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

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STUDIES ON SOmE B R I T I S H COLUMBIAN REPRESENTATIVES OF THE ERYTHROPELTIDACEAE (RHODOPHYCEAE, BANGIOPHYCIDAE)  by  DOUGLAS LEONARD B.Sc,  University  M BRIDE C  of B r i t i s h  Columbia* 1 9 6 8  A T H E S I S SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  t h e Department of Botany  We  accept t h i s  t h e s i s as  conforming t o the required  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA S e p t e m b e r , 1972  In p r e s e n t i n g an  this thesis  advanced degree at  the  Library  shall  in p a r t i a l  the U n i v e r s i t y  f o r s c h o l a r l y purposes may  of  representatives.  te  June S,  1972.  for  requirements  Columbia, reference  the Head o f my  It i s understood that  Botany  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  D a  be g r a n t e d by  permission.  Department of  British  the  Columbia  shall  not  be  I agree and  f o r e x t e n s i v e copying o f  t h i s t h e s i s f o r f i n a n c i a l gain  written  of  make i t f r e e l y a v a i l a b l e  I f u r t h e r agree t h a t p e r m i s s i o n  by h i s  fulfilment of  for  that  study.  this thesis Department  copying or  or  publication  allowed without  my  ii.  Supervisor: Dr. Kathleen  Cole  ABSTRACT Four species of the Erythropeltidaceae |smithora naiadurn Hollenberg, E r y t h r o t r i c h i a carnea  (Anderson)  (Dillwyn) J, Agardh, E r y t h r o t r i c h i a boryana  (Montange) Berthold and E r y t h r o t r i c h i a pulvinata Gardner^ were observed i n freshly c o l l e c t e d and cultured conditions using l i g h t and electron microscopic techniques. The North American P a c i f i c coast d i s t r i b u t i o n of these was  revised i n view of recent c o l l e c t i o n s i n B r i t i s h Columbia and  algae Alaska  by various workers. A study of their morphologies and l i f e h i s t o r i e s revealed new  information concerning production of asexual reproductive units (rnonospores)  from the basal attachment organs of Z. pulvinata and S_. naiadurn, At an ultrasfcructural l e v e l , many organelles i n the vegetative c e l l s of the Erythropeltidaceae examined were found to be s i m i l a r to those reported in other members of the Rhodophyceae. However, several i n t e r e s t i n g 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 s were noted. The c e l l u l a r shape was  remarkably  i r r e g u l a r , e x h i b i t i n g many cytoplasmic protrusions into the c e l l w a l l . The single lobed chloroplast possessed  a uniform lamellar arrangement and p r i m i t i v e  thylakoid stacks or bands. In addition, multivesicular bodies occurred within the cytoplasm  and i n the c e l l wall near the plasmalemma. There was  no  evidence  of any type of i n t e r c e l l u l a r connection. The vegetative c e l l u l t r a s t r u c t u r s of E. boryana and E_. pulvinata was  v i r t u a l l y i d e n t i c a l to S_. naiadurn but  E_, carnea exhibited fewer pyrenoid-traversing lamellae and a somewhat d i f f e r e n t c e l l wall morphology. Monospore d i f f e r e n t i a t i o n and release i n the Erythropeltidaceae was  found  to involve a number of s p e c i a l i z e d s u b c e l l u l a r a c t i v i t i e s . Concomitant with a rounding of the protoplast and reduction i n vacuolar area i n the vegetative c a l l , was  the accumulation  of two products o r i g i n a t i n g from dictyosornos  0  The possible functions of these products ara discussed i n r e l a t i o n to spore  iii release and attachment. Additional fine structural features of t h e developing monospore included an increased number of mitochondria and nuclear pores, a large amount of endoplasmic reticulum and an association between t h e chloroplast and the nuclear envelope. Upon release the monospore lacked a c e l l 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 c a l l 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 i s 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 c e l l s . Evidence of the process of gametogenesis and f e r t i l i z a t i o n in f_. boryana i s 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.  iv.  TABLE OF CONTENTS I.  1.  PREFACE.  II.  MATERIALS AND METHODS  I I I . DISTRIBUTION,  4.  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 *  I V . ULTRASTRUCTURE OF THE VEGETATIVE  o«e»oc««»©««ooo*o*e  • » o  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 , R e l e a s e and Degeneration.... .....o.........44. 0  b) G e r m i n a t i o n .  VI.  . . . . . • • • . « . . . . . . « e . « . > » o . . . . « . o . < . . » . s o « o a o » . . o . <  ULTRASTRUCTURAL EVIDENCE OF SEXUAL REPRODUCTION  VII.  VIII.  GENERAL DISCUSSION AND CONCLUSIONS  LITERATURE CITED  69 •  83.  94.  .....101.  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. P i 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). Fig. 14. Mature filament. Fig. 15. Monospore production. Fig. 16* 4-celled stage in culture. Fig. 17. Juvenile pad.  20«  vi  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) Fig. 6. Thylakoid  stack.  Fig. 7. Thylakoid  stack.  .....32.  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)... Fig. 3. Portion of c e l l . Fig. 4. Portion of pyrenoid. Fig, 5, Cell wall. Plate XIII. (E. bory Fig. 6. Light micrograph. Portion of filomant.  ....39.  vii. Fig. 7. Section through filament. 41.  Plate XIV. (jE. boryana) Fig. 8. Zone of attachment Fig. 9. ER-nuclear envelope association. Fig. 10. Portion of chloroplast lobe.  42.  Plate XV. (E_. pulvinata) 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) Fig. 8. Dictyosome. Fig. 9. Floridean starch grain. Fig. 10. Vacuole-liko structurs-ER Fig. 11. Dictyosome products.  association.  «59  a  viii. F i g . 12. Dictyo9ome products. F i g , 13, Release of dictyosome products. Plate XX, D i f f e r e n t i a t i n g monospore. (Smithor F i g . 14, Dictyosome. F i g . 15. Compact area of c e l l mall. F i g . 16. Liberation of monospore. F i g . 17. Thallus after spore release. F i g . 18, Remaining cytoplasm. F i g . 19. Light micrograph. Deciduous sorus, Plate XXI, Released monospore. (Smithora).... F i g . 20. Light micrograph. Released monospore F i g . 21. Section through released spore. Plate XXII. Released monospore, (Smithora) F i g . 22. Section through deciduous sorus, Plate XXIII. Released monospore. (Smithora).. F i g . 23. .Appressed chloroplast  lamellae.  F i g , 24. Appressed chloroplast  lamellae.  F i g . 25. Portion of pyrenoid. Plate XXIV. Released monospore. (Smithora) F i g . 26. Dictyosome. Fig. 27. Closely packed mitochondria. F i g . 28. Nucleus. F i g . 29. Plasmalemma. F i g . 30. Release of dictyosome product. Plate XXV. (£. boryana). F i g . 31. Unreleased monospore. Plate XXVI. (Erythrotrichia)  ix.  F i g . 32. F_. boryana. Portion of d i f f e r e n t i a t i n g monospore. F i g . 33. f_. carnea. Portion of d i f f e r e n t i a t i n g monospore. F i g . 34. F.. pulvinata. Portion of d i f f e r e n t i a t i n g monospor Plate XVII. Degenerating monospore. (Smithora)....,....... F i g . 1, Early stage of degeneration, Plate XVIII. Degenerating monospore. (Smithora) Fig, 2. Mitochondria, *  F i g . 3, Nucleus. F i g . 4. Chloroplast. F i g . 5. Portion of degenerating spore. Plate XXIX. Germinating monospore, (Smithora) F i g . 1. Light micrograph. Germinating spore. F i g . 2. Section through germinating spore. Plate XXX. Germinating monospore. (Smithora)  ,.  F i g . 3. Light rnicrographo Adhering germinating monospores. F i g . 4. Section through germinating sorus. Plate XXXI. Germinating monospore. (Smithora) F i g . 5. Light micrograph. Basal holdfast. F i g . 6. Dictyosome. F i g . 7. Area of c e l l wall i n i t i a t i o n . F i g . 8. Portion of developing pad. Plate XXXII. Germinating monospore, (Smithora) F i g , 9, Peripheral ER. F i g . 10. Peripheral ER. F i g . 11. C r y s t a l l i n e pyrenoid. Plate XXXIII. Germinating monospore. (Smithora) F i g , 12, C r y s t a l l i n e pyrenoid.  Plate XXXIV. Germinating monospore. (Smithora) Fig. 1 3 .  Vesicular units.  Fig. 1 4 .  Nuclear area.  Fig. 1 5 .  Nuclear area.  Fig, 1 6 ,  Paramural body.  8 2 .  F i g . 17. Spindle f i b r e s . Plate XXXV. Spermatangia.  8 9 c  (Smithora)  F i g . 1 . Light micrograph. Cross-section of sorus. F i g . 2 . Cross-section of sorus, Plate. XXXVI„ Spermatangia.  (Smithora)..........o.....................90.  F i g . 3 . Immature spermatangium. F i g . 4 , Immature spermatangia. F i g . 5 . Maturing spermatangium. Vesicle production. Plate. XXXVII. Spermatangia.  (Smithora)  o . . . . . . .  9 1 .  F i g . 6 , Release of v e s i c l e s . F i g . 7. C e l l wall protrusion. F i g , 8 . Chloroplast material. Plate XXXVIII. Spermatia. (Smithora)...  9 2 .  F i g . 9 . Liberated spermatium. Fig. 1 0 .  Liberated spermatia.  Fig. 1 1 .  Light micrograph. Liberated spermatia.  Plate XXXIX. (E. boryana) Fig. 1 .  9 3 .  Light micrograph. Spermatium attached to filament.  F i g . 2 . Spermatium attached to filament. F i g . 3 . Carpospore-like c e l l . F i g , 4. Attached spermatium. Plate XL Diagram of the evolution of growth types i n the Bangiophycidae,  1 0 0 ,  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 f a c i l i t i e s 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. The the  Rhodophyceae a r e t a x o n o m i c a l l y  Florideophycidae  group i s s u b d i v i d e d contains  PREFACE separated  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 into several orders,  Skuja  marine; filamentous  or nonfilamontous t h a l l i  attached  chloroplast with  located pyrenoid;  a centrally  pale, crescent-shaped c e l l s vegetative  species  genera represented  Erythrotriehia,  world  chosen f o r t h i s  ( D i l l w y n ) J , Agardh, E r y t h r o t r i e h i a boryana  the chosen p l a n t s i l l u s t r a t e  w i t h i n t h e group and p r o v i d e  naiadum  complexity.  f_. c a r n e a ,  morphological  the simplest, possesses a u n i s e r i a t e  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  by m u l t i s e r i a t e f l a t  attached  a maximum  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  a r i s i n g from a monostromatic d i s c - l i k e  The  There  of North America; E r y t h r o c l a d i a ,  a n d S m i t h o r a , The s p e c i e s  thalli  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  blades  coast  form.  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  thallus with a rhizoidal  basal  distinctive,  Hollenberg.  Collectively,  typified  monospores and/or  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  on t h e P a c i f i c  Porphyropsis  (Montange) B e r t h o l d ,  thalli  or p a r i e t a l  may become t h e d o m i n a n t a l g a l  were E r y t h r o t r i e h i a c a r n e a  structural  a single stellate  cells.  habitats a particular  variability  by a r h i z o i d a l o r  may be f o r m e d f r o m a s y m m e t r i c a l d i v i s i o n s o f  T h i s f a m i l y has a c h i e v e d  (Anderson)  The  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 ;  containing  study  latter  one o f w h i c h , t h e B a n g i a l e s ,  cushion-like holdfast; cells  four  tu/o s u b c l a s s e s ;  two f a m i l i e s ! t h e B a n g i a c e a e a n d t h e E r y t h r o p e l t i d a c e a e .  Erythropeltidaceae  are  Into  multiseriate flat  h o l d f a s t , E. p u l v i n a t a i s  o r i g i n a t i n g from a m u l t i s t r o m a t i c l a r g e r , monostromatic t o d i s t r o m a t i c  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  taxonomy o f t h e E r y t h r o p e l t i d a c e a e  (see P I ,  J).  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 t h i s s i t u a t i o n but his e f f o r t s have been severely c r i t i c i z e d by c e r t a i n 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 i s stressed." This s i t u a t i o n has undoubtedly been p r e c i p i t a t e d by such factors as the s c a r c i t y of many species, the epiphytic nature of others and the r e l a t i v e l y small s i z e of the majority of the representatives.  Refined  laboratory culture techniques and the use of the electron microscope have proved helpful i n surmounting such b a r r i e r s i n recent years. These tools were applied i n the present study i n an attempt to add to e x i s t i n g information on t h i s family.  3.  Plate I . Scale drawings of the species of Erythropeltidaceae used i n t h i s study: E r y t h r o t r i c h i a camea, E r y t h r o t r i c h i a boryana, E r y t h r o t r i c h i a 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°9 W.)» Sooke, Vancouver Island, e  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.). A l l species of Erythrotrichia were collected at the latter site. At these locations material i s most plentiful during the months of May to October in the lower and middle intertidal zones. Smithora i s 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., KNO350 mg,, Fe(EDTA)- 1 mg., TRIS- 500 mg., s o i l H20- 50 ml., vitamin 8122 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 t h a l l i 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 f i e l d or immediately  5. upon a r r i v a l at the l a b o r a t o r y , m a t e r i a l was f i x e d i n a s o l u t i o n 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) f o r 1 h r . , washed and followed by p o s t f i x a t i o n i n a mixture of 2% osmium t e t r o x i d e (v/v) and phosphate b u f f e r at pH 7.2 (1:1) f o r 1£ h r . F i x a t i o n and p o s t f i x a t i o n were c a r r i e d out at 4°C or at room temperature. After a thorough washing i n b u f f e r , the m a t e r i a l 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 ( L u f t , 1961) or Maraglas 655 (Spurlock, K a t t i n e and Freeman, 1963). A l t e r n a t i v e l y , the m a t e r i a l was embedded i n Spurrfs medium (Spurr, 1969) d i r e c t l y a f t e r the ethanol s e r i e s . Thin s e c t i o n s were cut using glass knives or a Ou Pont diamond k n i f e on a L.K.8. Ultratome  I . P o s t s t a i n i n g was c a r r i e d out f o r 20-30 min.  i n uranyl acetate (2% s o l . (w/v) i n 50% methanol (v/v)) and f o r 5-10 min. i n lead c i t r a t e (Reynolds, 1963). The prepared m a t e r i a l was examined using a H i t a c h i HU 11A e l e c t r o n microscope or an AEI 801 A e l e c t r o n microscope operating with an a c c e l e r a t i n g voltage of 50 KV. L i g h t microscopy was done with l i v i n g m a t e r i a l using a Wild 0120 l i g h t microscope. When transverse s e c t i o n s were r e q u i r e d , specimens were prepared with a f r e e z i n g 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, i n Blankinship and Keeler, 1892). The i n i t i a l d e f i n i t i v e account of the morphology of t h i s 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, c e l l s square or s l i g h t l y 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 l a t t e r structures as a r i s i n g i n the terminal parts of the blade. The "sporocarp"  (carposporangium) gives r i s e 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) c a r r i e d out more extensive investigations on "Porphyra" naiadum and c o r r e c t l y interpreted the pattern of asexual reproduction i n describing the development of mature plants from monospores. She made numerous unsuccessful attempts to culture these reproductive u n i t s . 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, a n t h e r i d i a l areas and c e l l d i v i s i o n but her figures and descriptions are inconclusive. At a l a t e r date, a morphological  re-evaluation c a r r i e d out by  (1959) resulted i n the placement of t h i s alga i n a new and a new  Hollenberg  genus, Smithora ,  family, Erythropeltidaceae, Subsequently, t h i s action has been  given biochemical  support by Rees and Conway (1962), Hollenberg's  description  of the plant i s as followsi "Plants epiphytic, with numerous obovate to cuneate and monostromatic blades a r i s i n g from a prostrate cushion-like perennial multistratose  7. base; c e l l s with a single s t e l l a t e chromatophore; plants with no r h i z o i d a l processes a r i s i n g from the lower c e l l s of the blades; carpospores . formed i n i r r e g u l a r , mostly terminal s o r i , i n packets of eight; spermatangia a r i s i n g i n i r r e g u l a r s o r i toward the middle portions of the blades as small c e l l s cut o f f externally from colored c e l l s of the l o c a l l y distx-omatic portions of the blades; plants reproducing asexually by means of i r r e g u l a r , terminal, monostromatic gelatinous s o r i which are released as a u n i t . " The account of carpospore formation i s e s s e n t i a l l y based on  Hus'  description. Hollenberg states: " . . . i t i s p r a c t i c a l l y 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 c o r r e l a t i o n of monospore release with periods of low t i d e s . Like Knox (1926), he  was  largely unsuccessful i n c u l t u r i n g the spores. Most recently, Richardson and Dixon  (1969) have detailed the presence  of a filamentous "conchocelis" stage i n the l i f e cycle of Smithora. However, the authors observed none of the reproductive structures described above and included no l i g h t micrograph  of their findings.  Observations and Discussion. D i s t r i b u t i o n : The previously recorded d i s t r i b u t i o n of Smithora naiadurn i s from northern B r i t i s h Columbia to I s l a Magdalena, Baja C a l i f o r n i a , Mexico (Dawson, 1961). However, from c o l l e c t i o n s by various workers, the following Alaskan specimens are recorded i n the phycological herbarium at the University of B r i t i s h 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 P a c i f i c coast d i s t r i b u t i o n f a r Smithora naiadurn i s from Kodiak Island, Alaska to I s l a Magdalena, 8aja C a l i f o r n i a , 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 . 1 9 6 9 ; UBC 40146, 1 8 . V . 1 9 6 9 ; UBC 4 0 2 4 8 - 4 0 2 4 9 , 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 l a c k R . : UBC 13200, 8 . V I 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 3 5 7 6 9 , 3 6 5 8 1 , 3 6 9 7 7 , 1 1 . V I I I . 1 9 6 9 . Cape S u t i l : UBC 1 5 7 4 1 , 1 5 8 7 9 - 1 5 8 8 2 , 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 B a y : UBC 3 8 2 9 5 , 3 8 3 0 2 - 3 8 3 0 3 , 3 0 . V I 1 1 . 1 9 6 8 . Garden I s . : UBC 1 0 9 2 5 - 1 0 9 2 7 , 1 1 0 0 6 , 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 1 0 9 2 0 1 0 9 2 1 , 1 3 3 1 4 - 1 3 3 1 5 , 7 . V I I . 1 9 5 9 . Nootka I s . : UBC 37512, 2 2 . V I 1 1 . 1 9 6 8 ; UBC 3 7 1 6 8 , 3 7 6 7 9 , 3 7 6 8 7 , 2 4 . VI11.1 968. Perez Rock": UBC 1 0 9 2 2 - 1 0 9 2 3 , 1 3 5 2 4 , 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 1 8 7 6 0 , 4 , V I I I . 1 9 6 8 ; UBC 1 8 7 6 1 , 7 . V I . 1 9 6 8 ; UBC 3 6 7 9 5 , 3 6 8 0 2 , 2 8 . X I . 1 9 6 8 ; UBC 4*2359, 1 6 , V . 1 9 6 9 ; UBC 3 9 0 1 1 , 3 9 0 2 2 , 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 J o s e f Bay: UBC 3 8 6 1 4 - 3 8 6 1 5 , 3 9 0 2 3 , 3 1 , V I I I . 1 9 6 8 . 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 . W h i f f e n s p i t : UBC 38796, 2 8 . I X . 1 9 6 8 ; UBC 3 9 0 1 0 , 2 5 . X . 1 9 6 8 ; UBC 3 9 1 8 5 , 3 9 3 2 9 , 2 2 . X I . 1 9 6 8 ; UBC 46065, 8 . X I I . 1 9 7 1 . W i n t e r H a r b o u r : UBC 1 1 0 2 1 , 3 1 . V . 1 9 5 9 , Woutuer I s . : UBC 1 0 9 2 4 , 1 1 2 5 6 , 2 1 . V . 1 9 5 9 . Y e l l o w B l u f f : UBC 3 7 2 8 2 , 3 7 5 0 5 , 3 7 5 1 1 , 2 1 . V I I I . 1 9 6 8 ; UBC 915 WS, 2 1 . V I I I . 1 9 6 8 . _  EAST COAST VANCOUVER ISLAND: Cluxewe R . i UBC 1 7 0 7 5 , 5 . V I I . 1 9 6 2 . Deer I s : UBC 3 5 5 7 6 - 3 5 5 7 7 , 9 . V I I I . 1 9 6 8 . D i s c o v e r y I s . : UBC 38797, 3 . I X , 1 9 6 8 . F a l s e Head: UBC 3 8 6 1 7 - 3 8 6 1 6 , 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 2 9 5 7 4 , 2 9 6 0 9 , 2 4 . V . 1 9 6 7 . Grassy I s . : UBC 36056, 3 7 2 7 6 , 3 6 6 1 3 , 2 0 . V I I I . . 1 9 6 8 . Quadra I s . ( s o u t h ) : UBC 4 3 7 9 3 , 1 0 . I X . 1 9 7 0 . 0  SCOTT ISLAND GROUP: Cox I s . : UBC 3 6 0 5 5 , 3 6 5 9 2 , 3 6 7 5 2 , T r i a n g l e I s . t UBC 19578, 7 . I X . 1 9 6 4 .  12.VIII.1968.  MAINLAND (JOHNSON STRAIT AREA): N e v i l l e P t . : UBC 1 5 9 6 3 , 2 9 . V I . 1 9 6 2 . PRINCE RUPERT AREA: Chatham C h a n n e l : UBC 1 5 1 1 0 - 1 5 1 1 1 , 8 . V I I . 1 9 6 2 . I s . : UBC 1 6 4 5 0 , 2 6 . V I . 1 9 6 3 .  Digby  QUEEN CHARLOTTE ISLANDS? Anthony I s . : UBC 1 6 5 5 1 , 1 6 5 5 4 , 1 9 . V I . 1 9 6 3 . Marchant R e e f , Graham I s „ : UBC 2 1 3 4 3 , 2 2 3 0 1 , 4 . V I I . 1 9 6 5 , S t r i a e I s . : UBC 1 6 2 4 0 , 1 6 2 8 2 , 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 e x a m i n a t i o n of many of t h e above h e r b a r i u m 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 t o November, 1 9 7 1 , I am i n g e n e r a l agreement w i t h H o l l e n b e r g ' s  (1959)  9.  r e s u l t s . However, no convincing evidence o f carpospores or neutral spores was obtained. There was a marked difference among winter populations of Smithora i n d i f f e r e n t areas. At the c o l l e c t i n g s i t e s on Vancouver Island (Sooke and Point No Point) the basal cushions remained throughout the winter, but i n Stanley Park a l l traces of the plant disappeared i n mid-November only to become established again the fallowing spring. The l a t t e r c o l l e c t i n g area has a lower s a l i n i t y  (near a freshwater outflow) and i s much more protected  than the other s i t e s . If Richardson and Dixon's conchocelis phase e x i s t s , perhaps i t serves as an overwintering mechanism i n c e r t a i n populations. "Spermatangia"  as described by Hollenberg (1959) were observed regularly  i n the f a l l months of each year. However, there was no convincing i n d i c a t i o n 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 i n any of the Bangiophycidae. Although they w i l l be referred t o as  "spermatangia/spermatia"  i n t h i s report, perhaps they may be likened to the/3-spores of Porphyra (Conway, 1964) which are formed i n a d i f f e r e n t manner. These structures w i l l be discussed further i n Section VI. Cultured Material: Limited success was obtained i n c u l t u r i n g Smithora. S t e r i l e blades could be cultured for periods up to four months. Monosporic blades would not survive beyond one month, although during t h i s period they would continue to d i f f e r e n t i a t e and release monDspores. These structures are usually released terminally but, i n culture, i s o l a t e d patches of precociously released spores could be observed toward the center of the sorus (PI. I l l , F i g . 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 r e a d i l y , either singly or i n masses, to form basal cushions (PI, I I I , F i g . 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 i s 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 i s diagrammatically illustrated in PI. IV. Recently, Harlin (1971) has shown that Smithora will grow in the f i e l d on polyethylene strips approximating the dimension  of Phyllospadix leaves.  Present results on cultured material in this laboratory also appear to indicate that this alga i s not dependent on i t s host for any nutritive material as was thought by some authors (e.g. Knox, 1926). However, i t suggests t h a t a delicate set of environmental conditions i s 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 I I . (flap of B r i t i s h Columbia showing c o l l e c t i o n s i t e s of Smithora naiadurn, E r y t h r o t r i c h i a carnea, E r y t h r o t r i c h i a boryana and E r y t h r o t r i c h i a pulvinata as recorded i n the phycological herbarium of the University  of B r i t i s h  Columbia by various workers. Legend. Acous P e n i n s u l a . . . . . . . . . . . . . . . . 2 3 .  Lawton  Am phi t r X t e  L i PPy  Anthony  P t o . « . . « o . o . o . o . . o . e 3 1 .  I s . o o . . o o o . 0 . 0 . 0 0 0 0 . . • . 5 «  B a m f i e l d . . . . . . . . . . . . . . . . . . . . . . . 3 5 .  Black River... 0 . . o . . Brooks Peninsula.... BUnSby  . . . . . . . . . . .  3 8 . 2  I S . , « o » . o o o . o . o e o o . . . o o o 7 o  2  .  P t . o . . . . . . . . . . . . . . . . . . . . . . 3 7 . Pt  . O 9 o o o . e . , o . o , a o o o » o . o » « . o o l 9 .  Lookout l S e o . o e o . . o o o . » o . . « . o . e o o o o 3 2 o Macquinna P t . . » . . . . . . . . . . . . . . . . . . . . 2 8 . Warchant Reef...... 0 . . . . . . . . . . . . o . . 3 . Mills P e n i n s u l a . . 0 . . . . . . . . . . . . . . . . . 3 6 . Neville P t o o e o o o . . o a o . * o . o o o o o . o e o » 4 6 . Nootka I s . . Q o o o . « o , . « . . o a . . o . . . . . . . 2 7 . P6reZ R O C k e . o e . . . . © # . o * o e . » e . o . o . « . 2 9 . Piper's Lagoon . 4 4 . Plover Is. 0 . . . . . . . . . . . . . . . . <• . . . 0 . 0 . 7 . POX nt NO P O X n t . « , o , o a e o « o « e . . . a . o « o 3 9 o Qlawdzeet-Bell Passage.............9. Quadra I s . o . » . . e e . o o o * e . . o . o . . . . « . . 4 5 e San Josef B a y . . . . . . . . . . . . . . . . . . . . . . 1 7 . Stanley Park.............. . 4 2 . Striae I s . . . . . . . . . . . . . . . . . . . . . . . . . 4 .  Cape S c o t t . o o o o o . o . . . 9 0 . 0 . 0 . . e » 1 3 . Cape S u t i l . o o o . o « » . . o o . . . o e e a o t t l r j . Chatham C h a n n e l . . . . . . . . . 0 . . . . 1 . Cluxewe R i v e r . . . . . . . . . . . . . . . . . . 4 7 . COX IS. ... o o o » . e e e . o f t e d . . o e o o o . 1 5 . Deer I s . . . . . . . . . . . . . . . . . . . . . . . . 4 9 . Dxgby I S o . . . . « « e o , o . . . . . . . . . . . e 2 . Discovery I s . . . . . . . . . . . . . . . . . . . 4 1 . Experiment Bight . . . . . . . . . . 1 2 . False Head......... . . . . . . 4 8 , Fisherman B a y . . . . . . . < • * , . . . « « e . 1 1 . Tofino...............«,,..•...*•*..30* Gabriola I s . . . . . . . . . 4 3 . Topknot P t . . 1 8 . Garden Is . . 0 . . . . 3 3 . Triangle I s , . . . . . . . . . . . . . . . . . . . . . . . 1 4 . Grassy I s . . . . . 2 5 . Ulhiffen s p i t . . . « . . .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 . 0  0  0  12.  PLATE I I I . Light micrographs.  Smithora  F i g . 1, Basal holdfast (bh) i n culture producing second generation monospores (arrow). F i g . 2. D i f f e r e n t i a t i n g monospore i n cultured f i r s t generation basal pad. vc denotes vegetative c e l l o F i g . 3. Second generation monospore i n c u l t u r e Fig. 4  0  e  2-celled stage of germinating monospore i n c u l t u r e .  F i g , 5, Young cultured basal holdfast. F i g . 6. Portion of mature blada showing d i f f e r e n t i a t i n g monospores (dm)  p  vegetative c e l l area (vc) and an i s o l a t e d area of precociously released monospores (rm).  13.  PLATE  IV.  Diagram of the p o s s i b l e 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 s t e p s of the schema.  IV  stage)  14.  b) E r y t h r o t r i e h i a Introduction. Some 36 s p e c i e s o f E r t h r o t r i c h i a Areschoug have been d e s c r i b e d  from  v a r i o u s p a r t s of t h e w o r l d . Because o f t h i s wide d i s t r i b u t i o n , an examination of each r e c o r d e d difficult.  s p e c i e s o r growth form would be, a t b e s t ,  extremely  T h i s s i t u a t i o n has r e s u l t e d i n taxonomic c o n f u s i o n and u n c e r t a i n t y .  H i s t o r i c a l l y , t h e f o l l o w i n g c h a r a c t e r i s e s have been used t o determine s p e c i e s o f E r y t h r o t r i e h i a ; c o l o u r , s i z e , form o f f i l a m e n t polysiphonous, branching  (monosiphonous,  ribbon-shaped, e t c . ) , type o f c h 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 ) ,  and host s p e c i f i c i t y . Numerous attempts have been made t o a s s i g n  some type o f n a t u r a l system o f c l a s s i f i c a t i o n t o t h e s e d i v i d e d t h e genus i n t o two groups; one f o r m i n g r i s e to f i l a m e n t s , the other forming f o l l o w e d by t h e secondary p r o c e s s  those  by a m u l t i c e l l u l a r  attached  which d i r e c t l y  attached  Hamel  (1882)  give  which g i v s r i s e t o b a s a l  of filament formation.  d i s t i n g u i s h e d t h r e e c a t e g o r i e s ; those basal c e l l ,  spores  spores  plants, Berthold  discs,  (1929)  by a s i n g l e , l o b e d o r unlobed  by a number o f r h i z o i d a l c e l l s  and those  attached  d i s c . Tanaka (1952) proposed a taxonomic b i s e c t i o n on  the b a s i s o f 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 r e c e n t l y , Heerebout (1968) has recommended t h s r e c o g n i t i o n o f o n l y t h r e e s p e c i e s , due t o t h B morphological was  variability  o f t h e s e p l a n t s i n c u l t u r e . U n f o r t u n a t e l y , he  unable t o examine r e p r e s e n t a t i v e s o f a l l d e s c r i b e d s p e c i e s and r e j e c t e d  many o n l y on t h e b a s i s o f p u b l i s h e d r e p o r t s . H i s d e s c r i p t i o n o f t h e genus and  key t o t h e s p e c i e s a r e as f o l l o w s : " E r y t h r o t r i e h i a , T h a l l u s e r e c t , filamentous or ribbon-shaped, o f t e n aiith a d i s c o r cushion-shaped attachment o r g a n . Filamentous t h a l l u s branched or unbranched, mono- or p o l y s i p h o n o u s . Ribbon-shaped t h a l l u s always monostromatic; unbranched. C e l l s b r i c k r e d , l e n g t h about 10-25 m i c r o n s . Chromatophore s t e l l a t e w i t h a d i s t i n c t p y r e n o i d . Asexual r e p r o d u c t i o n by monospores; l i f e c y c l e w i t h a c o n c h o c e l i s s t a g e . P i t c o n n e c t i o n s never seen.  Key t o t h e s p e c i e s . 1 a . T h a l l u s c o n s i s t i n g o f rows o f c e l l s a r r a n g e d i n one p l a n e , g i v i n g i t a r i b b o n - s h a p e d appearance. N e a r l y always w i t h a b a s a l d i s c , i n young s t a g e s sometimes o n l y a b a s a l d i s c i s present . E. b o r y a n a . 1b. T h a l l u s mono- o r p o l y s i p h o n o u s ,  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 a t t a c h e d by a b a s a l d i s c o r by s m a l l p r o t u b e r a n c e s of the basal c e l l carnea. 2b. T h a l l u s w i t h a l o n g b o r i n g r h i z o i d , composed o f 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 a t t a c h e d t o g a s t r o p o d s . . . _E. w e l w i t s c h i i . " Heerebout c o n s i d e r e d 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- o r p o l y s i p h o n y  and mode o f attachment t o be u n r e l i a b l e f o r taxonomic  p u r p o s e s . West (1966) has a l s o q u e s t i o n e d taxonomic c r i t e r i a .  the v a l i d i t y of using c e r t a i n  I n c o n t r a s t , Dangeard (1968,1969) l i s t s 34 s p e c i e s and  c o l o u r f u l l y d e s c r i b e s Heerebout's r e v i s i o n as f o l l o w s : "...sans doute en p r e m i e r e dans c e 'massacre' d'especes...". D e t a i l s o f c e r t a i n reproductive processes equally obscure.  i n E r y t h r o t r i e h i a are  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 o f t h e r e s u l t i n g daughter c e l l s forms a s p o r e .  I n one s p e c i e s  (E_. w e l w i t s c h i i )  an u n d i v i d e d 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 . R e p o r t s o f s e x u 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 described spermatia  b e i n g c u t 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 t o a f i l a m e n t a d j a c e n t t o t h e supposed carpogonium. Subsequent r e p o r t s o f such e v e n t s by Gardner (1927), B a a r d s e t h ( 1 9 4 1 ) 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 p r o c e s s . F o r example,  concerning  p o s t - f e r t i l i z a t i o n e v e n t s , B e r t h o l d (1882) d e s c r i b e s an u n d i v i d e d ,  fertilized  carpogonium b e i n g r e l e a s e d whereas Tanaka (1944) s t a t e s t h a t  fertilized  carpogonia  d i v i d e t o produce a "few" c a r p o s p o r e s .  Hserebout (1968)  16.  has reported the presence of a conchocelis phase of the l i f e cycle of E r y t h r o t r i c h i a which hB presumes to have grown from carpospores, although no d i r e c t evidence of this i s presented. Thus, i t i s obvious that there i s a need of c r i t i c a l research i n almost every phycological aspect of t h i s genus. Observations and Discussion. P a c i f i c Coast D i s t r i b u t i o n ^ : Three species of the genus E r y t h r o t r i c h i a are reported here according to the r e v i s i o n proposed by Heerebout (1968), with the exception that IE. pulvinata has been retained as a v a l i d species. The distinguishing morphological feature i s the presence of a r e l a t i v e l y large, monospore producing, multistromatic, basal holdfast. In addition, E_. pulvinata appears to occupy a s p e c i a l i z e d habitat (epiphytic on the u t r i c l e s of Codium f r a g i l e ) . In l i g h t of these findings, a re-examination of specimens of E r y t h r o t r i c h i a reported by various authors on the P a c i f i c coast of North America i s required i n order to obtain a more complete d i s t r i b u t i o n a l record of t h i s taxon, E r y t h r o t r i c h i a bogyana The previously recorded d i s t r i b u t i o n i s from Punta Baja to Bahia Asuncion, 8aja C a l i f o r n i a , Mexico (Dawson, 1961). BRITISH COLUMBIAN COLLECTION RECORDS. WEST COAST VANCOUVER ISLAND: Point No Point (Glacier P t , ) : UBC 1260 HIS, 2.VII.1971 (epiphytic on Phyllospadix s c o u l e r i and Smithora naiadurn ). E r y t h r o t r i c h i a carnaa The previously recorded d i s t r i b u t i o n i s from Monterey, C a l i f o r n i a to Golfo Dulse, Costa Rica; Clipperton Is. (Dawson, 1961) with a northward T h i s part of the t h e s i 3 i s based on an a r t i c l e by J.W. Markham, D.L. MnBride and P.R. Newroth which has been accepted f o r publication i n Syesi3. 1  17. extension by Norris and West (1967) at Shilshole Bay Marina, S e a t t l e , 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 p a c i f i c a ) ; UBC 972 WS, 27.V.1959 (epiphytic on C o r a l l i n a 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 (epiphytic on Lomentaria sp„).  895 WS,  2.VIII.1959  E r y t h r o t r i c h i a pulvinata The previously recorded d i s t r i b u t i o n i s from Middle Bay, Bahia  Oregon to  Asuncion, Baja C a l i f o r n i a , 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 P t . ) . UBC 1259 WS, 6.X.1971 (epiphytic on Codium f r a g i l e ) . F i e l d and Cultured Material;  Both E_. carnea and E. boryana were t y p i c a l  i n appearance (PI. V, F i g . 7,11), readily d i f f e r e n t i a t i n g and r e l e a s i n g monospores i n c u l t u r e . Upon germination,  the monospores of JE. carnea exhibit  a p o l a r i t y (PI. V, F i g . 8), forming one or two erect filaments (PI. V, F i g , 9). In E_. boryana monospores divide to produce a simple monostromatic basal disc (PI. V, F i g , 12) from which filaments are derived (PI. V, F i g . 11). E. carnea regularly exhibited i n s i t u monospore germination of branching boryana may  to give an appearance  (PI. V., F i g . 10) as described by Dixon and West (1967). E. also be attached to Smithora.(PI.  V, F i g . 13). Structures which  could be interpreted as spermatia and carpospores  were observed i n freshly  c o l l e c t e d E_. boryana and w i l l be discussed at length i n Section VI. Very few studies have been carried out on JE. pulvinata. In his o r i g i n a l description Gardner (1927) states that the pad may  be found without  the  filamentous t h a l l u s . In the population used for t h i s study, I have observed  18.  filamentous t h a l l i 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 i s also capable of producing these reproductive structures (PI. VI, Fig. 15). Upon germination in culture, they form a c e l l 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  JE. carnea. Mature filaments producing monospores (arrow) i n c u l t u r e .  0  F i g . 8. E_. carnea. Bipolar germination of cultured monospores. F i g . 9. E_, carnea. Juvenile filaments daveloping from one monospore i n culture ("tripolar" germination). Fig  0  10  o  E_„ carnea  0  In s i t u germination of cultured monospores.  F i g . 11. E_. boryana. Mature filaments and basal disc. F i g . 12. E_. boryana. Cultured basal pad. F i g . 13. _E. boryana. Freezing microtome section of association between E_. boryana and Smithora (h).  20  PLATE VI. E r y t h r o t r i e h i a pulvinata Pig. 14. Portion of mature filament. F i g . 15. Monospore production F i g . 16  0  (arrow) from basal • holdfast (bh) i n c u l t u r e .  4-celled stage of cultured, germinating monospore.  F i g , 17, Juvenile pad attached to u t r i c l e of Codium f r a g i l e , (h). F i g , 18. Mature pad growing i n culture on glass Petri dish. F i g , 19, Edge of cultured pad showing elongate c e l l u l a r shapes.  VI  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 i s 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 i s 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 i r r e g u l a r protoplast with a large chloroplast, mitochondria, endoplasmic reticulum, dictyosomes and a s i n g l e nucleus (PI. VII, F i g . 2). Additional structures within the c e l l include floridean starch granules, The c e l l wall, composed of two  vacuoles and many multivesicular bodies. or three d i s t i n c t layers, appears to  be s i m i l a r to that of Porphyra (Frei and Preston, 1964;  Bourne,1971) (PI.  VII, F i g . 2; PI. VIII, F i g . 5). According to F r e i and Preston (1964), the fibrous organization of these c e l l wall layers i s due to m i c r o f i b r i l s composed of/3-1,3 linked xylans. The layering e f f e c t seems to be due to a d i f f e r e n t organization of the m i c r o f i b r i l s with the outer layers being more compacted. A n o n f i b r i l l a r outermost layer which could be analagous to the mannan-containing c u t i c l B of Porphyra (Frei and Hanic and Craigie, 1969)  was  Preston,  also noted i n Smithora.  The shape of the protoplast i s much more variable than any reported i n the red algae, Pseudopodia-like extensions and are evident i n most c e l l s  1964;  previously  invaginations  (PI, VII, F i g . 2). Wall material seems to be  i s o l a t e d within the c e l l when the invaginations  are viewed i n cross-section.  The s i n g l e , lobed chloroplast, bounded by a double membrane, occupies most of the c e l l the lobes may  (PI. VII, F i g . 2; PI. VIII, F i g . 5). In some sections  appear as separate e n t i t i e s i s o l a t e d from the main body of  the chloroplast (PI. VII, F i g . 2; PI. VIII, F i g . 3). The arrangement of chloroplast lamellae i n younger c e l l s i s very regular, each i n d i v i d u a l thylakoid being orientated p a r a l l e l to the others (PI. VII, F i g . 2). There i s 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 c h a r a c t e r i s t i c of older c e l l s 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 pyrenoid  0  Fusion and  forking of lamellar edges often occur in these areas. Intermittent fusion of photosynthetic lamellae i s 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 1965). Lamellar spirals similar to those reported in 8  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 i s often penetrated by lamellae which appear swollen and frequently form common vesicles within i t s matrix VII, Fig. 2; PI. VIII, Fig. 5; PI.  (PI,  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 PI. 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 l o s t during preparation of the specimens. Typical red algal f l o r i d e a n starch occurs within the cytoplasm i n the form of e l l i p s o i d a l granules (PI. VII, F i g . 2; PI, VIII, F i g , 5). These granules vary i n staining i n t e n s i t y i n a manner s i m i l a r to that reported i n Porphyridium  (Gantt and Conti, 1965).  Mitochondria with tubular c r i s t a s are numerous and variable i n s i z e and shape (PI. VII, F i g . 2| PI, IX, F i g . 8} PI. X, F i g , 10). Infrequently, a mitochondrion i n a "doughnut" or r i n g formation was noted (PI. IX, F i g . 8). Other structures often occur within the centre of these atypical mitochondria. No extremely long or branched mitochondria s i m i l a r to those found i n Porphyridium  (Gantt and Conti, 1965) were observed i n Smithora.  Both rough and smooth endoplasmic reticulum occur i n these c e l l s , usually following the contour of the plasmalemma or the nuclear envelope. (PI. VII, F i g . 2; PI. VIII, F i g . 4; PI. X, F i g . 10). However, no connections between these e n t i t i e s have been found. In addition, what appears to be more densely s t a i n i n g ER i s seen intermittently i n the protoplast extensions (PI. IX, F i g . 8). One or more dictyosomes are often found i n a single c e l l u l a r cross-section (PI. VII, F i g . 2). They consist of the usual flattened cisternae and associated v e s i c l e s . No p a r t i c u l a r l o c a t i o n or function can be assigned to these organelles i n the vegetative c e l l . Younger vegetative c e l l s contain few well defined vacuoles. Howsver, i n older c e l l s these structures seem to increase i n s i z e and number (PI. VIII, F i g . 5; PI. X, F i g  e  13).  The nucleus i s t y p i c a l l y eucaryotic (PI. VII, F i g . 2; PI. VIII, F i g . 4), The evenly granular nucleoplasm and densely s t a i n i n g nucleolus are surrounded by a porous nuclear envelope. The nucleolus often occupies a peripheral p o s i t i o n i n the nucleus of Smithora. However, t h i s structure i s not necessarily orientated toward the chloroplast as reported i n 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 c e l l 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 i n plants which seem to originate from the plasmalemma should be termed plasmalemmasomes and those from within the cytoplasm, lomasomes. Plasmalemmasomes 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 i s often not clear. Lomasome-like structures have been reported previously in other red algae, e.g. Lomentaria (Bouck, 1962), Laurencia (Bisalputra et a l . , 195?) and Pseudoqloiophloea (Ramus, 1969). In addition, single vesicular structures were frequently noted in the c e l l wall near the plasmalemma (PI. X, Fig. 13). Similar structures were described in Laurencia (Bisalputra et a l . , 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 c e l l 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. Discussion. From time t o t i m e v a r i o u s a u t h o r s  have p r e s e n t e d  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 t h e Rhodophyta and o t h e r a l g a l groups ( S m i t h , 1 9 5 5 ) . 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 t h e B a n g i o p h y c i d a e , s i n c e t h i s group i s t h o u g h t t o p o s s e s s 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 o f s e x u a l r e p r o d u c t i o n i n some s p e c i e s , p r e s e n c e o f p h y c o b i l i n s and l a c k o f f l a g e l l a . However, u n t i l f u r t h e r r e s e a r c h i s c a r r i e d o u t any such p r o p o s a l 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 , t h e presence of unassociated the r e d algae  photosynthetic lamellae i s considered t y p i c a l of  ( G i b b s , 1960,1962a). T h i s has been used on o c c a s i o n as an  a d d i t i o n a l taxonomic c h a r a c t e r and c o u l d s u p p o r t  a proposed r e l a t i o n s h i p  between t h e r e d a l g a e and t h e Cyanophyta. W h i l e S m i t h o r a e x h i b i t s some o f t h e u l t r a s t r u c t u r a l f e a t u r e s c o n s i d e r e d t y p i c a l o f t h e 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 t h e 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 p o s s e s s i n g 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 s t a c k s w i t h i n t h e c h l o r o p l a s t . These bands appear t o be p r i m i t i v e s i n c e t h e y a r e by no means e x t e n s i v e and a r e a l m o s t 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 t h e s e s t r u c t u r e s occur randomly i n t h e c h l o r o p l a s t s  o f o l d e r as w e l l as younger c e l l s , they a r e n o t b e l i e v e d t o be i n v o l v e d i n c e l l d i v i s i o n . F r e q u a n t l y one end o f an i n n e r 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 s t a c k w h i l e t h e o t h e r end forms o r c o n t r i b u t e s t o a s w o l l e n w i t h i n t h e p y r e n o i d . T h i s may i n d i c a t e t h a t t h e bands a r e an i n t e g r a l of t h e c h l o r o p l a s t and p e r f o r m an i m p o r t a n t  vesicle part  f u n c t i o n . Consequently,, i t i s  e v i d e n t t h a t o t h e r members o f t h e Rhodophyta, i n p a r t i c u l a r t h a " l e s s advanced" members, s h o u l d be examined t o d e t e r m i n e t h e e x t e n t o f t h i s  27. banding phenomenon. Thus far there seem to be two s t r u c t u r a l types of red a l g a l chloroplasts depending upon the presence or absence of a pyrenoid. From l i g h t studies i t i s reported  microscopic  that c e r t a i n members of the Nemaliales and many of  the Bangiophycidaa possess pyrenoid-containing  chloroplasts ( F r i t s c h , 1945).  However, within this group the arrangement of the thylakoids i n r e l a t i o n to the chloroplast envelope seems to vary. Smithora displays numerous sheet-like photosynthetic  lamellae which tend to p a r a l l e l the contour of  the chloroplast envelope. Some members of the Nemaliales, e.g. Thorea (Bischoff, 1965)  and Acrochaetium (fflcBride, unpubl.) and the u n i c e l l u l a r  bangiophyte Rhodosorus (Giraud, 1963)  seem to possess a s i m i l a r chloroplast  structure. K y l i n i a (Gibbs, 1962a), Namalion (Gibbs, 1962a,1962b) and Rhodochorton (Witrakos, 1960), other members of the Nemaliales, may  also  possess t h i s feature although published micrographs are inconclusive. In contrast, Porphyra (Bourne, 1971), Porphyridium (Brody and Vatter, and others), Rhodella (Evans, 1970)  and Banqia (Honsell, 1963)  1959  have photo-  synthetic lamellae which terminate at the chloroplast envelope. The s i g n i f i c a n c e of t h i s well defined difference i n the u l t r a s t r u c t u r e of red a l g a l , pyrenoidcontaining 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 c e l l s s lomasomes and plasmalemmasomes. These authors suggest that lomasomes, which have been observed i n a large number of plants, may  be involved i n 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 w a l l . Various types of  these multivesicular bodies have been noted i n Smithora. Since formation of a thick supporting  c e l l wall i n t h i s alga would undoubtedly e n t a i l  28. important c e l l u l a r functions, i t mould seem that t h i s hypothesis i s not unreasonable. Ramus (1969) noted an abundance of lomasome-like bodies i n Pseudoqloiophloea associated with the formation  of c e l l wall material between  dividing c e l l s . It i s of i n t e r e s t that many more of these structures are observed i n older c e l l s of Smithora. Since Smithora's main method of reproduction seems to be vegetative  (portions of the monosporic t h a l l u s are released p e r i o d i c a l l y )  (Hollenberg, 1959), there i s a p o s s i b i l i t y that some of the above mentioned structures may  be involved i n 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 i n the fungus Rhizopus may  be associated with c e l l wall degradation.  Since  the  c e l l s of Smithora contain r e l a t i v e l y few vacuoles, another functional poss 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 s i n g l e v e s i c l e s which occur frequently i n the c e l l wall near the plasmalemma may  be involved  i n one or more of the functions discussed here. However, not u n t i l cytochemical and autoradiographic  techniques progress further can  explicit  functions be assigned to these various structures. It i s known that c e r t a i n members of the red algae are p a r a s i t i c ( F r i t s c h , 1945). The f a c t that Smithora i s an obligate epiphyte tends to arouse one's suspicions that a p a r a s i t i c r e l a t i o n s h i p may no obvious evidence of parasitism was  e x i s t . However,  observed i n sections through host  and epiphytic t i s s u e . Since t h i s alga has a well developed apparatus, t h i s i s not unexpected. Nevertheless,  photosynthetic  i t i s of i n t e r e s t that  Harlin (1971) has reported the possible occurrence of a nutrient transfer in this situation.  29  0  There have been several) l i g h t microscopic reports of p i t connections i n the Bangiophycidae  (see Dixon,, 1963 f o r review). Recent u l t r a s t r u c t u r a l  accounts of these structures i n the conchocelis phase of Porphyra (Lee and F u l t z , 1970| Bourne, Conway and Cole, 1970) and the conchocelis phase of Banqia (Sommerfeld and Leeper, 1970) have conclusively shown that t h i s c h a r a c t e r i s t i c can no longer be used to separate the rhodophycean subclasses. However, there i s no evidence of p i t connections i n Smithora. Each c e l l i s a separate e n t i t y , no connections of any type remain after d i v i s i o n .  30.  PLATE V I I . Smithora Fig„ 2. C r o s s - s e c t i o n  of a t y p i c a l  c h l o r o p l a s t envelope  (CE), p y r e n o i d  reticulum material  (ER). dictyosomes (CW),  DMA  (D)„  vegetative  cell  with a c h l o r o p l a s t (C)^,  (P), m i t o c h o n d r i a (ffl)» endoplasmic  f l o r i d e a n s t a r c h granules  pockets (white arrow) and o s m i o p h i l i c  a r r o w ) . Double arrow i n d i c a t e s a l a m e l l a r  (FS), c e l l  granules  wall  (black  stack.  V  'The d i s p a r i t y i n the types of n o t a t i o n s 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 s e c t i o n s of t h i s r e p o r t i s due t o the use of m a t e r i a l p r e v i o u s l y p u b l i s h e d by the author over a p e r i o d of time.  31.  PLATE  Will.  Smithora F i g . 3.  Chloroplast  F i g . 4.  Nucleus c o n s i s t i n g of nucleoplasm and  by  a nuclear  F i g . 5.  envelope  exhibiting a spiral  v e s i c u l a r bodies  v e s i c l e - l i k e lamellae  (Mv)  arrangement of a nucleolus  thylakoids.  (Nu)  surrounded  (NE).  Older c e l l with c h l o r o p l a s t and  containing swollen  and  lobe  centrally located (LV). Vacuoles  (V) and  are t y p i c a l of o l d e r c e l l s . T h y l a k o i d  f l o r i d e a n s t a r c h granules  (FS) are a l s o  present.  pyrenoid multi-  fusion  (arrow)  VIII  32.  PLATE  IX.  Smithora Fig.  6.  T h y l a k o i d s t a c k . Arrow i n d i c a t e s p e r i o d i c b r a n c h i n g  associated with stack Fig.  ?. T h y l a k o i d s t a c k . Note l a m e l l a r b r a n c h i n g  in  other m i t o c h o n d r i a  the c e n t r e . D a r k l y  an a d j a c e n t Fig.  9,  (Rffl) e x h i b i t i n g c r i s t a e and and  swollen  vesicle-  mitochondrial  a v e s i c u l a r s t r u c t u r e (ve) are l o c a t e d  stained ER-like material  cytoplasmic  (arrow) and  pyrenoid.  8. Ring shaped m i t o c h o n d r i o n  e n v e l o p e . Two  lamellae  formation.  l i k e l a m e l l a e w i t h i n the Fig.  of  (arrows) i s a l s o p r e s e n t  in  lobe.  P o i n t of j u n c t i o n between a l g a l c e l l  wall  (HUl). P a r t of a l g a l p r o t o p l a s t (Pr) i s a l s o shown.  (AW)  and  host c e l l  wall  IX  33.  PLATE X. Smithora F i g . 10. Section through portions of too neighbouring c e l l s  illustrating  multivesicular bodies (arrows) consisting mainly of spherical v e s i c l e s . Other structures include a mitochondrion, endoplasmic reticulum, lobe (CL) and c e l l wall  chloroplast with  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 v e s i c l e s . F i g . 13. Lamellar bodies (arrows) situated i n the c e l l wall and near vacuoles. F i g . 14© Numerous single v e s i c l e s i n 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 s p e c i e s used i n t h i s s t u d y were JE. c a r n e a ( 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 observation  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 t o Smithora  naiadurn ( S e c t i o n IVa) i s v e r y e v i d e n t . (PI.  Indeed, t h e c e l l w a l l o f £. boryana  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 t o  t h a t o f S m i t h o r a , c o n s i s t i n g o f 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 t r a n s p a r e n t  matrix.  However, t h e w a l l o f E_.  c a r n e a 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 t r a n s p a r e n t  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 t h e s u r f a c e o f 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 darkly s t a i n i n g l i n e i n the c e l l w a l l adjacent  t o t h e plasmalemma ( P I . X V I , F i g . 1 6 ) . T h i s l i n e i s p a r t i c u l a r l y i n r e g i o n s where t h e plasmalemma i s most c o n v o l u t e d  and c o u l d  noticeable  represent  an a r e a o f 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 o f Z. c a r n e a ( 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 t h a n t h a t o f E_. b o r y a n a ( 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 . 1 2 ) , The a c t u a l shape o f t h e p r o t o p l a s t v a r i e s presumably a c c o r d i n g  to the rate of c e l l d i v i s i o n  considerably,  (West, 1 9 6 6 ) .  A l l s p e c i e s o f 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 . 1 2 ) . S e c t i o n s t h r o u g h c h l o r o p l a s t m a t e r i a l show t h a t t h e m a j o r i t y o f t h y l a k o i d s a r e s i t u a t e d p a r a l l e l t o and f o l l o w t h e c o n t o u r o f t h e c h l o r o p l a s t e n v e l o p e (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 o f 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. C o l e i n t h e P r o c e e d i n g s o f t h e 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 plast lamellae  the only plant to regularly exhibit phycobilisomes on chloro(PI. XII, Fig.3). They are s i m i l a r to those described i n  Porphyridium (Gantt and Conti, 1965,1966? Gantt, Edwards and Conti, 1968). Ribosome-liks bodies were often observed between chloroplast lamellae Fig.  (PI. XIV,  10). In addition, possible DNA-containing areas (Bisalputra and Bisalputra,  1967)  and d r o p l e t - l i k e inclusions are scattered between the  A constant  lamellae,  feature of the chloroplast i s the presence of a c e n t r a l l y  located pyrenoid traversed by varying numbers of lamellae  (PI, XII, F i g . 3;  PI. XIII, F i g , 7} PI. XVI, F i g , 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 i n one s e c t i o n a l plane was  seldom greater than f i v e and usually these structures  ran i n a r e l a t i v e l y s t r a i g h t l i n e through the pyrenoid  (PI. XII, F i g . 3),  However, i n JE. boryana and JE. pulvinata there are large numbers of highly convoluted Fig.  lamellae within the pyrenoid matrix (PI. XIII, F i g . 7; PI.  12; PI. XVI, F i g . 14). The nucleus i s t y p i c a l l y eukaryotic  XIV,  XV,  (PI. XI, F i g . 2; PI. XII, F i g . 3;  PI,  F i g . 9; PI. XV, F i g , 12) and only very r a r e l y i s the outer membrane of the  nuclear envelope continuous with cisternae of ER  (PI. XIV,  F i g . 9). This  feature i s common i n 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, F i g . 16), vacuoles (PI, XI, F i g . 2;  XIII, F i g . 7; PI. XV,  PI.  F i g . 12) and f l o r i d e a n starch grains ( P i . XIV,  resemble those described i n the vegetative c e l l of Smithora (Section  F i g . 9) IVa).  An i n t r i g u i n g s t r u c t u r a l c h a r a c t e r i s t i c of a l g a l epiphytes, e s p e c i a l l y those which exhibit a host s p e c i f i c i t y , i s the zone of attachment. E_, pulvinata thus far appears to be exclusively epiphytic on the u t r i c l e s of Codium f r a g i l e (PI. XV,  F i g . 11) but t h i s zone shows no evidence of i n t e r a l g a l  36. protoplasmic association (PI, XVI, F i g , 13). The respective c e l l walls are merely joined i n a simple, smooth plana. In t h i s region, foreign objects are occasionally embedded i n the c e l l wall of the epiphyte. One such object bore a resemblance to a b a c t e r i a l c e l l  (PI. X V I , F i g . 15). JE. boryana i s  less host s p e c i f i c than JE. pulvinata. It i s of i n t e r e s t that E_. boryana i s epiphytic on Smithora since these algae are almost u l t r a s t r u c t u r a l l y i d e n t i c a l . In sections through the zone of attachment i t i s nearly impossible to d i f f e r e n t i a t e the respective c e l l walls (PI. XIV, F i g . 8). Discussion. An u l t r a s t r u c t u r a l comparison of c l o s e l y r e l a t e d a l g a l species has proven to be of taxonomic value i n c e r t a i n instances. In p a r t i c u l a r , "less advanced" red algae which have few d e f i n i t e morphological to u t i l i z e may  characteristics  lend themselves to this procedure. Evan's (1970) study of  Rhodella maculata has shown the usefulness of t h i s approach. The three species of E r y t h r o t r i c h i a chosen for t h i s study appear to i l l u s t r a t e a maximum morphological  v a r i a b i l i t y within the genus, but very  few concrete u l t r a s t r u c t u r a l differences were noted. The most constant difference observed was the form and smaller number of pyrenoid traversing lamellae i n 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 i n the u l t r a s t r u c t u r e 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 c e r t a i n species of D i n o f l a g e l l a t a . In addition, the u l t r a s t r u c t u r a l appearance of the c e l l wall i n JE. carnea seems to separate t h i s species from the others. West (1966) has also discussed c e r t a i n unusual features of the wall of E. carnea.  37. One must be extremely cautious i n the i n t e r p r e t a t i o n of i n t e r s p e c i f i c u l t r a s t r u c t u r a l differences as they could merely be due to variations i n habitat, season, age of tha plant, stage of the l i f e cycle, e t c . Indeed, Hori (1972) has shown u l t r a s t r u c t u r a l differences which occur between d i f f e r e n t stages i n the l i f e cycle of the same species of Monostroma. The presence of phycobilisomes i n I E . carnea i s i n t e r e s t i n g i n view of the general absence of these structures i n the other members of the Erythropeltidaceae examined. Gantt and Conti (1965) state that these s t r u c t ures are very s e n s i t i v e to f i x a t i o n conditions. However, a l l material used i n t h i s study was f i x e d i n the same manner. In f a c t , i n E_. carnea i t was possible to observe phycobilisomes i n one c e l l but not i n an adjacent c e l l of the same filament. If their presence depends on the q u a l i t y of chemical f i x a t i o n , one can only speculate why c e l l s i n the same filament of approximately the same age should react d i f f e r e n t l y to these procedures. Thus, E_. carnea, E_, boryana and Z, pulvinata represent d i s t i n c t species on a gross morphological l e v e l but show few u l t r a s t r u c t u r a l differences. In addition, the f i n e structure of the genus E r y t h r o t r i e h i a appears to be similar to that of Smithora suggesting that these genera are c l o s e l y related members of the Erythropeltidaceae. I t could also offer further support of Hollenberg's (1959) taxonomic reassignment of the l a t t e r genus.  38.  PLATE XI. E r y t h r o t r i c h i a carnea F i g . 1.  Light micrograph of portion of filament.  F i g . 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 c e n t r a l l y located pyrenoid vacuoles (V).  (P), nucleus (N)  and  XI  39.  PLATE X I I . Erythrotrichia Fig,  3,  carnea  Portion of c e l l showing nature of lamellae which traverse the  pyrenoid  (P) i n the chloroplast (C). Note the rough texture of the thylakoids  i n d i c a t i n g the presence of phycobilisomes.  Prominent nucleus (N) and  cell  wall (W) are also shown. Fig.  4, Portion of pyrenoid with swollen lamellae containing loosely  organized Fig.  fibrils.  5. C e l l 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 c e l l wall (W). chloroplast (C) with pyrenoid (P) and vacuoles (V).  XIII  41.  PLATE  XIV.  E r y t h r o t r i e h i a boryana Fig.  8, S e c t i o n t h r o u g h t h e zone of attachment between E. boryana  S m i t h o r a . 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  and  S_P denotes S m i t h o r a '  protoplast. Fig.  9. P o r t i o n of n u c l e u s (N) i l l u s t r a t i n g  ER-nuclear envelope c o n n e c t i o n  (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  F i g . 10.  P o r t i o n of c h l o r o p l a s t l o b e showing e l e c t r o n dense  bodies  (arrow).  grain. ribosome-like  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 c e l l wall.  XV  43.  PLATE XVI. E r y t h r o t r i e h i a pulvinata Fig  0  13. Electron micrograph i l l u s t r a t i n g attachment zone of alga. C a l l  wall of host, alga (Codium f r a g i l e ) (Hul) wall of E_. pulvinata (Ul) and 0  protoplast of E_„ pulvinata (EP) are shown. Fig.  14. Section through pyrenoid showing convoluted  Note the loosely organized f i b r i l s within the Fig,  lamellae.  15. Area of attachment with foreign objects embedded i n wall of E_„  pulvinata Fig,  traversing lamellae.  (W)„  16. Peripheral region of protoplast with mitochondria (M), a dictyosome  (D) and c e l l wall (W). Arrow indicates darkly s t a i n i n g material i n wall adjacent  to plasmalemma.  XVI  44.  V. ULTRASTRUCTURAL ASPECTS OF MONOSPORuGENESIS a) Monospore differentiation), release and degeneration. 1 Introduction. The d i f f e r e n t i a t i o n of whole vegetative c e l l s into spores i s a common means of asexual reproduction i n the Bangiophycidae (see Drew. 1956 for review). The term "monospore" has been applied to reproductive c e l l s produced i n t h i s manner, although there i s s t i l l some d i f f i c u l t y i n formulating acceptable term3 to distinguish between the d i f f e r e n t spore-like c e l l s evident i n t h i s group. During the present study, Smithora and E r y t h r o t r i c h i a were observed i n culture as well as i n f i e l d conditions and i t i s avident that monospore production constitutes one of the main methods of species propagation. A A discussion of l i g h t microscopic work on the production of these structures i n the Erythropeltidaceae i s presented i n Section III of this report. A majority of the u l t r a s t r u c t u r a l investigations c a r r i e d out on algal spore production have dealt with various aspects of chlorophycean genesis, e.g. Stiqsoclonium  zoosporo-  (Wanton, 1964), Qedoqonium (Hoffman, 1968j  Pickett-Heaps, 1971), Bulbochaete (Retallack and Butler, 1970), Tetracystis (Brown and Arnott, 1970)  and Enteromorpha (Evans and C h r i s t i e , 1970). In  the red algae, Peyriere (1969) has described c e r t a i n features of the tetrasporangium  of G r i f f i t h s i a and Tripodi (1971) has reported some 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 s 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 i s p a r t i a l l y based on a p u b l i c a t i o n by D.L. McBride and K. Cole i n Phycoloqia 10, 49-61 (1971), The text of the o r i g i n a l a r t i c l e has been brought up to date by i n c l u d i n g 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 i n Ochromonas (Chrysophyceae) and Palisano and Ulalne (1972) in Luqlena  (Euglanophyceae).  Observations. Monospore differentiation t The monosparogenous area of Smithora naiadurn i s sharply delimited from the vegetative thallus and i s usually observed as a band of more deeply pigmented, rounder cells (PI. XVII, Fig. 1), At a light microscope level, the cells i n this area appear to be undergoing mitotic division at a greater rate than the adjacent vegetative c e l l s  8  This, in conjunction with an apparent reduction i n vacuolar area, results in the differentiating cells appearing smaller than the vegetative cells in surface view, although the actual thickness of the thallus i s 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 c e l l 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 i n surface view. Like that of the vegetative c e l l , the nucleus of the developing monospore exhibits a typical eukaryotic structure (PI. XVIII, Fig.3). Perhaps the most conspicuous difference i s 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 i n many cases arranged i n 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, F i g . 6 ) S t r u c t u r a l l y they are s i m i l a r to those described 0  i n 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, F i g . 4 ) . At these regions a dense p r e c i p i t a t e of s t a i n  often occurred i n the prepared material. Ribosome-laden endoplasmic reticulum i s invariably observed adjacent to the nuclear envelope of the developing spore (PI. XVIII, F i g , 5). Often the cisternae may number 12 or more. Only a few cisternae were noted i n this position i n the vegetative c e l l . It i s of i n t e r e s t 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, F i g . 6 ) . Usually a few cisternae of predominantly smooth ER are associated with the plasmalemma (PI. XIX, F i g . Q). In addition, increased quantities of smooth and rough ER were observed i n other areas of the d i f f e r e n t i a t i n g monospore (PI, XVII, F i g . 2j PI. XVIII, F i g . 3), The mitochondria are t y p i c a l i n appearance;  possessing a double envelope,  tubular c r i s t a s and electron dense inclusions (PI, XVIII, Fig..5), However, there appears to be an increase i n the number of these organelles i n the developing spore. Mitochondria often appear i n the v i c i n i t y of dictyosomes, but no s t r i c t relationship such as that described i n C o r a l l i n a (Bailey and Bisalputra, 1970) and G r i f f i t h s i a (Peyriare, 1969) was observed i n t h i s material. The chloroplast of the developing monospore i s i r r e g u l a r l y lobed and exhibits single lamellae which are largely orientated p a r a l l e l to the chloroplast envelope  (PI. XVII, F i g , 2). It i s very s i m i l a r to that of the  vegetative c e l l except that the interlamellar spaces appear to enlarge  47.  somewhat a n d  acquire a granular  Many f l o r i d e a n s t a r c h and-are  generally larger  appearance.  grain3  are present i n the d i f f e r e n t i a t i n g  than those observed  XVIII, F i g , 3). Thin sectioning usually in  areas of s t a r c h  The  appears  Throughout development, in  v a r y i n g degrees  stacks early  give rise  ( P I , X V I I , F i g , 2 ) . The  distinctly  the c e n t r e of the c e l l substance  t o e m a n a t e f r o m i t . The  the dictyosomes  produce  ( P I , XIX,  irregular  coalesce shortly  formed  with their  vesicles  maturing  containing the  forming  and s m a l l v e s i c l e s  which  vesicles originating  after formation, resulting ( P I , X V I I , F i g . 2;  from  i n large, P I . XIX, F i g .  components such  as f l o r i d e a n s t a r c h  granules  ( P I . XIX,  ).  Toward t h e l a t t e r a second  substance  randomly  i n the c e l l ,  14).  is  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  9  spore.  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 these d e p o s i t s ( P I . XIX,  10). Occasionally c e l l u l a r  appear Fig.  Rough ER  first  F i g , 8), Occasionally  large f i b r i l l a r  membrane b o u n d d e p o s i t s w i t h i n t h e c e l l 11,12).  (1970) e x p e r i e n c e d  swollen cisternal  d i f f e r e n t p r o d u c t s . The  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 appear  line  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  of hypertrophy  a compacted f i b r i l l a r  plastic  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  the c e l l  t o two  (PI.  (Porphyridiales).  i n the development of the s p o r e . Large dictyosomes  faces toward  Fig.  i n f o l d i n g of the  ( P I . X V I I , F i g . 2 ) . E v i d o n t l y Evans  with Rhodella  dictyosome  resulted  g r a n u l e s . T h i s c a n be s e e n a s a n e l e c t r o n d e n s e  through these s t r u c t u r e s t h e same p r o b l e m  i n the v e g e t a t i v e c e l l  spores  stages of the accumulation of the f i b r i l l a r  begins to form.  Hypertrophied dictyosomes,  f o r m 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  These v e s i c l e s c o n t a i n l o o s e l y  organized f i b r i l s  transparent m a t r i x . In the stages immediately p r i o r  i n an  t o and  product,  located  ( P I , XX, F i g . electron after  spore  48  r e l e a s e the c e l l  i s f i l l e d w i t h these s t r u c t u r e s . Concomitant w i t h  the  d e p o s i t i o n of t h i s m a t e r i a l i s the marked enlargement and r o u n d i n g of cell.  In these l a t t e r s t a g e s of development both the d e n s e l y  v a c u o l e s and the s m a l l e r v e s i c l e s are p r e s e n t i n the c e l l Contrary to Hollenberg's  the  fibrillar  ( P I . XIX,  F i g . 11).  (1959) l i g h t m i c r o s c o p i c o b s e r v a t i o n , no  i n t e r c e l l u l a r c o n n e c t i o n s were observed between d e v e l o p i n g s p o r e s . In c o n t r a s t to  the v e g e t a t i v e c e l l , i t i s of i n t e r e s t t h a t very few  lomasoma-like  b o d i e s were o b s e r v e d . C o n c e n t r i c l a m e l l a r s t r u c t u r e s were a l s o few T h i s i s i n keeping w i t h r e s u l t s o b t a i n e d i n two (Gantt and C o n t i , 1965)  and  Batrachospermum  o t h e r r e d algaes  i n number. Porphyridium  (Brown and Uleier, 1970),  which i n d i c a t e t h a t t h e s e s t r u c t u r e s are p r e s e n t t o a g r e a t e r e x t e n t i n older, less active  cells.  P r i o r t o spore r e l e a s e the t h i c k n e s s o f the c e i l  wall i s considerably  l e s s than i n the v e g e t a t i v e t h a l l u s . W a l l p r o d u c t i o n has e v i d e n t l y not kept pace w i t h the enlargement of the monospore. The  area  immediately  a d j a c e n t t o the plasmalemma i s more compacted, p o s s i b l y due of  the e n l a r g e d spore or t o t h e a c c u m u l a t i o n  (see below) ( P I . XX,  XXV.  forms two  of r e l e a s e d f i b r i l l a r m a t e r i a l  F i g . 15).  The u l t r a s t r u c t u r a l (PI,  t o the p r e s s u r e  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  F i g . 31) are i d e n t i c a l t o those i n Smithora p r o d u c t s , each of which i s accumulated  : the  dictyosome  i n the cytoplasm  p r i o r to  spore r e l e a s e ( P I . XXVI, F i g . 32-34).  Monospore r e l e a s e : Immediately p r e c e d i n g and d u r i n g l i b e r a t i o n of the monospore, the v a c u o l e - l i k e s t r u c t u r e s m i g r a t e the c a l l (PI.  XIX,  (PI. XIX,  F i g . 12)  and s u b s e q u e n t l y  toward the p e r i p h e r y of  expal t h e i r f i b r i l l a r  contents  F i g , 1 3 ) . The mechanism of r e l e a s e i n v o l v e s a f u s i o n of the  vacuole  49. membrane to the plasmalemma. Occasionally a few of the smaller v e s i c l e s are released as w e l l , but the majority remain behind. The extrusion of a s i n g l e spore i s achieved by a breakage i n the c e l l wall on one or the other side of the t h a l l u s (PI. XX,  F i g . 1 6 , 1 7 ) , Since  the development of the c e l l s i n one p a r t i c u l a r sorus seems to be more or less synchronous, the sorus i s often released as a whole (PI, XX, F i g . 1 9 j PI, XXII, F i g . 2 2 ) . This could be the r e s u l t of a greater stress on the c e l l walls i n the proximal  area of the sorus. Such mass release appears to occur  more often than the release of s i n g l e spores. However, unless the e n t i r e sorus s e t t l e s immediately following release i t i s most probable that further wall d i s s o l u t i o n takes place r e s u l t i n g i n s i n g l e spores being liberated. There i s evidence that a small amount of cytoplasm remains i n the t h a l l u s after spore release (PI, XX, F i g . 1 7 , 1 8 ) , This material i n v a r i a b l y appears degenerate. Perhaps t h i s pocket of cytoplasm which contains a nucleus (PI.  XX, F i g . 1 8 ) , i s the r e s u l t of an asymmetric d i v i s i o n of the vegetative  protoplast p r i o r to d i f f e r e n t i a t i o n . It appears to be s i m i l a r to the pale, protoplasmic  remnant occurring i n t h i s manner and remaining after spore  release i n Membranella nitens (Hollenberg and Abbott, 1 9 6 8 ) . The released monospore i s s p h e r i c a l and approximates 15-25 microns i n diameter (PI, XXI, F i g . 2 0 , 2 1 ) . The spore i s bounded by a s i n g l e plasmalemma, the exterior of which may  be associated with a quantity of mucilage ( P i .  XXII, F i g . 22j PI. XXIV, F i g . 2 9 ) . No vestige of c e l l wall material from the parent t h a l l u s remains. This concurs with reports of naked monospores i n other red algae by various authors including Sommsrfeld and Nichols The monospore i s f i l l e d with the smaller, loosely f i b r i l l a r v e s i c l e s produced prior to release,, Many of these v e s i c l e s coalesce a f t e r spore  (1970).  50. l i b e r a t i o n to form larger, vacuole-like structures (PI. XXI, F i g . 21; PI. XXIII, F i g . 23). The chloroplast seems to be somewhat modified  i n the monospore.  Closely appressed lamellae often occur within the lobes giving the appearance of "pseudogranum-like" structures (PI. XXIII, F i g . 23,24). As many as 20 lamellae may be involved i n these formations.  Although these structures  could be associated with an altered p l a s t i d metabolism, i t i s perhaps more l i k e l y that they are due merely to the physical e f f e c t of pressure r e s u l t i n g from the accumulation of products i n the spore. In addition, thylakoid associations l i k e those reported i n tha vegetative c e l l were observed (PI. XXIII, F i g . 24). The pyrenoid i s characterized by a number of swollen traversing lamellae which often appear c l o s e l y appressed and frequently contain some osmiophilic droplets  (PI. XXIII, F i g . 25).  The notable lack of unoccupied cytoplasmic  matrix i n the monospore  severely r e s t r i c t s the d i s t r i b u t i o n of other organelles, mitochondria are often c l o s e l y packed and confined to a small area  (PI. XXIV, F i g . 27)„ The  nucleus i s usually surrounded by a layer of cytoplasm containing a small amount of ER (PI. XXIV, F i g . 28). Some dictyosome a c t i v i t y i s noted i n the mature monospore (PI. XXIV, Fig.  26). These organelles appear to be adding to the already abundant  vesicular material. Numerous f l o r i d e a n starch granules remain i n the spore and seem to be i s o l a t e d from the remaining b i t s of cytoplasm. Due to a lack of c e l l wall material, the d i f f e r e n t i a t e d monospore appears to be very d e l i c a t e . In many instances the pressure  of ths c e l l u l a r  contents i n combination with outside stimulus causes the plasmalemma to rupture, r e s u l t i n g i n a flow of vesicular contents out of the c e l l Fig.  (PI. XXI,  21). In addition, the plasmalemma i s frequently observed to bleb out  51. quantities of t h i s substance  ( P i , XXIV. F i g , 30).  Monospore degeneration; Although monospores w i l l germinate r e a d i l y under culture conditions, a c e r t a i n percentage of these structures undergo degeneration within several days a f t e r release. At a l i g h t microscopic l e v e l , the chief proclamation of this phenomenon i s the gradual loss of the c h a r a c t e r i s t i c red pigmentation of the spore, U l t r a s t r u c t u r a l l y there i s 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, F i g . 1). Abnormally  bloated mitochondria,  which often show indications of inner membrane disruption, are t y p i c a l of the degenerating spore (PI, XXVII, F i g . 1; PI, XXVIII, F i g . 2). Paraneural bodies (Marchant of the c e l l  and Robards, 1968)  are- increasingly evident near the periphery  (PI. XXVII, F i g . 1) and extensive, progressive vacuolation of  the cytoplasm occurs (PI. XXVII, F i g . 1;  PI. XXVIII, F i g . 2,3).  Disruption of the nuclear envelope and the subsequent breakdown of t h i s organelle may  signal the onset of c e l l death  (PI. XXVIII, F i g . 3),  In l a t e r stages, the chloroplast material i s almost completely disorganized. Lamellae are grossly distended and membrane destruction i s evident (PI. XXVIII, F i g . 4). F i n a l l y the plasmalemma breaks, possibly as the r e s u l t of uncontrolled osmotic pressures, and the spore r a p i d l y disintegrates. Numerous uncoordinated membrane fragments are c h a r a c t e r i s t i c of the c e l l u l a r debris (PI. XXVIII, F i g . 5). Discussion. In the f i e l d , the chief manifestation of the s u b c e l l u l a r changes involved i n the production of monospores i s a deeper pigmentation i n the d i f f e r e n t i a t i n g areas. The electron microscope has allowed a more detailed observation of the process. Not only i s there an increase i n dictyosome  52. a c t i v i t y but also a substantial increase i n the amount of ER and the number of f l o r i d e a n starch granules and mitochondria. Such changes would suggest that the production of monospores i n the Erythropeltidaceae examined i s 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 c e l l u l a r 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 i n the extensive production of two d i s t i n c t 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 i n large vacuole-like structures. Since substantial amounts of i t are expelled immediately prior to and during spore release, i t i s hypothesized that t h i s substance functions as a mucilaginous secretion, Knox (1926) noted that a large amount of mucilaginous matter i s associated with monospore release i n Smithora. Indeed, i t has been observed i n the current study that the mucilage i s produced i n copious amounts and could aid i n spore release by acting as a l u b r i c a n t . This could be c r i t i c a l  to the success of the process as l i b e r a t i o n takes place through  a small opening i n the c e l l w a l l . In addition, the mucilage would protect the f r a g i l e 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 c e l l s and mucilage production. The production of such a substance was also noted i n Porphyridium and Pseudooloiophloea by Ramus (1972), who  described i t as a polyanionic polysaccharide of high molecular weight. The second product associated with dictyosome a c t i v i t y i s contained  within smaller, more electron transparent v e s i c l e s , 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 t h i s compound i s secreted primarily after spore l i b e r a t i o n , i s produced i n large quantities and i s released very e a s i l y by rupturing the f r a g i l e , unprotected plasmalemma, i t i s hypothesized that i t acts as a cementl i k e 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 f a c t , not only do these spores s t i c k to the host plant securely, but also adhere to one another. So e f f e c t i v e i s t h i s b i o l o g i c a l cement, that i t i s very d i f f i c u l t to remove the s e t t l e d spores from p l a s t i c containers. Since Smithora and E r y t h r o t r i c h i a 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  t i d e and wave action could remove them from t h i s l o c a t i o n . Again, the timing of the secretion would be r e l a t i v e l y c r i t i c a l . Too early a release could cause the spores to adhere to the parent t h a l l u s thus r e s t r i c t i n g d i s p e r s a l . Wanton (1964) and Evans and C h r i s t i e (1970) have proposed a s i m i l a r function for the large accumulation of vesicular structures i n the zoospores of the green algae Stiqeoclonium and Enteromorpha r e s p e c t i v e l y . In recent studies on zoosporogenesis i n Oedoqonium (Chlorophyceae), Pickett-Heaps (1971) has also described two d i s t i n c t populations of dictyosomes, each concerned with the production of a d i f f e r e n t substance. In addition, Tripodi (1971 ) working with cystocarpic material of Polysiphonia, has t  noted two product accumulations which appear to be s t r u c t u r a l l y s i m i l a r t o those i n Smithora and E r y t h r o t r i c h i a . He suggests that the r o l e of these structures may  be involved with a reserve function, although he does not  r e l a t e any u l t r a s t r u c t u r a l 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, and more recently in Symbiodinium  1968)  (Dinophyceae)(Taylor, 1969) where a continuity  between the chloroplast matrix and the nucleoplasm was found. The c e l l wall of the parent thallus must undergo extensive changes to allow the monospores t o escape. The thickness of the c e l l wall i s definitely reduced but there must also be some loss of structural stability . Perhaps •\  the c e l l 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 i s the result of an enzymatic digestion, physical internal pressure or external environmental effects remains opBn to speculation. The shape of the released spore i s spherical indicating a certain amount of pressure i s exerted on the plasmalemma by the contents of the spore. However, some degree of f l e x i b i l i t y i s important to the structure in order to f a c i l i t a t e 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 c i r c u l 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 c r i t i c a l period of monospore reproduction appears to be the time between release from ths parent thallus and initiation of c e l l 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 i s 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 w i l l survive. It would be extremsly d i f f i c u l t to estimate the percentage of surviving spores and undoubtedly such a hypothetical proportion would vary considerably under different environmental conditions. It i s quite possible that in this study conditions of laboratory culture were responsible for a certain percentage of degeneration. There i s 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 i n 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) i n 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 i n conditions approaching those of the natural environment, i t i s d i f f i c u l t to speculate on the causes of this phenomenon. Certain studios have been carried out on scenescing plant material under different controlled conditions (e g„ De Vecchi., 1971), 0  but i t i s obvious that further research on such a r t i f i c i a l systems i s needed to permit the formulation of definite conclusions on the mechanisms of plant degeneration.  57.  PLATE  XVII.  Smithora. D i f f e r e n t i a t i n g monospore F i g . 1. Light microscopic and adjacent vegetative Fig.  surface view of proximal portion of sorus (s)  area (vg).  2. Section p a r a l l e l to surface of t h a l l u s i l l u s t r a t i n g d i f f e r e n t i a t i n g  c e l l with chloroplast lobes  (c)„ dictyosomes (d)„ endoplasmic reticulum  mitochondria (m), f l o r i d e a n starch grains ( f v l ) and surrounding c e l l wall (w).  ( f s ) , fibrous vacuole-like  (er)»  structures  XVII  58.  PLATE X V I I I . Smithora. D i f f e r e n t i a t i n g monospore F i g . 3. Portion of nucleus (n) with nucleolus (nu) and surrounding organelles. Arrow denotes DNA-containing area i n chloroplast. F i g . 4. Area of intimate association betaeen l i m i t i n g envelopes of nucleus (n) and chloroplast ( c ) . Arrow denotes p r e c i p i t a t e of s t a i n . F i g . 5. Portion of nucleus (n) with associated c i s t e r n a l stack of rough ER. A dictyosome (d) and a mitochondrion (m) are also l a b e l l e d . F i g . 6 . Portion of nucleus (n) i l l u s t r a t i n g porous nature of envelope (arrows) i n c e r t a i n r e s t r i c t e d areas. F i g , 7 . Tangential section through nuclear envelope i l l u s t r a t i n g sequence of pores and annular structure.  linear  XVIII  59.  PLATE XIX. Smithora. D i f f e r e n t i a t i n g monospore Fig,  B. Dictyosome (d) with large i r r e g u l a r fibrous v e s i c l e s (fv) forming  from maturing face. Note v e s i c l e s (arrow.)"and ER associated with forming face near c e l l w a l l . Fig.  9. Floridean starch grain (fs) included within fibrous vacuole-like  structure Fig.  10. Association between ER and fibrous vacuole-like structure  C e l l wall Fig.  (fvl).  (w) and a dictyosome (d) are also l a b e l l e d .  11. Smaller, more electron transparent  vacuole-like structures Fig,  (fvl).  ( f v l ) concurrently  12. Large, vacuole-like structures  v e s i c l e s (v) and large f i b r i l l a r present i n the developing spore.  ( f v l ) appressed to periphery of  protoplast near c e l l wall (w). Fig. 13. The vacuole-like structures ruptured  ( f v l ) releasing contents through  plasmalemma during l i b e r a t i o n of spore. Note presence of smaller  vesicles (v) and mitochondria (m).  XIX  60.  PLATE XX Smithora. D i f f e r e n t i a t i n g spore Fig.  14. Dictyosomes  vesicles Fig.  (d) p r o d u c i n g s m a l l e r , more e l e c t r o n t r a n s p a r e n t  ( v ) near c e l l w a l l (w).  15. Compacted a r e a o f c e l l w a l l  (w) i m m e d i a t e l y a d j a c e n t t o plasmalemma  (arrow) p r i o r t o s p o r e r e l e a s e . Fig.  16. C r o s s - s e c t i o n o f t h a l l u s showing monospore b e i n g l i b e r a t e d . Note  p r e s e n c e o f both t y p e s o f d e p o s i t s ( v , f v l ) and d i s t o r t i o n o f s p o r e d u r i n g r e l e a s e , ex i n d i c a t e s a r e a e x t e r n a l t o t h a l l u s . Fig.  17. C r o s s - s e c t i o n o f t h a l l u s a f t e r s p o r e l i b e r a t i o n . S i n g l e arrow  denotes r e m a i n i n g c y t o p l a s m . 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 . Fig,  18. Cytoplasm  nucleus  r e m a i n i n g i n t h a l l u s a f t e r s p o r e r e l e a s e . C e l l w a l l (w),  ( n ) , m i t o c h o n d r i a (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 a r e a e x t e r n a l t o t h a l l u s . Fig,  19, Photomicrograph  sorus ( s ) .  o f s p o r e s b e i n g l i b e r a t e d en mass (ms) and i n t a c t  XX  61.  PLATE XXI Smithora.  Released  monospore  F i g . 20. Photomicrograph of l i b e r a t e d monospore. Fig. 2 1 .  E l e c t r o n micrograph  chloroplast  (c) with p y r e n o i d  o f l i b e r a t e d monospore. Note c e n t r a l ,  lobed  (p) and c o a l e s c e d e l e c t r o n t r a n s p a r e n t  ( c v ) . S i n g l e arrow denotes u n p r o t e c t e d  plasmalemma and double  arrow  vesicles denotes  r e l e a s e of contents of e l e c t r o n transparent coalsced v e s i c l e s . A nucleus (n) i s a l s o l a b e l l e d .  XXI  62  PLATE XXII Smithora,, Released monospore F i g , 22, Section through released deciduous sorus consisting of c l o s e l y adhering monospores.  XXII  63.  PLATE XXIII S m i t h o r a . Released monospore F i g . 23. Lobe o f c h l o r o p l a s t w i t h c e n t r a l l y coalesced  association  F i g . 25. P o r t i o n traversing  appressed lamellae  and  electron transparent v e s i c l e s (cv).  F i g . 24. Lobe o f c h l o r o p l a s t thylakoid  located  with appressed l a m e l l a e  ( s i n g l e arrow).  of p y r e n o i d showing o s m i o p h i l i c  lamellae.  (double arrow) and  droplets  (od) w i t h i n  swollen  XXIII  64.  PLATE XXIV Smithora.  Released  monospore  F i g . 26. Dictyosome a c t i v i t y i n mature monospore  ( p r o d u c t i o n of e l e c t r o n  transparent v e s i c l e s ( v ) ) . F i g . 27. Four c l o s e l y packed m i t o c h o n d r i a . A f l o r i d e a n s t a r c h g r a n u l e and c o a l e s c e d v e s i c l e s F i g . 28. Nucleus amount of ER  (cv) a r e a l s o shown.  (n) and n u c l e o l u s  (nu) o f mature monospore. Note s m a l l  (arrow) nsxt to the n u c l e a r  envelops.  F i g . 29. S i n g l e , u n p r o t e c t e d plasmalemma o f mature s p o r e , ex denotes a r e a e x t e r n a l to spore luhich may i n c l u d e m u c i l a g i n o u s m a t e r i a l . F i g , 30. Release  of v e s i c u l a r c o n t e n t s  ex denotes a r e a e x t e r n a l t o s p o r e .  (v) by a b l a b b i n g of the plasmalemma.  XXIV  65,  PLATE XXV E r y t h r o t r i c h i a boryana F i g . 31. U n r e l e a s a d monospore w i t h i n with pyrenoid  (p), nucleus  c e l l wall  (w) showing c h l o r o p l a s t (c)  (n) and f l o r i d e a n s t a r c h g r a i n s  (fs)„  XXV  66.  PLATE XXVI Erythrotriehia F i g . 32. E_. boryana. Portion of d i f f e r e n t i a t i n g 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 ) o r i g i n a t i n g from the dictyosome (single arrow). Prominent nucleus (n) surrounded by ER i s also shown. F i g . 33. E_„ carnea. Portion of d i f f e r e n t i a t i n g monospore. Note f i b r i l l a r vacuole-like structures  ( f v l ) and dictyosome (arrow).  F i g . 34, IE, pulvinata. Description as f o r F i g , 33,  XXVI  67.  PLATE XXVII Smithora. F i g . 1.  Degenerating  Monospore i n e a r l y s t a g e s o f d e g e n e r a t i o n  chloroplast  l a m e l l a e (arrow), s w o l l e n m i t o c h o n d r i a  ( f s ) and paramural b o d i e s  (double  arrow).  monospore exhibiting (m)  B  swollen  floridean  starch  XXVII  68  PLATE XXVIII Smithora.  Degenerating  monospore  F i g . 2. S w o l l e n m i t o c h o n d r i a w i t h 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 d e g e n e r a t i o n .  Note  numerous v a c u o l e s ( v ) . F i g . 4.  D i s o r g a n i z a t i o n and s w e l l i n g of c h l o r o p l a s t  F i g . 5. C e l l u l a r  lamellae.  d i s i n t e g r a t i o n a f t e r plasmalemma breakage. Note  membranous elements  (arrow).  remaining  XXVIII  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 i n v e s t i g a t i o n s of t h i s nature dealing w i t h the Rhodophyceae, d e t a i l e d s t u d i e s 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 a l s o being c a r r i e d out on zoospore germination i n Qedoqonium (Chlorophyceae) (Pickett-Heaps, 1971) and r e c e n t l y Quatrano germination i n Fucus  (1972) has i n v e s t i g a t e d zygote  (Phaeophyceae).  Smithora provides an i d e a l m a t e r i a l f o r 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 c o n d i t i o n s . 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 w a l l and s e v e r a l vacuoles ( P I . 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 pyrenoidcontaining c h l o r o p l a s t (PI. XXIX, F i g . 2; P I . XXX, F i g . 4 ) , a nucleus ( P I . XXXI, F i g . 7,8; P I . XXXIV, F i g . 14), dictyosomes (PI. XXXI, F i g . 6; P I . XXXII, F i g . 9 ) , endoplasmic reticulum ( P I . XXXI, F i g . 7; PI. XXXII, F i g . 9,10), mitochondria (PI. XXIX, F i g . 2; P I . XXXI, F i g . 7; P I . XXXII, F i g . 10; PI. XXXIV, F i g . 14) and soma f l o r i d e a n s t a r c h (PI. XXXIV, F i g . 14). The two methods of basal h o l d f a s t formation are evident under the l i g h t microscope as w e l l as the e l e c t r o n microscope. A s i n g l e spore forms a c e l l w a l l ( P I . XXIX, F i g . 1,2) and through successive c s l l d i v i s i o n s produces a basal holdfast ( P I . XXXI, F i g . 5 ) . A l t e r n a t i v e l y , an i d e n t i c a l ''This s e c t i o n 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 i n d i v i d u a l  c e l l walls which eventually contribute to a common c e l l wall (PI. Fig.  XXX,  3,4), Wall construction appears to be i n i t i a t e d i n the area of the c e l l  adjacent  to the peripherally situated nucleus (PI. XXXI, F i g . 7), but  eventually i t i s formed continuously  around the c e l l  (although  be unrelated, l o c a l i z e d areas of d i f f e r e n t thicknesses)  there  may  (PI, XXXI, F i g . 8).  The bonding of adjacent c e l l walls i s suggested by the observation  that  folds occur between t h i n sectioned c e l l s only i n areas where the walls are not c l o s e l y interwoven (PI, XXX,  F i g . 4j PI, XXXI, F i g , 8).  The germinating monospore i s distinguished by a large increase i n tha peripheral ER which appears to ba extremely active ( P i , XXXII, F i g . 9). Cisternae are often swollen, associated with large numbers of ribosomes and found i n close proximity 10). Dictyosomes may  to the convoluted  plasamlamma (PI. XXXII, F i g ,  also be present i n these areas (PI. XXXII, F i g . 9).  It i s possible that both the ER and the dictyosomes are involved i n the synthesis of wall material which i s a primary a c t i v i t y of the c e l l at this time. Dictyosomes a l 3 o appear to play an important r o l a i n vacuole  formation.  A la?ge number of these organelles are situated with their maturing faces and associated v e s i c l e s i n close proximity to the enlarging vacuoles (PI. XXXI, F i g . 6). An association between dictyosomes and vacuoles has also been noted i n the red alga Batrachospermum (Brown and Weier, 1970). The lobed, pyrenoid-containing  chloroplast i s s t r u c t u r a l l y unchanged  from that reported i n the newly released monospore (Section Va) i n possessing areas of c l o s e l y appressed lamellae  (PI. XXIX, F i g . 2} PI. XXXIV, F i g . 14).  However, the number of such areas appears to be somemhat reduced. Typical f l o r i d e a n 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 i s 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 i s 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 i s 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. i n diameter. The angle of intersection in the cross-hatched pattern i s 70-75°. In addition, electron translucent areas, probably representing included traversing lamellae and the ribosomelike particles referred to by Holdsworth (1968) were observed (PI. XXXIII, Fig. 12). The nucleus i s eukaryotic i n appearance and i s 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 o f t e n appear d e v o i d o f s t r u c t u r e d m a t e r i a l c o u l d a l s o c e n t r e s o f s p i n d l e f i b r e o r g a n i z a t i o n which have been d e s t r o y e d by procedures  be  fixation  ( P I . XXXIV, F i g . 1 5 ) . B o r d e r i n g 3uch areas i s a h i g h c o n c e n t r a t i o n  o f n u c l e a r pores i n d i c a t i n g a p o s s i b l e n u c l e a r p o l a r i t y a t t h i s e a r l y of d i v i s i o n . La Cour and W e l l s  (1972) have shown a more i r r e g u l a r  stage  distribution  of pores i n prophase as compared to i n t e r p h a s e s t a g e s of c e r t a i n h i g h e r p l a n t s . Two  types of v e s i c u l a r u n i t s of unknown f u n c t i o n were observed  regularly  i n the cytoplasm o f the g e r m i n a t i n g monospore. The f i r s t  type i s i n v a r i a b l y  a s s o c i a t e d im groups w i t h the ER s u r r o u n d i n g the nucleus  ( P I . XXXIV, F i g . 1 4 ) .  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  c o n s i s t of s e v e r a l  ( P I . XXXIV, F i g . 1 3 ) . These u n i t s appear t o  coneentric, membranous spheres bounding a c e n t r a l more  e l e c t r o n t r a n s p a r e n t a r e a . 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 s t r u c t u r e which e x i s t s i n l a r g e a g g r e g a t i o n s i n the c y t o p l a s m  (PI.  XXXIV, F i g . 1 5 ) . These e c c e n t r i c a l l y l o c a t e d s t r u c t u r e s s u p e r f i c i a l l y dictyosome  v e s i c l e s but a r e seldom seen i n the v i c i n i t y  Paraneural bodies  (fflarchant and Robards, 1968)  are few  resemble  of these o r g a n e l l e s .  i n number  (PI. XXXIV,  F i g . 16) and are u n l i k e the above mentioned v e s i c u l a r u n i t s which a r e more regular i n structure. Discussion. From o b s e r v a t i o n s on the methods of h o l d f a s t f o r m a t i o n i n S m i t h o r a it  i s e v i d e n t t h a t w i t h i n a p o p u l a t i o n of r e l e a s e d monospores a c e r t a i n  number w i l l b e g i n development from u n i c e l l u l a r s t a g e s w h i l e the w i l l b e g i n development from m u l t i c e l l u l a r  remainder  s t a g e s o f v a r y i n g degrees  of  c o m p l e x i t y . T h i s mechanism c o u l d a i d i n d e s y n c h r o n i z i n g the development of a g e n e r a t i o n of monospores. Thus, the p o p u l a t i o n would r e a c h r e p r o d u c t i v e m a t u r i t y over a l o n g e r p e r i o d of t i m e , t e n d i n g t o minimize  the  possibility  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 i n the time required to complete an asexual reproductive c y c l e . However, t h i s mechanism would also reduce the number of plants developing  from a given number of  spores.  The nature of wall production i n a l g a l 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 i n the production wall material or wall precursors. Positive cytochemical obtained  for the presence of polysaccharides  Thompson and Preston  of  tests have been  i n these organelles. In addition,  (1968) have suggested that proteins may  be an important  s t r u c t u r a l component i n c e r t a i n a l g a l c e l l walls. Thus, one might expect a germinating spore to be a c t i v e l y involved i n the production  of proteins  with enzymatic or s t r u c t u r a l functions as well as the carbohydrate moity. Indeed, Quatrano (1968,1972) has shown that r h i z o i d 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 s i m i l a r 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 d i r e c t l y from the ER across the plasmalemma into the c e l l w a l l . This mechanism has also beers postulated to occur i n Oedoqonium (Pickett-Heaps,  1971). The  hypothesis  that the ER acts as a f i n a l synthesis and packaging point of wall material i s a t t r a c t i v e but must be based on further studies. In the germinating monospore the dictyosome complement i s capable of carrying out several d i f f e r e n t functions simultaneously.  This p r i n c i p l e o f  74. " d i v i s i o n of labour" allows some of these organelles to be involved i n vacuole construction while the remainder  appear to take part i n production  of wall material,, In the developing zoospore of Oedoqonium Pickett-Heaps (1971) also noted two d i s t i n c t populations of dictyosomes, each apparently with a d i f f e r e n t function. In t h i s context, i t i s also i n t e r e s t i n g to note a change i n roles of the dictyosome  populations i n a monospore from the  d i f f e r e n t i a t i n g state, through the f r e e - f l o a t i n g condition to germination. As spore d i f f e r e n t i a t i o n 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 t h a l l u s and for a short period a f t e r , the production of an adhesive material occurs (Section Va). Prio^ to s e t t l i n g , the dictyosome populations are l e s s active, but upon germination appear to become involved i n vacuole formation and production of  a wall material. Evans and C h r i s t i e (1970) reported a s i m i l a r  set of changes i n the germinating zoospore of Enteromorpha. The c r y s t a l l i n e matrix i s an i n t e r e s t i n g , i f not regular, feature of the pyrenoid. C r y s t a l l i n e pyrenoids have been reported previously i n brown algae (Evans, 1966), diatoms (Holdsworth, 1968; Taylor, 1972), a d i n o f l a g e l l a t e  (Kowallik, 1969)  Coombs et a l . ,  1968;  and a green alga (Bertagnolli  and Nadakavukaren, 1970). From these studies i t i s evident that a c r y s t a l l i n e matrix i s not present i n a l l pyrenoids of a p a r t i c u l a r algal population or, i f present, may  be apparent i n p a r t i c u l a r regions only. In f a c t , one  report d e t a i l s t h i s s t r u c t u r a l property i n the diatom Navicula only after c o l c h i c i n e treatment  (Coombs et a l . , 1968). In t h i s laboratory, studies on  freshly c o l l e c t e d vegetative material, monosporogenous material and spermatangial s o r i have shown no evidence of c r y s t a l l i n e pyrenoids. However, i n ths present study monospores subjected to culture conditions have t h i s  75. s t r u c t u r a l property* I t i s i n t s r a s t i n g to nots that other i l l u s t r a t e d reports have also been made using cultured material. Holdstuorth (1971) has recently reported the presence of proteinaceous components i n the pyrenoid o f Ereinosphaera  (Chlorophyceae) which appear to  have s i m i l a r properties to c e r t a i n enzymes i n the carbon f i x a t i o n pathway of photosynthesis. Thus, i t might be of i n t e r e s t to note the structure of pyrenoids possessing c r y s t a l l i n e matrices under d i f f e r e n t photosynthetic conditions. Such controlled experiments may shed some l i g h t on the formation of these s t r u c t u r e s . Perhaps this phenomenon i s a modification preparatory to withstanding adverse environmental  conditions, or even one of the f i r s t  steps i n c e l l degeneration. Indeed, Ragatli, Weintraub and Lo (1970) have described "pseudocrystalline" structures i n the chloroplasts of excised degenerating leaf material of Nicotiana which are very s i m i l a r to the c r y s t a l l i z e d pyrenoid of Smithora. They suggest t h i s may represent a highly ordered mechanism for coping with s t a r v a t i o n . The substructure of the c r y s t a l l a t t i c e i n Smithora monospores i s comparable to that reported i n the diatom Achnanthes (Holdsworth, 1968) and the green u n i c e l l C h l o r a l l a (Bertagnolli and Nadakavukaren, 1970). However, the mean centre to centre distance between p a r a l l e l subunit rows i s d i f f e r e n t . In both the aforementioned  algae this spacing was found to  be 8.0 nm. while i n Smithora the distance i s 12.5 nm. In the d i n o f l a g e l l a t e Prorocentrum, Kowallik (1969) reported a distance of 12.2 nm. between the centres of adjacent subunits. The s i g n i f i c a n c e of these differences i s not yet clear although i t could indicate important variations i n pyrenoid composition between c e r t a i n algae. U n t i l recently, microtubular spindle f i b r e s have not been demonstrated i n red algae (Hommersand and Searles, 1971). But now reports of these  76. structures by Chapman, Chapman and Lang (1971) i n Porphyridium and MacDonald Phycol. i n press) i n Membranoptera indicate that they are probably involved i n c e l l d i v i s i o n i n t h i s group. Their presence i n Smithora further strengthens t h i s 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 f i n e s t r u c t u r a l changes early i n the germination of the monospore which appear to be t y p i c a l of t h i s process. There i s a marked reduction i n f l o r i d e a n  starch content, a notable increase  i n peripheral rough ER and an increase i n vacuolar area. It i s conceivable that these changes are i n t e g r a l l y associated with the process of c e l l wall construction.  77.  PLATE XXIX. Smithora. Fig.  1.  Fig.  2 . E l e c t r o n micrograph  wall  (cw), v a c u o l e s  L i g h t micrograph  Germinating  monospore  of a g e r m i n a t i n g monospore. of a g e r m i n a t i n g monospore i l l u s t r a t i n g  ( v ) . a pyrenoid  i n d i c a t e s a r e a o f appressed  (p) and c h l o r o p l a s t l o b e s  chloroplast lamellae.  a cell  ( c l ) . Arrow  XXIX  78,  PLATE Smithora., Fig.  3 . L i g h t micrograph  XXX  Germinating  monospore  o f a number of a d h e r i n g , g e r m i n a t i n g monospores.  F i g . 4 . E l e c t r o n micrograph  of a d e v e l o p i n g b a s a l  h o l d f a s t formed  from  numerous s p o r e s . Arrows i n d i c a t e an a r e a of common c e l l w a l l between two s p o r e s . Note t h a t dark f o l d l i n e s  do not pass between j o i n e d  spores.  XXX  79.  PLATE XXXI Smithora Fig.  5.  L i g h t micrograph  0  Germinating  o f a mature b a s a l h o l d f a s t  monospore (bh) a t t a c h e d t o a h o s t  plant (h). Fig.  6. A dictyosome  (d) w i t h a s s o c i a t e d v e s i c l e s i n c l o s e p r o x i m i t y t o an  enlarging vacuole ( v ) . Fig.  7 . P o r t i o n of g e r m i n a t i n g s p o r e i l l u s t r a t i n g area o f c e l l w a l l i n i t i a t i o n  (cw) a d j a c e n t t o n u c l e u s ( n ) . Fig.  8. P o r t i o n of a d e v e l o p i n g pad i l l u s t r a t i n g  wall  (cw) and a v a c u o l e  a nucleus  ( n ) , common c e l l  ( v ) . Arrow i n d i c a t e s an a r e a o f t h i c k e n i n g c e l l  wall.  XXXI  80.  PLATE XXXII S m i t h o r a . Germinating monospore F i g . 9« A t a n g e n t i a l s e c t i o n through a p o r t i o n of a monospore showing hypertrophied  peripheral  ER and dictyosomes ( d ) .  F i g . 1 0 . Rough ER w i t h c i s t e r n a e i n c l o s e a s s o c i a t i o n w i t h the plasmalemma. A mitochondrion  (m). a v a c u o l e  (v) and t h e c e l l w a l l a r e a l s o  F i g . 1 1 , A p o r t i o n of a c h l o r o p l a s t and  a cleaved,  labelled.  (c) showing s i n g l e l a m e l l a e  angular c r y s t a l l i n e p y r e n o i d ( c p ) .  (arrow)  XXXII  81.  PLATE XXXIII Smithora. Germinating monospore F i g . 12  0  Section through a c r y s t a l l i n e pyrenoid i l l u s t r a t i n g the nature of  the c r y s t a l l a t t i c e . Note the included traversing lamella ( t l ) and like particles  (arrow).  ribosome-  XXXIII  82.  PLATE XXXIV Smithora. Germinating monospore Fig.  13. A group of vesicular units i n association with the  ER.  F i g . 114. Portion of a c e l l showing the location of the vesicular units near the nucleus (n). A nucleolus (nu)„ ER, a f l o r i d e a n starch grain (fs) and appressed chloroplast lamellae (arrow) are also marked. Fig.  15. Section through a nuclear area (n) showing an aggregation of  v e s i c l e s (ve) . Note the unstructured area of cytoplasm 1  porous nuclear envelope  (a) adjacent to the  (arrow).  Fig.  16. Paraneural body near c e l l w a l l .  Fig.  17. Microtubular spindle f i b r e s r a d i a t i n g toward the nucleus (n). Note  the tangential sections of nuclear pores (single arrow), ER, and the extranuclear l o c a t i o n of the spindle f i b r e organizing centre (double arrow).  XXXIV  63 . VI. ULTRASTRUCTURAL EVIDENCE OF SEXUAL REPRODUCTION Introduction. Perhaps one of the most i n t e r e s t i n g and important aspects of studies on red algae are the descriptions of sexual reproduction. Although  this  process i s taell documented i n many members of the Florideophycidas, i t i s poorly known i n most Bangiophycidae. During t h i s study, the only of sexual reproduction i n the Erythropeltidaceae was of "spermatia"  (Hollenberg,, 1959)  evidence  the regular production  i n Smithora and sporadic i n d i c a t i o n s of  f e r t i l i z a t i o n i n E r y t h r o t r i c h i a boryana. It must ba stressed that i n t h i s report terms describing sexual reproduction (e.g. spermatia,  carpospora,  etc.) are used i n their most tentative sense duo to the lack of information on t h e i r s p e c i f i c function i n t h i s family. As i s shown by the various c o n f l i c t i n g reports of sexual reproduction i n the Erythropeltidaceae, the s i z e of the plants and, more s p e c i f i c a l l y , the u n i c e l l u l a r nature of the reproductive "organs"  hinders documentation using conventional l i g h t  microscopic techniques. On the other hand, the seemingly transient nature of t h i s process  ( f e r t i l i z a t i o n , i n p a r t i c u l a r ) provides a formidable  barrier to electron microscopic i n v e s t i g a t i o n . In addition, many such algae tend to display only asexual reproduction i n laboratory conditions. These are indeed perplexing problems which have undoubtedly caused some of the present confusion surrounding sexual reproduction i n these plants. Observations. S p e r m a t i a q e n e 3 i s i n Smithora; Spermatia may  be produced extensively i n the  medial distromatic portion of the mature blade i n the f a l l of the year  c  At a l i g h t microscopic l e v e l , each vegetative c e l l appears to undergo an asymmetric d i v i s i o n p a r a l l e l to the surface of the t h a l l u s , r e s u l t i n g i n the production of numerous, small (3-5 microns), pale c e l l s  (PI. XXXV,  84.  Fig. 1). At an u l t r a s t r u c t u r a l l e v e l , i n i t i a t i o n of t h i s process involves the migration of the usually e c c e n t r i c a l l y located nucleus toward the end of the c e l l nearest to the surface of the t h a l l u s (PI. XXXV, F i g . 2), allowing the formation of unequal daughter c e l l s . After d i v i s i o n i t appears that the nucleus of the larger c e l l migrates toward the opposite end of the protoplast (PI.  XXXV, F i g . 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) l i g h t microscope observation) and no pyrenoid (PI. XXXVI, F i g , 3). Evidently only one or two chloroplast lobes have been included during the unequal d i v i s i o n . However, a t y p i c a l nucleus (PI. XXXV, F i g . 2j PI. XXXVI, Fig.  3,5), mitochondria (PI. XXXVI, F i g , 3; PI. XXXVII, F i g . 8 ) , ER (PI.  XXXVI, F i g . 3), dictyosomes  (PI. XXXVl, F i g . 4,5), f l o r i d e a n starch grains  (PI,  XXXVI, F i g . 3; PI. XXXVII, F i g , 8) and perhaps a few small vacuoles  (PI,  XXXVI, F i g . 3) are usual components of the spermatangium. Dictyosomes appear to play an important r o l e i n the maturation of these  structures. Soon after c e l l d i v i s i o n these organelles begin to hypertrophy (PI.  XXXVI, F i g . 4 ) and produce numerous vesicles containing a f i b r i l l a r  substance  (PI. XXXVI, F i g . 5) reminiscent of that i n the d i f f e r e n t i a t i n g  monospore (Section Va), However, the contents of the spermatangial v e s i c l e s appear more compacted with a c e n t r a l , electron dense core of highly compressed fibrils  (PI. XXXVI, F i g . 5j PI. XXXVII, F i g . 6,7). These structures are then  released into the c e l l wall by a process s i m i l a r to reverse pinocytosis involving a fusion of the vesicular membrane with the plasmalemma (PI. XXXVII, F i g . 6). The material accumulates  between the spermatangial protoplast  and the c e l l wall, p a r t i c u l a r l y at the margin nearest the plane of the  85.  thallus ( P I . XXXVII, F i g . 6 , 7 ) . Thus, the surface  of a mature sorus  exhibits many i r r e g u l a r protrusions of c e l l wall material at s i t e s of spermatiagenesis ( P I . XXXV, F i g . 2; P I . XXXVII, F i g . 7 ) . Eventually the c e l l wall ruptures and the naked spermatium i s released  ( P I . XXXVIII, F i g ,  9 , 1 0 ) . At this point i t i s evident that the material produced by the dictyosomes i s l i q u i d or semiliquid i n nature as a large amount appears to flow from the t h a l l u s after c e l l  liberation..  Under the l i g h t microscope the released spermatium i s almost colourless due to the lack of extensive chloroplast material It i s u l t r a s t r u c t u r a l l y  i d e n t i c a l to the unreleased  ( P I . XXXVIII, F i g . 1 1 ) . c e l l with a c e r t a i n  amount of vesicular material appearing to remain after release ( P I . XXXVIII, Fig.  9 ) . Numerous attempts to document their function were u n f r u i t f u l .  Evidence of f e r t i l i z a t i o n i n E r y t h r o t r i c h i a boryana; The presence of "spermatia" adhering to filaments of E_. boryana was noted under the l i g h t microscope during two separate c o l l e c t i o n s i n the f a l l of 1 9 7 1 . These attached structures are v i r t u a l l y colourless and possess a c e l l wall ( P I . XXXIX, F i g . 1 ) . It i s evident that the spermatium d i d not preserve well under the f i x a t i o n procedure used for electron microscopy ( P I . XXXIX, F i g , 4 ) . However, considering the nature of the loosely organized  c e l l wall and the  presence of t y p i c a l f l o r i d e a n starch granules, these pale c e l l s are c e r t a i n l y red algal and most probably originated i n d i f f e r e n t filaments of E_. boryana. Mitochondria,  dictyosomes and vacuoles are additional d i s c e r n i b l e structures  ( P I . XXXIX, F i g , 4 ) . No nuclear material was observed but this may have been destroyed  by f i x a t i o n procedures. Of i n t e r e s t i s the short c e l l u l a r " s t a l k "  or "foot" present  i n the area of attachment ( P I , XXXIX, F i g . 1 , 2 , 4 ) . D i r e c t l y  beneath this point, the underlying c e l l usually appears to have undergone an unequal d i v i s i o n s i m i l a r to that i n the spermatangial sorus of Smithora,  86.  with  the r e s u l t i n g s m a l l c e l l b e i n g  This "carpospore-like" c e l l m a t e r i a l , mitochondria, XXXIX, F i g , 3 ) , No  subsequent stages  time of o b s e r v a t i o n .  No  i d e n t i c a l to the  a nucleus,  dictyosomes, ER  a l t h o u g h t h i s c o u l d be due  of any  contains  virtually  and  spermatangium.  a s m a l l amount of c h l o r o p l a s t  f l o r i d e a n starch grains  of v e s i c l e p r o d u c t i o n  (PI.  were noted  to the p a r t i c u l a r s t a g e of development at  t r i c h o g y n e - l i k e s t r u c t u r e s or i n t e r c e l l u l a r  the connections  type were o b s e r v e d . Discussion,  I t i s apparent t h a t the r e l e a s e of dictyosome o r i g i n a t e d v e s i c l e s i n t o the c e l l w a l l i s an i n t e g r a l p a r t of the mechanism of s p e r m a t i a l i n Smithora. When a l a r g e amount of t h i s m u c i l a g a - l i k e e x t e r n a l to the p r o t o p l a s t , the w a l l appears to b u r s t pressure.  It may  a s s o c i a t e d with personal  the r e l e a s e of s p e r m a t i a i n o t h e r  U l t r a s t r u c t u r a l observation  contact."  " f i n g e r " from the u n d e r l y i n g  still  (Section  i t was  on the o u t s i d e of the f i l a m e n t was  As i s shown i n P I .  red algae  not  may  (Neushul,  M.,  postulated  sorus  graphically  e s t a b l i s h i n g or l o s i n g release a  p r o j e c t i n t o the  c l a s s i c example of a t r i c h o g y n e .  protoplasmic pore  I f the spermatium i s  near the e n t r a n c e to the pore a f a l s e i m p r e s s i o n  i m p a r t e d . Thus, p r e v i o u s  a l s o been  determined whether  XXXV ( F i g . 2 ) , a f t e r c e l l vegetative c e l l  on  Va).  of the mature s p e r m a t a n g i a l  Drew's (1956) statements "...  g i v i n g a f a l s e but  to p h y s i c a l  some e f f e c t  be l i k e n e d to t h a t  to a i d i n e r y t h r o p e l t i d a c e a n monospore r e l e a s e  the s m a l l c e l l  as i f due  of the w a l l . M u c i l a g e - l i k e m a t e r i a l has  communication). T h i s mechanism may  illustrates  m a t e r i a l i s accumulated  a l s o be p o s s i b l e t h a t t h i s m a t e r i a l has  the s t r u c t u r a l s t a b i l i t y  liberation  of f e r t i l i z a t i o n i s  r e p o r t s 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. S i n c e the p r o d u c t i o n  and  r e l e a s e of s p e r m a t i a i s a r e g u l a r f e a t u r e of  87.  Smithora. i t i s probable that they play an important role i n the l i f e cycle of the plant. These structures must carry out t h e i r function r e l a t i v e l y rapidly due to t h e i r apparent f r a g i l i t y .  However, thay did not survive i n  culture, thus, i t i s not possible at t h i s time to d i r e c t l y implicate spermatia i n the sexual process of Smithora. At a l i g h t microscope l e v e l the evidence of f e r t i l i z a t i o n  i n J E , boryana  i s indeed i n t r i g u i n g . In c l a s s i c a l terms the observations related here would represent a s i t u a t i o n after f e r t i l i z a t i o n  p r i o r to carpospora release.  The production of carpospores i n J E . boryana would than be i d e n t i c a l to spermatiagenesis as described by Berthold (1882) i n I E . c i l i a r i s and Hollenberg (1959) i n Smithora. It i s d i f f i c u l t  to d i r e c t l y dispute this hypothesis  since, i n t h i s 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 s e l e c t i v e l y release nuclear material. This i s unlikely i n view of the nature of c e l l fusion i n 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 i n the process of release. The formation of a spermatial c e l l wall has been reported to occur i n other red algae during the f r e e - f l o a t i n g state or after adhering to the carpogonial area (see F r i t s c h , 1945 f o r review). An a l t e r n a t i v e , and perhaps more a t t r a c t i v e , explanation of the s i t u a t i o n l  p  L* boryana would involve the induction of female gametogenesis by the  attached spermatium and subsequent Induction of gametogenesis,  fusion with the carpospore-like c e l l .  possibly the r e s u l t of a chemical stimulus,  has been described i n other a l g a l species (Coleman, 1962). In addition, t h i s mechanism would require de novo synthesis of a pyrenoid which has been postulated to occur i n the zoospores of the green algae Qedogonium  88. .  (Hoffman, 1968) and T e t r a c v s t i s (Broyjn and A r n o t t , 1970). If t h i s general  hypothesis  mare t o prove t e n a b l e , t h i s phenomenon  might be c l a s s i f i e d as a type o f isogamous r e p r o d u c t i o n . to note t h a t t h i s c a t e g o r y  of r e p r o d u c t i o n  It i s interesting  i s c h a r a c t e r i s t i c of s i m p l e  r e p r e s e n t a t i v e s o f t h e Chlorophyceae and the Phaeophyceaa. such a t h e o r y must be f u r t h e r s u b s t a n t i a t e d b e f o r e  Nevertheless,  detailed presentation.  89o  PLATE XXXV S m i t h o r a . Spermatangia F i g * 1,  L i g h t micrograph of a c r o s s - s e c t i o n of a s p e r m a t a n g i a l  F i g . 2.  E l e c t r o n micrograph of a c r o s s - s e c t i o n of a s p e r m a t a n g i a l  Note i r r e g u l a r s u r f a c e  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  protoplasm  the  (arrow) and  d i f f e r e n t p o s i t i o n s of the  nuclei  sorus. sorus.  vegetative  (n).  XXXV  90  PLATE X X X v I Smithora., Fig.  3 o Immature  material (nu),  spermatangium i l l u s t r a t i n g  (c), floridean starch  vacuoles  ( v ) and  Spermatangia s m a l l amount o f  g r a i n s ( f s ) , ER,  mitochondria  4.  Immature s p e r m a t a n g i a  Fig.  5.  M a t u r i n g spermatangium w i t h c e n t r a l l y  (arrow).  (n) w i t h n u c l e o l u s  (m).  Fig.  dictyosomes  a nucleus  chloroplast  w i t h dictyosomes  beginning to hypertrophy located nucleus  (n)  (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  (arrow).  and substance  XXXVI  91.  PLATE XXXVII Smithora. Spermatangia F i g . 6. C e l l releasing contents of dictyosome derived v e s i c l e s (arrow) into c e l l w a l l . 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 i n the d i r e c t i o n of the arrow.  F i g , 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  92.  PLATE XXXVIII Smithora. Spermatia Fig.  9c Newly l i b e r a t e d , naked spermatium containing a nucleus (n),  f l o r i d e a n starch grains ( f s ) , ER, some chloroplast material mitochondria  (arrow),  (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, f r e s - f l o a t i n g  spermatia.  Fig.  11. Light micrograph of released, pale  spermatia.  XXXVIII  93.  PLATE XXXIX E r y t h r o t r i e h i a boryana. Fig  0  Fig.  1. Light micrograph of spermatium attached to filament. 2. Electron micrograph of spermatium attached to filament. Note  carpospore-like c e l l and "foot" of attached Fig.  3. Carpospore-like  (c),  f l o r i d e a n starch grains ( f s ) , ER,  cell.  c e l l containing a nucleus (n), chloroplast material dictyosomss (arrow) and  mitochondria  (double arrow). Fig.  4. Attached spermatium e x h i b i t i n g c e l l wall ( w ) , f l o r i d e a n starch grains  ( f s ) , a mitochondrion (m) and vacuoles  (v).  XXXIX  94. V I I . GENERAL DISCUSSION AND  CONCLUSIONS  A_ p r o p o s a l on t h e e v o l u t i o n o f .gr_gujth_ t y p e s i n t h e B a n g i o p h y c i d a e s  From  u l t r a s t r u c t u r a l o b s e r v a t i o n s on t h e E r y t h r o p e l t i d a c e a e r e p o r t e d here and from p u b l i s h e d r e s u l t s on o t h e r members of t h e s u b c l a s s  Bangiophycidae  i t i s e v i d e n t t h a t t h e r e a r e two d i s t i n c t l y d i f f e r e n t t y p e s of r e d 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 b e s t 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 l o b e ( P I . X L ) . In t h e f i r s t  type,  many of t h e p h o t o s y n t h e t i c l a m e l l a e t e r m i n a t e a t t h e c h l o r o p l a s t  envelope,  i n d i c a t i n g t h e absence o f a p e r i p h e r a l t h y l a k o i d . In t h e p r e s e n t 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 d e s i g n a t e d t y p e I. f o l l o w i n g bangiophycean genera p o s s e s s c h l o r o p l a s t s o f t h i s Porphyridium  (Brady and V a t t e r , 1959}  Sarda and L a c o u r l y , 1970}  N e u s h u l , 1970}  R h o d e l l a (Evans, 1 9 7 0 ) , Banqia Cole, unpubl.), Porphyra Akiyama, 1968}  category?  S p e e r , Dougherty and Jones,  Gantt and C o n t i , 1965,1966} G a n t t , Edwards and C o n t i , 1968} Wehrmeyer, 1971}  ( H o n s e l l , 1963}  ( G i b b s , 1960}  1964}  Guerin-Dumartrait,  Ramus, 1972),  Sommerfeld and L e e p e r ,  Ueda, 1961}  Yokomura, 1967}  1970}  Kito  and  Bourne, Conway and C o l e , 1970}  Lee  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  and F u l t z , 1970}  Kazama and F u l l e r , 1970}  The  Bourne,  1971).  o c c u r i n t h e f o l l o w i n g genera: Rhodosorus ( G i r a u d , 1 9 6 3 ) , (fflcBride, u n p u b l . ) , E r y t h r o t r i e h i a (see s e c t i o n s I V , V , V I ) , (McBride, unpubl.), Smithora  Erythrocladia Goniotrichum  (see s e c t i o n s I V , V , V I ) .  On t h e b a s i s of t h i s w e l l d e f i n e d d i f f e r e n c e , i t i s p o s s i b l e t o c o n s t r u c t a p r o p o s a l on t h e e v o l u t i o n of growth t y p e s i n t h e B a n g i o p h y c i d a e Those m u l t i c e l l u l a r genera i n t h e " P o r p h y r i d i u m " l i n e s i d e r e d t o be members of t h e f a m i l y Bangiaceae  (PI, XL).  (type I) are con-  i n the order Bangiales  (presence o f r h i z o i d a l p r o c e s s e s i n the l o w e r c e l l s ; c a r p o s p o r a n g i a  and  95. spermatangia producing  numerous carpospores  and spermatia r e s p e c t i v e l y ) .  The m u l t i c e l l u l a r genera of the "Rhodosorus" l i n e  (type II) are members  of the family Erythropeltidaceae i n the order Bangiales or the family Goniotrichaceae  i n the order Goniotrichales (most lack r h i z o i d a l  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 i n the scheme because t h e i r chloroplast structure i s unknown. It would be e s p e c i a l l y valuable to examine such rare plants as the u n i c e l l u l a r Rhodospora and Chroothece and the m u l t i c e l l u l a r Asterocystis and Porphyropsis. Since the creation of an evolutionary theory must rely on fundamental s i m i l a r i t i e s and differences, the value of the scheme as outlined would l i e i n the presumably very basic, g e n e t i c a l l y c o n t r o l l e d c h a r a c t e r i s t i c s of chloroplast structure. The question arises as to which evolutionary l i n e could have given r i s e to the Florideophycidae  ( i f indeed only one l i n e gave r i s e to t h i s  group). Evidence i n favor of the Rhodosorus l i n e i s appealing. Chloroplasts of type II are found i n c e r t a i n members of the florideophycean order, Nemaliales  e.g. Acrochaetium sp. (McBride, unpubl.) and Thorea r i e k e i  (Bischoff, 1965). Most other members of the Florideophycidae contain d i s c - l i k e chloroplasts which exhibit a peripheral thylakoid. Possibly these structures could be derived more e a s i l y from a type II c h l o r o p l a s t . Evidence i n favor of florideophycean o r i g i n from the Porphyridium  l i n e includes  the presence of p i t connections  i n the conchocelis phase of Porphyra  (Bourne, Conway and Cole, 1970;  Lee and F u l t z , 1970)  and Banqia (Sommerfeld  and Leeper, 1970)* However,, p i t - l i k e structures are a common feature of c e r t a i n Ascomycetes and have been reported i n some blue-green  algae  96. (Butler and Allsopp, 1972), suggesting that they could have arisen  independ-  ently several times. The problem of the o r i g i n of the red algae i s s t i l l somewhat vexing due i n most part to the lack of f o s s i l i z e d forms. Klein and Cronquist (1967) have reviewed the various arguments f o r and against a phylogenetic association between the blua-green and the red algae. A discussion of t h i s type i s beyond the scope of this report. However, i t i s generally agreed that the u l t r a s t r u c t u r a l and physiological c h a r a c t e r i s t i c s of the alga Cyanidium (e.g.  Hirose, 1958;  Allen, 1959;  Bogorad and Mullens, 1962;  Rosen and Siegesmund, 1961;  Mercer,  Seckbach, 1971) indicate an intermediate position  between the two groups. In t h i s context, i t must be stressed that tha evolutionary proposal presented hare does not suggest direct l i n k s among the algae described. It i s r e a l i s e d that present day forms are the r e s u l t of yaars of evolutionary stress and s e l e c t i o n and can bs used only as examples of  growth types.  Changes i n the number and function o_f subcellular components during the course of the l i f e cycle; D i s t i n c t changes i n the number and the amount of subcellular components were noted during the d i f f e r e n t phases i n the l i f e cycle of the plants. In some cases, concomitant with these changss, a difference i n function was observed. Using the vegetative c e l l as a standard f o r comparison,  these changes are outlined below;  1) . Nucleus- there i s an increase i n the number of nuclear pores during monospore d i f f e r e n t i a t i o n , 2) , Dictyosome- these organelles hypertrophy at the onset of monospore d i f f e r e n t i a t i o n and become involved i n 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 i n Smithora, dictyosomes  also hypertrophy  and  produce a f i b r i l l a r product. 3) . Endoplasmic  reticulum- monospore d i f f e r e n t i a t i o n r e s u l t s i n an increase  i n perinuclear ER while subsequent spore gsrmination i s t y p i f i e d by a substantial increase i n peripheral ER which may be involved i n c e l l wall formation. 4) . Mitochondria- these organelles increase i n number during monospore d i f f e r e n t i a t i o n . Swelling and inner membrane disruption occur during spore degeneration. 5) . Chloroplast- lamellae form "pseudogranum-like" structures during monosporogenesis and swell during degeneration. 6) . Vacuoles- a loss of these structures occurs during monosporogenesis with subsequent reformation upon monospore germination. A substantial increase i n the number of vacuoles i s noted during spore degeneration, 7) , Floridean starch grains- these structures increase i n number and s i z e during monospore d i f f e r e n t i a t i o n but undergo a rapid decrease upon monospore germination, 8) . Multivesicular bodies and concentric lamellar bodies- there i s an increase i n the number of both these structures i n older c e l l s and a large number of the former i n degenerating spores.  Conclusions, New  information has been recorded on ths d i s t r i b u t i o n , l i f e h i s t o r i e s  and u l t r a s t r u c t u r e of four species of Erythropeltidaceae found i n tha coastal waters of B r i t i s h Columbia, North American P a c i f i c coast d i s t r i b u t i o n : E r y t h r o t r i e h i a carnea- Piper's Lagoon, Vancouver Is., B r i t i s h Columbia to Colfo Dulse, Costa Ricaj Clipporton Is.  98.  E r y t h r o t r i e h i a boryana- Point N  0  Point (Glacier P t . ) , Vancouver Is.,  B r i t i s h Columbia to Bahio. Ascuncio'n, Baja C a l i f o r n i a , Mexico. E r y t h r o t r i e h i a pulvinata- Bamfield, Vancouver Is., B r i t i s h Columbia to Bahxa Asuncion, Baja C a l i f o r n i a , Mexico. Smithora naiadum- Cape Chiniak, Kodiak Is., Alaska to I s l a Magdalena, Baja C a l i f o r n i a , Mexico. Life histories; Monospores are produced from the basal attachment organs of E r y t h r o t r i e h i a pulvinata and Smithora naiadum. Ultrastructure; Vegetative c e l l - A s i m i l a r vegetative u l t r a s t r u c t u r e i s found among the species examined. S_. naiadum, E_. boryana and E_, pulvinata exhibit an i r r e g u l a r protoplast, a pyrenoid containing chloroplast with s i n g l e uniform lamellae, multivesicular bodies and other t y p i c a l l y rhodophycean s u b c e l l u l a r components. In addition, the chloroplast lamellae of Smithora are occasiona l l y associated i n primitive bands or stacks. IE. carnea i s somewhat d i f f e r e n t than the above species, notably i n c e l l wall and pyrenoid u l t r a s t r u c t u r e . Monospore d i f f e r e n t i a t i o n , release and degeneration- The formation and release of monospores i s dependent on the a c t i v i t i e s of the dictyosome. These organelles produce two substances, one of which may  be involved i n spore  release, the other i n spore attachment. A large amount of perinuclear ER, an increased number of mitochondria and a large number of nuclear pores are t y p i c a l of the d i f f e r e n t i a t i n g monospore. The naked, released spores possess i n t e r e s t i n g "pseudogranum-like" lamellar associations, A c e r t a i n percentage of spores appear to undergo an organized degeneration i n c u l t u r e . Monospore germination- In the s e t t l e d monospore, the formation of a c a l l wall  99.  occurs adjacent to the nuclear region. The germinating  spore i s t y p i f i e d  by an increase i n vacuolar area and a large amount of peripheral ER which may be involved i n c e l l wall formation. Microtubular spindle f i b r e s and a c r y s t a l l i n e matrix i n the pyrenoid are i r r e g u l a r l y present i n 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 i n the Erythropeltidaceae, the u l t r a s t r u c t u r a l account of. spermatiagenesis  i n Smithora i s of i n t e r e s t . The dictyosome plays an important  role i n the maturation  of these pale c e l l s . In addition, the u l t r a s t r u c t u r a l  evidence of f e r t i l i z a t i o n i n £, boryana suggests the possible occurrence of an induction phenomenon. The u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s of the erythropeltidacean chloroplast and other published data on chloroplast u l t r a s t r u c t u r e i n the Bangiophycidae have allowed the presentation of a b i l a t e r a l scheme on the evolution of growth types i n t h i s subclass. In order to obtain a more complete•understanding of the b i o l o g i c a l aspects of the Erythropeltidaceae i t i s evident that there are a number of a d d i t i o n a l areas which must be studied. However, u n t i l the formulation of a culture technique which allows the laboratory observation of the complete l i f e cycle of a p a r t i c u l a r representative, t h i s goal w i l l be d i f f i c u l t to a t t a i n . This i s p a r t i c u l a r l y applicable i n 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 a i d i n the a p p l i c a t i o n of current electron microscopic cytochemical and autoradiographic techniques  i n an e f f o r t to  conclusively describe the s u b c e l l u l a r mechanisms of sporogenesis.  100.  PLATE XL A b i l a t e r a l schsme on the e v o l u t i o n of growth types i n t h e Bangiophycidae p r i m a r i l y based on t h e d i f f e r e n c e i n c h l o r o p l a s t s t r u c t u r e shown i n t h e lower p o r t i o n of t h a p l a t e .  X L  multiseriate, filamentous (Banciia sp.)  bladed (Smithora)  bladed ( Porphyra)  uniseriate, filamentous (Bangia sp.)  multiseriate, filamentous (Erythrotrichia  prostrate, disc-like (Erythrocladia)  unicellular (Porphyridium, Rhodella)  unicellular (Rhodosorus)  Cyanidium-like unicell.?  TYPE I  TYPE II  branched, filamentous (Goniotrichum)  uniseriate, filamentous (Erythrotrichia sp75  101.  VII. LITERATURE CITED A l l e n , M.B. 1959. S t u d i e s w i t h Cyanidium c a l d a r i u m , c h l o r o p h y t e . Arch. W i k r o b i o j . 32: 270-277.  an anomalously pigmented  B a a r d s e t h , E. 1941. The marine algae of T r i s t a n de Cunha. 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