cape S c o t t . o o o o o . o . . . 9 0 . 0 . 0 . . e » 1 3 . Nootka I s . . Q o o o . « o , . « . . o a . . o . . . . . . . 2 7 . Cape S u t i l . o o o . o « » . . o o . . . o e e a o t t l r j . P6reZ R O C k e . o e . . . . © # . o * o e . » e . o . o . « . 2 9 . Chatham C h a n n e l . . 0 . . . . . . . 0 . . . . 0 1 . Piper's Lagoon . 4 4 . Cluxewe R i v e r . . . . . . . . . . . . . . . . . . 4 7 . Plover Is. 0 . . . . . . . . . . . . . . . . <• . . . 0 . 0 . 7 . COX IS. ... o o o » . e e e . o f t e d . . o e o o o . 1 5 . POX nt NO P O X n t . « , o , o a e o « o « e . . . a . o « o 3 9 o Deer I s . . . . . . . . . . . . . . . . . . . . . . . . 4 9 . Qlawdzeet-Bell Passage............ .9. Dxgby I S o . . . . « « e o , o . . . . . . . . . . . e 2 . Quadra I s . o . » . . e e . o o o * e . . o . o . . . . « . . 4 5 e Discovery I s . . . . . . . . . . . . . . . . . . . 4 1 . San Josef B a y . . . . . . . . . . . . . . . . . . . . . . 1 7 . Experiment Bight . . . . . . . . . . 1 2 . Stanley Park.............. . 4 2 . False Head......... . . . . . . 4 8 , Striae I s . . . . . . 0 . . . . . . . . . . . . . . . . . . . 4 . Fisherman B a y . . . . . . . < • * , . . . « « e . 1 1 . T o f i n o . . . . . . . . . . . . . . . « , , . . • . . . * • * . . 3 0 * Gabriola Is.... . . . . . 4 3 . Topknot P t . . 1 8 . Garden Is . . 0 . . . . 3 3 . Triangle Is,....... . . . . . . . . . . . . . . . . 1 4 . Grassy Is..... 2 5 . Ulhiffen spit... « . . . 4 0 . Guise Bay. . . . . . . . . 1 6 . Winter Harbour.. 2 0 , Hedley I s . . . . . . . . . . . . . . . . . . . . . . 8 . Wouwer Is.. ....«..•»•*... 0 . . . . . . . . . . 3 4 . Hope Is.... 6 . Yellow Bluff . . . . 2 6 . Lawn Pt. 2 1 . 12. PLATE III. Light micrographs. Smithora Fig. 1, Basal holdfast (bh) in culture producing second generation monospores (arrow). Fig. 2. Differentiating monospore in cultured f i r s t generation basal pad. vc denotes vegetative cello Fig. 3. Second generation monospore in culture e Fig. 4 0 2-celled stage of germinating monospore in culture. Fig, 5, Young cultured basal holdfast. Fig. 6. Portion of mature blada showing differentiating monospores (dm)p vegetative c e l l area (vc) and an isolated area of precociously released monospores (rm). 13. PLATE IV. Diagram of the possible l i f e c y c l e of Smithora naiadum. Dotted l i n e s i n d i c a t e poorly documented steps of the schema. IV stage) 14. b) E r y t h r o t r i e h i a Introduction. Some 36 species of E r t h r o t r i c h i a Areschoug have been described from various parts of the world. Because of t h i s wide d i s t r i b u t i o n , an examination of each recorded species or growth form would be, at best, extremely d i f f i c u l t . This s i t u a t i o n has resu l t e d i n taxonomic confusion and uncertainty. H i s t o r i c a l l y , the following c h a r a c t e r i s e s have been used to determine species of E r y t h r o t r i e h i a ; colour, s i z e , form of filament (monosiphonous, polysiphonous, ribbon-shaped, e t c . ) , type of ch l o r o p l a s t ( p a r i e t a l or s t e l l a t e ) , branching and host s p e c i f i c i t y . Numerous attempts have been made to assign some type of natural system of c l a s s i f i c a t i o n to these plants, Berthold (1882) divided the genus i n t o two groups; one forming spores which d i r e c t l y give r i s e to filaments, the other forming spores which givs r i s e to basal d i s c s , followed by the secondary process of filament formation. Hamel (1929) distinguished three categories; those attached by a s i n g l e , lobed or unlobed basal c e l l , those attached by a number of r h i z o i d a l c e l l s and those attached by a m u l t i c e l l u l a r d i s c . Tanaka (1952) proposed a taxonomic b i s e c t i o n on the basis of p a r i e t a l or s t e l l a t e c h l o r o p l a s t s , More recent l y , Heerebout (1968) has recommended ths recognition of only three species, due to t h B morphological v a r i a b i l i t y of these plants i n c u l t u r e . Unfortunately, he was unable to examine representatives of a l l described species and rejected many only on the basis of published reports. His d e s c r i p t i o n of the genus and key to the species are as follows: " E r y t h r o t r i e h i a , Thallus erect, filamentous or ribbon-shaped, often aiith a di s c or cushion-shaped attachment organ. Filamentous t h a l l u s branched or unbranched, mono- or polysiphonous. Ribbon-shaped t h a l l u s always monostromatic; unbranched. C e l l s b r i c k red, length about 10-25 microns. Chromatophore s t e l l a t e with a d i s t i n c t pyrenoid. Asexual reproduction by monospores; l i f e c y c l e with a conchocelis stage. P i t connections never seen. Key to the s p e c i e s . 1a. T h a l l u s c o n s i s t i n g of rows of c e l l s arranged i n one plane, g i v i n g i t a ribbon-shaped appearance. Nearly always w i t h a b a s a l d i s c , i n young stages sometimes only a b a s a l d i s c i s present . E. boryana. 1b. T h a l l u s mono- or polysiphonous, c e l l rows r a d i a l l y arranged...2. 2a. T h a l l u s o f t e n attached by a bas a l d i s c or by sm a l l protuberances of the b a s a l c e l l carnea. 2b. T h a l l u s w i t h a long boring r h i z o i d , composed of h y a l i n e c e l l s , always growing on R a l f s i a t h a l l i attached to gastropods... _E. w e l w i t s c h i i . " Heerebout considered such c h a r a c t e r i s t i c s as c h l o r o p l a s t morphology, mono- or polysiphony and mode of attachment to be u n r e l i a b l e f o r taxonomic purposes. West (1966) has a l s o questioned the v a l i d i t y of using c e r t a i n taxonomic c r i t e r i a . In c o n t r a s t , Dangeard (1968,1969) l i s t s 34 spe c i e s and c o l o u r f u l l y d e s c r ibes Heerebout's r e v i s i o n as f o l l o w s : "...sans doute en premiere dans ce 'massacre' d'especes...". D e t a i l s of c e r t a i n r e p r o d u c t i v e processes i n E r y t h r o t r i e h i a are eq u a l l y obscure. Asexual r e p r o d u c t i o n i s accomplished p r i m a r i l y through monosporogenesis, whereby a v e g e t a t i v e c e l l undergoes a d i v i s i o n and one of the r e s u l t i n g daughter c e l l s forms a spore. In one species (E_. w e l w i t s c h i i ) an undivided v e g e t a t i v e c e l l may be r e l e a s e d as a u n i t . Reports of sexu a l r e p r o d u c t i o n have been s p o r a d i c . B e r t h o l d (1882) f i r s t d e s cribed spermatia being cut o f f from a v e g e t a t i v e c e l l , r e l e a s e d and a t t a c h i n g to a f i l a m e n t adjacent to the supposed carpogonium. Subsequent r e p o r t s of such events by Gardner (1927), Baardseth(1941) and Tanaka (1944, 1952) have shed l i t t l e a d d i t i o n a l l i g h t on t h i s process. For example, concerning p o s t - f e r t i l i z a t i o n events, B e r t h o l d (1882) describes an und i v i d e d , f e r t i l i z e d carpogonium being r e l e a s e d whereas Tanaka (1944) s t a t e s t h a t f e r t i l i z e d carpogonia d i v i d e to produce a "few" carpospores. Hserebout (1968) 16. has reported the presence of a conchocelis phase of the l i f e cycle of Erythrotrichia which hB presumes to have grown from carpospores, although no direct evidence of this i s presented. Thus, i t is obvious that there is a need of c r i t i c a l research in almost every phycological aspect of this genus. Observations and Discussion. Pacific Coast Distribution^: Three species of the genus Erythrotrichia are reported here according to the revision proposed by Heerebout (1968), with the exception that IE. pulvinata has been retained as a valid species. The distinguishing morphological feature is the presence of a relatively large, monospore producing, multistromatic, basal holdfast. In addition, E_. pulvinata appears to occupy a specialized habitat (epiphytic on the utricles of Codium fr a g i l e ) . In light of these findings, a re-examination of specimens of Erythrotrichia reported by various authors on the Pacific coast of North America is required in order to obtain a more complete distributional record of this taxon, Erythrotrichia bogyana The previously recorded distribution i s from Punta Baja to Bahia Asuncion, 8aja California, Mexico (Dawson, 1961). BRITISH COLUMBIAN COLLECTION RECORDS. WEST COAST VANCOUVER ISLAND: Point No Point (Glacier Pt,): UBC 1260 HIS, 2.VII.1971 (epiphytic on Phyllospadix scouleri and Smithora naiadurn ). Erythrotrichia carnaa The previously recorded distribution i s from Monterey, California to Golfo Dulse, Costa Rica; Clipperton Is. (Dawson, 1961) with a northward 1This part of the t h e s i 3 i s based on an article by J.W. Markham, D.L. MnBride and P.R. Newroth which has been accepted for publication in Syesi3. 17. extension by Norris and West (1967) at Shilshole Bay Marina, Seattle, Washington. BRITISH COLUMBIAN COLLECTION RECORDS. WEST COAST VANCOUVER ISLAND: Bamfield: UBC 648 WS_, 30.VII.1969 (epiphytic on Callithamnion pikeanum). Garden Is.: UBC 964 WS, 27.V.1959 (epiphytic on Polysiphonia pacifica); UBC 972 WS, 27.V.1959 (epiphytic on Corallina vancouveriensis). Point No Point (Glacier Pt.) UBC 1260 WS, 8.VII.1971 (epiphytic on Codium f r a g i l e ) . EAST COAST VANCOUVER ISLAND: Piper's Lagoon: UBC 895 WS, 2.VIII.1959 (epiphytic on Lomentaria sp„). Erythrotrichia pulvinata The previously recorded distribution i s from Middle Bay, Oregon to Bahia Asuncion, Baja California, Mexico (Dawson, 1961). BRITISH COLUMBIAN COLLECTION RECORDS. WEST COAST VANCOUVER ISLAND: Bamfield: UBC 41754, 28.VII.1969? UBC  41671,647 WS, 29.VII.1969 (epiphytic on Codium f r a g i l e ) . Point No Point (Glacier Pt.). UBC 1259 WS, 6.X.1971 (epiphytic on Codium f r a g i l e ) . Field and Cultured Material; Both E_. carnea and E. boryana were typical in appearance (PI. V, Fig. 7,11), readily differentiating and releasing monospores in culture. Upon germination, the monospores of JE. carnea exhibit a polarity (PI. V, Fig. 8), forming one or two erect filaments (PI. V, Fig, 9). In E_. boryana monospores divide to produce a simple monostromatic basal disc (PI. V, Fig, 12) from which filaments are derived (PI. V, Fig. 11). E. carnea regularly exhibited in situ monospore germination to give an appearance of branching (PI. V., Fig. 10) as described by Dixon and West (1967). E. boryana may also be attached to Smithora.(PI. V, Fig. 13). Structures which could be interpreted as spermatia and carpospores were observed in freshly collected E_. boryana and w i l l be discussed at length in Section VI. Very few studies have been carried out on JE. pulvinata. In his original description Gardner (1927) states that the pad may be found without the filamentous thallus. In the population used for this study, I have observed 1 8 . filamentous thalli only once (PI. VI, Fig. 14), although the basal cushions appear to thrive. As was suggested in the discussion on Smithora (Section III), a delicate set of environmental conditions may be required for blade formation and perhaps this relatively exposed site (Point No Point) does not provide them. Dawson (1953) described monospore production in JE. pulvinata and Hollenberg (1971) reasserted the fact that thsy are formed in the usual manner. Results in this laboratory indicate that „ as in Smithora, the basal cushion is also capable of producing these reproductive structures (PI. VI, Fig. 15). Upon germination in culture, they form a cell wall and divide (PI, VI, Fig. 16,17) to produce a large, multicellular holdfast (PI. VI, Fig. 18). The cells at the edge of the pad are elongated, possibly due to a greater rate of division (PI. VI, Fig. 19). Evidence of additional reproductive structures and filament formation was not obtained. 19. PLATE V. ERYTHROTRICHIA Fig, 7 0 JE. carnea. Mature filaments producing monospores (arrow) in culture. Fig. 8. E_. carnea. Bipolar germination of cultured monospores. Fig. 9. E_, carnea. Juvenile filaments daveloping from one monospore in culture ("tripolar" germination). F i g 0 10o E_„ carnea 0 In situ germination of cultured monospores. Fig. 11. E_. boryana. Mature filaments and basal disc. Fig. 12. E_. boryana. Cultured basal pad. Fig. 13. _E. boryana. Freezing microtome section of association between E_. boryana and Smithora (h). 20 PLATE VI. Erythrotriehia pulvinata Pig. 14. Portion of mature filament. Fig. 15. Monospore production (arrow) from basal • holdfast (bh) in culture. Fig. 16 0 4-celled stage of cultured, germinating monospore. Fig, 17, Juvenile pad attached to utricle of Codium fragile, (h). Fig, 18. Mature pad growing in culture on glass Petri dish. Fig, 19, Edge of cultured pad showing elongate cellular shapes. V I 21. IV. ULTRASTRUCTURE OF THE VEGETATIVE CELL Introduction. Recently there has been a growing interest in ultrastructural details concerning members of the Rhodophyceae. Much of the research has been done with the larger and "more advanced" subclass Florideophycidae. There appear to be published reports describing six genera of the "less advanced" -Bangiophycidae. Of these, Porphyridium. a unicellular form, has drawn much attention (Brody and Vatter, 1959} Speer, Dougherty and Jones, 1964; Gantt and Conti, 1965,1966; Gantt, Edwards and Conti, 1968; Guerin-Dumartrait, Sarda and Lacourly, 1970; Neushul, 1970; Wehrmeyer, 1971; Chapman, Chapman and Lang, 1971; Ramus, 1972), Porphyra has also been investigated by a seriss of authors (Gibbs, 1960; Ueda, 1961; Yokomura, 1967; Kito and Akiyama, 1968; Kazama and Fuller, 1970; Bourne, Conway and Cole, 1970; Loe and Fultz, 1970; Bourne, 1971; Cole, 1972). Evans (1970) described a new genus, Rhoriella, primarily on the basis of electron microscopy. A certain amount of information is also available on Banqia (Honsell, 1963; Sommerfield and Leeper, 1970), Rhodosorus (Giraud, 1963) and Compsopoqon, a freshwater form (Nichols, Ridgway and Bold, 1966). To my knowledge there are no published ultrastructural accounts dealing with members of the Erythropeltidaceae. a) Smithora^ Observations. Vegetative cells of Smithora are approximately 10 microns in diameter although some monostromatic areas of the thallus may contain larger cells* ^This portion of the thesis is based on a publication by D.L. McBride and K. Cole in Phycoloqia 8, 177-186 (1969). The text of the original article has been brought up to date by including subsequent references where appropriate. 22. Each consists of a thick c a l l wall and an irregular protoplast with a large chloroplast, mitochondria, endoplasmic reticulum, dictyosomes and a single nucleus (PI. VII, Fig. 2). Additional structures within the c e l l include floridean starch granules, vacuoles and many multivesicular bodies. The c e l l wall, composed of two or three distinct layers, appears to be similar to that of Porphyra (Frei and Preston, 1964; Bourne,1971) (PI. VII, Fig. 2; PI. VIII, Fig. 5). According to Frei and Preston (1964), the fibrous organization of these c e l l wall layers is due to microfibrils composed of/3-1,3 linked xylans. The layering effect seems to be due to a different organization of the microfibrils with the outer layers being more compacted. A nonfibrillar outermost layer which could be analagous to the mannan-containing c u t i c l B of Porphyra (Frei and Preston, 1964; Hanic and Craigie, 1969) was also noted in Smithora. The shape of the protoplast i s much more variable than any previously reported in the red algae, Pseudopodia-like extensions and invaginations are evident in most cells (PI, VII, Fig. 2). Wall material seems to be isolated within the c e l l when the invaginations are viewed in cross-section. The single, lobed chloroplast, bounded by a double membrane, occupies most of the c e l l (PI. VII, Fig. 2; PI. VIII, Fig. 5). In some sections the lobes may appear as separate entities isolated from the main body of the chloroplast (PI. VII, Fig. 2; PI. VIII, Fig. 3). The arrangement of chloroplast lamellae in younger cells i s very regular, each individual thylakoid being orientated parallel to the others (PI. VII, Fig. 2). There is one thylakoid which follows the contour of the chloroplast envelope (peripheral thylakoid). However, at no time was the chloroplast envelope continuous with any of the thylakoids. The same general chloroplast structure i s also characteristic of older cells but the lamellae do not appear as 23. smooth or a 9 regularly parallel (PI, VIII, Fig. 5), Thylakoid associations such as those reported in other algal groups (Kirk and Tilney-Bassett, 1967) have not been noted previously in the Rhodophyta. Thus, a most interesting feature of the chloroplast of Smithora is a stacked arrangement of varying numbers of lamellae in certain restricted areas (PI, VII, Fig. 2% PI. IX, Fig. 6,7). The lamellar stacks are almost exclusively formed by an overlapping of thylakoid edges. This results in a narrow, localized stack usually situated near t h E pyrenoid0 Fusion and forking of lamellar edges often occur in these areas. Intermittent fusion of photosynthetic lamellae is found throughout the chloroplast of Smithora (PI. VIII, Fig.,5). However, this lamellar fusion rarely occurs in the regular arrangement seen in Porphyridium (Gantt and Conti 8 1965). Lamellar spirals similar to those reported in Porphyridium (Gantt and Conti, 1965) were also noted in Smithora (PI. VIII, Fig. 3). The centrally located pyrenoid is similar to that reported in other red algal species (Gibbs, 1962a). It is often penetrated by lamellae which appear swollen and frequently form common vesicles within its matrix (PI, VII, Fig. 2; PI. VIII, Fig. 5; P I. IX, Fig. 7). Numerous electron transparent areas, sometimes containing a f i b r i l l a r material, are scattered throughout the chloroplast between the thylakoids (PI. VII, Fig. 2 j P I . VIII, Fig, 5). These structures have been interpreted as localized areas of DNA and have been found in other red algal species, e.g. Laurencia (Bisalputra and Bisalputra, 1967) and Porphyra (Yokomura, 1967). Osmiophilic droplets are also frequently observed between the lamellae (PI . VII, Fig, 2; P I . VIII, Fig. 5), No convincing evidence indicating the presence of phycobilisomes has been found although these structures 24. could have been lost during preparation of the specimens. Typical red algal floridean starch occurs within the cytoplasm in the form of ellipsoidal granules (PI. VII, Fig. 2; PI, VIII, Fig, 5). These granules vary in staining intensity in a manner similar to that reported in Porphyridium (Gantt and Conti, 1965). Mitochondria with tubular cristas are numerous and variable in size and shape (PI. VII, Fig. 2| PI, IX, Fig. 8} PI. X, Fig, 10). Infrequently, a mitochondrion in a "doughnut" or ring formation was noted (PI. IX, Fig. 8). Other structures often occur within the centre of these atypical mitochondria. No extremely long or branched mitochondria similar to those found in Porphyridium (Gantt and Conti, 1965) were observed i n Smithora. Both rough and smooth endoplasmic reticulum occur in these c e l l s , usually following the contour of the plasmalemma or the nuclear envelope. (PI. VII, Fig. 2; PI. VIII, Fig. 4; PI. X, Fig. 10). However, no connections between these entities have been found. In addition, what appears to be more densely staining ER i s seen intermittently in the protoplast extensions (PI. IX, Fig. 8). One or more dictyosomes are often found in a single cellular cross-section (PI. VII, Fig. 2). They consist of the usual flattened cisternae and associated vesicles. No particular location or function can be assigned to these organelles in the vegetative c e l l . Younger vegetative cells contain few well defined vacuoles. Howsver, in older cells these structures seem to increase in size and number (PI. VIII, Fig. 5; PI. X, F i g e 13). The nucleus i s typically eucaryotic (PI. VII, Fig. 2; PI. VIII, Fig. 4), The evenly granular nucleoplasm and densely staining nucleolus are surrounded by a porous nuclear envelope. The nucleolus often occupies a peripheral position in the nucleus of Smithora. However, this structure i s not necessarily orientated toward the chloroplast as reported in Porphyridium (Gantt and Conti, 25. 1965). Lomasome-like bodies were frequently observed in Smithora as membrane bound aggregations of vesicles within the cytoplasm (PI. X, Fig. 10,12) and as groups of vesicles being released into the cell wall (PI. X, Fig, 10,11). The vesicles themselves vary in size and are bounded by a single membrane. Marchant and Robards (1968) suggest that those multivesicular bodies in plants which seem to originate from the plasmalemma should be termed plasmalemmasomes and those from within the cytoplasm, lomasomes. Plasmalemma-somes are usually associated with tubular vesicles in Smithora (PI. X, Fig.11), while the lomasomes seem to be composed mainly of aggregations of spherical vesicles (PI, X, Fig. 10,12). However, the distinction between these two types of multivesicular bodies is often not clear. Lomasome-like structures have been reported previously in other red algae, e.g. Lomentaria (Bouck, 1962), Laurencia (Bisalputra et al., 195?) and Pseudoqloiophloea (Ramus, 1969). In addition, single vesicular structures were frequently noted in the cell wall near the plasmalemma (PI. X, Fig. 13). Similar structures were described in Laurencia (Bisalputra et al., 1967) and the green alga Chara (Barton, 1965). In older cells various types of whorled lamellar bodies were seen as well (PI. X, Fig. 13). These have also been noted in older cells of Porphyridium (Gantt and Conti, 1965), Polysiphonia (Rawlence and Taylor, 1972) and Batrachospermum (Brown and Weier, 1970). Sections were made at the junction of tha basal portion of Smithora and the host tissue, which yielded no evidence of cytoplasmic connections between the individual cells of the host and the epiphyte. The respective cell walls seem to be merely cemented together in a smooth plane. In addition, there were no intercellular ccnnections within ths alga i t s e l f . 26. D i s c u s s i o n . From time t o time v a r i o u s authors have presented evidence supporting a p h y l o g e n e t i c a l r e l a t i o n s h i p between the Rhodophyta and other a l g a l groups (Smith, 1955). The t h e o r i e s which have been proposed d i r e c t l y i m p l i c a t e the Bangiophycidae, s i n c e t h i s group i s thought to possess c e r t a i n p r i m i t i v e c h a r a c t e r i s t i c s i n common w i t h l e s s advanced a l g a l groups. These i n c l u d e : l a c k of sexual r e p r o d u c t i o n i n some s p e c i e s , presence of p h y c o b i l i n s and l a c k of f l a g e l l a . However, u n t i l f u r t h e r research i s c a r r i e d out any such proposal w i l l remain i n s e c u r e . U l t r a s t r u c t u r a l s t u d i e s c o u l d be e s p e c i a l l y v a l u a b l e i n t h i s r e s p e c t . Indeed, one f i n e s t r u c t u r a l c h a r a c t e r i s t i c , the presence of unassociated photosynthetic l a m e l l a e i s considered t y p i c a l of the red algae (Gibbs, 1960,1962a). This has been used on occasion as an a d d i t i o n a l taxonomic char a c t e r and cou l d support a proposed r e l a t i o n s h i p between the red algae and the Cyanophyta. While Smithora e x h i b i t s some of the u l t r a s t r u c t u r a l f e a t u r e s considered t y p i c a l of the Rhodophyceae ( t h i c k , l a y e r e d c e l l w a l l and f l o r i d e a n s t a r c h s t o r e d o u t s i d e the c h l o r o p l a s t ) , i t i s unique t h u s f a r i n possessing r e l a t i v e l y narrow, l o o s e l y a s s o c i a t e d . t h y l a k o i d stacks w i t h i n the c h l o r o p l a s t . These bands appear to be p r i m i t i v e s i n c e they are by no means extensive and are almost e x c l u s i v e l y r e s t r i c t e d to l a m e l l a r edges. Because these s t r u c t u r e s occur randomly i n the c h l o r o p l a s t s of o l d e r as w e l l as younger c e l l s , they are not b e l i e v e d to be i n v o l v e d i n c e l l d i v i s i o n . Frequantly one end of an inner t h y l a k o i d p a r t i c i p a t e s i n a l a m e l l a r stack w h i l e the other end forms or c o n t r i b u t e s to a swollen v e s i c l e w i t h i n the pyrenoid. This may i n d i c a t e t h a t the bands are an i n t e g r a l p a r t of the c h l o r o p l a s t and perform an important f u n c t i o n . Consequently,, i t i s evident t h a t other members of the Rhodophyta, i n p a r t i c u l a r tha " l e s s advanced" members, should be examined to determine the extent of t h i s 27. banding phenomenon. Thus far there seem to be two structural types of red algal chloroplasts depending upon the presence or absence of a pyrenoid. From light microscopic studies i t i s reported that certain members of the Nemaliales and many of the Bangiophycidaa possess pyrenoid-containing chloroplasts (Fritsch, 1945). However, within this group the arrangement of the thylakoids in relation to the chloroplast envelope seems to vary. Smithora displays numerous sheet-like photosynthetic lamellae which tend to parallel the contour of the chloroplast envelope. Some members of the Nemaliales, e.g. Thorea (Bischoff, 1965) and Acrochaetium (fflcBride, unpubl.) and the unicellular bangiophyte Rhodosorus (Giraud, 1963) seem to possess a similar chloroplast structure. Kylinia (Gibbs, 1962a), Namalion (Gibbs, 1962a,1962b) and Rhodochorton (Witrakos, 1960), other members of the Nemaliales, may also possess this feature although published micrographs are inconclusive. In contrast, Porphyra (Bourne, 1971), Porphyridium (Brody and Vatter, 1959 and others), Rhodella (Evans, 1970) and Banqia (Honsell, 1963) have photo-synthetic lamellae which terminate at the chloroplast envelope. The significance of this well defined difference in the ultrastructure of red algal, pyrenoid-containing chloroplasts w i l l be discussed i n Section VII. Warchant and Robards (1968) describe two types of multivesicular (paraneural) bodies associated with plant cellss lomasomes and plasmalemmasomes. These authors suggest that lomasomes, which have been observed in a large number of plants, may be involved in transport of c e l l wall precursors across the plasmalemmma. They also propose that the plasmalemmasome i s concerned with secondary modifications of the c e l l wall. Various types of these multivesicular bodies have been noted in Smithora. Since formation of a thick supporting c e l l wall in this alga would undoubtedly entail 28. important cellular functions, i t mould seem that this hypothesis i s not unreasonable. Ramus (1969) noted an abundance of lomasome-like bodies in Pseudoqloiophloea associated with the formation of c e l l wall material between dividing c e l l s . It i s of interest that many more of these structures are observed in older cells of Smithora. Since Smithora's main method of reproduction seems to be vegetative (portions of the monosporic thallus are released periodically) (Hollenberg, 1959), there is a possibility that some of the above mentioned structures may be involved in transport of catabolic enzymes capable of acting on c e l l wall material. This function would obviously be very important to the plant. Hawker and Gooday (1969) also proposed that lomasomes in the fungus Rhizopus may be associated with c e l l wall degradation. Since the cells of Smithora contain relatively few vacuoles, another functional pos-s i b i l i t y of these paraneural bodies which could be entertained i s the transport of metabolic waste from the c e l l . In addition, the single vesicles which occur frequently in the c e l l wall near the plasmalemma may be involved in one or more of the functions discussed here. However, not until cyto-chemical and autoradiographic techniques progress further can explicit functions be assigned to these various structures. It i s known that certain members of the red algae are parasitic (Fritsch, 1945). The fact that Smithora is an obligate epiphyte tends to arouse one's suspicions that a parasitic relationship may exist. However, no obvious evidence of parasitism was observed in sections through host and epiphytic tissue. Since this alga has a well developed photosynthetic apparatus, this i s not unexpected. Nevertheless, i t i s of interest that Harlin (1971) has reported the possible occurrence of a nutrient transfer in this situation. 2 9 0 There have been several) light microscopic reports of pit connections in the Bangiophycidae (see Dixon,, 1963 for review). Recent ultrastructural accounts of these structures in the conchocelis phase of Porphyra (Lee and Fultz, 1970| Bourne, Conway and Cole, 1970) and the conchocelis phase of Banqia (Sommerfeld and Leeper, 1970) have conclusively shown that this characteristic can no longer be used to separate the rhodophycean subclasses. However, there i s no evidence of pit connections in Smithora. Each c e l l i s a separate entity, no connections of any type remain after division. 30. PLATE VII. Smithora Fig„ 2. Cross-section of a t y p i c a l vegetative c e l l with a chl o r o p l a s t (C)^, chlo r o p l a s t envelope (CE), pyrenoid (P), mitochondria (ffl)» endoplasmic reticulum (ER). dictyosomes (D)„ f l o r i d e a n starch granules (FS), c e l l wall material (CW), DMA pockets (white arrow) and osmiophilic granules (black arrow). Double arrow indicates a lamellar stack. V 'The d i s p a r i t y i n the types of notations used to l a b e l i l l u s t r a t i o n s i n d i f f e r e n t sections of t h i s report i s due to the use of material previously published by the author over a period of time. 31. PLATE W i l l . Smithora F i g . 3. Chloroplast lobe e x h i b i t i n g a s p i r a l arrangement of thylakoids. F i g . 4. Nucleus c o n s i s t i n g of nucleoplasm and a nucleolus (Nu) surrounded by a nuclear envelope (NE). F i g . 5. Older c e l l with chl o r o p l a s t and c e n t r a l l y located pyrenoid containing swollen v e s i c l e - l i k e lamellae (LV). Vacuoles (V) and m u l t i -v e s i c u l a r bodies (Mv) are t y p i c a l of older c e l l s . Thylakoid fusion (arrow) and f l o r i d e a n starch granules (FS) are also present. VIII 32. PLATE IX. Smithora F i g . 6 . Thylakoid stack. Arrow i n d i c a t e s p e r i o d i c branching of lamellae associated with stack formation. F i g . ?. Thylakoid stack. Note lamellar branching (arrow) and swollen v e s i c l e -l i k e lamellae within the pyrenoid. F i g . 8. Ring shaped mitochondrion (Rffl) e x h i b i t i n g c r i s t a e and mitochondrial envelope. Two other mitochondria and a v e s i c u l a r structure (ve) are located i n the centre. Darkly stained ER-like material (arrows) i s also present i n an adjacent cytoplasmic lobe. F i g . 9, Point of junction between a l g a l c e l l wall (AW) and host c e l l wall (HUl). Part of a l g a l protoplast (Pr) i s also shown. IX 33. PLATE X. Smithora Fig. 10. Section through portions of too neighbouring cells i l l u s t r a t i n g multivesicular bodies (arrows) consisting mainly of spherical vesicles. Other structures include a mitochondrion, endoplasmic reticulum, chloroplast with lobe (CL) and c e l l wall material. Fig, 11. Multivesicular bodies (arrows) consisting of many elongate tubular vesicles associated with the plasmalemma (PI). Fig. 12. A larger multivesicular body consisting mainly of spherical vesicles. Fig. 13. Lamellar bodies (arrows) situated in the c e l l wall and near vacuoles. Fig. 14© Numerous single vesicles in the c e l l wall near the plasmalemma. Arrows indicate vesicles s t i l l attached to the plasmalemma. X 34. b) E r y t h r o t r i c h i a ^ Observations. The species used i n t h i s study were JE. carnea ( P I . X I , F i g . 1 ) , E. boryana ( P I . X I I I , F i g . 6) and E. p u l v i n a t a ( P I . XV, F i g . 1 1). At f i r s t o b s e r v a t i o n the u l t r a s t r u c t u r a l s i m i l a r i t y of these p l a n t s to Smithora  naiadurn (Section IVa) i s very e v i d e n t . Indeed, the c e l l w a l l of £. boryana ( P I . X I I I , F i g . 7) and E. p u l v i n a t a ( P I . XV, F i g . 12) appears i d e n t i c a l to t h a t of Smithora, c o n s i s t i n g of p r o g r e s s i v e l y more compacted s t r u c t u r a l f i b r i l s w i t h i n an e l e c t r o n transparent m a t r i x . However, the w a l l of E_. carnea d i f f e r s somewhat i n c o n t a i n i n g e l e c t r o n transparent areas i n the outermost l a y e r s as w e l l as a l o o s e l y f i b r i l l a r m a t e r i a l on the su r f a c e of the f i l a m e n t ( P I . X I I , F i g . 5 ) . JE. p u l v i n a t a e x h i b i t s an i n t e r e s t i n g mod-i f i c a t i o n i n possessing a d a r k l y s t a i n i n g l i n e i n the c e l l w a l l adjacent to the plasmalemma (P I . XVI, F i g . 16). This l i n e i s p a r t i c u l a r l y n o t i c e a b l e i n regions where the plasmalemma i s most convoluted and co u l d represent an area of h i g h l y compressed s t r u c t u r a l f i b r i l s . The p r o t o p l a s t of Z. carnea ( P I . X I , F i g . 2) i s much more r e g u l a r i n o u t l i n e than t h a t of E_. boryana ( P I . X I I I , F i g . 7) and E. p u l v i n a t a ( P I . XV, F i g . 12), The a c t u a l shape of the p r o t o p l a s t v a r i e s c o n s i d e r a b l y , presumably according to the r a t e of c e l l d i v i s i o n (West, 1966). A l l s pecies of E r y t h r o t r i c h i a examined f e a t u r e a c e n t r a l , s t e l l a t e c h l o r o p l a s t ( P I . X I , F i g . 2; P I . X I I I , F i g . 7; P I . XV, F i g . 12). Secti o n s through c h l o r o p l a s t m a t e r i a l show t h a t the m a j o r i t y of t h y l a k o i d s are s i t u a t e d p a r a l l e l to and f o l l o w the contour of the c h l o r o p l a s t envelope (PI. X I , F i g . 2; P I . X I I , F i g , 3; P I . X I I I , F i g . 7; P I . XV, F i g . 1 2). ^ P a r t s of t h i s s e c t i o n w i l l appear i n an a r t i c l e by D.L. McBride and K. Cole i n the Proceedings of the V l l t h I n t e r n a t i o n a l Seaweed Symposium. Saoporo 1972. 35. £. carnea was the only plant to regularly exhibit phycobilisomes on chloro-plast lamellae (PI. XII, Fig.3). They are similar to those described in Porphyridium (Gantt and Conti, 1965,1966? Gantt, Edwards and Conti, 1968). Ribosome-liks bodies were often observed between chloroplast lamellae (PI. XIV, Fig. 10). In addition, possible DNA-containing areas (Bisalputra and Bisalputra, 1967) and droplet-like inclusions are scattered between the lamellae, A constant feature of the chloroplast i s the presence of a centrally located pyrenoid traversed by varying numbers of lamellae (PI, XII, Fig. 3; PI. XIII, Fig, 7} PI. XVI, Fig, 14) which often contain a f i b r i l l a r material (PI. XII, Fig.4). In E_. carnea the number of traversing lamellae seen in one sectional plane was seldom greater than five and usually these structures ran in a relatively straight line through the pyrenoid (PI. XII, Fig. 3), However, in JE. boryana and JE. pulvinata there are large numbers of highly convoluted lamellae within the pyrenoid matrix (PI. XIII, Fig. 7; PI. XV, Fig. 12; PI. XVI, Fig. 14). The nucleus i s typically eukaryotic (PI. XI, Fig. 2; PI. XII, Fig. 3; PI, XIV, Fig. 9; PI. XV, Fig, 12) and only very rarely is the outer membrane of the nuclear envelope continuous with cisternae of ER (PI. XIV, Fig. 9). This feature i s common in many other plants (Ledbetter and Porter, 1970). Remaining c e l l organelles and inclusions such as mitochondria (PI. XVI, Fig, 16), dictyosomes (PI. XVI, Fig. 16), vacuoles (PI, XI, Fig. 2; PI. XIII, Fig. 7; PI. XV, Fig. 12) and floridean starch grains (Pi. XIV, Fig. 9) resemble those described in the vegetative c e l l of Smithora (Section IVa). An intriguing structural characteristic of algal epiphytes, especially those which exhibit a host spe c i f i c i t y , is the zone of attachment. E_, pulvinata thus far appears to be exclusively epiphytic on the utricles of Codium fragile (PI. XV, Fig. 11) but this zone shows no evidence of interalgal 36. protoplasmic association (PI, XVI, Fig, 13). The respective c e l l walls are merely joined in a simple, smooth plana. In this region, foreign objects are occasionally embedded in the c e l l wall of the epiphyte. One such object bore a resemblance to a bacterial c e l l (PI. X V I , Fig. 15). JE. boryana i s less host specific than JE. pulvinata. It i s of interest that E_. boryana i s epiphytic on Smithora since these algae are almost ultrastructurally identical. In sections through the zone of attachment i t is nearly impossible to differentiate the respective c e l l walls (PI. XIV, Fig. 8). Discussion. An ultrastructural comparison of closely related algal species has proven to be of taxonomic value in certain instances. In particular, "less advanced" red algae which have few definite morphological characteristics to u t i l i z e may lend themselves to this procedure. Evan's (1970) study of Rhodella maculata has shown the usefulness of this approach. The three species of Erythrotrichia chosen for this study appear to i l l u s t r a t e a maximum morphological va r i a b i l i t y within the genus, but very few concrete ultrastructural differences were noted. The most constant difference observed was the form and smaller number of pyrenoid traversing lamellae in the chloroplast of E_. carnea as compared with I E . boryana and JE. pulvinata. Cole (1971) has reported such a v a r i a b i l i t y among the conchocelis phases of three species of Porphyra. Hori (1971) has noted differences in the ultrastructure of the thylakoid system within the pyrenoid among species of ftonostroma (Chlorophyceae). Dodge and Crawford (1971) also noted a d i f f e r -ent pyrenoid structure among certain species of Dinoflagellata. In addition, the ultrastructural appearance of the c e l l wall in JE. carnea seems to separate this species from the others. West (1966) has also discussed certain unusual features of the wall of E. carnea. 37. One must be extremely cautious in the interpretation of interspecific ultrastructural differences as they could merely be due to variations in habitat, season, age of tha plant, stage of the l i f e cycle, etc. Indeed, Hori (1972) has shown ultrastructural differences which occur between different stages in the l i f e cycle of the same species of Monostroma. The presence of phycobilisomes in I E . carnea i s interesting in view of the general absence of these structures in the other members of the Erythropeltidaceae examined. Gantt and Conti (1965) state that these struct-ures are very sensitive to fixation conditions. However, a l l material used i n this study was fixed in the same manner. In fact, in E_. carnea i t was possible to observe phycobilisomes in one c e l l but not in an adjacent c e l l of the same filament. If their presence depends on the quality of chemical fixation, one can only speculate why cells in the same filament of approximately the same age should react differently to these procedures. Thus, E_. carnea, E_, boryana and Z, pulvinata represent distinct species on a gross morphological level but show few ultrastructural differences. In addition, the fine structure of the genus Erythrotriehia appears to be similar to that of Smithora suggesting that these genera are closely related members of the Erythropeltidaceae. It could also offer further support of Hollenberg's (1959) taxonomic reassignment of the latter genus. 38. PLATE XI. Erythrotrichia carnea Fig. 1. Light micrograph of portion of filament. Fig. 2. Longitudinal section through filament i l l u s t r a t i n g c e l l wall chloroplast (C) with centrally located pyrenoid (P), nucleus (N) and vacuoles (V). XI 3 9 . PLATE XII. Erythrotrichia c a r n e a Fig, 3 , Portion of c e l l showing nature of lamellae which traverse the pyrenoid (P) in the chloroplast (C). Note the rough texture of the thylakoids indicating the presence of phycobilisomes. Prominent nucleus (N) and c e l l wall (W) are also shown. Fig. 4, Portion of pyrenoid with swollen lamellae containing loosely organized f i b r i l s . Fig. 5. Cell wall containing electron transparent areas (T) and f i b r i l l a r material (arrow) on outer surface. XII 40. PLATE XIII. Erythrotrichia boryana Fig, 6 . Light micrograph of portion of filament. Fig. 7. Longitudinal section through filament showing cell wall (W). chloroplast (C) with pyrenoid (P) and vacuoles (V). XIII 4 1 . PLATE XIV. E r y t h r o t r i e h i a boryana F i g . 8, S e c t i o n through the zone of attachment between E. boryana and Smithora. EP denotes E r y t h g o t r i c h i a p r o t o p l a s t and S_P denotes Smithora' p r o t o p l a s t . F i g . 9. P o r t i o n of nucleus (N) i l l u s t r a t i n g ER-nuclear envelope connection (arrow). FS i n d i c a t e s f l o r i d e a n s t a r c h g r a i n . F i g . 10. P o r t i o n of c h l o r o p l a s t lobe showing e l e c t r o n dense r i b o s o m e - l i k e bodies (arrow). 42. PLATE XV. Erythrotrichia pulvinata Fig. 11. Light micrograph of basal cushion of plant attached to utricle of Codium fragile. Fig. 12. Section through basal part of plant with chloroplast (C), pyrenoid (P), nucleus (N), vacuoles (V) and cell wall. X V 43. PLATE XVI. Erythrotriehia pulvinata F i g 0 13. Electron micrograph i l l u s t r a t i n g attachment zone of alga. Call wall of host, alga (Codium fragile) (Hul)0 wall of E_. pulvinata (Ul) and protoplast of E_„ pulvinata (EP) are shown. Fig. 14. Section through pyrenoid showing convoluted traversing lamellae. Note the loosely organized f i b r i l s within the lamellae. Fig, 15. Area of attachment with foreign objects embedded in wall of E_„ pulvinata (W)„ Fig, 16. Peripheral region of protoplast with mitochondria (M), a dictyosome (D) and c e l l wall (W). Arrow indicates darkly staining material in wall adjacent to plasmalemma. XV I 44. V. ULTRASTRUCTURAL ASPECTS OF MONOSPORuGENESIS a) Monospore differentiation), release and degeneration. 1 Introduction. The differentiation of whole vegetative c e l l s into spores is a common means of asexual reproduction in the Bangiophycidae (see Drew. 1956 for review). The term "monospore" has been applied to reproductive cells produced in this manner, although there is s t i l l some d i f f i c u l t y in formul-ating acceptable term3 to distinguish between the different spore-like cells evident in this group. During the present study, Smithora and Erythrotrichia were observed in culture as well as in f i e l d conditions and i t is avident that monospore production constitutes one of the main methods of species propagation. A A discussion of light microscopic work on the production of these structures in the Erythropeltidaceae i s presented in Section III of this report. A majority of the ultrastructural investigations carried out on algal spore production have dealt with various aspects of chlorophycean zoosporo-genesis, e.g. Stiqsoclonium (Wanton, 1964), Qedoqonium (Hoffman, 1968j Pickett-Heaps, 1971), Bulbochaete (Retallack and Butler, 1970), Tetracystis (Brown and Arnott, 1970) and Enteromorpha (Evans and Christie, 1970). In the red algae, Peyriere (1969) has described certain features of the tetrasporangium of G r i f f i t h s i a and Tripodi (1971) has reported some fine structural characteristics of the cystocarp of Polysiphonia. To my knowledge there are no published electron microscopic accounts of monosporogenesis in the Bangiophycidae. ^This portion of the thesis is partially based on a publication by D.L. McBride and K. Cole in Phycoloqia 10, 49-61 (1971), The text of the original article has been brought up to date by including recent references where appropriate. 45. Ultrastructural observations on the aging and degeneration of algal cells have been sparse,, Schuster, Hershenov and Aaronson (1968) have described aspects of this phenomenon in Ochromonas (Chrysophyceae) and Palisano and Ulalne (1972) in Luqlena (Euglanophyceae). Observations. Monospore differentiation t The monosparogenous area of Smithora naiadurn is sharply delimited from the vegetative thallus and is usually observed as a band of more deeply pigmented, rounder cells (PI. XVII, Fig. 1), At a light microscope level, the cells in this area appear to be undergoing mitotic division at a greater rate than the adjacent vegetative cells 8 This, in conjunction with an apparent reduction in vacuolar area, results in the differentiating cells appearing smaller than the vegetative cells in surface view, although the actual thickness of the thallus is greater in the sporulating areas. At an ultrastructural lavel, the most obvious manifestations of the transition to the monospore are the loss of the very irregular protoplast outline exhibited in the vegetative cell and the reduction in vacuolar area (PI, XVII, Fig. 2). As the spore matures, the shape of the protoplast progresses from regularly oblong to almost spherical and increases in size from 10-2Q microns to 20-30 microns in surface view. Like that of the vegetative cell, the nucleus of the developing monospore exhibits a typical eukaryotic structure (PI. XVIII, Fig.3). Perhaps the most conspicuous difference is the presence of a larger number of pores in the nuclear envelope of the developing spore. When viewed in tangential section the pores are circular and in many cases arranged in a linear order. (PI. XVIII, Fig. 7), like those described in Bumilleria (Xanthophyceaa) (Massalski and Leedale, 1969). Also, as reported by the above authors, the nuclear pores appear to be more concentrated in certain areas of the nuclear 46. envelope (PI. XVIII, Fig. 6) 0 Structurally they are similar to those described in pea seedlings, consisting of several central granules and a surrounding annulus composed of numerous subannuli (Yoo and Bayley, 1967). Intimate associations between the chloroplast and the nuclear envelope were frequently observed (PI. XVIII, Fig. 4). At these regions a dense precipitate of stain often occurred in the prepared material. Ribosome-laden endoplasmic reticulum i s invariably observed adjacent to the nuclear envelope of the developing spore (PI. XVIII, Fig, 5). Often the cisternae may number 12 or more. Only a few cisternae were noted in this position in the vegetative c e l l . It i s of interest that the distance between the nuclear envelope and the CR i s greater at the areas of the nuclear envelope which exhibit a large number of pores (PI. XVIII, Fig. 6). Usually a few cisternae of predominantly smooth ER are associated with the plasmalemma (PI. XIX, Fig. Q). In addition, increased quantities of smooth and rough ER were observed in other areas of the differentiating monospore (PI, XVII, Fig. 2j PI. XVIII, Fig. 3), The mitochondria are typical in appearance; possessing a double envelope, tubular cristas and electron dense inclusions (PI, XVIII, Fig..5), However, there appears to be an increase i n the number of these organelles in the developing spore. Mitochondria often appear in the vicinity of dictyosomes, but no s t r i c t relationship such as that described i n Corallina (Bailey and Bisalputra, 1970) and G r i f f i t h s i a (Peyriare, 1969) was observed in this material. The chloroplast of the developing monospore is irregularly lobed and exhibits single lamellae which are largely orientated parallel to the chloroplast envelope (PI. XVII, Fig, 2). It is very similar to that of the vegetative c e l l except that the interlamellar spaces appear to enlarge 47. somewhat and a c q u i r e a g r a n u l a r a p p e a r a n c e . Many f l o r i d e a n s t a r c h grain3 a r e p r e s e n t i n t h e d i f f e r e n t i a t i n g s p o r e s and-are g e n e r a l l y l a r g e r t h a n t h o s e o b s e r v e d i n t h e v e g e t a t i v e c e l l ( P I . X V I I I , F i g , 3 ) . T h i n s e c t i o n i n g u s u a l l y r e s u l t e d i n f o l d i n g o f t h e p l a s t i c i n a r e a s o f s t a r c h g r a n u l e s . T h i s can be s e e n as an e l e c t r o n dense l i n e t h r o u g h t h e s e s t r u c t u r e s ( P I . X V I I , F i g . 2 ) . E v i d o n t l y Evans (1970) e x p e r i e n c e d t h e same prob l e m w i t h R h o d e l l a ( P o r p h y r i d i a l e s ) . The d i c t y o s o m e a p p e a r s t o be v a r y a c t i v e i n t h e d i f f e r e n t i a t i n g s p o r e . Throughout development, t h e c e l l c o n t a i n s a l a r g e number o f t h e s e o r g a n e l l e s i n v a r y i n g d e g r e e s o f h y p e r t r o p h y ( P I , X V I I , F i g , 2 ) . The s w o l l e n c i s t e r n a l s t a c k s g i v e r i s e t o two d i s t i n c t l y d i f f e r e n t p r o d u c t s . The f i r s t i s formed e a r l y i n t h e development o f t h e s p o r e . L a r g e d i c t y o s o m e s w i t h t h e i r m a t u r i n g f a c e s t o w a r d t h e c e n t r e o f t h e c e l l p r o d u c e i r r e g u l a r v e s i c l e s c o n t a i n i n g a compacted f i b r i l l a r s u b s t a n c e ( P I , X I X , F i g , 8 ) , O c c a s i o n a l l y t h e f o r m i n g f a c e s o f t h e s e o r g a n e l l e s a r e a s s o c i a t e d w i t h ER and s m a l l v e s i c l e s w h i c h appear t o emanate from i t . The l a r g e f i b r i l l a r v e s i c l e s o r i g i n a t i n g f r o m t h e d i c t y o s o m e s c o a l e s c e s h o r t l y a f t e r f o r m a t i o n , r e s u l t i n g i n l a r g e , membrane bound d e p o s i t s w i t h i n t h e c e l l ( P I , X V I I , F i g . 2; P I . X I X , F i g . 1 1 , 1 2 ) . Rough ER i s f r e q u e n t l y a s s o c i a t e d w i t h t h e s e d e p o s i t s ( P I . X I X , F i g . 1 0 ) . O c c a s i o n a l l y c e l l u l a r components s u c h as f l o r i d e a n s t a r c h g r a n u l e s appear t o become i n c o r p o r a t e d w i t h i n t h e v a c u o l s - l i k e s t r u c t u r e s ( P I . X I X , F i g . 9 ) . Toward t h e l a t t e r s t a g e s o f t h e a c c u m u l a t i o n o f t h e f i b r i l l a r p r o d u c t , a s e c o n d s u b s t a n c e b e g i n s t o f o r m . H y p e r t r o p h i e d d i c t y o s o m e s , l o c a t e d randomly i n t h e c e l l , f orm s m a l l e r , more s p h e r i c a l v e s i c l e s ( P I , XX, F i g . 1 4 ) . These v e s i c l e s c o n t a i n l o o s e l y o r g a n i z e d f i b r i l s i n an e l e c t r o n t r a n s p a r e n t m a t r i x . I n t h e s t a g e s i m m e d i a t e l y p r i o r t o and a f t e r s p o r e 48 release the c e l l i s f i l l e d with these s t r u c t u r e s . Concomitant with the deposition of t h i s material i s the marked enlargement and rounding of the c e l l . In these l a t t e r stages of development both the densely f i b r i l l a r vacuoles and the smaller v e s i c l e s are present i n the c e l l (PI. XIX, F i g . 11). Contrary to Hollenberg's (1959) l i g h t microscopic observation, no i n t e r c e l l u l a r connections were observed between developing spores. In contrast to the vegetative c e l l , i t i s of i n t e r e s t that very few lomasoma-like bodies were observed. Concentric lamellar structures were also few i n number. This i s i n keeping with r e s u l t s obtained i n two other red algaes Porphyridium (Gantt and Conti, 1965) and Batrachospermum (Brown and Uleier, 1970), which i n d i c a t e that these structures are present to a greater extent i n older, l e s s a c t i v e c e l l s . P r i o r to spore release the thickness of the c e i l wall i s considerably l e s s than i n the vegetative t h a l l u s . Wall production has evidently not kept pace with the enlargement of the monospore. The area immediately adjacent to the plasmalemma i s more compacted, p o s s i b l y due to the pressure of the enlarged spore or to the accumulation of released f i b r i l l a r material (see below) (PI. XX, F i g . 15). The u l t r a s t r u c t u r a l d e t a i l s of monospore d i f f e r e n t i a t i o n i n E r y t h r o t r i c h i a (PI, XXV. F i g . 31) are i d e n t i c a l to those i n Smithora : the dictyosome forms two products, each of which i s accumulated i n the cytoplasm p r i o r to spore release (PI. XXVI, F i g . 32-34). Monospore release: Immediately preceding and during l i b e r a t i o n of the monospore, the vacuole-like structures migrate toward the periphery of the c a l l (PI. XIX, F i g . 12) and subsequently expal t h e i r f i b r i l l a r contents (PI. XIX, F i g , 13). The mechanism of release involves a f u s i o n of the vacuole 49. membrane to the plasmalemma. Occasionally a few of the smaller vesicles are released as well, but the majority remain behind. The extrusion of a single spore i s achieved by a breakage in the c e l l wall on one or the other side of the thallus (PI. XX, Fig. 16,17), Since the development of the c e l l s in one particular sorus seems to be more or less synchronous, the sorus i s often released as a whole (PI, XX, Fig. 19j PI, XXII, Fig. 22). This could be the result of a greater stress on the c e l l walls in the proximal area of the sorus. Such mass release appears to occur more often than the release of single spores. However, unless the entire sorus settles immediately following release i t i s most probable that further wall dissolution takes place resulting in single spores being liberated. There i s evidence that a small amount of cytoplasm remains in the thallus after spore release (PI, XX, Fig. 17,18), This material invariably appears degenerate. Perhaps this pocket of cytoplasm which contains a nucleus (PI. XX, Fig. 18), i s the result of an asymmetric division of the vegetative protoplast prior to differentiation. It appears to be similar to the pale, protoplasmic remnant occurring in this manner and remaining after spore release in Membranella nitens (Hollenberg and Abbott, 1968). The released monospore is spherical and approximates 15-25 microns in diameter (PI, XXI, Fig. 20,21). The spore i s bounded by a single plasmalemma, the exterior of which may be associated with a quantity of mucilage (Pi. XXII, Fig. 22j PI. XXIV, Fig. 29). No vestige of c e l l wall material from the parent thallus remains. This concurs with reports of naked monospores in other red algae by various authors including Sommsrfeld and Nichols (1970). The monospore i s f i l l e d with the smaller, loosely f i b r i l l a r vesicles produced prior to release,, Many of these vesicles coalesce after spore 50. liberation to form larger, vacuole-like structures (PI. XXI, Fig. 21; PI. XXIII, Fig. 23). The chloroplast seems to be somewhat modified in the monospore. Closely appressed lamellae often occur within the lobes giving the appearance of "pseudogranum-like" structures (PI. XXIII, Fig. 23,24). As many as 20 lamellae may be involved in these formations. Although these structures could be associated with an altered plastid metabolism, i t i s perhaps more lik e l y that they are due merely to the physical effect of pressure resulting from the accumulation of products in the spore. In addition, thylakoid associations l i k e those reported in tha vegetative c e l l were observed (PI. XXIII, Fig. 24). The pyrenoid i s characterized by a number of swollen traversing lamellae which often appear closely appressed and frequently contain some osmiophilic droplets (PI. XXIII, Fig. 25). The notable lack of unoccupied cytoplasmic matrix in the monospore severely restricts the distribution of other organelles, mitochondria are often closely packed and confined to a small area (PI. XXIV, Fig. 27)„ The nucleus i s usually surrounded by a layer of cytoplasm containing a small amount of ER (PI. XXIV, Fig. 28). Some dictyosome activity i s noted in the mature monospore (PI. XXIV, Fig. 26). These organelles appear to be adding to the already abundant vesicular material. Numerous floridean starch granules remain in the spore and seem to be isolated from the remaining bits of cytoplasm. Due to a lack of c e l l wall material, the differentiated monospore appears to be very delicate. In many instances the pressure of ths cellular contents in combination with outside stimulus causes the plasmalemma to rupture, resulting in a flow of vesicular contents out of the c e l l (PI. XXI, Fig. 21). In addition, the plasmalemma is frequently observed to bleb out 51. quantities of this substance (Pi, XXIV. Fig, 30). Monospore degeneration; Although monospores w i l l germinate readily under culture conditions, a certain percentage of these structures undergo degeneration within several days after release. At a light microscopic level, the chief proclamation of this phenomenon is the gradual loss of the characteristic red pigmentation of the spore, Ultrastructurally there is an ordered breakdown of each organelle system. I n i t i a l l y , ER and dictyosomes become less active and inconspicuous, Chloroplast lamellae appear to swell and become disorganized (PI. XXVII, Fig. 1). Abnormally bloated mitochondria, which often show indications of inner membrane disruption, are typical of the degenerating spore (PI, XXVII, Fig. 1; PI, XXVIII, Fig. 2). Paraneural bodies (Marchant and Robards, 1968) are- increasingly evident near the periphery of the c e l l (PI. XXVII, Fig. 1) and extensive, progressive vacuolation of the cytoplasm occurs (PI. XXVII, Fig. 1; PI. XXVIII, Fig. 2,3). Disruption of the nuclear envelope and the subsequent breakdown of this organelle may signal the onset of c e l l death (PI. XXVIII, Fig. 3), In later stages, the chloroplast material is almost completely disorganized. Lamellae are grossly distended and membrane destruction i s evident (PI. XXVIII, Fig. 4). Finally the plasmalemma breaks, possibly as the result of uncontrolled osmotic pressures, and the spore rapidly disintegrates. Numerous uncoordinated membrane fragments are characteristic of the cellular debris (PI. XXVIII, Fig. 5). Discussion. In the f i e l d , the chief manifestation of the subcellular changes involved in the production of monospores is a deeper pigmentation i n the differentiating areas. The electron microscope has allowed a more detailed observation of the process. Not only i s there an increase in dictyosome 52. activity but also a substantial increase in the amount of ER and the number of floridean starch granules and mitochondria. Such changes would suggest that the production of monospores in the Erythropeltidaceae examined is not merely a release of rounded vegetative c e l l s , but involves a complex, active metamorphosis. However, an exact explanation of a l l the cellular a c t i v i t i e s must be based on extended physiological and cytochemical studies. It i s evident that the formation of monospores depends greatly on the dictyosome. This organelle i s implicated in the extensive production of two distinct products during sporogenesis which may play an important role i n the release and attachment of the spore. The f i r s t product, a compacted f i b r i l l a r substance, i s aggregated in large vacuole-like structures. Since substantial amounts of i t are expelled immediately prior to and during spore release, i t is hypothesized that this substance functions as a mucilaginous secretion, Knox (1926) noted that a large amount of mucilaginous matter is associated with monospore release in Smithora. Indeed, i t has been observed i n the current study that the mucilage is produced in copious amounts and could aid in spore release by acting as a lubricant. This could be c r i t i c a l to the success of the process as liberation takes place through a small opening in the c e l l wall. In addition, the mucilage would protect the fragile spore after release. The vacuolar structures containing i t bear a resemblance to the electron opaque spheres described by Bouck (1962) in the gland c e l l s of Lomentaria baileyana. Bouck suggested an association between the gland cells and mucilage production. The production of such a substance was also noted in Porphyridium and Pseudooloiophloea by Ramus (1972), who described i t as a polyanionic polysaccharide of high molecular weight. The second product associated with dictyosome activity i s contained within smaller, more electron transparent vesicles, the greater percentage 53. of which are formed after production of the larger, more densely f i b r i l l a r structures. Since this compound is secreted primarily after spore liberation, i s produced in large quantities and i s released very easily by rupturing the fragile, unprotected plasmalemma, i t is hypothesized that i t acts as a cement-like substance essential to the success of the reproductive process. The a b i l i t y of the monospores of Smithora to adhere to various objects was mentioned by Knox (1926), In fact, not only do these spores stick to the host plant securely, but also adhere to one another. So effective i s this biological cement, that i t i s very d i f f i c u l t to remove the settled spores from plastic containers. Since Smithora and Erythrotrichia are epiphytic, thB advantage of such a substance would be tremendous. The cement would f a c i l i t a t e a rapid, secure attachment of the spores to the host plant before tide and wave action could remove them from this location. Again, the timing of the secretion would be relatively c r i t i c a l . Too early a release could cause the spores to adhere to the parent thallus thus restricting dispersal. Wanton (1964) and Evans and Christie (1970) have proposed a similar function for the large accumulation of vesicular structures in the zoospores of the green algae Stiqeoclonium and Enteromorpha respectively. In recent studies on zoosporogenesis in Oedoqonium (Chlorophyceae), Pickett-Heaps (1971) has also described two distinct populations of dictyosomes, each concerned with the production of a different substance. In addition, Tripodi (1971 )t working with cystocarpic material of Polysiphonia, has noted two product accumulations which appear to be structurally similar to those in Smithora and Erythrotrichia. He suggests that the role of these structures may be involved with a reserve function, although he does not relate any ultrastructural events leading to carpospore release. The large amount of rough ER i n the developing spore suggests an 54. increase in metabolic rate, particularly in regard to protein synthesis. In a classical sense, the larger number of nuclear pores could facilitate transport of messenger RNA to the nearby rough ER f o r efficient translation i n t o manufactured products. In addition, the precipitate found at areas of the nuclear envelope in close proximity to the chloroplast envelope could be the result of a transfer or buildup of a particular compound or compounds. Associations between the nuclear envelope and the chloroplast envelope surrounding the pyrenoid have been reported in Rhodslia (Evans, 1970) and Asteromonas (Prasinophyceae) (Peterfi and Manton, 1968). Nuclear-chloroplast associations have been shown in other algae (reviewed in Cols and Lin, 1968) and more recently in Symbiodinium (Dinophyceae)(Taylor, 1969) where a continuity between the chloroplast matrix and the nucleoplasm was found. The cell wall of the parent thallus must undergo extensive changes to allow the monospores to escape. The thickness of the cell wall is definitely reduced but there must also be some loss of structural stability . Perhaps •\ the cell walls i n the vicinity of the monospore undergo an actual decomposition or degradation to permit the release of whole sori or individual spores. Whether this change is the result of an enzymatic digestion, physical internal pressure or external environmental effects remains opBn to speculation. The shape of the released spore is spherical indicating a certain amount of pressure is exerted on the plasmalemma by the contents of the spore. However, some degree of flexibility is important to the structure in order to facilitate an easier release from the thallus. Plate XX (Fig. 16) illustrates the stress which must be exerted on the spore as i t squeezes through the narrow passage in the wall. In contrast to the differentiating monospore, the spore on release seems very quiescent. Organelles aro crowded into small spaces between 55. copious amounts of vesicular product. Presumably this would restrict circul-ation of metabolic precursors and the organelles themselves, resulting in decreased synthetic activity prior to attachment of the mature monospore. The consistency of the ultrastructural characteristics of monosporogenesis in Smithora and Erythrotriehia indicate that such subcellular activities are probably carried out by a l l members of the Erythropeltidaceae. Indeed, studies of other spore producing algae (Evans and Christie, 1970; Pickett-Heaps, 1971; Tripodi, 1971) have indicated that certain facets of this process may be common. The most critical period of monospore reproduction appears to be the time between release from ths parent thallus and initiation of cell wall construction. Released algal spores are subjected to numerous environmental hazards which must be overcome to ensure the success of the reproductive process. As exemplified by the erythropeltidacean monospore, these structures are often fragile and easily destroyed by outside influences, A biological mechanism which is thought to compensate for this involves the production of larger numbers of spores, presumably on the premise that the greater V v the number released, the more that will survive. It would be extremsly difficult to estimate the percentage of surviving spores and undoubtedly such a hypothetical proportion would vary considerably under different environmental conditions. It is quite possible that in this study conditions of laboratory culture were responsible for a certain percentage of degenerat-ion. There is l i t t l e known of the ultrastructural aspects of degeneration in algae, although a considerable amount of research has been carried out with the phenomena of acenescence and death in higher plants, Ths mitochondrial component appears to be particularly susceptible to ultrastructural changss 56. in the degenerating monospore. Similar mitochondrial swelling and disruption of the inner membrane have been noted in scenescing wheat leaves (Shaw and Manocha, 1965), chemically treated oat roots (Hanchey, Wheeler and Luke, 1966) and scenescing oat leaves (De Vecchi, 1971). Nuclear breakdown (Shaw and Manocha ,1965) and cytoplasmic vesiculation (Ragetli, Weintraub and Lo, 1970) in scenescing leaf cells are also similar to that reported in the present study. In addition, my observations tend to agree with Hanchey, Wheeler and Luke's (1968) suggestion that large numbers of paramural bodies may be indicative of calls destined to undergo disintegration. Unfortunately, when studying degeneration in conditions approaching those of the natural environment, i t is difficult to speculate on the causes of this phenomenon. Certain studios have been carried out on scenescing plant material under different controlled conditions (e0g„ De Vecchi., 1971), but i t is obvious that further research on such ar t i f i c i a l systems is needed to permit the formulation of definite conclusions on the mechanisms of plant degeneration. 57. PLATE XVII. Smithora. Differentiating monospore Fig. 1. Light microscopic surface view of proximal portion of sorus (s) and adjacent vegetative area (vg). Fig. 2. Section parallel to surface of thallus i l l u s t r a t i n g differentiating c e l l with chloroplast lobes (c)„ dictyosomes (d)„ endoplasmic reticulum (er)» mitochondria (m), floridean starch grains (fs), fibrous vacuole-like structures (fvl) and surrounding c e l l wall (w). XVII 58 . PLATE X V I I I . Smithora. Differentiating monospore Fig. 3. Portion of nucleus (n) with nucleolus (nu) and surrounding organelles. Arrow denotes DNA-containing area in chloroplast. Fig. 4. Area of intimate association betaeen limiting envelopes of nucleus (n) and chloroplast (c). Arrow denotes precipitate of stain. Fig. 5. Portion of nucleus (n) with associated cisternal stack of rough ER. A dictyosome (d) and a mitochondrion (m) are also labelled. Fig. 6 . Portion of nucleus (n) i l l u s t r a t i n g porous nature of envelope (arrows) in certain restricted areas. Fig, 7 . Tangential section through nuclear envelope i l l u s t r a t i n g linear sequence of pores and annular structure. X V I I I 5 9 . PLATE XIX. Smithora. Differentiating monospore Fig, B. Dictyosome (d) with large irregular fibrous vesicles (fv) forming from maturing face. Note vesicles (arrow.)"and ER associated with forming face near c e l l wall. Fig. 9. Floridean starch grain (fs) included within fibrous vacuole-like structure ( f v l ) . Fig. 10. Association between ER and fibrous vacuole-like structure ( f v l ) . Cell wall (w) and a dictyosome (d) are also labelled. Fig. 11. Smaller, more electron transparent vesicles (v) and large f i b r i l l a r vacuole-like structures (fvl) concurrently present in the developing spore. Fig, 12. Large, vacuole-like structures (fvl) appressed to periphery of protoplast near c e l l wall (w). Fig. 13. The vacuole-like structures (fvl) releasing contents through ruptured plasmalemma during liberation of spore. Note presence of smaller vesicles (v) and mitochondria (m). X I X 60. PLATE XX Smithora. D i f f e r e n t i a t i n g spore F i g . 14. Dictyosomes (d) producing s m a l l e r , more e l e c t r o n transparent v e s i c l e s (v) near c e l l w a l l (w). F i g . 15. Compacted area of c e l l w a l l (w) immediately adjacent to plasmalemma (arrow) p r i o r to spore r e l e a s e . F i g . 16. C r o s s - s e c t i o n of t h a l l u s showing monospore being l i b e r a t e d . Note presence of both types of deposits (v, f v l ) and d i s t o r t i o n of spore during r e l e a s e , ex i n d i c a t e s area e x t e r n a l to t h a l l u s . F i g . 17. C r o s s - s e c t i o n of t h a l l u s a f t e r spore l i b e r a t i o n . S i n g l e arrow denotes remaining cytoplasm. Double arrow denotes break i n c e l l w a l l (w) through which a monospore was r e l e a s e d . F i g , 18. Cytoplasm remaining i n t h a l l u s a f t e r spore r e l e a s e . C e l l w a l l (w), nucleus (n), mitochondria (m) and c h l o r o p l a s t m a t e r i a l (c) are l a b e l l e d . ex denotes area e x t e r n a l to t h a l l u s . F i g , 19, Photomicrograph of spores being l i b e r a t e d en mass (ms) and i n t a c t sorus ( s ) . X X 6 1 . PLATE XXI Smithora. Released monospore F i g . 20. Photomicrograph of l i b e r a t e d monospore. F i g . 2 1 . Electron micrograph of l i b e r a t e d monospore. Note c e n t r a l , lobed chlo r o p l a s t (c) with pyrenoid (p) and coalesced electron transparent v e s i c l e s (cv). Single arrow denotes unprotected plasmalemma and double arrow denotes release of contents of electron transparent coalsced v e s i c l e s . A nucleus (n) i s also l a b e l l e d . XXI 62 PLATE XXII Smithora,, Released monospore Fig, 22, Section through released deciduous sorus consisting of closely adhering monospores. XXII 63. PLATE XXIII Smithora. Released monospore F i g . 23. Lobe of chloroplast with c e n t r a l l y located appressed lamellae and coalesced electron transparent v e s i c l e s (cv). F i g . 24. Lobe of chlor o p l a s t with appressed lamellae (double arrow) and thylakoid a s s o c i a t i o n (single arrow). F i g . 25. Portion of pyrenoid showing osmiophilic droplets (od) within swollen t r a v e r s i n g lamellae. XXIII 64. PLATE XXIV Smithora. Released monospore F i g . 26. Dictyosome a c t i v i t y i n mature monospore (production of electron transparent v e s i c l e s ( v ) ) . F i g . 27. Four c l o s e l y packed mitochondria. A f l o r i d e a n starch granule and coalesced v e s i c l e s (cv) are also shown. F i g . 28. Nucleus (n) and nucleolus (nu) of mature monospore. Note small amount of ER (arrow) nsxt to the nuclear envelops. F i g . 29. Single, unprotected plasmalemma of mature spore, ex denotes area external to spore luhich may include mucilaginous m a t e r i a l . F i g , 30. Release of v e s i c u l a r contents (v) by a blabbing of the plasmalemma. ex denotes area external to spore. XXIV 65, PLATE XXV E r y t h r o t r i c h i a boryana F i g . 31. Unreleasad monospore within c e l l wall (w) showing c h l o r o p l a s t (c) with pyrenoid (p), nucleus (n) and f l o r i d e a n starch grains (fs)„ XXV 66. PLATE XXVI Erythrotriehia Fig. 32. E_. boryana. Portion of differentiating monospore within c e l l wall (w) i l l u s t r a t i n g nature of the two products (cv, f v l ) originating from the dictyosome (single arrow). Prominent nucleus (n) surrounded by ER i s also shown. Fig. 33. E_„ carnea. Portion of differentiating monospore. Note f i b r i l l a r vacuole-like structures (fvl) and dictyosome (arrow). Fig. 34, IE, pulvinata. Description as for Fig, 33, XXVI 67. PLATE XXVII Smithora. Degenerating monospore F i g . 1. Monospore i n early stages of degeneration e x h i b i t i n g swollen chlo r o p l a s t lamellae (arrow), swollen mitochondria (m) B f l o r i d e a n starch (fs) and paramural bodies (double arrow). X X V I I 68 PLATE XXVIII Smithora. Degenerating monospore F i g . 2. Swollen mitochondria with i n d i s t i n c t c r i s t a e (arrow). Note numerous vacuoles (v). F i g . 3. Nuclear breakdown i n d i c a t i n g advanced spore degeneration. Note numerous vacuoles (v). F i g . 4. Disorganization and swelling of chl o r o p l a s t lamellae. F i g . 5. C e l l u l a r d i s i n t e g r a t i o n a f t e r plasmalemma breakage. Note remaining membranous elements (arrow). X X V I I I 69. b) Monospore Germination i n Smithora.'* Introduction. Studies on the u l t r a s t r u c t u r a l events during a l g a l spore germination have been r e l a t i v e l y few i n number. Although there appears to be no published investigations of t h i s nature dealing with the Rhodophyceae, detailed studies have been presented on zoospore germination i n the green algae Stiqeoclonium (Wanton, 1964) and Enteromorpha (Evans^and C h r i s t i e , 1970). Work i s also being carried out on zoospore germination i n Qedoqonium (Chlorophyceae) (Pickett-Heaps, 1971) and recently Quatrano (1972) has investigated zygote germination i n Fucus (Phaeophyceae). Smithora provides an i d e a l material for such research as large numbers of spores w i l l germinate i n r e l a t i v e synchrony under laboratory conditions. Observations. At a l i g h t microscopic l e v e l , monospore germination i s marked by the appearance of a c e l l wall and several vacuoles (PI. XXIX, F i g . 1). At an u l t r a s t r u c t u r a l l e v e l , the germinating spore also possesses a pyrenoid-containing chloroplast (PI. XXIX, Fig. 2; PI. XXX, F i g . 4), a nucleus (PI. XXXI, F i g . 7,8; PI. XXXIV, Fig. 14), dictyosomes (PI. XXXI, F i g . 6; PI. XXXII, F i g . 9), endoplasmic reticulum (PI. XXXI, F i g . 7; PI. XXXII, F i g . 9,10), mitochondria (PI. XXIX, F i g . 2; PI. XXXI, F i g . 7; PI. XXXII, F i g . 10; PI. XXXIV, F i g . 14) and soma floridean starch (PI. XXXIV, F i g . 14). The two methods of basal holdfast formation are evident under the l i g h t microscope as well as the electron microscope. A single spore forms a c e l l wall (PI. XXIX, Fig. 1,2) and through successive c s l l d i v isions produces a basal holdfast (PI. XXXI, F i g . 5). Alt e r n a t i v e l y , an i d e n t i c a l ''This section i s based on an a r t i c l e by D,L. McBride and K. Cole i n Phycolooia 11; 181-191 (1972). 70. holdfast may ba formed by many spores adhering and secreting individual c e l l walls which eventually contribute to a common c e l l wall (PI. XXX, Fig. 3,4), Wall construction appears to be i n i t i a t e d in the area of the c e l l adjacent to the peripherally situated nucleus (PI. XXXI, Fig. 7), but eventually i t i s formed continuously around the c e l l (although there may be unrelated, localized areas of different thicknesses) (PI, XXXI, Fig. 8). The bonding of adjacent c e l l walls is suggested by the observation that folds occur between thin sectioned cells only in areas where the walls are not closely interwoven (PI, XXX, Fig. 4j PI, XXXI, Fig, 8). The germinating monospore is distinguished by a large increase in tha peripheral ER which appears to ba extremely active (Pi, XXXII, Fig. 9). Cisternae are often swollen, associated with large numbers of ribosomes and found in close proximity to the convoluted plasamlamma (PI. XXXII, Fig, 10). Dictyosomes may also be present in these areas (PI. XXXII, Fig. 9). It i s possible that both the ER and the dictyosomes are involved in the synthesis of wall material which is a primary activity of the c e l l at this time. Dictyosomes al3o appear to play an important rola i n vacuole formation. A la?ge number of these organelles are situated with their maturing faces and associated vesicles in close proximity to the enlarging vacuoles (PI. XXXI, Fig. 6). An association between dictyosomes and vacuoles has also been noted in the red alga Batrachospermum (Brown and Weier, 1970). The lobed, pyrenoid-containing chloroplast i s structurally unchanged from that reported i n the newly released monospore (Section Va) i n possessing areas of closely appressed lamellae (PI. XXIX, Fig. 2} PI. XXXIV, Fig. 14). However, the number of such areas appears to be somemhat reduced. Typical floridean starch grains are conspicuously few i n number. 71. Conditions of laboratory culture may have some bearing on the cellular content of floridean starch (Burton, H», personal communication). It is also conceivable that the amount of reserve substance may be dependent on the particular metabolic stats of the c e l l . Two types of pyrenoid structure are represented in germinating monospore material. A typical, centrally located pyrenoid possessing irregular traversing lamellae is the most regular feature (PI. XXIX, Fig. 2; PI. XXX, Fig. 4). However, a small percentage of cells exhibit a definite crystallization of the pyrenoids. These "pyrenoids" are somewhat reduced in size and many appear to have cleaved, resulting in two or three angular crystalline bodies (PI. XXXII, Fig. 11). The crystal lattice is formed from linked subunits which appear in a parallel-line or in a cross-hatched pattern depending upon the plans of sectioning (PI. XXXIII, Fig, 12). The parallel lines of both configurations have a centre to centre spacing of 12.5 nm. with each subunit measuring 6,0-7,0 nm. in diameter. The angle of intersection in the cross-hatched pattern is 70-75°. In addition, electron translucent areas, probably representing included traversing lamellae and the ribosome-like particles referred to by Holdsworth (1968) were observed (PI. XXXIII, Fig. 12). The nucleus is eukaryotic in appearance and is surrounded by numerous cisternae of rough endoplasmic reticulum (PI. XXXIV, Fig. 14). No connections between the nuclear envelope and surrounding complement of ER were noted. Spindle fibres were occasionally observed in nuclear areas preparing for karyokinesis (PI. XXXIV, Fig. 17). Unfortunately the microtubular elements were not preserved isell under the fixation conditions employed. The fibres appear to originate externally to the nuclear envelope and radiate towards the nucleus from a spindle organizing centre. Areas adjacent to the nuclear 72. envelope which often appear devoid of structured material could also be centres of spindle f i b r e organization which have been destroyed by f i x a t i o n procedures (PI. XXXIV, F i g . 15). Bordering 3uch areas i s a high concentration of nuclear pores i n d i c a t i n g a possible nuclear p o l a r i t y at t h i s early stage of d i v i s i o n . La Cour and Wells (1972) have shown a more i r r e g u l a r d i s t r i b u t i o n of pores i n prophase as compared to interphase stages of c e r t a i n higher p l a n t s . Two types of v e s i c u l a r units of unknown function were observed r e g u l a r l y i n the cytoplasm of the germinating monospore. The f i r s t type i s i n v a r i a b l y associated im groups with the ER surrounding the nucleus (PI. XXXIV, F i g . 14). Each u n i t i s s p h e r i c a l or s l i g h t l y e l l i p s o i d a l i n shape and measures .05-.07 microns i n diameter (PI. XXXIV, F i g . 13). These units appear to co n s i s t of several coneentric, membranous spheres bounding a c e n t r a l more electron transparent area. The second type of v e s i c l e i s a s p h e r i c a l , membrane bound structure which e x i s t s i n large aggregations i n the cytoplasm (PI. XXXIV, F i g . 15). These e c c e n t r i c a l l y located structures s u p e r f i c i a l l y resemble dictyosome v e s i c l e s but are seldom seen i n the v i c i n i t y of these organelles. Paraneural bodies (fflarchant and Robards, 1968) are few i n number (PI. XXXIV, F i g . 16) and are unlike the above mentioned v e s i c u l a r units which are more regular i n s t r u c t u r e . Discussion. From observations on the methods of holdfast formation i n Smithora i t i s evident that within a population of released monospores a c e r t a i n number w i l l begin development from u n i c e l l u l a r stages while the remainder w i l l begin development from m u l t i c e l l u l a r stages of varying degrees of complexity. This mechanism could a i d i n desynchronizing the development of a generation of monospores. Thus, the population would reach reproductive maturity over a longer period of time, tending to minimize the p o s s i b i l i t y 73. of adverse environmental conditions destroying a "crop" of released monospores. Another possible advantage of the development of a holdfast from numerous spores would be a decrease in the time required to complete an asexual reproductive cycle. However, this mechanism would also reduce the number of plants developing from a given number of spores. The nature of wall production in algal c e l l s has been investigated by a number of authors including Brown (1969), Brown et a l . (1970), Jordan (1970), Pickett-Heaps (1971) and Pickett-Heaps and Fowke (1970). In these and other studies the dictyosome has been implicated in the production of wall material or wall precursors. Positive cytochemical tests have been obtained for the presence of polysaccharides in these organelles. In addition, Thompson and Preston (1968) have suggested that proteins may be an important structural component in certain algal c e l l walls. Thus, one might expect a germinating spore to be actively involved in the production of proteins with enzymatic or structural functions as well as the carbohydrate moity. Indeed, Quatrano (1968,1972) has shown that rhizoid formation i n Fucus zygotes i s dependent on protein synthesis. In germinating monospores of Smithora, peripherally located rough ER appears to become associated with the plasmalemma. A similar peripherally located network has also been reported i n Porphyridium (Gantt and Conti, 1965) and i n Rhodella (Evans, 1970). In Smithora i t appears that material could be transferred directly from the ER across the plasmalemma into the c e l l wall. This mechanism has also beers postulated to occur in Oedoqonium (Pickett-Heaps, 1971). The hypothesis that the ER acts as a f i n a l synthesis and packaging point of wall material is attractive but must be based on further studies. In the germinating monospore the dictyosome complement i s capable of carrying out several different functions simultaneously. This principle of 74. "division of labour" allows some of these organelles to be involved in vacuole construction while the remainder appear to take part in production of wall material,, In the developing zoospore of Oedoqonium Pickett-Heaps (1971) also noted two distinct populations of dictyosomes, each apparently with a different function. In this context, i t i s also interesting to note a change in roles of the dictyosome populations in a monospore from the differentiating state, through the free-floating condition to germination. As spore differentiation occurs the dictyosomes are concerned with the manufacture of large deposits of a material probably of a mucilaginous nature (Section Va). As the spore nears the time of release from the thallus and for a short period after, the production of an adhesive material occurs (Section Va). Prio^ to settling, the dictyosome populations are less active, but upon germination appear to become involved in vacuole formation and production of a wall material. Evans and Christie (1970) reported a similar set of changes in the germinating zoospore of Enteromorpha. The crystalline matrix i s an interesting, i f not regular, feature of the pyrenoid. Crystalline pyrenoids have been reported previously in brown algae (Evans, 1966), diatoms (Holdsworth, 1968; Coombs et a l . , 1968; Taylor, 1972), a dinoflagellate (Kowallik, 1969) and a green alga (Bertagnolli and Nadakavukaren, 1970). From these studies i t i s evident that a crystalline matrix i s not present in a l l pyrenoids of a particular algal population or, i f present, may be apparent in particular regions only. In fact, one report details this structural property in the diatom Navicula only after colchicine treatment (Coombs et a l . , 1968). In this laboratory, studies on freshly collected vegetative material, monosporogenous material and sperm-atangial sori have shown no evidence of crystalline pyrenoids. However, in ths present study monospores subjected to culture conditions have this 75. structural property* It i s intsrasting to nots that other illustrated reports have also been made using cultured material. Holdstuorth (1971) has recently reported the presence of proteinaceous components in the pyrenoid of Ereinosphaera (Chlorophyceae) which appear to have similar properties to certain enzymes in the carbon fixation pathway of photosynthesis. Thus, i t might be of interest to note the structure of pyrenoids possessing crystalline matrices under different photosynthetic conditions. Such controlled experiments may shed some light on the formation of these structures. Perhaps this phenomenon i s a modification preparatory to withstanding adverse environmental conditions, or even one of the f i r s t steps in c e l l degeneration. Indeed, Ragatli, Weintraub and Lo (1970) have described "pseudocrystalline" structures in the chloroplasts of excised degenerating leaf material of Nicotiana which are very similar to the crystal-li z e d pyrenoid of Smithora. They suggest this may represent a highly ordered mechanism for coping with starvation. The substructure of the crystal l a t t i c e in Smithora monospores i s comparable to that reported in the diatom Achnanthes (Holdsworth, 1968) and the green unicell Chloralla (Bertagnolli and Nadakavukaren, 1970). However, the mean centre to centre distance between parallel subunit rows is different. In both the aforementioned algae this spacing was found to be 8.0 nm. while in Smithora the distance i s 12.5 nm. In the dinoflagellate Prorocentrum, Kowallik (1969) reported a distance of 12.2 nm. between the centres of adjacent subunits. The significance of these differences i s not yet clear although i t could indicate important variations i n pyrenoid composition between certain algae. Until recently, microtubular spindle fibres have not been demonstrated in red algae (Hommersand and Searles, 1971). But now reports of these 76. structures by Chapman, Chapman and Lang (1971) in Porphyridium and MacDonald Phycol. i n press) in Membranoptera indicate that they are probably involved in c e l l division in this group. Their presence in Smithora further strengthens this view. However, i t i s evident that much more study i s needed to f u l l y elucidate this process of c e l l division. In conclusion there are several fine structural changes early in the germination of the monospore which appear to be typical of this process. There i s a marked reduction in floridean starch content, a notable increase in peripheral rough ER and an increase in vacuolar area. It i s conceivable that these changes are integrally associated with the process of c e l l wall construction. 77. PLATE XXIX. Smithora. Germinating monospore F i g . 1. Light micrograph of a germinating monospore. F i g . 2 . Electron micrograph of a germinating monospore i l l u s t r a t i n g a c e l l wall (cw), vacuoles (v). a pyrenoid (p) and ch l o r o p l a s t lobes ( c l ) . Arrow indi c a t e s area of appressed c h l o r o p l a s t lamellae. XXIX 78, PLATE XXX Smithora., Germinating monospore F i g . 3 . Light micrograph of a number of adhering, germinating monospores. F i g . 4 . Electron micrograph of a developing basal holdfast formed from numerous spores. Arrows i n d i c a t e an area of common c e l l wall between two spores. Note that dark f o l d l i n e s do not pass between joined spores. X X X 7 9 . PLATE XXXI Smithora 0 Germinating monospore F i g . 5. Light micrograph of a mature basal holdfast (bh) attached to a host plant (h). F i g . 6. A dictyosome (d) with associated v e s i c l e s i n c l o s e proximity to an enlarging vacuole (v). F i g . 7 . Portion of germinating spore i l l u s t r a t i n g area of c e l l wall i n i t i a t i o n (cw) adjacent to nucleus (n). F i g . 8. Portion of a developing pad i l l u s t r a t i n g a nucleus (n), common c e l l wall (cw) and a vacuole (v). Arrow ind i c a t e s an area of thickening c e l l w a l l . X X X I 80. PLATE XXXII Smithora. Germinating monospore F i g . 9« A tangential s e c t i o n through a portion of a monospore showing hypertrophied peripheral ER and dictyosomes (d). F i g . 1 0 . Rough ER with cisternae i n close a s s o c i a t i o n with the plasmalemma. A mitochondrion (m). a vacuole (v) and the c e l l wall are also l a b e l l e d . F i g . 1 1 , A portion of a ch l o r o p l a s t (c) showing s i n g l e lamellae (arrow) and a cleaved, angular c r y s t a l l i n e pyrenoid (cp). XXXII 81. PLATE XXXIII Smithora. Germinating monospore Fig. 12 0 Section through a crystalline pyrenoid i l l u s t r a t i n g the nature of the crystal l a t t i c e . Note the included traversing lamella (tl) and ribosome-like particles (arrow). XXXIII 82. PLATE XXXIV Smithora. Germinating monospore Fig. 13. A group of vesicular units in association with the ER. Fig. 114. Portion of a c e l l showing the location of the vesicular units near the nucleus (n). A nucleolus (nu)„ ER, a floridean starch grain (fs) and appressed chloroplast lamellae (arrow) are also marked. Fig. 15. Section through a nuclear area (n) showing an aggregation of vesicles (ve)1. Note the unstructured area of cytoplasm (a) adjacent to the porous nuclear envelope (arrow). Fig. 16. Paraneural body near c e l l wall. Fig. 17. Microtubular spindle fibres radiating toward the nucleus (n). Note the tangential sections of nuclear pores (single arrow), ER, and the extranuclear location of the spindle fibre organizing centre (double arrow). XXXIV 63 . VI. ULTRASTRUCTURAL EVIDENCE OF SEXUAL REPRODUCTION Introduction. Perhaps one of the most interesting and important aspects of studies on red algae are the descriptions of sexual reproduction. Although this process i s taell documented in many members of the Florideophycidas, i t is poorly known in most Bangiophycidae. During this study, the only evidence of sexual reproduction in the Erythropeltidaceae was the regular production of "spermatia" (Hollenberg,, 1959) in Smithora and sporadic indications of f e r t i l i z a t i o n in Erythrotrichia boryana. It must ba stressed that in this report terms describing sexual reproduction (e.g. spermatia, carpospora, etc.) are used in their most tentative sense duo to the lack of information on their specific function in this family. As is shown by the various conflicting reports of sexual reproduction in the Erythropeltidaceae, the size of the plants and, more spec i f i c a l l y , the unicellular nature of the reproductive "organs" hinders documentation using conventional light microscopic techniques. On the other hand, the seemingly transient nature of this process ( f e r t i l i z a t i o n , in particular) provides a formidable barrier to electron microscopic investigation. In addition, many such algae tend to display only asexual reproduction in laboratory conditions. These are indeed perplexing problems which have undoubtedly caused some of the present confusion surrounding sexual reproduction in these plants. Observations. Spermatiaqene3is in Smithora; Spermatia may be produced extensively i n the medial distromatic portion of the mature blade in the f a l l of the year c At a light microscopic level, each vegetative c e l l appears to undergo an asymmetric division parallel to the surface of the thallus, resulting in the production of numerous, small (3-5 microns), pale cells (PI. XXXV, 8 4 . Fig. 1). At an ultrastructural level, i n i t i a t i o n of this process involves the migration of the usually eccentrically located nucleus toward the end of the c e l l nearest to the surface of the thallus (PI. XXXV, Fig. 2), allowing the formation of unequal daughter c e l l s . After division i t appears that the nucleus of the larger c e l l migrates toward the opposite end of the protoplast (PI. XXXV, Fig. 2). The newly formed spermatangium i s not only smaller than the vegetative c e l l but contains l i t t l e chloroplast material (contrary to Hollenberg"s (1959) light microscope observation) and no pyrenoid (PI. XXXVI, Fig, 3). Evidently only one or two chloroplast lobes have been included during the unequal division. However, a typical nucleus (PI. XXXV, Fig. 2j PI. XXXVI, Fig. 3,5), mitochondria (PI. XXXVI, Fig, 3; PI. XXXVII, Fig. 8 ) , ER (PI. XXXVI, Fig. 3), dictyosomes (PI. XXXVl, Fig. 4,5), floridean starch grains (PI, XXXVI, Fig. 3; PI. XXXVII, Fig, 8) and perhaps a few small vacuoles (PI, XXXVI, Fig. 3) are usual components of the spermatangium. Dictyosomes appear to play an important role in the maturation of these structures. Soon after c e l l division these organelles begin to hypertrophy (PI. XXXVI, Fig. 4 ) and produce numerous vesicles containing a f i b r i l l a r substance (PI. XXXVI, Fig. 5) reminiscent of that in the differentiating monospore (Section Va), However, the contents of the spermatangial vesicles appear more compacted with a central, electron dense core of highly compressed f i b r i l s (PI. XXXVI, Fig. 5j PI. XXXVII, Fig. 6,7). These structures are then released into the c e l l wall by a process similar to reverse pinocytosis involving a fusion of the vesicular membrane with the plasmalemma (PI. XXXVII, Fig. 6). The material accumulates between the spermatangial protoplast and the c e l l wall, particularly at the margin nearest the plane of the 8 5 . thallus ( P I . XXXVII, Fig. 6,7). Thus, the surface of a mature sorus exhibits many irregular protrusions of c e l l wall material at sites of spermatiagenesis ( P I . XXXV, Fig. 2; P I . XXXVII, Fig. 7 ) . Eventually the c e l l wall ruptures and the naked spermatium i s released ( P I . XXXVIII, Fig, 9,10). At this point i t i s evident that the material produced by the dictyosomes i s liquid or semiliquid in nature as a large amount appears to flow from the thallus after c e l l liberation.. Under the light microscope the released spermatium is almost colourless due to the lack of extensive chloroplast material ( P I . XXXVIII, Fig. 1 1 ) . It is ultrastructurally identical to the unreleased c e l l with a certain amount of vesicular material appearing to remain after release ( P I . XXXVIII, Fig. 9 ) . Numerous attempts to document their function were unfruitful. Evidence of f e r t i l i z a t i o n in Erythrotrichia boryana; The presence of "spermatia" adhering to filaments of E_. boryana was noted under the light microscope during two separate collections in the f a l l of 1971. These attached structures are virtually colourless and possess a c e l l wall ( P I . XXXIX, Fig. 1 ) . It is evident that the spermatium did not preserve well under the fixation procedure used for electron microscopy ( P I . XXXIX, Fig, 4 ) . However, considering the nature of the loosely organized c e l l wall and the presence of typical floridean starch granules, these pale cells are certainly red algal and most probably originated in different filaments of E_. boryana. Mitochondria, dictyosomes and vacuoles are additional discernible structures ( P I . XXXIX, Fig, 4 ) . No nuclear material was observed but this may have been destroyed by fixation procedures. Of interest i s the short cellular "stalk" or "foot" present in the area of attachment ( P I , XXXIX, Fig. 1,2,4). Directly beneath this point, the underlying c e l l usually appears to have undergone an unequal division similar to that in the spermatangial sorus of Smithora, 86. with the r e s u l t i n g small c e l l being v i r t u a l l y i d e n t i c a l to the spermatangium. This "carpospore-like" c e l l contains a nucleus, a small amount of chloroplast material, mitochondria, dictyosomes, ER and f l o r i d e a n starch grains (PI. XXXIX, F i g , 3), No subsequent stages of v e s i c l e production were noted although t h i s could be due to the p a r t i c u l a r stage of development at the time of observation. No trichogyne-like structures or i n t e r c e l l u l a r connections of any type were observed. Discussion, It i s apparent that the release of dictyosome o r i g i n a t e d v e s i c l e s into the c e l l wall i s an i n t e g r a l part of the mechanism of spermatial l i b e r a t i o n i n Smithora. When a large amount of t h i s mucilaga-like material i s accumulated external to the protoplast, the wall appears to burst as i f due to p h y s i c a l pressure. It may also be possible that t h i s material has some e f f e c t on the s t r u c t u r a l s t a b i l i t y of the w a l l . Mucilage-like material has also been associated with the release of spermatia i n other red algae (Neushul, M., personal communication). This mechanism may be likened to that postulated to a i d i n erythropeltidacean monospore release (Section Va). U l t r a s t r u c t u r a l observation of the mature spermatangial sorus g r a p h i c a l l y i l l u s t r a t e s Drew's (1956) statements "... i t was not determined whether the small c e l l on the outside of the filament was e s t a b l i s h i n g or l o s i n g contact." As i s shown i n P I . XXXV (Fig. 2), a f t e r c e l l release a protoplasmic " f i n g e r " from the underlying vegetative c e l l may p r o j e c t into the pore giving a f a l s e but c l a s s i c example of a trichogyne. If the spermatium i s s t i l l near the entrance to the pore a f a l s e impression of f e r t i l i z a t i o n i s imparted. Thus, previous reports of f e r t i l i z a t i o n i n t h i s group must be re-examined. Since the production and release of spermatia i s a regular feature of 8 7 . Smithora. i t i s probable that they play an important role in the l i f e cycle of the plant. These structures must carry out their function relatively rapidly due to their apparent f r a g i l i t y . However, thay did not survive in culture, thus, i t i s not possible at this time to directly implicate spermatia in the sexual process of Smithora. At a light microscope level the evidence of f e r t i l i z a t i o n in J E , boryana is indeed intriguing. In classical terms the observations related here would represent a situation after f e r t i l i z a t i o n prior to carpospora release. The production of carpospores in J E . boryana would than be identical to spermatiagenesis as described by Berthold (1882) in I E . c i l i a r i s and Hollenberg (1959) in Smithora. It i s d i f f i c u l t to directly dispute this hypothesis since, in this report, a nucleus was not observed in; the attached c e l l . However, because i t otherwise appears to possess a complete set of cytoplasmic components, some mechanism would be needed to selectively release nuclear material. This is unlikely in view of the nature of c e l l fusion in other organisms. Because cf the presence of a c e l l wall, i t i s also unlikely that the attached c e l l i s in the process of release. The formation of a spermatial c e l l wall has been reported to occur in other red algae during the free-floating state or after adhering to the carpogonial area (see Fritsch, 1945 for review). An alternative, and perhaps more attractive, explanation of the situation l p L* boryana would involve the induction of female gametogenesis by the attached spermatium and subsequent fusion with the carpospore-like c e l l . Induction of gametogenesis, possibly the result of a chemical stimulus, has been described in other algal species (Coleman, 1962). In addition, this mechanism would require de novo synthesis of a pyrenoid which has been postulated to occur in the zoospores of the green algae Qedogonium 88. . (Hoffman, 1968) and T e t r a c v s t i s (Broyjn and Arnott, 1970). If t h i s general hypothesis mare to prove tenable, t h i s phenomenon might be c l a s s i f i e d as a type of isogamous reproduction. It i s i n t e r e s t i n g to note that t h i s category of reproduction i s c h a r a c t e r i s t i c of simple representatives of the Chlorophyceae and the Phaeophyceaa. Nevertheless, such a theory must be further substantiated before d e t a i l e d presentation. 89o PLATE XXXV Smithora. Spermatangia Fig * 1, Light micrograph of a c r o s s - s e c t i o n of a spermatangial sorus. F i g . 2. Electron micrograph of a c r o s s - s e c t i o n of a spermatangial sorus. Note i r r e g u l a r surface of t h a l l u s , a f i n g e r - l i k e p r o j e c t i o n of vegetative protoplasm (arrow) and the d i f f e r e n t p o s i t i o n s of the n u c l e i (n). XXXV 90 PLATE XXXvI Smithora., S p e r m a t a n g i a F i g . 3 o Immature spermatangium i l l u s t r a t i n g s m a l l amount of c h l o r o p l a s t m a t e r i a l ( c ) , f l o r i d e a n s t a r c h g r a i n s ( f s ) , ER, a n u c l e u s (n) w i t h n u c l e o l u s ( n u ) , v a c u o l e s (v) and m i t o c h o n d r i a (m). F i g . 4. Immature s p e r m a t a n g i a w i t h d i c t y o s o m e s b e g i n n i n g t o h y p e r t r o p h y ( a r r o w ) . F i g . 5. M a t u r i n g spermatangium w i t h c e n t r a l l y l o c a t e d n u c l e u s (n) and d i c t y o s o m e s (d) p r o d u c i n g numerous v e s i c l e s c o n t a i n i n g a f i b r i l l a r s u b s t a n c e ( a r r o w ) . X X X V I 91. PLATE XXXVII Smithora. Spermatangia Fig. 6. Cell releasing contents of dictyosome derived vesicles (arrow) into c e l l wall. Note buildup of material around protoplast. Fig, 7 . A large quantity of the f i b r i l l a r material has been released into the c e l l wallo Note the protrusion of wall material in the direction of the arrow. Fig, 8 . Portion of a maturing spermatangium i l l u s t r a t i n g nature of chloroplast material (c), A mitochondrion (m) and the nucleus (n) are also marked. XXXVII 9 2 . PLATE XXXVIII Smithora. Spermatia Fig. 9c Newly liberated, naked spermatium containing a nucleus (n), floridean starch grains (fs), ER, some chloroplast material (arrow), mitochondria (m) and some remaining f i b r i l l a r material (Double arrow). Note the nearby protoplast of the vegetative c e l l (vc) and the copious amount of f i b r i l l a r material between the two c e l l s . Fig. 10. Released, fres-floating spermatia. Fig. 11. Light micrograph of released, pale spermatia. X X X V I I I 93. PLATE XXXIX Erythrotriehia boryana. Fig 0 1. Light micrograph of spermatium attached to filament. Fig. 2. Electron micrograph of spermatium attached to filament. Note carpospore-like c e l l and "foot" of attached c e l l . Fig. 3. Carpospore-like c e l l containing a nucleus (n), chloroplast material (c), floridean starch grains (fs), ER, dictyosomss (arrow) and mitochondria (double arrow). Fig. 4. Attached spermatium exhibiting c e l l wall ( w ) , floridean starch grains (fs), a mitochondrion (m) and vacuoles (v). XXXIX 94. VII. GENERAL DISCUSSION AND CONCLUSIONS A_ proposal on the e v o l u t i o n of .gr_gujth_ types i n the Bangiophycidaes From u l t r a s t r u c t u r a l observations on the E r y t h r o p e l t i d a c e a e reported here and from p u b l i s h e d r e s u l t s on other members of the subclass Bangiophycidae i t i s evident t h a t there are two d i s t i n c t l y d i f f e r e n t types of red a l g a l , p y r e n o i d - c o n t a i n i n g c h l o r o p l a s t s . The s t r u c t u r a l d i f f e r e n c e i s best shown i n a c r o s s - s e c t i o n of a c h l o r o p l a s t lobe ( P I . XL). In the f i r s t type, many of the photosynthetic l a m e l l a e terminate at the c h l o r o p l a s t envelope, i n d i c a t i n g the absence of a p e r i p h e r a l t h y l a k o i d . In the present d i s c u s s i o n , c h l o r o p l a s t s e x h i b i t i n g t h i s s t r u c t u r e w i l l be designated type I. The f o l l o w i n g bangiophycean genera possess c h l o r o p l a s t s of t h i s category? Porphyridium (Brady and V a t t e r , 1959} Speer, Dougherty and Jones, 1964} Gantt and C o n t i , 1965,1966} Gantt, Edwards and C o n t i , 1968} Guerin-Dumartrait, Sarda and L a c o u r l y , 1970} Neushul, 1970} Wehrmeyer, 1971} Ramus, 1972), Rhodella (Evans, 1970), Banqia ( H o n s e l l , 1963} Sommerfeld and Leeper, 1970} Cole, unpubl.), Porphyra (Gibbs, 1960} Ueda, 1961} Yokomura, 1967} K i t o and Akiyama, 1968} Kazama and F u l l e r , 1970} Bourne, Conway and Cole, 1970} Lee and F u l t z , 1970} Bourne, 1971). A l t e r n a t i v e l y , type I I c h l o r o p l a s t s e x h i b i t a p e r i p h e r a l t h y l a k o i d and occur i n the f o l l o w i n g genera: Rhodosorus (Giraud, 1963), E r y t h r o c l a d i a (fflcBride, unpubl.), E r y t h r o t r i e h i a (see s e c t i o n s IV,V,VI), Goniotrichum (McBride, unpubl.), Smithora (see s e c t i o n s IV,V,VI). On the b a s i s of t h i s w e l l d efined d i f f e r e n c e , i t i s p o s s i b l e to c o n s t r u c t a proposal on the e v o l u t i o n of growth types i n the Bangiophycidae ( P I , XL). Those m u l t i c e l l u l a r genera i n the "Porphyridium" l i n e (type I) are con-s i d e r e d to be members of the f a m i l y Bangiaceae i n the order Bangiales (presence of r h i z o i d a l processes i n the lower c e l l s ; carposporangia and 95. spermatangia producing numerous carpospores and spermatia respectively). The multicellular genera of the "Rhodosorus" line (type II) are members of the family Erythropeltidaceae in the order Bangiales or the family Goniotrichaceae in the order Goniotrichales (most lack rhizoidal processes and although knowledge of sexual reproductive processes i s incomplete, where known, one spermatium i s produced per spermatangium). Unfortunately, not a l l Bangiophycidae can be presently included in the scheme because their chloroplast structure i s unknown. It would be especially valuable to examine such rare plants as the unicellular Rhodospora and Chroothece and the multicellular Asterocystis and Porphyropsis. Since the creation of an evolutionary theory must rely on fundamental similarities and differences, the value of the scheme as outlined would l i e in the presumably very basic, genetically controlled characteristics of chloroplast structure. The question arises as to which evolutionary line could have given rise to the Florideophycidae ( i f indeed only one line gave rise to this group). Evidence in favor of the Rhodosorus line i s appealing. Chloroplasts of type II are found in certain members of the florideophycean order, Nemaliales e.g. Acrochaetium sp. (McBride, unpubl.) and Thorea riekei (Bischoff, 1965). Most other members of the Florideophycidae contain disc-like chloroplasts which exhibit a peripheral thylakoid. Possibly these structures could be derived more easily from a type II chloroplast. Evidence in favor of florideophycean origin from the Porphyridium line includes the presence of pit connections in the conchocelis phase of Porphyra (Bourne, Conway and Cole, 1970; Lee and Fultz, 1970) and Banqia (Sommerfeld and Leeper, 1970)* However,, p i t - l i k e structures are a common feature of certain Ascomycetes and have been reported in some blue-green algae 96. (Butler and Allsopp, 1972), suggesting that they could have arisen independ-ently several times. The problem of the origin of the red algae i s s t i l l somewhat vexing due in most part to the lack of fossilized forms. Klein and Cronquist (1967) have reviewed the various arguments for and against a phylogenetic association between the blua-green and the red algae. A discussion of this type i s beyond the scope of this report. However, i t is generally agreed that the ultrastructural and physiological characteristics of the alga Cyanidium (e.g. Hirose, 1958; Allen, 1959; Rosen and Siegesmund, 1961; Mercer, Bogorad and Mullens, 1962; Seckbach, 1971) indicate an intermediate position between the two groups. In this context, i t must be stressed that tha evolutionary proposal presented hare does not suggest direct links among the algae described. It is realised that present day forms are the result of yaars of evolutionary stress and selection and can bs used only as examples of growth types. Changes in the number and function o_f subcellular components during the  course of the l i f e cycle; Distinct changes in the number and the amount of subcellular components were noted during the different phases in the l i f e cycle of the plants. In some cases, concomitant with these changss, a difference in function was observed. Using the vegetative c e l l as a standard for comparison, these changes are outlined below; 1) . Nucleus- there is an increase in the number of nuclear pores during monospore differentiation, 2) , Dictyosome- these organelles hypertrophy at the onset of monospore differentiation and become involved in the formation of two products, one of which continues to be produced after spore release. Upon monospore germination these organelles are concerned with c e l l wall and vacuole formation. 97. During spermatiagenesis in Smithora, dictyosomes also hypertrophy and produce a f i b r i l l a r product. 3) . Endoplasmic reticulum- monospore differentiation results in an increase in perinuclear ER while subsequent spore gsrmination i s typified by a substantial increase in peripheral ER which may be involved in c e l l wall formation. 4) . Mitochondria- these organelles increase in number during monospore differentiation. Swelling and inner membrane disruption occur during spore degeneration. 5) . Chloroplast- lamellae form "pseudogranum-like" structures during mono-sporogenesis and swell during degeneration. 6) . Vacuoles- a loss of these structures occurs during monosporogenesis with subsequent reformation upon monospore germination. A substantial increase in the number of vacuoles is noted during spore degeneration, 7) , Floridean starch grains- these structures increase in number and size during monospore differentiation but undergo a rapid decrease upon monospore germination, 8) . Multivesicular bodies and concentric lamellar bodies- there i s an increase in the number of both these structures in older c e l l s and a large number of the former in degenerating spores. Conclusions, New information has been recorded on ths distribution, l i f e histories and ultrastructure of four species of Erythropeltidaceae found in tha coastal waters of British Columbia, North American Pacific coast distribution: Erythrotriehia carnea- Piper's Lagoon, Vancouver Is., British Columbia to Colfo Dulse, Costa Ricaj Clipporton Is. 98. Erythrotriehia boryana- Point N 0 Point (Glacier Pt.), Vancouver Is., British Columbia to Bahio. Ascuncio'n, Baja California, Mexico. Erythrotriehia pulvinata- Bamfield, Vancouver Is., British Columbia to Bahxa Asuncion, Baja California, Mexico. Smithora naiadum- Cape Chiniak, Kodiak Is., Alaska to Isla Magdalena, Baja California, Mexico. Life histories; Monospores are produced from the basal attachment organs of Erythrotriehia  pulvinata and Smithora naiadum. Ultrastructure; Vegetative c e l l - A similar vegetative ultrastructure i s found among the species examined. S_. naiadum, E_. boryana and E_, pulvinata exhibit an irregular protoplast, a pyrenoid containing chloroplast with single uniform lamellae, multivesicular bodies and other typically rhodophycean subcellular components. In addition, the chloroplast lamellae of Smithora are occasion-ally associated i n primitive bands or stacks. IE. carnea i s somewhat different than the above species, notably i n c e l l wall and pyrenoid ultrastructure. Monospore differentiation, release and degeneration- The formation and release of monospores i s dependent on the activities of the dictyosome. These organelles produce two substances, one of which may be involved in spore release, the other in spore attachment. A large amount of perinuclear ER, an increased number of mitochondria and a large number of nuclear pores are typical of the differentiating monospore. The naked, released spores possess interesting "pseudogranum-like" lamellar associations, A certain percentage of spores appear to undergo an organized degeneration in culture. Monospore germination- In the settled monospore, the formation of a c a l l wall 99. occurs adjacent to the nuclear region. The germinating spore i s typified by an increase i n vacuolar area and a large amount of peripheral ER which may be involved in c e l l wall formation. Microtubular spindle fibres and a crystalline matrix in the pyrenoid are irregularly present in these c a l l s . Sexual reproduction- Because gamatogenesis and f e r t i l i z a t i o n are poorly documented in the Erythropeltidaceae, the ultrastructural account of. spermatiagenesis in Smithora i s of interest. The dictyosome plays an important role in the maturation of these pale c e l l s . In addition, the ultrastructural evidence of f e r t i l i z a t i o n in £, boryana suggests the possible occurrence of an induction phenomenon. The ultrastructural characteristics of the erythropeltidacean chloroplast and other published data on chloroplast ultrastructure in the Bangiophycidae have allowed the presentation of a bilateral scheme on the evolution of growth types i n this subclass. In order to obtain a more complete•understanding of the biological aspects of the Erythropeltidaceae i t i s evident that there are a number of additional areas which must be studied. However, un t i l the formulation of a culture technique which allows the laboratory observation of the complete l i f e cycle of a particular representative, this goal w i l l be d i f f i c u l t to attain. This i s particularly applicable in regard to sexual reproduction. It i s also evident that electron microscope studies w i l l be a prerequisite to proper documentation of such phenomena. A dependable culture system would also aid in the application of current electron microscopic cytochemical and autoradiographic techniques in an effort to conclusively describe the subcellular mechanisms of sporogenesis. 100. PLATE XL A b i l a t e r a l schsme on the evolution of growth types i n the Bangiophycidae p r i m a r i l y based on the di f f e r e n c e i n c h l o r o p l a s t structure shown i n the lower portion of tha p l a t e . X L multiseriate, filamentous (Banciia sp.) bladed ( Porphyra) uniseriate, filamentous (Bangia sp.) multiseriate, bladed filamentous (Smithora) (Erythrotrichia branched, filamentous (Goniotrichum) unicellular (Porphyridium, Rhodella) prostrate, disc-like (Erythrocladia) uniseriate, filamentous (Erythrotrichia sp75 unicellular (Rhodosorus) Cyanidium-like unicell.? TYPE I TYPE II 101. VII. LITERATURE CITED A l l e n , M.B. 1959. Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch. Wikrobioj. 32: 270-277. 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