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A field, culture, and cytological study of Porphyra gardneri, Porphyra nereocystis and Porphyra thuretii… Hawkes, Michael William 1978

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A FIELD, CULTURE, AND CYTOLOGICAL STUDY OF POR PHYRA GARDNEHI, PQRPHYRA IEREOCYSTIS AND PORPHYRA THURETII (BHODOPHYTA, BANGIOPHYCIDAE) by MICHAEL WILLIAM HAWKES . Sc. (Honours), U n i v e r s i t y of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Botany Department) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1978 © MICHAEL WILLIAM HAWKES, 197 8 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f< an advanced degree at the U n i v e r s i t y o f B r i t i s h C o lum b i a , I a g r e e tha the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . MICHAEL W. HAWKES Department o f BOTANY  The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 9 MAY 1978 i i A b i o l o g i s t s h o u l d s t r i v e t o be as d i v e r s e i n outlook as Humboldt and Darwin, to observe with the i n t e n s i t y of A g a s s i z , but above a l l , at r i s k of being thought an i c o n o c l a s t , to be ever wary of dogma. i i i Research S u p e r v i s o r : Dr. Robert F, Scagel A B S T R A C T A f i e l d , c u l t u r e and c y t o l o g i c a l i n v e s t i g a t i o n has been made of the l i f e h i s t o r i e s o f Porphyra g a r d n e r i (Smith e t Bollenberg) Hawkes, P. n e r e o c y s t i s Anderson and P. t h u r e t i i S e t c h e l l e t Dawson, three s u p e r f i c i a l l y s i m i l a r e p i p h y t i c red algae (Bhodophyceae, B a n g i a l e s ) . Porphyra g a r d n e r i was found from February through November, but i s b a s i c a l l y a s p r i n g - e a r l y summer s p e c i e s , gpiphyra n e r e o c y s t i s i s an autumn-winter s p e c i e s and £• t h u r e t i i i s a w i n t e r - e a r l y s p r i n g s p e c i e s . The r e p o r t e d number of host s p e c i e s and g e o g r a p h i c a l d i s t r i b u t i o n i s i n c r e a s e d f o r a l l three s p e c i e s . A l i g h t microscope study of spermatogenesis and carposporogenesis was undertaken t o c h a r a c t e r i z e the spermatangium and carpogonium and to f o l l o w t h e i r d i v i s i o n seguence. The spermatangium i n Porphyra g a r d n e r i and probably P. t h u r e t i i i s formed by the production of a new w a l l l a y e r around a transformed v e g e t a t i v e c e l l . In c o n t r a s t , no new w a l l l a y e r i s formed by P. n e r e o c y s t i s spermatangia. making them d i f f i c u l t to d i s t i n g u i s h from v e g e t a t i v e c e l l s . The carpogonium o f P. g a r d n e r i and P. t h u r e t i i i s e a s i l y r ecognized because i t d i f f e r e n t i a t e s b i p o l a r p r o t o t r i c h o g y n e s . In P. n e r e o c y s t i s the carpogonium does not form p r c t c t r i c h o g y n e s and can only be d i s t i n g u i s h e d from v e g e t a t i v e c e l l s by i t s e l o n g a t e , r e c t a n g u l a r shape., i v On the b a s i s o f t h e s e o b s e r v a t i o n s and r e p o r t s i n the l i t e r a t u r e I suggest t h a t t h e s p e c i e s of Porphyra can be separated i n t o a t l e a s t two groups based on t h e i r spermatangial and c a r p o g o n i a l morphology; the more advanced group has carpogonia with p r o t o t r i c h o g y n e s and c l e a r l y d e f i n e d spermatangia due t o the d e p o s i t i o n of a new w a l l l a y e r , whereas the more p r i m i t i v e group l a c k s these f e a t u r e s . The f i r s t d i v i s i o n i n both spermatogenesis and carposporogenesis i s p e r i c l i n a l . T h i s i s d i s c u s s e d with r e s p e c t to the d i v i s i o n sequences r e p o r t e d by other workers. The t r a n s f e r of P o r p h y r e l l a G a r d n e r i Smith e t Hollenberq to Porphyra g a r d n e r i i s e x p l a i n e d on the b a s i s of the observed mode o f carpogonium formation and c a r p o s p o r a n g i a l d i v i s i o n . Of the t h r e e s p e c i e s s t u d i e d , only Porphyra g a r d n e r i produces monospores. Under a l l photoperiod and temperature regimes t e s t e d monospores germinated i n t o the f o l i o s e phase. At an u l t r a s t r u c t u r a l l e v e l monosporogenesis i n Porjphjfra g a r d n e r i i s c h a r a c t e r i z e d by the production of abundant s m a l l and l a r g e f i b r o u s v e s i c l e s . The economic i m p l i c a t i o n s o f monospore production are d i s c u s s e d . F i e l d o b s e r v a t i o n s of Porphyra g a r d n e r i suqqest t h a t photoperiod i s an important f a c t o r i n v o l v e d i n i n d u c i n q the formation of spermatangia and carpogonia. A c o n c h o c e l i s phase i s reported f o r the f i r s t time f o r RQ.I2hlJL<* 2§£^aS£i and Porphyra t h u r e t i i . Under a l l photoperiod and temperature reqimes t e s t e d carpospores qerminated i n t o the c o n c h o c e l i s phase. In P. s a r d n e r i and P. t h u r e t i i s h o r t days favoured V c c n c h o s p o r a n q i a l branch f o r m a t i o n ; however, no such p h o t o p e r i o d i c e f f e c t was observed f o r P. n e r e o c y s t i s . , Although my r e s u l t s were i n c o n c l u s i v e , r e p o r t s i n the l i t e r a t u r e i n d i c a t e t h a t temperature i s a c r i t i c a l f a c t o r i n ccnchospore r e l e a s e . Amoeboid movement of conchospores i s reported f o r E°I£J3J£§ t h u r e t i i and d i s c u s s e d with r e s p e c t t o the occurrence of t h i s phenomenon i n the Rhodophyta g e n e r a l l y . The epiphyte-*host* attachment zone o f Porphyra g a r d n e r i and P. n e r e o c y s t i s was examined with the l i g h t microscope. The b a s a l r h i z o i d s of P. g a r d n e r i penetrate deep i n t o the medulla of the Laminaria blade, whereas the r h i z o i d s o f P. n e r e p c y s t j s make only a s u p e r f i c i a l p e n e t r a t i o n o f t h e N e r e o c v s t i s s t i p e . The spermatia of jgorphyra g a r d n e r i and P. n e r e o c y s t i s were examined u l t r a s t r u c t u r a l l y and found t o have many s i m i l a r i t i e s with F l o r i d e o p h y c i d a e spermatia. The occurrence of f e r t i l i z a t i o n i n the l i f e h i s t o r y of Porphyra g a r d n e r i i s documented with the l i g h t and e l e c t r o n microscopes. A d i p l o i d chromosome number 2n=8 i s r e p o r t e d f o r the carpospores and v e g e t a t i v e c e l l s of the c o n c h o c e l i s phase °f £• g a r d n e r i . T h i s i s the f i r s t unequivocal r e p o r t of se x u a l r e p r o d u c t i o n i n the genus Porphyra. Suspected spermatia a t t a c h e d t o the carpogonia and l i n k e d to them v i a f e r t i l i z a t i o n c a n a l s have been observed i n P.* n e r e o c y s t i s and P. t h u r e t i i sugqestinq t h a t s e x u a l r e p r o d u c t i o n a l s o occurs i n t h e i r l i f e h i s t o r i e s . Reports of sexual r e p r o d u c t i o n i n other members of the Banqiophycidae are evaluated i n l i g h t of my o b s e r v a t i o n s . v i TABLE OF CONTENTS AESTRACT i i i TABLE OF CONTENTS v i LIST OF TABLES ••••••••*•«••••••••* * * ••*•*«***•••••«*••**«* XX LIST OF FIGURES x i i LIST OF APPENDICES XXV ACKNOWLEDGEMENTS ... . xxvi GENERAL INTRODUCTION 1 TERMINOLOGY ......................... . . .................... 4 PART I - FIELD AND HERBARIUM STUDIES ...................... 9 I n t r o d u c t i o n ........................................... 9 M a t e r i a l s and methods .................................. 14 Resul t s ................................................ 17 *• R.Q££fel£.§ g a r d n e r i ................................... 17 1. Seasonal occurrence of epiphyte and 'Host* ....... 17 2. *Host* s p e c i e s ................................... 20 3. A r t i f i c i a l host experiment ....................... 21 4. Ge o g r a p h i c a l d i s t r i b u t i o n ........................ 21 B» Porphyra n e r e o c y s t i s ................................ 23 1. Seasonal occurrence of epiphyte and 'Host 1 ....... 23 2. V e r t i c a l d i s t r i b u t i o n ............................ 26 3. 'Host 1 s p e c i e s 26 4. A r t i f i c i a l host experiment ....................... 26 5. Geographical d i s t r i b u t i o n ........................ 27 C. Porphyra t h u r e t i i ................................... 28 1. Seasonal occurrence .............................. 28 C A EPOSPOROGEN ESIS • • • 2. 1 H o s t * s p e c i e s ... 3. A r t i f i c i a l •Host* experiment .......... 4. Geographical d i s t r i b u t i c n ............, D i s c u s s i o n .... .... ... . . *• Porphyra g a r d n e r i ..... E, Porphyra n e r e o c y s t i s . , C. Porphyra t h u r e t i i ,,.,. FART I I - SPERMATOGENESIS AND I n t r o d u c t i o n ............. M a t e r i a l s and methods .... S€SUltS * • * * • • • a * * * # • • * • • • A. Porphyra g a r d n e r i ..... 1. S perraatogenesis .•.. 2. Carposporogenesis .. 3. Examination o f type B. Porphyra n e r e o c y s t i s .. 1. Spermatogenesis .... 2. Carposporogenesis .. C. Porphyra t h u r e t i i ..... 1. Spermatogenesis .... 2. Carposporogenesis .. D i s c u s s i o n .. • • •.......... PABT I I I - CULTURE STUDIES I n t r o d u c t i o n ........ • •... M a t e r i a l s and methods .... Res u l t s ... * . . . *..•"•.. *.. A. Porphyra g a r d n e r i ..... 1. Monospores ......... l o c a l i t y p l a n t s ... v i i i 2. Spermatangium and carpogcniuw formation .......... 95 3. Carpospore germination - the c o n c h o c e l i s phase ... 95 4. Conchcsporangial branch formation 97 5. Conchospore r e l e a s e 97 B « <i!PJE£lLY££ n e r e o c y s t i s ................................ 98 1. Carpospore germination - the c o n c h o c e l i s phase ... 98 2. Ccnchosporangial branch fo r m a t i o n ................ 100 3. Conchospore r e l e a s e 100 C RQI2k2L& llJM£gtii ......v............................ 101 1. Carpospore germination - the c o n c h o c e l i s phase ... 101 2. Ccnchosporangial branch formation ................ 103 3. Conchospore r e l e a s e .............................. 103 D i s c u s s i o n ............................................. 104 1. Monospores . 104 2. Spermatangium and carpogonium formation .......... 105 3. E f f e c t of photoperiod on monospore and carpospore germination •..................................... 106 4..The c o n c h o c e l i s phase ............................ 107 5. Conchosporangial branches 111 6. Conchospore r e l e a s e .* 113 7. Ccnchospores ....... .....•........................ 116 PAST IV - CYTCLCGICAL STUDIES 128 General i n t r o d u c t i o n ..... ....•......................... 128 M a t e r i a l s and methods .................................. 128 A. Epiphyte-* Host' attachment .......................... 131 I n t r o d u c t i o n ........................................... 131 B e s u l t s ....... .. 132 1* Porphyra g a r d n e r i .............. ..... ............. 132 i x 2. Porphyra n e r e o c y s t i s ...........«... .............. 132 D i s c u s s i o n ....................... ...................... 133 B . Monosporogenesis ........... .. ... ... ... .......... ... • 134 I n t r o d u c t i o n ..............».«•»....»»» .................. 134 BesuIts . '...".*...... . ....."'..« •• * ...... .. •......... .<**.'.«*•* 134 D i s c u s s i o n .....•»• .• '•*...... . .... . .... . . ...... . ... . . ...... . . 136 C . Sexual r e p r o d u c t i o n i n Porphyra g a r d n e r i ............ 137 I n t r o d u c t i o n ........................................... 137 1* The spermatiutn ................................... 139 2. Reports o f f e r t i l i z a t i o n ......................... 141 3. Reports of f u n g a l i n f e c t i o n of Porphyra .......... 143 R e s u l t s .......... ................................. . . ... 146 1. Spermatogenesis - e l e c t r o n microscopy ............ 146 2. F e r t i l i z a t i o n - l i g h t microscopy ................. 147 3. F e r t i l i z a t i o n - e l e c t r o n microscopy .............. 148 4. Chromosome counts 150 5. Feulgen s t a i n i n g .....*....*........*. ..**....•... 150 D i s c u s s i o n * •••••>'•'•"» »•'..". »'«»«»«>».•••»•». «•'...•* ....... ...•« •••.. 152 1. Spermatogenesis .................................. 152 2. F e r t i l i z a t i o n ...••... . •. • •..... ..... ...•• 155 D. Evidence of s e x u a l r e p r o d u c t i o n i n Porphyra n e r e o c y s t i s and Porphyra t h u r e t i i ... ....... 160 I n t r o d u c t i o n .. ................................... ...... 160 R e s u l t s ... v.'. . ... «•.... . ' . - . •'. .......... . .• «•.'••.... 160 1. Porphyra n e r e o c y s t i s ...................... ....... 160 2. Porphyra t h u r e t i i ...................... .......... 161 D i s c u s s i o n ........... .... .................. .. ........ . . 162 GEN ERA! SUMMARY . . . . . . . . . .... . . . . . . . . . ... .V . . . . . . . . . . . . . . . . , 209 X II11BATDEE CITED 212 APPENDIX I ........... ......................... .......... .. 234 APPENDIX I I ......................... ........... ........... 236 x i L I S T OF T A B L E S I . Herbaria from which specimens were examined ...... 38 I I . Host p l a n t s of Porphyra g a r d n e r i 39 I I I . Biomass (grams dry weight) of Porphyra n e r e o c y s t i s per v e r t i c a l meter of N e r e o c y s t i s s t i pe ............................... • 40 IV, Host p l a n t s of Porphyra t h u r e t i i ................. 41 V. Annual v a r i a t i o n i n d u r a t i o n o f d a y l i g h t {hr. and min.) on the 21st day of the month at 35°N and 48° 50.1«N (study s i t e ) ................. 42 VI. Summary of the s p e c i e s of Porphyra which possess p r o t o t r i c h o g y n e s , and those which do not 74 VII. Annual t r e n d i n s u r f a c e water temperature (°C) at the study s i t e (48° 50.1»N, 125° 11.1»W) and Amphitrite Point (48° 55.2*N, 125° 32.2fW) ... 119 V I I I . The s p e c i e s of Porphyra f o r which d i p l o i d chromosome numbers have been r e p o r t e d f o r the carpospores or v e g e t a t i v e c e l l s of the c o n c h o c e l i s phase 163 x i i LIST OF FIGORES 1a. Monosporic t h a l l i cf Porphyra g a r d n e r i growing on the blade margins of Laminaria s e t c h e l l i i . The type specimen (DS 306401 i n UC) ............... 44 1b. A 2.7 m# f e r t i l e specimen of Porphyra n e r e o c y s t i s c o l l e c t e d a t the study s i t e (UBC 57180) .................................. 44 1c. The l e c t o t y p e of Porphjrra tju£§tii (UC 791973) .... 44 2a. The f i e l d work was c a r r i e d out i n Bar k l e y Sound on the west c o a s t of Vancouver I s l a n d ( i n s e t ) . The study s i t e was l o c a t e d o f f Diana I s l a n d ( c i r c l e d ) , near the Bamfield Marine S t a t i o n (from Canadian Hydrographic S e r v i c e C h a r t #L-3001) ... 46 2b. A e r i a l photograph of Diana I s l a n d and the study s i t e . The d i r e c t i o n o f the open P a c i f i c Ocean i s i n d i c a t e d by the arrow, (from B. C. .Government a i r photograph #BC 7238-182) ..... 46 3a. C h a r a c t e r i s t i c heart-shaped new blade growth of Laminaria s e t c h e l l i i (UBC 54895) .................. 48 3b. E a r l y stage i n the s p l i t t i n g of the blade o f L. s e t c h e l l i i (UBC 57204) ......................... 48 3c. Mature, l a c e r a t e d blade of L. s e t c h e l l i i with t h a l l i of Porphyra g a r d n e r i along i t s margins (UBC~54896) ........................... 48 3d. Monosporic t h a l l i of P. jgarjner_i {UBC 54819) ...... 48 Porphyra g a r d n e r i 4a. T h a l l u s of Porphyra g a r d n e r i which has spermatangia, carpogonia and young carposporangia a l o n g i t s l a t e r a l margins. Monospores are s t i l l being r e l e a s e d along the d i s t a l margin (UBC 54821) 50 4b. Old, l a c e r a t e d , and e p i p h y t i z e d t h a l l u s of Laminaria s e t c h e l l i i with a few t h a l l i o f Porphyra g a r d n e r i on i t s margins (arrows). The new Laminaria blade i s j u s t beginning t o grow (UBC 57205) .............................. 50 4c-e. M o r p hological v a r i a t i o n i n t h a l l i of P. g a r d n e r i which are spermatangial and c a r p o s p o r a n g i a l . Note the c h a r a c t e r i s t i c p a t t e r n formed by the spermatangia along the x i i i l a t e r a l margins, f i g u r e 4c: UBC 54138, from B o t a n i c a l Beach; f i g u r e s 4d ana 4es UBC 54891, from Cape Beale ................................... 50 4f. T h a l l u s of Pi gairdneri from the type l o c a l i t y . T h i s p l a n t i s r e l e a s i n g monospores along i t s d i s t a l margin ana spermatia ana carpospores along i t s l a t e r a l margins (UBC 54818) ............. 50 Porphyra n e r e o c y s t i s 5a. Young v e g e t a t i v e t h a l l i are c h a r a c t e r i s t i c a l l y long ana s t r a p shaped, t a p e r i n g g r a a u a l l y to a po i n t (DBC 57187) 52 5b. The formation of spermatangia begins a t the l a t e r a l margins and proceeds b a s a l l y . These r e g i o n s of the t h a l l u s are e a s i l y recognized by t h e i r pale yellow t o white c o l o u r (OBC 57188) ..... 52 5c. as a r e s u l t o f spermatium l i b e r a t i o n , the l a t e r a l spermatangial margins begin t o erode. T h i s s t a r t s a t the apex and proceeds b a s a l l y . The remaining a p i c a l p o r t i o n of the t h a l l u s c o n s i s t s of v e g e t a t i v e c e l l s and carposporangia (UBC 57184) 52 5d. Spermatangia forming marginal and submarginal s t r e a k s which are o r i e n t e d i n an apex to base d i r e c t i o n (UBC 57180) ....... 52 5e. The t i p s of o l d e r t h a l l i often have a s l i g h t l y mottled appearance caused by abundant carposporangia (UBC 57192) ........................ 52 Porphyra t h u r e t i i 6a. M o r p h o l o g i c a l v a r i a t i o n i n t h a l l i which were growing on the s t i p e of N e r e o c y s t i s luetkeana i n Barkley Sound. Note the c h a r a c t e r i s t i c r u f f l e d margins (UBC 57200) ....................... 54 6b. The p r o d u c t i o n of spermatangia begins i n the a p i c a l marginal r e g i o n of the t h a l l u s (UBC 52109) ....................................... 54 6c,d. On o l d e r t h a l l i s h o r t s t r e a k s of spermatangia form submarginally. Note the c h a r a c t e r i s t i c p a t t e r n that r e s u l t s (UBC 57201 and UC 95596 r e s p e c t i v e l y ) 54 6e. Specimen from the Monterey P e n i n s u l a which resembles specimens from Barkley Sound i n s i z e and spermatangial p a t t e r n • 54 x i v Porphyra g a r d n e r i - spermatogenesis, l i g h t microscopy 7a. V e g e t a t i v e c e l l with l a r g e c e n t r a l pyrenoid and c h l o r o p l a s t . The nucleus i s p e r i p h e r a l l y l o c a t e d .;,........................................ 77 7b. New w a l l l a y e r (arrow) l a i d down around spermatangium p r i c r t o the f i r s t d i v i s i o n ......... 77 7c. The f i r s t d i v i s i o n of the spermatangium i s p e r i c l i n a l ........................................ 77 7d-f. Subseguent d i v i s i c n s of the spermatangium ......... 77 7g« Mature spermatangium 4 spermatia deep i n ^ jr&nsv*sxT3*3 ssc"bxon • • • * * •* • • *» • • * * ••*•••••*•*•*•*•«• *7 *7 7h. S u r f a c e view of an immature spermatangium ......... 77 7 i . S u r f a c e view of a mature spermatangium ............ 77 Porphyra g a r d n e r i 8a. Spermatogenesis. The d i v i s i o n sequence l e a d i n g to a mature spermatangium of 64 spermatia. Note the p r o d u c t i o n of a new w a l l l a y e r ( i n d i c a t e d by a t h i c k e r l i n e ) p r i o r to d i v i s i o n , and the absence of any • c r u c i a t e * d i v i s i o n s ............... 79 8b. Carposporogenesis. D i v i s i o n seguence which produced 2, 4 or 8 carpospores per carposporangium. The upper carposporangium c o n t a i n i n g 4 carpospores i s most obvious i n s u r f a c e view and i s shown i n F i g u r e s 9g and 9h. The d i v i s i o n pathway i n d i c a t e d by s o l i d arrows i s most common . , , . . « ^ .......'........*........ .< 79 Porphyra j a r d n e r i - carposporogenesis 9a. Surface view of v e g e t a t i v e c e l l s .................. 81 9b. Surface view of spermatangia (lower l e f t ) , c a rpogonia and carp o s p o r a n g i a ..................... 81 9c. Transverse s e c t i o n of the t h a l l u s showing two carpogonia. Note the b i p o l a r p r o t o t r i c h o g y n e s and attached spermatium (arrow) ................... 81 9d. T r a n s v e r s e s e c t i o n showing two c l o s e l y appressed carpogonia. Note att a c h e d spermatium (arrow) ..................... •..... ........... ..... 81 9e, Transverse s e c t i o n showing t h a t the f i r s t d i v i s i o n o f t h e caposporangium i s p e r i c l i n a l ...... 81 9f . Transverse s e c t i o n showing the o r i e n t a t i o n of XV the t h i r d and f o u r t h d i v i s i o n s of the carposporangium. Shown i n s u r f a c e v i e s i n F i g u r e s 9g and 9h. See a l s o F i g u r e 8a ............. 81 9g. Su r f a c e view of carposporangium t h a t has undergone three d i v i s i o n s and c o n t a i n s 4 carpospores. See a l s o F i g u r e 8a ................... 81 9h. As i n F i g u r e 9g, but a d i f f e r e n t plane of f o c u s ... 81 9 i . Transverse s e c t i o n through a carposporangium c o n t a i n i n g 8 carpospores (only 4 v i s i b l e ) ......... 81 9 j . Surface view of a carposporangium c o n t a i n i n g 8 carpospores (only the fop 4 are v i s i b l e ) .......... 81 Porphyra n e r e o c y s t i s - spermatogenesis 9k. Surface view of the f o l i o s e phase showing v e g e t a t i v e c e l l s with a s i n g l e s t e l l a t e c h l o r o p l a s t ....................................... 81 91. Surface view cf spermatangia which have undergone a few d i v i s i o n s ......................... 81 Porphyra n e r e o c y s t i s - spermatogenesis 10a. Transverse s e c t i o n through an immature spermatangium ..................................... , 83 10b. Surface view of mature spermatangial r e g i o n of the t h a l l u s . I n d i v i d u a l spermatangia are not c l e a r l y d e l i m i t e d ................................. 83 10c. Transverse s e c t i o n through a mature spermatangium which i s 8 spermatia deep ........... 83 Porphyra n e r e o c y s t i s - carposporogenesis 10d. Transverse s e c t i o n through a v e g e t a t i v e c e l l r e g i o n o f the t h a l l u s . Note the s i n g l e s t e l l a t e c h l o r o p l a s t , c e n t r a l pyrenoid and l a t e r a l nucleus ............................. .............. 83 1.0e. Transverse s e c t i o n through a carpogdnium. Note i t s s l i g h t l y more elongate shape compared t o the v e g e t a t i v e c e l l s 83 10f. Transverse s e c t i o n showing two c l o s e l y appressed carpogonia. Note the spermatium (arrow) and f e r t i l i z a t i o n c a n a l 83 10g. Transverse s e c t i o n of two carposporangia shewing t h a t the f i r s t d i v i s i o n i s p e r i c l i n a l ..... 83 10h. Transverse s e c t i o n of two carposporangia. The x v i one on the l e f t i s a t the stage shown i n s u r f a c e view i n Fig u r e 10i, whereas the one on the r i g h t i s a t the stage shown i n Figure 10j. T h i s d i v i s i o n seguence i s the same as t h a t shown f o r Porphyra g a r d n e r i i n Figure 8b 83 10i. Surface view of a carposporangium c o n t a i n i n g 4 carpospores ....................................... 83 10j. Surface view of a carposporangium c o n t a i n i n g 8 (only the top 4 are v i s i b l e ) . See a l s o F i g u r e 10h 83 10k. Surface view of mature carposporangia. I t i s d i f f i c u l t t o determine the boundaries of a s i n g l e carposporangium ............................ 83 101. Transverse s e c t i o n showing two c l o s e l y appressed ca r p o s p o r a n g i a which have undergone more than one d i v i s i o n i n the plane o f the t h a l l u s ........................................... 83 11a. Porphyra n e r e o c y s t i s , t r a n s v e r s e s e c t i o n through a r e g i o n of mature c a r p o s p o r a n g i a . I t i s d i f f i c u l t t o determine the o r i g i n a l boundaries o f a s i n g l e carposporangium ............ 85 Porphyra t h u r e t i i • - spermatogenesis 11b,c. S u r f a c e view showing e a r l y d i v i s i o n s of the spermatangia ...................... ..............., 85 11d. Transverse s e c t i o n through an immature spermatangium ..................................... 85 11e. Mature spermatangium with s u r f a c e dimensions a/4, b/4 and c o n t a i n i n g a t o t a l o f 64 spermatia. ........................................ 85 11f. T r a n sverse s e c t i o n through a mature spermatangium. ..................................., ., 85 Porphyra t h u r e t i i - carposporogenesis 11g. Transverse s e c t i o n through a carpogonium which appears t o have very s l i g h t b i p o l a r p r o t o t r i c h o g y n e s ................. ....... 85 11h. Transverse s e c t i o n showing that the f i r s t d i v i s i o n of the carposporangium i s p e r i c l i n a l ..... 85 11i . T r a n sverse s e c t i o n of a carposporangium t h a t has undergone second and t h i r d a n t i c l i n a l d i v i s i o n s . A s u r f a c e view i s shown i n F i g u r e 11j ........................................ 85 x v i i 11j. S u r f a c e view of a carposporangium which has undergone three d i v i s i o n s 35 11k. Surface view of a mature carposporangium c o n t a i n i n g 8 carpospores (only the top 4 are v i s i b l e ) . See f i g u r e 111 .......................... 85 111. Transverse s e c t i o n of a mature carposporangium .... 85 Porphyra g a r d n e r i 12a. Mcnospores being r e l e a s e d along the margin of a t h a l l u s c o l l e c t e d at the study s i t e ............... 121 12b-f. Various stages i n the development of the f o l i o s e t h a l l u s from a monospore. Note the long r h i z o i d a 1 protuberances ........................... 121 12g. Released carpospores 121 12h, Carpospore germination to g i v e the c o n c h o c e l i s phase ............................................. 121 1 2 i . S i n g l e s p o r e - l i k e s w e l l i n g on the c o n c h o c e l i s f i l a m e n t ............................................ 121 12j. Conchosporangial branches ......................... 121 Por£h_y.ra g a r d n e r i 13a. C o n c h o c e l i s f i l a m e n t s , conchosporangial branch (lower r i g h t ) and r e l e a s e d conchospore (arrow) .... 123 13b. B i p o l a r s p o r e l i n g r e s u l t i n g from conchospore germination .... 123 13c. C h a r a c t e r i s t i c p i n n a t e l y branched morphology of the c o n c h o c e l i s phase growing i n o y s t e r s h e l l ..... 123 Porphyra n e r e o c y s t i s 13d. Beleased carpospores .............................. 123 13e. Carpospore germination to give the c o n c h o c e l i s phase ............................................. 123 13f. B a s a l s p o r e - l i k e s w e l l i n g of the c o n c h o c e l i s phase ............................................. 123 13g. A p i c a l s p o r e - l i k e s w e l l i n g of the c o n c h o c e l i s phase ............................................. 123 13h. I r r e g u l a r and beaded-branch types ................. 123 13i. S g u a r e - c e l l e d branch .............................. 123 x v i i i 13 j . Small c c n c h o s p o r a n g i a l branch connected t o the v e g e t a t i v e f i l a m e n t by a s g u a r e - c e l l e d branch ..... 123 13k. Conchosporangial branches 123 Porphyra n e r e o c y s t i s 14a. Closeup of conchosporangial branches showing the s i n g l e s t e l l a t e c h l o r c p l a s t i n each ccnchosporangium ................................ 125 14b. P i t plugs (arrows) between concbosporangia i n the c c n c h o s p o r a n g i a l branch ( t r e a t e d with chromosome f i x a t i v e and s t a i n ) .................... . 125 14c. D i v i s i o n s w i t h i n the conchosporangial branch ...... 125 14d. C h a r a c t e r i s t i c pinnate branching of the c o n c h o c e l i s phase growing i n o y s t e r s h e l l . Note the i r r e g u l a r s w e l l i n g s ........................... 125 Porphyra t h u r e t i i 14e. Released carpospores .............................. 125 14f. Carpospore germination to give the c o n c h o c e l i s phase ............................................. 125 14g. V e g e t a t i v e c o n c h o c e l i s f i l a m e n t s , beaded branches and young c o n c h o s p o r a n g i a l branch (arrow) ....................... .... ................ 125 14h. C l u s t e r of conchosporangial branches showing s i n g l e s t e l l a t e c h l o r o p l a s t i n each conchosporangium .................................. 125 14i . Candelabra-shaped c l u s t e r of conchosporangial branches showing t h e i r p o s i t i v e phototropism ...... 125 14j. C cnchosporangial branch with conchospores ......... 125 14k. Released conchospores ............................. 125 Porphyra t h u r e t i i 15a-d. Conchospores e x h i b i t i n g amoeboid change of shape and movement. F i g u r e s 15a and 15b taken 90 seconds a p a r t . F i g u r e s 15c and 15d taken 45 seconds apart ...................................... 127 15e. Young b i p o l a r s p o r e l i n g r e s u l t i n g from conchospore germination. Note r h i z o i d a l protuberance ...................................... 127 xix 151,g. O l d e r , u n i s e r i a t e s p o r e l i n g s s t a r t i n g to form b a s a l pad ................................... 127 15h. B i s e r i a t e stage of s p o r e l i n g growth 127 1 5 i , j . O lder s p o r e l i n g s . Note the b a s a l r h i z o i d a l pad in F i g u r e 15 j . ............................... 127 16a. Transverse s e c t i o n of the blade margin o f Laminarja s e t c h e l l i i and the base of Porphyra g a r d n e r i . The r h i z o i d a l f i l a m e n t s can be seen p e n e t r a t i n g i n t o the medulla of the Lamiuaria blade .............................. 166 16 fe. Closeup of the r h i z o i d a l p e n e t r a t i o n i n t o the medulla .............. ... .......... .......... 166 16c. Transverse s e c t i o n through a N e r e o c y s t i s s t i p e and the Porphyra n e r e o c y s t i s attached to i t ....... 166 16d. Closeup of the c e n t r a l r e g i o n of the attachment d i s c ................................ .. ............ 166 17. Tranverse s e c t i o n o f Porphyra g a r d n e r i t h a l l u s , through a young monosporangium. Note dictyosomes, s m a l l f i b r o u s v e s i c l e s and l a y e r of f i b r o u s m a t e r i a l being formed on the o u t s i d e of the plasma membrane ............................. 168 18. Transverse s e c t i o n o f Porphyra g a r d n e r i t h a l l u s through a monosporangium which has developed beyond the stage shown i n F i g u r e 17. Small f i b r o u s v e s i c l e s are abundant and l a r g e f i b r o u s v e s i c l e s are beginning to form .................... 170 19a. Dictyosome a c t i v i t y producing s m a l l f i b r o u s v e s i c l e s 172 19b. Closeup of a monosporangial w a l l showing the depositon of f i b r o u s m a t e r i a l along i t s i n n e r s u r f a c e (arrow) ................................... 172 19c. Monosporangium i n the f i n a l stages of d i f f e r e n t i a t i o n p r i o r t o monospore l i b e r a t i o n . Both s m a l l and l a r g e f i b r o u s v e s i c l e s are abundant 172 Porphyra g a r d n e r i - spermatogenesis, e l e c t r o n microscopy (transverse s e c t i o n s ) 20a. V e g e t a t i v e c e l l o f the f o l i o s e t h a l l u s ............ 174 20b. The f i r s t d i v i s i o n o f the spermatangium i s n e a r l y complete. Note the new w a l l l a y e r XX {arrow) that has been l a i d down i n the t r a n s i t i o n from a v e g e t a t i v e c e l l ................. 174 20c,d. L a t e r d i v i s i o n s t a g e s of the spermatangium ........ 174 Porphyra g a r d n e r i - spermatogenesis, e l e c t r o n microscopy {transverse s e c t i o n s ) 21a. F i n a l d i v i s i o n s i n t h e formation of a mature spermatangium. Note the pro d u c t i o n of l a r g e Jl lb £ O U S V6 S I C JL*3S • * • • • • • • • • • • • • *• ••. •* '*. • • • * • • • • • • • #•- 1V 6 21b. Spermatium from a mature spermatangium. I t c o n t a i n s a nuc l e u s , mitochondria, reduced c h l o r o p l a s t , s e v e r a l s m a l l and one l a r g e f i b r o u s v e s i c l e . Note s m a l l f i b r o u s v e s i c l e emptying i t s contents i n t o the l a r g e f i b r o u s v e s i c l e {arrow) 176 21c. Two spermatia i n a spermatargium near the r e l e a s i n g margin. The l a r g e f i b r o u s v e s i c l e has j u s t become e x t r a c y t o p l a s m i c . ..................... 176 22. Mature spermatium j u s t p r i o r t o r e l e a s e . Note the nucleus with h i g h l y condensed chromatin and no n u c l e a r membrane, reduced c h l o r o p l a s t with numerous p l a s t o g l o b u l i , mitochondrion and abundant s m a l l f i b r o u s v e s i c l e s ................... 178 £oi£kxia. g a r d n e r i - f e r t i l i z a t i o n , l i g h t microscopy Transverse s e c t i o n s 23a. Spermatium att a c h e d t o the prot o t r i c h o g y n e of the carpogonium 180 23b. Two spermatia with f e r t i l i z a t i o n c a n a l s going i n t o the carpogonium .............................. 180 23c. Spermatia attached t o both p r o t o t r i c h o g y n e s (note f e r t i l i z a t i o n c a n a l s ) ....................... 180 24a,b. Two scanning e l e c t r o n micrographs of the t h a l l u s s u r f a c e o f Porphyra j a r d n e r i showing s e v e r a l spermatia attached to the p r o t o t r i c h o g y n e s .................................. 182 Por£hy.ra g a r d n e r i - f e r t i l i z a t i o n , e l e c t r o n microscopy (tr a n s v e r s e s e c t i o n s ) 25a. Low m a g n i f i c a t i o n view of a spermatium att a c h e d t c the p r o t o t r i c h o g y n e 184 25b-d. Three examples of spermatia attached to the x x i p r o t o t r i c h o g y n e p r i o r t o the formation of the f e r t i l i z a t i o n c a n a l . They c o n t a i n a c h a r a c t e r i s t i c reduced c h l o r o p l a s t , mitochondria and non-membrane bound n u c l e a r m a t e r i a l .......................................... 184 Porphyra g a r d n e r i - f e r t i l i z a t i o n 26. Transverse s e c t i o n through a carpogonium and two spermatia which have t r a n s f e r r e d t h e i r n u c l e a r m a t e r i a l down the f e r t i l i z a t i o n c a n a l i n t o the carpogonium .............................. 186 Porphyra g a r d n e r i - f e r t i l i z a t i o n 27. Transverse s e c t i o n through a carpogonium showing both p r o t o t r i c h o g y n e s , c e n t r a l pyrenoid and l a t e r a l n ucleus. The presence of a s m a l l d e p o s i t o f w a l l m a t e r i a l (arrow) i n the pr o t o t r i c h o g y n e below the spermatium i n d i c a t e s t h at a f e r t i l i z a t i o n c a n a l has been formed, although i t i s not v i s i b l e i n the plane of s e c t i o n . Suspected Cyanophyta are a l s o v i s i b l e on the t h a l l u s s u r f a c e (double arrow) ............. 188 Porphyra g a r d n e r i - f e r t i l i z a t i o n ( t r a n s verse s e c t i o n s ) 28a,b. Two spermatia which have t r a n s f e r r e d t h e i r n u c l e a r m a t e r i a l down the f e r t i l i z a t i o n c a n a l i n t o the carpogonium, but which s t i l l have the remains of a c h l o r o p l a s t l e f t i n them ............. 190 28c,d. P r o t o t r i c h o g y n e r e g i o n of the carpogonium with a reduced c h l o r o p l a s t which i s thought t o have come from the spermatium during the t r a n s f e r of nu c l e a r m a t e r i a l .................................. 190 Porphyra g a r d n e r i - f e r t i l i z a t i o n 29. Transverse s e c t i o n through a carpogonium which has the remains of a spermatium attached to the p r o t o t r i c h o g y n e , and what may be the h i g h l y condensed s p e r m a t i a l n u c l e a r m a t e r i a l (arrow) ..... 192 Porphyra g a r d n e r i - carp c s p o r o g e n e s i s (transverse s e c t i o n s ) 30a. F e r t i l i z e d carpogonium showing the two daughter n u c l e i r e s u l t i n g from the f i r s t d i v i s i o n of the f u s i o n nucleus. The f e r t i l i z a t i o n c a n a l i s s t i l l p r esent although the spermatium has d i s i n t e g r a t e d ................ ........................ 194 x x i i 30b. The f i r s t d i v i s i o n o f the carposporangium i s p e r i c l i n a l and produces 2 carpospores (note the f e r t i l i z a t i o n c a n a l s t i l l present i n the wall) ..... 194 P2rj3hy_ra g a r d n e r i - chromosome counts 31a. Spermatium, n=4 ................................... 196 31b. Veg e t a t i v e c e l l of the f o l i o s e t h a l l u s , n=4 ....... 196 31c,d. Carpospore, 2n=8 196 31e,f. V e g e t a t i v e c e l l of the c o n c h o c e l i s phase, 2n=8 .... 196 Porphyra g a r d n e r i - Feulgen s t a i n i n g ( t r a n s v e r s e s e c t i o n s ) 32a. Mature spermatangium, most spermatia s t i l l have n u c l e i i n a d i f f u s e i n t e r p h a s e s t a t e (arrow) ...... 198 32b. Mature spermatangium r i g h t on the r e l e a s i n g margin. The n u c l e a r m a t e r i a l i s h i g h l y condensed (arrow) ................................. 198 32c. Seleased spermatia with h i g h l y condensed nucl e a r m a t e r i a l (dark dots) 198 Porphyra g a r d n e r i - Feulgen s t a i n i n g ( t r a n s v e r s e s e c t i o n s ) 33a. Carpogonium and attached spermatium which c o n t a i n s condensed n u c l e a r m a t e r i a l . No f e r t i l i z a t i o n c a n a l i s present .................... 200 33b. Carpogcnium and c a r p o g o n i a l nucleus l a t e r a l l y l o c a t e d . The attached spermatium c o n t a i n s n u c l e a r m a t e r i a l i n a d i f f u s e s t a t e , and a f e r t i l i z a t i o n c a n a l i s present .................... 200 33c,d. Two examples of spermatia c o n t a i n i n g condensed n u c l e a r m a t e r i a l i n c l o s e a s s o c i a t i o n with the f e r t i l i z a t i o n c a n a l ............................... 200 33e. Carpogonium with attached empty spermatium and a densely s t a i n i n g mass of s p e r m a t i a l n u c l e a r m a t e r i a l i n the carpogonium c l o s e to the f e r t i l i z a t i o n c a n a l ............................... 200 3 3 f - i . S e v e r a l examples of carpogonia with empty spermatia on t h e i r o u t e r w a l l s and from 1-5 densely s t a i n i n g masses of s p e r m a t i a l n u c l e a r m a t e r i a l i n s i d e 200 x x i i i 3 3 j . Carpogonium with l a r g e , suspected f u s i o n nucleus (note empty spermatium and f e r t i l i z a t i o n canal) .............................. 200 33k. The f i r s t d i v i s i o n of the f u s i o n nucleus p r i o r to c y t o k i n e s i s (see a l s o F i g u r e 30a) .............. 200 331. Two carpospores produced by the f i r s t d i v i s i o n o f the carposporangium (see a l s o F i g u r e 30b) ...... 200 33tt-o. Subseguent stages of d i v i s i o n t o produce 4 (Figure 33m) or 8 (F i g u r e 33o) carpospores ........ 200 34. Diagram summarizing the known morphological and c y t o l o g i c a l l i f e h i s t o r y c f Porphyra g a r d n e r i . I t i s suspected that meiosis occurs i n t h e conchosporangium at the time of conchospore for m a t i o n ......................................... 202 35. Porphyra n e r e o c y s t i s spermatium i n a spermatangium near the r e l e a s i n g margin. The spermatium c o n t a i n s abundant s m a l l and l a r g e f i b r o u s v e s i c l e s , a reduced c h l o r o p l a s t , mitochondria, and c e n t r a l nucleus ................. 204 Porphyra n e r e o c y s t i s 36a,b. Transverse s e c t i o n of the f o l i o s e t h a l l u s through carpogonia and suspected spermatia forming f e r t i l i z a t i o n c a n a l s ...................... 206 36c. E l e c t r o n micrograph o f a t r a n s v e r s e s e c t i o n through a suspected spermatium and f e r t i l i z a t i o n c a n a l ............................... 206 Porphyra t h u r e t i i 36d. Transverse s e c t i o n of the f o l i o s e t h a l l u s through a carpogonium and suspected spermatium ...., 206 36e. Transverse s e c t i o n o f the f o l i o s e t h a l l u s through a carposporangium that has undergone the f i r s t d i v i s i o n . A suspected spermatium and f e r t i l i z a t i o n c a n a l a re present ................... 206 37a. Porphyra n e r e o c y s t i s spermatia, n=3 ............... 208 37b. Porphyra n e r e o c y s t i s v e g e t a t i v e c e l l s of the f o l i o s e t h a l l u s , n=3 .............................. 208 37c. Porphyra t h u r e t i i spermatium, n=2 ................. 208 37d,e Porphyra n e r e o c y s t i s carpospore, 2n=6. (only 5 chromosomes v i s i b l e ) .............................. 208 xx i v 37f,g Porphyra n e r e o c y s t i s conchosporangial branch, 2n=6 .7 . . . . . . . . 2 0 8 XXV LIST OF APPENDICES I. Porphyra c o l l e c t i n g s t a t i o n s and dates of c o l l e c t i o n s ........................................ 234 I I . R e p r e s e n t a t i v e specimens of Porphyra g a r d n e r i , P. n e r e o c y s t i s and P. t h u r e t i i ..................... 236 x x v i ACKNOWLEDGEMENTS I am indebted t o Dr. fl. F. Scagel f o r use of the c u l t u r e f a c i l i t i e s , a d v i c e and generous f i n a n c i a l support (NEC Grant A-4471) throughout t h i s study. N a t i o n a l Research C o u n c i l of Canada Postgraduate s c h o l a r s h i p s have enabled me to c a r r y out t h i s work. Dr. K, Cole i s g r a t e f u l l y acknowledged f o r p r o v i d i n g me with a c c e s s to her l a b o r a t o r y equipment used i n pre p a r i n g my specimens f o r t h e c y t o l o g i c a l s t u d i e s . I would l i k e to express my a p p r e c i a t i o n to Dr. G. C. Hughes f o r i n f o r m a t i o n on f u n g a l i n f e c t i o n s of Porphyra. t o Dr. T. F. Mumford J r . , f o r p r o v i d i n g me with some of the p e r t i n a n t l i t e r a t u r e , and t o Dr. R. E. Foreman and Dr. P. G. H a r r i s o n f o r suggesting improvements i n the manuscript. S p e c i a l thanks go to C h r i s Tanner f o r h i s u n f a i l i n g a s s i s t a n c e on SCOBA d i v e s t o c o l l e c t specimens. The comradeship which developed on our t r i p s to the sea and to the mountains w i l l not be f o r g o t t e n . The e x c e l l e n t t e c h n i c a l a d v i c e on e l e c t r o n microscopy and photography given t o me by l a s z l o Veto i s a p p r e c i a t e d . Thanks are a l s o extended to David Walker f o r i n t r o d u c i n g me to the JB-4 methacrylate technique. Dr. I. A. Abbott provided me with e x c e l l e n t f a c i l i t i e s and a s s i s t a n c e d u r i n g my stay a t Hopkins Marine S t a t i o n , and permitted me t o examine G. M. Smith herbarium specimens i n her care. Thank you t o Dr. G. J . Hollenberg f o r l e n d i n g me the holotype of P o r p h y r e l l a c a l i f o r n i c a and specimens of Porphyra g a r d n e r i ; t o Dr. P. C. S i l v a f o r the l o a n o f the holotype of P o r p h y r e l l a g a r d n e r i : t o Dr. M. J . Wynne f o r specimens from Amchitka I s l a n d ; and t o R. Setzer f o r the l o a n x x v i i of specimens o f Porphyra g a r d n e r i . My parents i n t e r e s t i n my s t u d i e s has been an i n s p i r a t i o n . The encouragement and p a t i e n t understanding of Denise Bonin has made the task of w r i t i n g t h i s t h e s i s t o l e r a b l e . 1 GENERAL INTRODUCTION De s p i t e the g r e a t amount of i n v e s t i g a t i o n t h a t the genus Porphyra has r e c e i v e d , t h e r e i s not a s i n g l e s p e c i e s f o r which the complete l i f e h i s t o r y , both morphological and c y t o l o g i c a l , i s known. H i g i t a ' s (1967b) work on Porphyra yezognsis Ueda i s the most thorough study t o date. U n f o r t u n a t e l y he d i d not present a d e t a i l e d documentation of the f e r t i l i z a t i o n process, which i s the major f e a t u r e of the Porphyra l i f e h i s t o r y r e g u i r i n g v e r i f i c a t i o n . One of my primary o b j e c t i v e s i n t h i s study was to r e c t i f y t h i s s i t u a t i o n by attempting t o answer the much d i s p u t e d g u e s t i o n of t h e occurrence of s e x u a l r e p r o d u c t i o n i n the genus. The other main o b j e c t i v e was to c h a r a c t e r i z e 3 s u p e r f i c i a l l y s i m i l a r e p i p h y t i c , monostromatic Porphyra s p e c i e s by o b t a i n i n g as much i n f o r m a t i o n about t h e i r l i f e h i s t o r i e s and b a s i c b i o l o g y as p o s s i b l e . Three approaches to these problems were taken. The f i r s t was a f i e l d study t o determine the seasonal occurrence of the f o l i o s e phase and the time of spore formation. The second was a c u l t u r e study of spore f o r m a t i o n and the e f f e c t of s e l e c t e d environmental f a c t o r s on spore formation and germination. The t h i r d approach was a c y t o l o g i c a l study to o b t a i n chromosome counts f o r a l l spore types and l i f e h i s t o r y stages to see i f t h e r e was any change i n the p l o i d y l e v e l , thus i n d i r e c t l y i n d i c a t i n g the occurrence of f e r t i l i z a t i o n and m e i o s i s . A l i g h t and e l e c t r o n microscope study of sporogenesis by the 2 f o l i o s e t h a l l u s was undertaken because I f e l t t h a t i t might gi v e d i r e c t evidence o f the occurrence of f e r t i l i z a t i o n . I n i t i a l l y I began working with Porphyra n e r e o c y s t i s Anderson. I t i s one of t h e l a r g e s t Porphyra s p e c i e s , and i s r e p o r t e d l y very p a l a t a b l e (Hus, 1902). I t t h e r e f o r e seemed to be a p l a n t with p o t e n t i a l commercial v a l u e . I t s e p i p h y t i c h a b i t a l s o i n t r i g u e d me, and I was c u r i o u s t o see i f any s p e c i a l l i f e h i s t o r y m o d i f i c a t i o n s had been made to adapt i t to i t s •host* p l a n t , N e r e o c y s t i s luetkeana (Mertens) P o s t e l s et Buprecht. During the f i r s t year o f my f i e l d s t u d i e s I became aware of Porphyra g a r d n e r i (Smith e t Hollenberg) Hawkes*, which s u p e r f i c i a l l y resembles Porphyra n e r e o c y s t i s and had been co n s i d e r e d by Gardner ( i n : C o l l i n s e t a l . 1919) and K y l i n (1941) to be a dwarf or young form o f t h i s s p e c i e s . In view of t h i s , and because no l i f e h i s t o r y i n f o r m a t i o n had been published on t h i s a l g a s i n c e i t was d e s c r i b e d by Smith and Hollenberg (1943), I decided to i n c l u d e i t i n my study. T h i s was a f o r t u n a t e d e c i s i o n because i t proved to be the most convenient p l a n t t o work with. As a r e s u l t , the major part of t h i s t h e s i s d e a l s with Porphyra g a r d n e r i . During the second year of my f i e l d o b s e r v a t i o n s I c o l l e c t e d Porphyria t h u r e t i i S e t c h e l l et Dawson on the s t i p e of M§£§°cy.stis. T h i s was the f i r s t time t h a t t h i s s p e c i e s had l l h e n I began my i n v e s t i g a t i o n , t h i s p l a n t was known as P o r p h y r e l l a g a r d n e r i Smith et Hollenberg. As a r e s u l t of my ob s e r v a t i o n s of the mode of carpogonium formation and d i v i s i o n (to be d i s c u s s e d i n Part I I ) I t r a n s f e r r e d t h i s s p e c i e s t o the genus Porphyra (Hawkes, 1977b). 3 been found north of Oregon (Conway et a l . 1975). Because so l i t t l e was known about th e ecology and l i f e h i s t o r y of t h i s a l g a I decided to i n c l u d e i t i n ay study. I have presented the t h e s i s i n f o u r p a r t s : P a r t I - F i e l d and herbarium s t u d i e s ; P a r t I I - Spermatogenesis and carposporogenesis; Part I I I - C u l t u r e s t u d i e s ; and P a r t IV-C y t o l o g i c a l s t u d i e s . P a r t I I i s one aspect o f the c y t o l o g i c a l s t u d i e s which I have presented s e p a r a t e l y , near the beginning of the t h e s i s , because i t i s e s s e n t i a l i n j u s t i f y i n g the t r a n s f e r of P o r p h y r e l l a aardjneri to the genus Porphyra. 4 TERMINOLOGY The morphological l i f e h i s t o r y o f most Porphyra s p e c i e s c o n s i s t s of an a l t e r n a t i o n between a f o l i o s e t h a l l u s and a fil a m e n t o u s phase. I w i l l be r e f e r r i n g t o these l i f e h i s t o r y stages as the f o l i o s e t h a l l u s or phase, and the c o n c h o c e l i s phase, r e s p e c t i v e l y . There i s a p l e t h o r a of terminology which has been a p p l i e d to the r e p r o d u c t i v e s t r u c t u r e s of Porphyra. In her c l a s s i c work on the Bangiophycidae Drew (1956) presented a spore c l a s s i f i c a t i o n scheme which i n c l u d e d Porphyra. Richardson (1972) proposed a scheme s p e c i f i c a l l y f o r Bangia and Porphyra. As a r e s u l t of my o b s e r v a t i o n (Hawkes, 1977a, 1978) that sexual r e p r o d u c t i o n occurs i n the l i f e h i s t o r y of Porphyra g a r d n e r i . a reassessment and s t a n d a r d i z a t i o n o f terminology was necessary. The f o l l o w i n g i s a summary c f the terms I have used f o r the r e p r o d u c t i v e s t r u c t u r e s and spore types which I encountered i n the 3 Porph,yra s p e c i e s I s t u d i e d . For a d e t a i l e d summary of the o r i g i n a l sources of these terms as w e l l as other terminology t h a t has been a p p l i e d to the r e p r o d u c t i v e s t r u c t u r e s of the f o l i o s e phase see Conway et a l . (1975, p. 189) . F o l i o s e Phase Spermatangium - produces the spermatia by a s e r i e s of p e r i c l i n a l and a n t i c l i n a l d i v i s i o n s . Synonymous 5 with J2> -spore mother c e l l (Conway e t a l . 1975), packet (Hus, 1902), ana antheridium (Hus, 1902; K u r o g i , 1972). Spermatium - the male gamete. Haploid. Synonymous with ^ - s p o r e (Conway et a l . 1975), and a n t h e r z o i d s (Hus, 1902). Cargogonium - formed by the t r a n s f o r m a t i o n of a v e g e t a t i v e c e l l , may or may not produce p r o t o t r i c h o g y n e s . Synonymous with mother c e l l s (Drew, 1956) and -spore mother c e l l s (Conway e t a l . 1975; Hawkes, 1977b). l E o t o t r i c h o g y n e - r e c e p t i v e protuberance of the carpogonium. Length v a r i e s with the s p e c i e s and not a l l s p e c i e s produce them. Honcstromatic s p e c i e s produce 2 per carpogonium, whereas d i s t r o m a t i c s p e c i e s produce only one. Carposporangium - f o l l o w i n g f e r t i l i z a t i o n the carpogonium becomes the carposporangium. Produces the carpospores by a s e r i e s of p e r i c l i n a l and a n t i c l i n a l d i v i s i o n s . The p o s s i b i l i t y e x i s t s t h a t t h i s can occur without f e r t i l i z a t i o n . Synonymous with packet, sporocarps (Hus, 1902), c y s t o c a r p s (Kurogi, 1972), and packets of O^-spores (Conway e t a l . 1975; Hawkes, 1977b). 6 Carpospore - r e l e a s e d from the carposporangium and germinates i n t o the c o n c h o c e l i s phase. D i p l o i d , although h a p l o i d carpospores may form i n some cases. Syncnymous with ^ - s p o r e s (Conway g t a l . 1975; Hawkes, 1977b). Monos£ore - a s e x u a l spore produced by the f o l i o s e phase of s e v e r a l Porphyra s p e c i e s by the t r a n s f o r m a t i o n of a v e g e t a t i v e c e l l without any d i v i s i o n s . F o l l o w i n g r e l e a s e a monospore germinates back i n t o the f o l i o s e phase. Synonymous with n e u t r a l spore (Kurogi, 1961). In some of the Japanese l i t e r a t u r e (eg. Kur o g i , 1953b) the conchospores were r e f e r r e d t o as monospores. The term monospore has a l s o been a p p l i e d t o spores produced by the c o n c h o c e l i s phase which germinate to give more c o n c h o c e l i s phase (Conway and C o l e , 1977). Conchocelis Phase QgSghoseorangial branch - t y p i c a l l y an u n i s e r i a t e branch which i s wider than the v e g e t a t i v e c o n c h o c e l i s f i l a m e n t s and c o n s i s t s of a l i n e a r s e r i e s of conchospor angia. C h a r a c t e r i z e d by a t h i c k c e l l w a l l compared to the v e g e t a t i v e f i l a m e n t s . Each conchosporangium c o n t a i n s a s i n g l e s t e l l a t e c h l o r o p l a s t . The a v a i l a b l e evidence suggests t h a t 7 the conchosporangium i s the s i t e of meiosis (Giraud and Magne, 1968; K i t o , 1974). Conchosporangial branch i s synonymous with monosporangial branch {Kurogi, 1953b). Conchospores - formed by 2-4 d i v i s i o n s of the conchosporangium i n some s p e c i e s ( M i g i t a and Abe, 1966; M i g i t a , 1967b; 1974). I t i s s t i l l not c l e a r whether only one conchospore per conchosporangium i s produced by some s p e c i e s . R e f e r r e d to as monospores i n some of the Japanese l i t e r a t u r e (eg. Kurogi, 1953b) . A b r i e f j u s t i f i c a t i o n o f my s e l e c t i o n of terms f o r the r e p r o d u c t i v e s t r u c t u r e s formed by the f o l i o s e phase i s i n of Porphyra g a r d n e r i are homologous with t h e i r c o u n t e r p a r t s i n the F l o r i d e o p h y c i d a e . In view of t h i s , and i n an attempt t o be c o n s i s t e n t with p r e v i o u s work (Papenfuss, 1955), and to st a n d a r d i z e the terminology i n the two s u b c l a s s e s , the terms spermatium, spermatangium, carpogonium and carpospores have been used. D i f f e r e n c e s i n the p o s i t i o n and mode of fo r m a t i o n of the spermatia and carpospores have been pointed to as c r i t e r i a c h a r a c t e r i z i n g the Bangiophycidae and F l o r i d e o p h y c i d a e ( F r i t s c h , 1945, p. 437; Drew, 1951). In the former, s e v e r a l spermatia are produced per spernatangium, whereas i n the l a t t e r o nly one i s produced. A f t e r f e r t i l i z a t i o n i n Porphyra g a r d n e r i the e n t i r e zygote d i v i d e s to produce s e v e r a l carpospores per order. F u n c t i o n a l l y the spermatia, carpogonia and carpospores 8 carposporangium, but i n the F l o r i d e o p h y c i d a e the zygote d i r e c t l y or i n d i r e c t l y g i v e s r i s e t o the gonimoblast whose f i l a m e n t s then produce a s i n g l e carpospore per carposporangium. I t should be noted t h a t the d e t a i l s of spermatium production r e a l l y only apply t o t h e Bangiaceae, and a p p a r e n t l y not a l l Bangiophycidae. For example, i n Smithora o n l y one spermatium per spermatangium i s produced. I t i s f e l t t h a t these d i f f e r e n c e s are not o f s u f f i c i e n t magnitude to warrant c r e a t i n g new terms f o r the r e p r o d u c t i v e s t r u c t u r e s i n Porphyra. The s l i g h t protuberances of the carpogonium found i n most s p e c i e s cf Porphyra (Table VI) have been r e f e r r e d to as pseudo-trichogynes (Dangeard, 1927), p r o t o t r i c h o g y n e s (Tseng and Chang, 1955), 1 t r i c h o g y n e - l i k e * (Conway and C o l e , 1973) and t r i c h o g y n e s (Kunieda, 1939). In Porphyra g a r d n e r i these s p e c i a l i z e d protuberances of the carpogonium a c t as the r e c e p t i v e s i t e f o r the spermatia and seem t o be r e s p o n s i b l e f o r the s p e c i f i c i t y o f spermatium attachment. F u n c t i o n a l l y they are homologous with the t r i c h o g y n e s of the F l o r i d e o p h y c i d a e ; however, because they are not as w e l l developed the term p r c t c t r i c h o g y n e would seem most a p p r o p r i a t e . 9 PART I - FIELD AND HERBARIUM STUDIES I n t r o d u c t i o n Before attempting any c u l t u r e work or c y t o l o g i c a l i n v e s t i g a t i o n I f e l t t h a t i t was of the utmost importance to f a m i l i a r i z e myself with P o o ^ l i a aardjae_ri, £.. n e r e o c y s t i s . and £• t h u r e t i i i n the f i e l d . The purpose of the f i e l d study was to determine the seasonal occurrence, type and time of spore f o r m a t i o n , * host' s p e c i e s , and g e o g r a p h i c a l d i s t r i b u t i o n f o r each s p e c i e s . I hoped that t h i s i n f o r m a t i o n would enable me to f i x t h a l l i a t the r i g h t time f o r c y t o l o g i c a l study of chromosome numbers and s e x u a l r e p r o d u c t i o n . Furthermore, because these 3 s p e c i e s are e p i p h y t e s , I thought t h a t some knowledge of the sea s o n a l development of the • host{s)« would be e s s e n t i a l to a complete understanding of t h e i r l i f e h i s t o r i e s . The f o l l o w i n g i s a b r i e f c h a r a c t e r i z a t i o n of the 3 s p e c i e s and a summary of p r e v i o u s i n v e s t i g a t i o n s of them. Porphyra g a r d n e r i i s a f o l i o s e , monostromatic red a l g a which grows e p i p h y t i c a l l y on s e v e r a l members of the L a m i n a r i a l e s {Figure 1a). In the o r i g i n a l d e s c r i p t i o n of i t . Smith and Hollenberg (1943, as P o r p h yr e l i a ga i d p e r i ) reported t h a t i n e a r l y summer t h a l l i from Point Joe on the Monterey P e n i n s u l a , C a l i f o r n i a were e i t h e r v e g e t a t i v e or producing monospores. By l a t e summer spermatia and spores they t e n t a t i v e l y c a l l e d carpospores were being produced. The spermatia were s a i d t o occur i n packets as i s t y p i c a l of Porpjjxra, but the carpospores were r e p o r t e d to occur s i n g l y . 10 Smith and Hollenberg (1943, p. -.215) s t a t e d , "The carpogonia are formed by a c e l l d i v i s i o n i n which t h e r e i s a c u r v i n g w a l l , g u i t e s i m i l a r to the c u r v i n g c e l l w a l l s producing the moncspores of E r y t h r o t r i c h i a and the c e l l s thus formed are l i b e r a t e d s i n g l y . 1 ' These s i n g l e spores formed by an unequal c e l l d i v i s i o n were the b a s i s f o r e s t a b l i s h i n g the genus f o r p h y r e l l a (Bangiophycidae), with P o r p h y r e l l a g a r d n e r i as the type s p e c i e s (holotype, G. M. Smith 39-12, DS 306401 i n UC) . The f a t e of these spores was not determined by Smith and Hollenberg, Subsequent workers have r e p o r t e d the occurrence of Porphyra j a r d n e r i (as P o r p h y r e l l a gardneri) elsewhere i n C a l i f o r n i a (Smith, 1944; Abbott and Hol l e n b e r g , 1976), i n Oregon (Doty, 1947; Markham and C e l e s t i n o , 1976), Washington and B r i t i s h Columbia (S c a g e l , 1957, 1973; Widdowscn, 1974) and Alaska (Wynne, 1972; Li n d s t r c m , 1977), However, no f u r t h e r i n f o r m a t i o n concerning the l i f e h i s t o r y of t h i s a l g a has been p u b l i s h e d . Porphyra n e r e o c y s t i s i s a monostroaatic member of the Bangiophycidae which grows e p i p h y t i c a l l y on the s t i p e of Ner e o c y s t i s l u e t k e a n a (Mertens) P o s t e l s e t Buprecht. I t i s one of the l a r g e s t s p e c i e s of Porphyra (Figure 1b), reaching up to 3,7 i i n l e n g t h and 0.7 m i n width (UC 96517, from Esguimalt, Vancouver I s l a n d ) . I t was f i r s t r e p o r t e d by Anderson (1891, name o n l y ; d e s c r i p t i o n p u b l i s h e d i n B l a n k i n s h i p and K e e l e r , 1892) based on m a t e r i a l from Santa Cruz or the F a r a l l o n I s l a n d s , C a l i f o r n i a . No holotype e x i s t s . Hus (1900) noted t h a t P y r o p i a c a l i f o r n i c a J . Agardh i s a synonym of 11 22IEhl£.* n e r e o c y s t i s . Porphyra n e r e o c y s t i s i s the only s p e c i e s I s t u d i e d which has been i n v e s t i g a t e d by other workers to any extent. I t has been r e p o r t e d i n C a l i f o r n i a (Howe, 1893; K y l i n , 1941*; Smith, 1944; Abbott and H o l l e n b e r g , 1976), Oregon {Doty, 1947), Washington {Kylin, 1925; Krishnamurtby, 1972; Mumford, 1973a), B r i t i s h Columbia ( C o l l i n s , 1913; Scagel, 1957, 1973; wlddcwson, 1974; Conway e t a l . , 1 975) and Alaska ( S e t c h e l l and Gardner, 1903; Johansen, 1971; Lindstrom, 1977). Hus (1902) was the f i r s t t o comment on the p a l a t a b i l i t y of P. n e r e o c y s t i s and i t s use by the Chinese i n C a l i f o r n i a . The commercial p o t e n t i a l of the p l a n t has r e c e n t l y r e c e i v e d e x t e n s i v e i n v e s t i g a t i o n by Woessner (1974) and Woessner e t a l . (1977). The c o n c h o c e l i s phase has been c u l t u r e d by Krishnamurthy (1969a), Mumford (1973a), Conway e t a l . (1?75) and Conway and Cole (1977). Conway and Cole (1975) b r i e f l y r e p o r t e d on the u l t r a s t r u c t u r e of the v e g e t a t i v e c e l l of both the f o l i o s e t h a l l u s and the c o n c h o c e l i s phase. Despite a c o n s i d e r a b l e amount of study, the l i f e h i s t o r y of t h i s alga i s s t i l l not w e l l known. For example, t h e r e are c o n f l i c t i n g r e p o r t s of i t s s e a s o n a l occurrence. Anderson ( i n B l a n k i n s h i p and K e e l e r , 1892) only i n d i c a t e d t h a t i t " . . . i s seldom found u n t i l past midsummer, when the long stems of N e r e o c y s t i s are well grown." Smith (1944) and Abbott and * K y l i n mistakenly r e f e r r e d specimens of Porphyra g a r d n e r i t o Porphyra n e r e o c y s t i s 12 Hollenberg (1976) reported that the f o l i o s e phase i s an annual and i s u s u a l l y found from November to June. Both Hoessner e t a l . (1977) and A r a s a k i (1974) agree t h a t i t i s a winter s p e c i e s . I n c o n t r a s t , Conway e£ al,. (1975) r e p o r t e d the season cf occurrence as summer through winter, and Conway and Cole (1977) i n d i c a t e d i t i s summer. There are al s o c o n f l i c t i n g r e p o r t s of the degree of host s p e c i f i c i t y e x h i b i t e d by P. n e r e o c y s t i s . Anderson (in B l a n k i n s h i p and K e e l e r , 1892) i n d i c a t e d t h a t i t was most f r e q u e n t l y found on N e r e o c y s t i s . although not e x c l u s i v e l y U n i t e d to i t . Hus (1902) re p o r t e d i t growing on rocks. Abbott and Ho l l e n b e r g (1976) i n d i c a t e d that i t i s o c c a s i o n a l y found on other L a m i n a r i a l e s , but Soessner (1974) f e l t i t was e x c l u s i v e t o N e r e o c y s t i s . Porphyra t h u r e t i i i s another monpstromatic, e p i p h y t i c Porphyra about which very l i t t l e i s known, p a r t i c u l a r l y i t s range of host p l a n t s o r i t s s e a s o n a l occurrence. Paul C. S i l v a has i n d i c a t e d (on a note a t t a c h e d to the l e c t o t y p e , 10 D e c , 1971) t h a t the o r i g i n a l p u b l i c a t i o n o f P. t h u r e t i i was intended to be i n Dawson (1944) which was published i n J u l y 1944. However, the s p e c i e s was a c t u a l l y p u b l i s h e d f i r s t by Smith (1944) who i n c l u d e d i t i n flaring Algae Of The Monterey Pe n i n s u l a C a l i f o r n i a , which appeared e a r l y i n 1944. Smith d i d not c i t e a type c o l l e c t i o n but s t a t e d t h a t the type l o c a l i t y was P a c i f i c Grove. Dawson (1944) designated S e t c h e l l #5161 as the type, i n d i c a t i n g t h a t i t was from P a c i f i c Grove; however, ac c o r d i n g t o the l a b e l on specimens of that number i n the UC herbarium, and t o S e t c h e l l ' s f i e l d book, h i s #5161 was 13 c o l l e c t e d at Carmel Bay. A p l a n t from t h i s 29 Hay 1900 c o l l e c t i o n (DC 791973) was designated as the l e c t o t y p e by P. C. S i l v a i n September 1949 (Figure 1c). I t was found growing e p i p h y t i c a l l y on G r a c i l a r i a s j o e s t e d t i i K y l i n . Dawson (1944) noted t h a t i n a d d i t i o n to the type l o c a l i t y , t he s p e c i e s was a l s o known from Santa Cruz and Monterey Bay. Hus (1902) r e p o r t e d t h i s s p e c i e s under the name Porphyra l e u c p s t i c t a Thuret. Porphyra t h u r e t i i was subsequently r e p o r t e d from Oregon (Doty, 1947). I t has r e c e n t l y been rep o r t e d i n B r i t i s h Columbia f o r the f i r s t t i m e 1 ( Conway et a l . 1975), based on a c o l l e c t i o n I made a t my study s i t e i n Barkley Sound, Vancouver I s l a n d . Dawson (1952) reported P. t h u r e t i i from s e v e r a l l o c a t i o n s i n Mexico, and i n d i c a t e d t h a t the p l a n t s T a y l o r (1945) c o l l e c t e d i n Costa B i c a and r e f e r r e d to as Porphyra naiadum Andersen , were a c t u a l l y P. t h u r e t i i . Both Smith (1944) and Dawson (1944) r e p o r t e d t h a t P. t h u r e t i i grows on G r a c i l a r i a s j o e s t e d t i i or on rocks. Da«scn (1952) s t a t e d t h a t the p l a n t s from Mexico were s a x i c o l o u s or r a r e l y e p i p h y t i c . The p l a n t r e p o r t e d by Conway et a l . (1975) was growing on t h e s t i p e of j£ieoc£stis luetkeana. The season of occurrence has been r e p o r t e d as the s p r i n g (March to May) (Hus, 1902, as P. l e u c o s t i c t a ; Smith, 1944; *Scagel (1973) recorded P. t h u r e t i i from B a r k l e y Sound, but Conway et a l . (1975) i n d i c a t e d t hat these specimens are probably more c o r r e c t l y i d e n t i f i e d as Porphyra abbottae Krishnamurthy and P. f u c i c o l a Krishnamurthy. 14 Dawson, 1944), and summer ( Conway and Cole , 1977). M a t e r i a l s And Methods Most of the f i e l d work was c a r r i e d out near the Bamfield Marine S t a t i o n on the west c o a s t of Vancouver I s l a n d , B r i t i s h Columbia (Figure 2a). A study s i t e o f f Diana I s l a n d i n B a r k l e y Sound (48° 50.1»N, 125° 11.1*w) was s e l e c t e d (Figure 2b) because i t i s semi-exposed t o t h e open P a c i f i c Ocean and has a s h e l t e r e d channel between i t and Diana I s l a n d , where a boat can be landed under most c o n d i t i o n s throughout the year. A •Zodiac* i n f l a t a b l e boat was used f o r a l l f i e l d work. At the study s i t e f . g a r d n e r i reached o p t i m a l development ° n Laminaria s e t c h e l l i i S i l v a , Most o b s e r v a t i o n s were made on specimens c o l l e c t e d from t h i s *host* from May 1974 through May 1977. Other s i t e s i n Barkley Sound where Porphyra g a r d n e r i was c o l l e c t e d were: Ross I s l e t s , Leach I s l e t , Execution Rock, and Cape Beale. See Appendix I f o r a complete l i s t of my c o l l e c t i n g s t a t i o n s , t h e i r l o c a t i o n , and dates of c o l l e c t i o n . Porphyra n e r e o c y s t i s was v a r i a b l e i n i t s abundance a t the study s i t e from year t o year so i t was necessary t o c o l l e c t i t at other s i t e s i n the Barkley Sound area as w e l l . These i n c l u d e d the kelp beds o f f : A g u i l a r Point, Cable Beach, Second Beach, and Leach I s l e t . At the study s i t e P. n e r e o c y s t i s was observed from June 1974 through A p r i l 1977., RQLSklE.!* t h u r e t i i was not observed at the study s i t e u n t i l 1975 and was never abundant enough to make a d e t a i l e d monthly 15 study of i t . Recently ( A p r i l 1977) i t was found i n abundance at Leach I s l e t , a much more exposed l o c a t i o n i n B a r k l e y Sound. Porphyra g a r d n e r i c o u l d be c o l l e c t e d on low t i d e s (0.5 m or l e s s ) , whereas P. n e r e o c y s t i s and P. t h u r e t i i c o u l d be obtained at a l l but the h i g h e s t t i d e s simply by p u l l i n g up a ! § I § 2 C V . s t i s p l a n t . Despite t h i s , SCUBA was f r e g u e n t l y used and proved i n v a l u a b l e f o r making in s i t u o b s e r v a t i o n s and c o l l e c t i n g f r e s h m a t e r i a l f o r chromosome counts and e l e c t r o n microscopy. Porphyra g a r d n e r i a l s o grows on s e v e r a l s u b t i d a l hosts which c o u l d be reached only by using SCUBA. SCUEA was a l s o needed to anchor experiments u s i n g a r t i f i c i a l n e r e o c y s t i s p l a n t s and an i n s i t u c u l t u r e chamber f o r c o n c h o c e l i s f i l a m e n t s . When c o l l e c t i o n s were made a t the study s i t e , s u r f a c e seawater temperature was measured. Hhen the o p p o r t u n i t y arose, specimens were c o l l e c t e d from other l o c a l i t i e s . In the case c f Porphyra g a r d n e r i these were: B r i t i s h Columbia: Langara I s l a n d , Queen C h a r l o t t e I s l a n d s ; Gordon I s l a n d s , Queen C h a r l o t t e I s l a n d s ; Tree I s l e t s ; Brooks P e n i n s u l a ; Grassy I s l a n d , Kyuguot Sound; Hot S p r i n g s Cove; and B o t a n i c a l Beach, Port Renfrew. Oregon: H a r r i s Beach. C a l i f o r n i a : Ano Nuevo I s l a n d ; P o i n t Joe and Pescadero P o i n t , Monterey P e n i n s u l a ; and Mission P o i n t , Carmel. L o c a l i t i e s o u t s i d e the Barkley Sound area where Porphyra n e r e o c y s t i s was c o l l e c t e d i n c l u d e d : B r i t i s h Columbia: Langara I s l a n d , Queen C h a r l o t t e I s l a n d s ; Bolkus I s l a n d s , S k i n c u t t l e I n l e t , Queen C h a r l o t t e I s l a n d s ; Tree I s l e t s ; Hope I s l a n d ; Grassy I s l a n d , Kyuguot Sound; B o t a n i c a l Beach; Whiffen S p i t , Sooke; and South Pender I s l a n d . C a l i f o r n i a : Pebble Beach, Monterey P e n i n s u l a . 16 Other s i t e s i n B r i t i s h Columbia where Porphyra t h u r e t i i was c o l l e c t e d i n c l u d e d : B e n n e l l Sound, Queen C h a r l o t t e I s l a n d s and V o l c a n i c Cove, Kyuguot Channel, Vancouver I s l a n d . The purpose of o b s e r v i n g these other s i t e s was to see i f there sere any pop u l a t i o n d i f f e r e n c e s i n the time and type of spore formation. The f i e l d study o f Porphyra g a r d n e r i a t i t s type l o c a l i t y {Point Joe, Monterey Peninsula) was e s s e n t i a l i n order t o confirm t h a t the p l a n t s a t the study s i t e were the same taxon. at the study s i t e and the Bolkus I s l a n d s , Queen C h a r l o t t e I s l a n d s , where abundant Porphyra n e r e o c y s t i s was encountered, s e v e r a l N e r e o c y s t i s p l a n t s were c o l l e c t e d i n order to get a g u a n t i t a t i v e assessment o f the v e r t i c a l d i s t r i b u t i o n of P. n e r e o c y s t i s along the s t i p e . S t a r t i n g a t the pneumatocyst, samples were taken at 1 m i n t e r v a l s down t h e s t i p e . A l l the £• n e r e o c y s t i s p l a n t s i n each v e r t i c a l meter were c o l l e c t e d and d r i e d at 70°C f o r 1 week and a dry weight was taken. A s e r i e s o f a r t i f i c i a l host experiments was run to see i f i t was the host p l a n t i t s e l f , or j u s t the unique p o s i t i o n i n the environment which i t occupies t h a t the epiphyte r e q u i r e s . Three types of a r t i f i c i a l N e r e o c y s t i s s t i p e s were made, us i n g manila rope (3-strand, 15 mm diameter), v i n y l t u b i n g ( F i s h e r brand, 11 mm o u t s i d e diameter) and s u r g i c a l rubber ( F i s h e r brand, 11 mm o u t s i d e diameter) f o r t h e ' s t i p e * . At one end the * s t i p e * was lashed to a 4 1 s e a l e d p l a s t i c b o t t l e f o r f l o a t a t i o n . The ' p l a n t s * were then anchored to rock c l i m b i n g p i t o n s (Cassin - I t a l y , 250 mm long with r i n g 47 mm diameter) which were d r i v e n i n t o c r e v i c e s i n the rocky bottom at a depth of 7-10 m. Polypropylene rope (13 mm diameter) was used to t i e 17 the •plants* t o the p i t o n r i n g . These a r t i f i c i a l N e r e o c y s t i s s t i p e s were put out i n the f i e l d i n September 1974 and August 1975. A r t i f i c i a l Lam i n a r i a - l i k e p l a n t s were made from heavy nylon mesh cut i n t o s t r i p s . They were a l s o anchored using p i t o n s , and were p l a c e d i n the L a j j - n a r i a zone at approximately the 0.4 m t i d e l e v e l . In a d d i t i o n to the f i e l d study, specimens i n numerous h e r b a r i a (Table I) were examined to supplement f i e l d o b s e r v a t i o n s and to c o n f i r m t h a t I had c o r r e c t l y i d e n t i f i e d the s p e c i e s being s t u d i e d . R e s u l t s A., Poi£hyra g a r d n e r i 1. Seasonal Occurrence Of Epiphyte And *Host* A b r i e f c o n s i d e r a t i o n of the annual growth and decay of Laminaria s e t c h e l l i i i s e s s e n t i a l to an understanding of the establishment and seasonal occurrence o f Porphyra g a r d n e r i . At the study s i t e the blade of L. s e t c h e l l i i was worn back almost to the s t i p e by November. During l a t e November or e a r l y December, the meristematic t r a n s i t i o n zone commenced forming a new blade, which was i n i t i a l l y a c h a r a c t e r i s t i c h eart shape (Figure 3a). Growth was r a p i d and w i t h i n a month the Made began t o s p l i t d i s t a l l y ( F i g u r e 3b), T h i s s p l i t t i n g continued as the blade grew and r e s u l t e d i n the formation of the t y p i c a l 18 l a c e r a t e d blade by February or Harch (Figure 3 c ) . During the f i r s t months of growth the new blade was r e l a t i v e l y f r e e of ep i p h y t e s . Growth of diatoms and e c t o c a r p a l e a n epiphytes became obvious by e a r l y summer; they appeared f i r s t on the d i s t a l p o r t i o n s of the blade which c o n t a i n e d the o l d e s t t i s s u e . During the autumn the blade became i n c r e a s i n g l y e p i p h y t i z e d and worn away. This fclade e r o s i o n r e s u l t e d i n the disappearance o f a s u i t a b l e s u b s t r a t e f o r I2r.Dhy.ra aardner,i. The f i r s t t h a l l i of Porphyra g a r d n e r i appeared a t the end of February a t the study s i t e . T h e i r d i s t r i b u t i o n was very s t r i k i n g , being r e s t r i c t e d to the margins of t h e Laminaria blade (Figure 3c). The number of P. g a r d n e r i t h a l l i c o n tinued to i n c r e a s e r a p i d l y throughout the s p r i n g , and the margins of l a i n a r i a s e t c h e l l i i soon became densely l i n e d with the plant (Figure 1a). These t h a l l i of Porphyra g a r d n e r i had a c h a r a c t e r i s t i c l o o s e g e l a t i n o u s appearance along t h e i r d i s t a l margin (Figure 3d) due t o a massive p r o d u c t i o n and r e l e a s e of monospores (to be d i s c u s s e d i n Part I I I ) . The f i r s t t h a l l i to produce spermatangia were observed during l a t e A p r i l ( F i g u r e 4a). They were e a s i l y recognized by the f i n e , c o l o u r l e s s patchwork margin t h a t the spermatangia form. About a month a f t e r the appearance of the spermatangia the l a t e r a l submarginal r e g i o n s became s l i g h t l y more deeply pigmented than the c e n t r a l v e g e t a t i v e r e g i o n of the t h a l l u s , due t o the pr o d u c t i o n of carposporangia (to be d i s c u s s e d i n P a r t s I I and I I I ) . T h a l l i growing i n the s p r i n g and e a r l y summer can reach a 19 l e n g t h of 130 mm and a width of 60 mm, but t y p i c a l l y they are 20-70 mm long and 10-30 mm wide. The l a r g e s t t h a l l i occurred on E i s e n i a arjborea Areschoug and Laminaria s e t c h e l l i i which were growing s u b t i d a l l y . L a t e r i n the summer the t h a l l i were g e n e r a l l y much s m a l l e r than those found e a r l i e r i n the season. The number of t h a l l i d e c l i n e d i n the autumn as the L. s e t c h e l l i i blade eroded. At t h i s time s e v e r a l s m a l l t h a l l i appeared on the s u r f a c e of the Laminaria blade i n s t e a d of being r e s t r i c t e d t o the margins. As long as o l d L a m i n a r i a t h a l l u s i s present Porphyra g a r d n e r i w i l l c o n t i n u e to grow ( F i g u r e 4b). Most !• g a r d n e r i was gone by September-October, but I c o l l e c t e d some as l a t e as November. The t h a l l u s morphology and r e p r o d u c t i o n of JSIfiilXEii g a r d n e r i at the other s i t e s where i t was c o l l e c t e d i n B r i t i s h Columbia were s i m i l a r t o those observed a t the study s i t e ( F i g u r e s 4c, 4d, and 4e). Specimens from B o t a n i c a l Beach (Figure 4c) tended to have more r u f f l e d margins than study s i t e p l a n t s but were s i m i l a r i n a l l other r e s p e c t s . Because these s i t e s were v i s i t e d only once i t i s i m p o s s i b l e t o make comparisons of the seasonal occurrence or r e p r o d u c t i o n . The f i e l d study of the Monterey p l a n t s , combined with c u l t u r e and c y t o l o g i c a l work (to be d i s c u s s e d i n P a r t s I I , I I I , and I V ) , c o n c l u s i v e l y demonstrated that they a r e the same taxon which i s present at the study s i t e . However, there were d i f f e r e n c e s i n the abundance of t h a l l i , and i n the time of spermatangium and carpogonium formation between the p l a n t s at these two s i t e s . Porphyria g a r d n e r i was more abundant a t P o i n t 20 Joe (type l o c a l i t y ) i n November than i t was a t the study s i t e . Also the t h a l l i at P o i n t Joe (Figure 4f) as w e l l as a t Pescadero P o i n t and M i s s i o n P o i n t were g e n e r a l l y much s m a l l e r (30 mm long average, up t o 70 mm) than t h a l l i from f a r t h e r north. An examination o f herbarium specimens from C a l i f o r n i a , Oregon, Washington, B r i t i s h Columbia and Alaska, i n s e v e r a l h e r b a r i a (CSUH, DS, GJH, GMS, RS, UBC, UC, UCSC) confirmed these o b s e r v a t i o n s . Most t h a l l i c o l l e c t e d at Point Joe i n J u l y 1976 were monosporic, t h i s being confirmed by c u l t u r e work done a t the Hopkins Marine S t a t i o n . Some t h a l l i had spermatangial l a t e r a l and monosporic d i s t a l margins, whereas others were spermatangial, c a r p o s p o r a n g i a l and monosporic. The frequency of carposporangia i s low compared t o more northern p o p u l a t i o n s . The predominance of monosporic t h a l l i i n J u l y i s i n sharp c o n t r a s t t o the s i t u a t i o n a t the study s i t e where most t h a l l i were c a r p o s p o r a n g i a l and s p e r m a t a n g i a l . An examination of herbarium specimens c o l l e c t e d i n the Monterey area r e v e a l e d t h a t they were almost always monosporic. In f a c t , the type specimen i s e x c l u s i v e l y monosporic, a c u r i o u s f a c t c o n s i d e r i n g that the genus P o r p h y r e l l a was based on the d e t a i l s cf carpogonium formation and carpospore r e l e a s e . 2. *Host» Species Porphyra g a r d n e r i i s not r e s t r i c t e d to one host, having been observed on f i f t e e n l a m i n a r i a l e a n and one rhodophycean alqae (Table I I ) . S a x i c o l o u s specimens were not found. Throughout i t s range i t reaches optimal development on the 21 margins of Laminaria s e t c h e l l i i c r other Laminaria s p e c i e s . The •host* s p e c i e s grow i n the t i d a l range between approximately +1.4 m to -3.0 m. Porph yra g a r d n e r i i s s m a l l (10-20 mm long) on 'host's' l i k e Egregia m e n z i e s i i {Turner) fireschoug, which are i n the upper p a r t of t h i s range. In the e a r l y s p r i n g months P. g a r d n e r i can be found i n abundance on the • l e a f l e t s ' of E g r e g i a . but by May no t h a l l i were found on t h i s 'host'. Porphyra g a r d n e r i t h a l l i on E i s e n i a arbqrea were g e n e r a l l y l a r g e r and became r e p r o d u c t i v e e a r l i e r than those on Laminaria s e t c h e l l i i . On a l l the 'host' s p e c i e s , P. g a r d n e r i was t y p i c a l l y a s s o c i a t e d with the t h a l l u s margins. One e x c e p t i o n to t h i s t r e n d was the host P c s t e l s i a palmaeformis Ruprecht, where the t h a l l i of P. g a r d n e r i were growing on abraded p o r t i o n s of the s t i p e . 3. A r t i f i c i a l Host Experiment None of the attempts t o grow Porphyra g a r d n e r i on the a r t i f i c i a l host p l a n t was s u c c e s s f u l . 4. G e o g r a p h i c a l D i s t r i b u t i o n From my own c o l l e c t i o n s , herbarium specimens and r e p o r t s i n the l i t e r a t u r e i t i s c l e a r t h a t Porphyra g a r d n e r i has a wide d i s t r i b u t i o n . Scagel (1957) and Dawson (1961) r e p o r t e d i t (as P o r p h y r e l l a gardneri) to extend from northern B r i t i s h Columbia to the Monterey P e n i n s u l a . S p a r l i n g (1971, as P o r p h y r e l l a gardneri) extended the southern range to Hazard 22 Canyon, San L u i s Obispo County, C a l i f o r n i a (35° 18'N, 120° 53*»). Specimens on the l e a f l e t s of E q r e g i a l a e v i g a t a S e t c h e l l <AHF 77063) from Punta Banda, Baja C a l i f o r n i a , Mexico (31° 44»N, 116° 44'H) have been examined and are r e f e r r e d t o Porphyra g a r d n e r i . T h i s apparently i s o l a t e d p o p u l a t i o n r e p r e s e n t s a g r e a t e x t e n s i o n of the southern l i m i t of £• g a r d n e r i . Punta Banda f a l l s w i t h i n one of the areas o f c o l d water u p w e l l i n g d e s c r i b e d by Dawson (1951) along the Baja C a l i f o r n i a c o ast. S e v e r a l other t y p i c a l l y more nor t h e r n , c c c l e r water algae are found there as w e l l (Abbott and North, 1972). Hynne (1972) r e p o r t e d Por.phy.ra g a r d n e r i (as P o r p h y r e l l a gardneri) from Amchitka I s l a n d i n the A l e u t i a n I s l a n d s , Alaska. C o l l e c t i o n s deposited i n the U n i v e r s i t y cf B r i t i s h Columbia P h y c c l o g i c a l Herbarium extend the range w e l l i n t o northern Alaska and west along the A l e u t i a n I s l a n d s to Murder P o i n t , A t t n I s l a n d (52° 48»N, 173° 11»£, UBC 7949). Specimens of Porphyra n e r e o c y s t i s r e p o r t e d by Zinova* (1940) on Laminaria l o n g i p e s Bory from Bering I s l a n d i n the Commander Is l a n d s (55° 12 * N, 165° 58»E) are r e f e r r a b l e t o Porphyra g a r d n e r i and r e p r e s e n t i t s western d i s t r i b u t i o n a l l i m i t . (A specimen c o l l e c t e d by E. Kardakova on 30 January 1930, and deposited i n the B o t a n i c a l I n s t i t u t e of the Academy of Science o f U.S.S.H. i n Leningrad has been examined. UBC 56455 i s a photograph of t h i s specimen). No Porphyra s p e c i e s e p i p h y t i c on Laminaria margins have been reported f o r the Sea 1 A u t h o r * s name t r a n s l i t e r a t e d on the o r i g i n a l as Sinova. 23 of Okhotsk (Zinova, 1954) , Sakhalin I s l a n d (Vozzhinskaya, 1964), or the K u r i l I s l a n d s (Nagai, 1941). Tokida (1960) and Oh mi (1963) do not mention any Japanese s p e c i e s of Porphyra on Laminaria, and Dr. M. Kurogi ( p e r s o n a l communication) has confirmed t h i s . Porphyra g a r d n e r i i s a l s o absent f r c o the S t r a i t of Georgia, B r i t i s h Columbia. A l i s t o f r e p r e s e n t a t i v e specimens of P. g a r d n e r i from throughout t h i s g e o g r a p h i c a l range i s given i n Appendix I I . E» Porphyra n e r e o c y s t i s 1. Seasonal Occurrence Of Epiphyte And 'Host* N e r e o c y s t i s luetkeana i s an annual, although a few p l a n t s can s u r v i v e f o r as long as 18 months. I t f i r s t appeared at the study s i t e at the beginning of March. The p l a n t s were e s s e n t i a l l y e p i p h y t e - f r e e d u r i n g the period of r a p i d s t i p e growth. In June and J u l y , growth of the bryozoan Membranippra megfaranacea was noted on the laminae and pneumatocyst. Diva stenoehyl,la. S e t c h e l l et Gardner, U. f e n e s t r a t a P o s t e l s e t Buprecht and s p e c i e s of Enteromorpha appeared on the pneumatocyst d u r i n g August. By J u l y most o f the N e r e o c y s t i s p l a n t s had reached the s u r f a c e and s t i p e growth was much reduced. The s t i p e s showed s i g n s of a b r a s i o n , but were not e p i p h y t i z e d . Once N e r e o c y s t i s reached the end of i t s s t i p e growth phase, establishment of e p i p h y t e s was r a p i d . By l a t e September t o e a r l y October a b a c t e r i a l and diatom 'scum* f o l l o w e d by e c t o c a r p a l e a n epiphytes became e s t a b l i s h e d on the s t i p e . 24 Throughout the autumn and winter the p l a n t s became p r o g r e s s i v e l y more h e a v i l y e p i p h y t i z e d . Breakdown and e r o s i o n of the s t i p e weakened the p l a n t s and thereby c o n t r i b u t e d to the almost complete e l i m i n a t i o n of the N e r e o c y s t i s beds during the winter and s p r i n g . A few p l a n t s , which s u r v i v e d the winter storms i n t a c t , were e a s i l y recognized by t h e i r dense c o a t i n g of e p i p h y t e s . -At the study s i t e Porphyra n e r e o c y s t i s f i r s t appeared on the N e r e o c y s t i s s t i p e s i n l a t e November t o e a r l y December. I t grew on the upper p o r t i o n of the s t i p e which f l o a t s cn the s u r f a c e at low t i d e . The young t h a l l i have a c h a r a c t e r i s t i c l o n g , narrow ribbon shape which slowly t a p e r s to a point (Figure 5a}., The margins are s t r a i g h t , and without w r i n k l e s or f o l d s . Large, mature t h a l l i up to 2.7 m long (Figure 1b) were found i n January and February. During January, the l a t e r a l margins of the t h a l l i became c o l o u r l e s s due to the production of spermatangia (Figure 5b). These marginal spermatangial s t r i p s v a r i e d i n width from 10 mm t o 90 mm. They began t o erode i n the d i s t a l r e g i o n o f t h e t h a l l u s and continued to erode i n a basal d i r e c t i o n , l e a v i n g a d i s t a l t i p of v e g e t a t i v e c e l l s and carposporangia (Figure 5 c ) . Long, narrow, submarginal s t r e a k s of spermatangia a l s o formed, and were j o i n e d i n p l a c e s to the marginal s t r i p s (Figure 5d). Spermatangia were never observed i n s m a l l microscopic patches as reported by Humford (1973a) and Conway et a l . (1975). By the end of January or e a r l y February, carpogonia and carposporangia began forming i n the d i s t a l and l a t e r a l submarginal areas o f the t h a l l u s (to be d i s c u s s e d i n P a r t I I ) . 25 In e l d e r t h a l l i the c a r p o s p o r a n g i a o f t e n gave the d i s t a l t i p s a mottled appearance (Figure 5e). P01jp.h7.ra n e r e o c y s t i s l a s t e d from November to J u l y or August i n the f i e l d , I have c o l l e c t e d i t i n abundance i n the s p r i n g and e a r l y summer when i t i s always a s s o c i a t e d with a N e r e o c y s t i s p l a n t from the p r e v i o u s year. A number of other Porphyra s p e c i e s were a l s o observed on the s t i p e of N e r e o c y s t i s along with Porphyra n e r e o c y s t i s . These i n c l u d e d : Porpfryra g a r d n e r i (UBC 55942), P. miniata <C. Agardh) C. Agardh (OBC 57219), P. p e r f o r a t a J . Agardh (OBC 57222), £• s m i t h i i Hollenberg g t Abbott {OBC 57220), and £• t h u r e t i i (OBC 57201) . Porphyra n e r e o c y s t i s appeared i n the S t r a i t of Georgia approximately two months e a r l i e r than i t d i d a t the study s i t e . New t h a l l i were c o l l e c t e d as e a r l y as September at Sidney, Vancouver I s l a n d (OBC 1434) and South Pender I s l a n d (OBC 57194), and i n October a t F r i d a y Harbor, San Juan I s l a n d , Washington (OBC 57195). Porphyra n e r e o c y s t i s a l s o appeared e a r l i e r i n C a l i f o r n i a than i t d i d a t the study s i t e (J. Woessner, per s o n a l communication, August 1S77). I c o l l e c t e d young t h a l l i i n November at the Monterey P e n i n s u l a (OBC 57196). At the other s i t e s a l o n g the outer c o a s t of B r i t i s h Columbia the appearance of t h a l l i and t h e i r r e p r o d u c t i v e s t a t u s at the time of c o l l e c t i o n suggests t h a t they have a seasonal occurrence s i m i l a r to the study s i t e p l a n t s . 26 2. V e r t i c a l D i s t r i b u t i o n Porphyra n e r e o c y s t i s v a r i e s i n i t s s i z e and abundance depending on i t s p o s i t i o n on the N e r e o c y s t i s s t i p e . Host p l a n t s are c o n c e n t r a t e d on t h e upper 3 m of s t i p e , with the l a r g e s t p l a n t s t y p i c a l l y being i n the f i r s t 1-2 m {Table I I I ) . At the lower end of the d i s t r i b u t i o n the t h a l l i are s m a l l (10-100 mm) and v e g e t a t i v e , another c u r i o u s f e a t u r e i s t h a t on most s t i p e s the t h a l l i are l i m i t e d to one s i d e of the s t i p e . 3. *Host» Species Porphyra n e r e o c y s t i s was found e x c l u s i v e l y on the s t i p e of N e r e p s y s t i s with the e x c e p t i o n o f a few t h a l l i found growing on the h e a v i l y e p i p h y t i z e d blades of M a c r o c y s t i s i n t e g r i f c l i a Sory (UBC 57197). Although t h e r e a r e r e p o r t s i n the l i t e r a t u r e t h a t P. n e r e o c y s t i s can grow s a x i c o l o u s l y , I never observed t h i s . 4. A r t i f i c i a l Host Experiment The best m a t e r i a l t e s t e d f o r i m i t a t i n g a N e r e o c y s t i s s t i p e was s u r g i c a l rubber. The ' p l a n t * I put out i n the f i e l d i n 1975 l a s t e d 14 months. I t supported a m a g n i f i c e n t growth of algae, some o f which i n c l u d e d : Jilya s t e n o p h y l l a . fiollenbergia s u b u l a t a (Harvey) S o l l a s t o n , A l a r i a margjnata P o s t e l s e t Huprecht and Desmarestia l i g u l a t a var. l i g u l a t a ( L i g h t f o c t ) lamouroux. In November* a few s m a l l (30-40 mm long) t h a l l i appeared on the a r t i f i c i a l s t i p e (UBC 57221). They had the t y p i c a l shape of young P. n e r e p c v s t i s . but none of them grew beyond t h i s stage, making p o s i t i v e i d e n t i f i c a t i o n 27 i m p o s s i b l e . 5. Geographical D i s t r i b u t i o n From my own c o l l e c t i o n s , r e p o r t s i n the l i t e r a t u r e and herbarium specimens, the known d i s t r i b u t i o n of Forphyra n e r e o c y s t i s i s : S t a r a y a Bay, Onalaska I s l a n d , A l e u t i a n I s l a n d s , Alaska (53° 37.4»N, 165° 30.6«W, UBC 26776), nor t h e a s t to Box P o i n t , Montague I s l a n d , Alaska (59° 58'N, 147° 22'H, UBC 25836) and south to San L u i s Obispo County, C a l i f o r n i a <35° 10«M, 120° 45*8) (Dawson, 1961). D r i f t specimens are o c c a s i o n a l l y found i n southern C a l i f o r n i a (Abbott and Hollenberg, 1976). Zinova (1940) reported t h a t Porphyra n e r e o c y s t i s grows on Laminaria l c n g i p e s and N e r e o c y s t i s luetkeana a t B e r i n g I s l a n d , Commander i s l a n d s , U.S.S.E. (55° 12«N, 165° 58*E) . I have been able to examine these specimens, which are d e p o s i t e d i n the B o t a n i c a l I n s t i t u t e of the Academy of Science of U.S.S.B. i n Leningrad. As I i n d i c a t e d e a r l i e r , the specimen from Laminaria l c n g i p e s i s a c t u a l l y Porphyra g a r d n e r i : however, the specimen from N e r e o c y s t i s i s r e f e r a b l e t o P. n e r e o c y s t i s (UBC 57206, i s a c o l o u r s l i d e of t h i s p l a n t ) . Some workers (P. A. Lebednik , p e r s o n a l communication) f e e l t h a t t h i s r e p o r t of N e r e o c y s t i s l u e t k e a n a i n the Commander I s l a n d s was unknowingly based on d r i f t specimens. I f e e l that t h i s would be worth r e - i n v e s t i g a t i n g because Zinova (1940) s p e c i f i c a l l y s t a t e d t h a t N e r e o c y s t i s grows i n the s u b - l i t t o r a l zone and forms " t h i c k copses which make i t d i f f i c u l t or i m p o s s i b l e f o r a boat t o pass through." I have attemped t o o b t a i n f u r t h e r i n f o r m a t i o n on 28 t h i s p o i n t , but without s u c c e s s . A l i s t of r e p r e s e n t a t i v e specimens of Porphyra n e r e o c y s t i s from throughout i t s g e o g r a p h i c a l range i s given i n Appendix I I . C. Por£hy_ra t h u r e t i i 1. Seasonal Occurrence T h i s l i t t l e known al g a was f i r s t encountered at the study s i t e i n February 1975 on the s t i p e of N e r e o c y s t i s luetkeana along with Porphyra n e r e o c y s t i s . The one f e r t i l e p l a n t from t h i s c o l l e c t i o n (UBC 52019) was i d e n t i f i e d by Dr. T. F. Mumford, J r . I have subsequently c o l l e c t e d t h i s s p e c i e s on s e v e r a l o c c a s i o n s . Porphyra t h u r e t i i was c o l l e c t e d i n January, but i t was not abundant u n t i l March or e a r l y A p r i l . I t was not encountered a f t e r May. The most c h a r a c t e r i s t i c f e a t u r e i s the s t r o n g l y r u f f l e d margin (Figure 6a), which r e a d i l y d i s t i n g u i s h e s v e g e t a t i v e t h a l l i from P. n e r e o c y s t i s . S u p e r f i c i a l l y the r u f f l e d margins g i v e P. t h u r e t i i a resemblance to P. m i c i a t a ; however, the two can r e a d i l y be separated on the b a s i s of t h e i r monostromatic and d i s t r o r o a t i c c o n s t r u c t i o n s , r e s p e c t i v e l y . A few t h a l l i producing spermatangia were observed i n January, but most d i d not do so u n t i l February or March. The spermatangia are f i r s t n o t i c e a b l e as a t h i n , c o l o u r l e s s marginal band a t the t i p of the t h a l l u s (Figure 6b). T h i s band g r a d u a l l y i n c r e a s e d i n l e n g t h b a s a l l y , and on o l d e r t h a l l i patches and s h o r t s t r e a k s of spermatangia formed at the t h a l l u s apex and i n the l a t e r a l submarginal areas ( F i g u r e s 6c and 6d). 29 The s p e c i f i c time cf the i n i t i a l stages of carpogonium formation i s not known. F e r t i l e t h a l l i were c o l l e c t e d i n January, but most d i d not form carposporangia u n t i l February or March. Porphyra t h u r e t i i i n the Barkley Sound area i s t y p i c a l l y 150-220 mm long and 50-80 mm wide, although t h a l l i up t o 390 mm by 105 mm d i d o c c u r . Herbarium specimens (DC, GMS) of P. t h u r e t i i from the Monterey Peni n s u l a region were found to be g e n e r a l l y l a r g e r (up to 770 mm long and 230 mm wide) and have l e s s pronounced marginal r u f f l i n g than the specimens from Barkley Sound ( F i g u r e s 2c and 6d). I t should be noted t h a t the Monterey p l a n t s were c o l l e c t e d l a t e r i n the year (May-June) and had been f l o a t i n g i n the d r i f t , so t h i s may be an e x p l a n a t i o n f o r t h e i r l a r g e r s i z e . Specimens c o l l e c t e d i n A p r i l were more t y p i c a l of the s i z e o f t h a l l u s I have observed i n B a r k l e y Sound (Figure 6e). 2. *Host' Species Porphyra t h u r e t i i occurs on 7 d i f f e r e n t host s p e c i e s , and i n one case was found on r o c k , c o n f i r m i n g the r e p o r t s of Dawson (1944) and Smith (1944). I t has been observed on 1 s e a g r a s s , 3 phaeophycean and 3 rhodophycean algae (Table IV). In the Barkley Sound r e g i o n P. t h u r e t i i was observed growing e p i p h y t i c a l l y only cn members o f the L a m i n a r i a l e s . In c o n t r a s t , specimens from the Monterey r e g i o n were e p i p h y t i c on members of the Hhodophyceae. 30 3. A r t i f i c i a l •Host* Experiment One p l a n t of Porehyra- t h u r e t i i (UBC 57199) which appeared on the a r t i f i c i a l N e r e o c y s t i s p l a n t grew to 146 mm i n l e n g t h and reached r e p r o d u c t i v e m a t u r i t y . 4. Geographical D i s t r i b u t i o n In B r i t i s h Columbia, Porphyra t h u r e t i i has been c o l l e c t e d at B e n n e l l Sound, Queen C h a r l o t t e I s l a n d s (53° 22.65*N, 132° 30.7*H, UBC 55302), V o l c a n i c Cove, Kyuguot Channel, Vancouver I s l a n d (49« 58.7'N, 127° 13.9»W, UBC 54446) and two s i t e s i n Barkley Sound, Vancouver I s l a n d (48° 50.1»N, 125° 11. V B , OBC 52019; 48° 49.8»N, 125° 14.4*B, OBC 57200) . Doty (1947) r e p o r t e d i t once a t Chetco Cove i n Oregon (42° 00*N, 124° 18»W). In C a l i f o r n i a , Porphyra t h u r e t i i i s known only from Santa Cruz (36° 58*N, 122° -00*9) (Dawson, 1944), the Monterey Pe n i n s u l a (36° 30* N, 121° 57*H, UC 95596) and Carmel Bay (36° 33*N, 121°56«1, OC 791973). Dawson (1952) rep o r t e d P. t h u r e t i i from s e v e r a l l o c a t i o n s i n P a c i f i c Baja C a l i f o r n i a , Mexico. These specimens, d e p o s i t e d i n the h e r b a r i a of UC and AHF, were examined and i n my o p i n i o n r e p r e s e n t a d i f f e r e n t taxon because of t h e i r s m a l l s i z e , shape, and spermatangium and carposporangium d i s t r i b u t i o n . A r e p r e s e n t a t i v e specimen l i s t of Porphyra t h u r e t i i i s given i n Appendix I I . 3 1 D i s c u s s i o n A. Porphyra g a r d n e r i Although i t has been found at the study s i t e throughout 10 months o f the year, Porphyra g a r d n e r i i s a spring-summer s p e c i e s . I t s monospore c y c l e r e s u l t s i n the r e - e s t a b l i s h m e n t of the p o p u l a t i o n to some extent i n the autumn, e n a b l i n g i t to p e r s i s t longer than most Porphyra s p e c i e s . The predominance of the monospore c y c l e i n p l a n t s from Monterey may enable the f o l i o s e phase to e x i s t year-round i n t h i s a r ea. F c r t h e r seasonal s t u d i e s are r e g u i r e d t o confirm t h i s suggestion. I t has been noted t h a t the time and freguency of spermatium and spore f o r m a t i o n v a r i e s with l a t i t u d e . C u l t u r e s t u d i e s of Porphyra (Iwasaki, 1961) and Bangia (Richardscn and Dixon, 1968; Dixon and Bichardson, 1969; Sommerfeld and N i c h o l s , 1973) have shown t h a t photoperiod i s important i n c o n t r o l l i n g monospore and carpospore formation. T h e r e f o r e i t seems reasonable to suggest t h a t v a r i a t i o n i n the times cf spermatangium and carpogonium formation i s the r e s u l t o f the annual photoperiod v a r i a t i o n with l a t i t u d e . In Table V the d u r a t i o n of d a y l i g h t on the 21st day of each month i s compared f o r 35°N l a t i t u d e and 48° 50.1»N (study s i t e ) . Data f o r 35°N have been o b t a i n e d from the Smithsonian M e t e o r o l o g i c a l Tables ( L i s t , 1966), whereas s t u d y - s i t e data have been taken from d a i l y s u n r i s e and sunset t a b l e s f o r V i c t o r i a , B r i t i s h Columbia (National Eesearch C o u n c i l , A s t r o p h y s i c s Branch, 1974). L a t i t u d e 35°N i s used because i t i s the c l o s e s t l a t i t u d e t o 36° 30 * N (Monterey P e n i n s u l a , type l o c a l i t y ) f o r which data are 32 given. There i s approximately a 10 minute d i f f e r e n c e i n the d u r a t i o n of d a y l i g h t between these two l a t i t u d e s on the 21st June, but t h i s i s a c c u r a t e enough f o r the purposes of the present d i s c u s s i o n . At the study s i t e spermatangia f i r s t appeared when the daylength was approximately 14 hr. Carpogonia, then carposporangia, formed a s h o r t time a f t e r t h i s when a s l i g h t l y l o n g e r daylength was reached, estimated t o be between 14 hr 30 min and 15 hr. As w i l l be seen from Table V, t h i s d u r a t i o n of d a y l i g h t i s on l y j u s t reached at 3 5°N on 21st June. T h i s s t r o n g l y suggests t h a t photoperiod i s r e s p o n s i b l e f o r the low freguency of carpogonium formation i n the Monterey p l a n t s . Whether t h i s i s a t r u l y p h o t o p e r i o d i c or j u s t a p h o t o s y n t h e t i c e f f e c t w i l l be d i s c u s s e d i n Part I I I . Higher seawater temperature may a l s o be a f a c t o r . Sommerfeld and N i c h o l s (1973) found t h a t i n Bangia a higher temperature favoured p r o d u c t i o n of monospores r a t h e r than carpospores, The v a r i a t i o n i n t h a l l u s s i z e of Porphyra g a r d n e r i with v e r t i c a l p o s i t i o n on the shore i s an i n t e r e s t i n g phenomenon which may be the r e s u l t of growth i n h i b i t i o n by prolonged exposure, Kurogi (1961) noted t h a t growth of Porphyra yezoensis was more r a p i d a t lower i n t e r t i d a l l e v e l s . As a r e s u l t of my study, P. g a r d n e r i i s now known from a c o n s i d e r a b l e number of •host* s p e c i e s , a l l but one o f which are i n the L a m i n a r i a l e s . Whether t h e r e i s a requirement f o r a s p e c i f i c compound produced by the Lam i n a r i a l e s i s not known. The s t r i k i n g marginal d i s t r i b u t i o n of P. g a r d n e r i on the blades of Laminaria s e t c h e l l i i i s probably the r e s u l t of the 33 t e a r i n g of the Laminaria blade producing a wound s i t e i n which spore germination and r h i z o i d attachment can occur. T h i s a l s o appears to be the case f o r the ether 'host* p l a n t s , where P. g a r d n e r i i s found growing on areas of the t h a l l u s where there has been wounding caused by a b r a s i o n or some other means. In her recent work on the red a l g a l p a r a s i t e H a r v e y e l l a m i r a b i l i s (Beinsch) Schmitz e t Beinke, Goff . (1975) found t h a t spore e s t a b l i s h m e n t depended on the presence of a wound area on the host p l a n t caused by g r a z i n g isopods and amphipods. The absence o f e p i p h y t e s on young Laminaria blades may be due t o r a p i d growth and s l o u g h i n g o f f of s u r f a c e m a t e r i a l or production of exudates; some of which (e.g. p h e n o l i c s ) are known t o be t o x i c ( C r a i g i e and Mclachlan, 1964; HcLachlan and C r a i g i e , 1966; S i e b u r t h and Jensen, 1969) The phenomenon of epiphytism and host s p e c i f i c i t y i s an i n t r i g u i n g one. H a r l i n (1973a) demonstrated t h a t i t was the unigue p o s i t i o n t h a t the host p l a n t occupies i n the environment which the o b l i g a t e epiphyte Smithora naiadum (Ander son ) Hollenberg r e g u i r e s . The f a c t that Porphyra g a r d n e r i was not found on rock and would not grow on the a r t i f i c a l Laminaria suggests that i t may have a p h y s i o l o g i c a l reguirement f o r the host. 34 E. Porphyra n e r e o c y s t i s My f i e l d s t u d i e s i n d i c a t e t h a t Porphyra n e r e o c y s t i s i s a winter s p e c i e s . T h i s i s i n agreement with the r e p o r t s o f Smith (1944), Abbott and Hollenberg (1976) and Woessner et a l . (1977). The r e p o r t s o f Conway et a l . (1975) t h a t i t occurs from summer through winter and of Conway and Cole (1977) t h a t i t i s a summer s p e c i e s are a t t r i b u t a b l e to two phenomena. The f i r s t i s t h a t because Porphyra n e r e o c y s t i s i s a s u b t i d a l p l a n t i t i s not *burnt o f f * d u r i n g the s p r i n g months as happens to ether winter Porphyra s p e c i e s (Mumford, 1975). As a r e s u l t i t can p e r s i s t w e l l i n f o the summer provided t h a t i t s host p l a n t i s not c a r r i e d away. Without c o l l e c t i n g a t a s i n g l e s i t e year round i t i s easy to get a d i f f e r e n t concept c f the seasonal occurrence. The second phenomenon which has caused c o n f u s i o n i s the much e a r l i e r appearance of P. n e r e o c y s t i s i n the S t r a i t of Georgia and v i c i n i t y than on the outer coast. In the S t r a i t i t i s an autumn p l a n t . When I f i r s t encountered i t t h i s e a r l y I thought I was d e a l i n g with a d i f f e r e n t s p e c i e s ; however, subseguent study has not supported t h i s c o n c l u s i o n . J u s t what c o n d i t i o n s i n the S t r a i t o f Georgia r e g i o n a r e r e s p o n s i b l e f o r a l t e r i n g the seasonal occurrence of P. n e r e o c y s t i s are not c l e a r , but they are probably i n v o l v e d with t r i g g e r i n g conchospore r e l e a s e (to be d i s c u s s e d i n P a r t I I I ) . Most workers (e.g. B i r d et a l . 1972) have i n d i c a t e d that the seawater temperature i s a c r i t i c a l f a c t o r r e q u i r e d f o r ccnchcspcre r e l e a s e . Parsons (1965) has noted t h a t , because of the s t a b i l i z i n g e f f e c t of freshwater i n p u t from the Fraser 35 R i v e r , the s p r i n g phytoplankton bloom occurs 4-6 weeks e a r l i e r i n the S t r a i t of Georgia than on the outer coast. Perhaps the f a c t o r s i n v o l v e d here are a l s o a f f e c t i n g P. n e r e o c y s t i s . The N e r e o c y s t i s p l a n t s i n the S t r a i t a l s o appear e a r l i e r i n the year and begin to senesce e a r l i e r than outer coast p l a n t s . Another d i f f e r e n c e between the S t r a i t and the c u t e r c o a s t i s t h a t the low t i d e s i n the s p r i n g and summer occur i n the middle of the day i n the S t r a i t as opposed to e a r l y i n the morning on the c u t e r c o a s t ( Krishnamurthy, 1969b; Humford, 1975). T h i s r e s u l t s i n higher d e s i c c a t i o n and i n s o l a t i o n f o r p l a n t s f l o a t i n g on the s u r f a c e a t low t i d e i n the S t r a i t . ESlEhlE* n e r e o c y s t i s i n the S t r a i t a l s o becomes r e p r o d u c t i v e e a r l i e r than i t does on the outer c o a s t . Porphyra n e r e o c y s t i s i s remarkably »host* s p e c i f i c , being r e s t r i c t e d e s s e n t i a l l y t o N e r e o c y s t i s l u e t k e a n a . I have never observed i t growing on rocks and agree with Mumford (1973a) th a t such r e p o r t s are probably m i s i d e n t i f i c a t i o n s of fofihXEa l i f i i a t a . S e v e r a l specimens i n the UBC herbarium repo r t e d to be s a x i c c l o u s i n m i d - i n t e r t i d a l t i d e pools were found to be P. m i n i a t a . The young t h a l l i f i g u r e d by Conway et a l . (1975, F i g . 19a, UBC 33206) as P. n e r e o c j j s t i s are r e f e r r a b l e t o P. m i n i a t a . M i s i d e n t i f i c a t i o n of P. g a r d n e r i and P. miniata as being P. n e r e o c y s t i s has been r e s p o n s i b l e f o r r e p o r t s o f P. n e r e o c y s t i s on hosts other than N e r e o c y s t i s . For example, K y l i n (1941) mistook P. g a r d n e r i on Pterygophora c a l i f o r n i c a Ruprecht f o r a young or dwarf form of P. n e r e o c y s t i s . fly s t u d i e s have a l s o shown t h a t a number of other Porphyra s p e c i e s are capable of growing on the s t i p e of 36 N e r e o c y s t i s and could p o t e n t i a l l y be mistaken f o r P. n e r e o c y s t i s . Porphyra n e r e o c y s t j s was found only on N e r e o c y s t i s which had terminated i t s r a p i d s t i p e growth. Such p l a n t s were not encountered u n t i l the autumn and winter and were c h a r a c t e r i z e d by a heavy l a y e r of diatom and e c t o c a r p a l e a n e p i p h y t e s . As with P. g a r d n e r i . i t i s f e l t t h a t a wound on the s t i p e , caused by a b r a s i o n or g r a z i n g i s important f o r conchospore establishment. The reason f o r P. n e r e o c y s t i s u s u a l l y being r e s t r i c t e d to one s i d e of the s t i p e becomes obvious when the k e l p bed i s observed a t low t i d e . At t h i s time, the upper 3-5 m of the s t i p e i s f l o a t i n g at the s u r f a c e . The h a l f of the s t i p e t h a t i s exposed to the a i r experiences a great amount of d e s i c c a t i o n , which would tend to i n h i b i t the growth of !!• S§£§2£2§tis. The e p i p h y t i c h a b i t of Pot£byr§ n e r e o c y s t j s has obvious advantages. The p l a n t i s on a s u b s t r a t e which i s r e l a t i v e l y f r e e from g r a z i n g , and which keeps the plant c o n s t a n t l y submerged but near the s u r f a c e and adequate i l l u m i n a t i o n . 37 C. f o r g h y r a t h u r e t i i My f i e l d o b s e r v a t i o n s i n d i c a t e t h a t Porphyra t h u r e t i i i s a w i n t e r - e a r l y s p r i n g s p e c i e s , as has been repo r t e d by Smith (1944) and Dawson (1944). At present the d i s t r i b u t i o n of P. t h u r e t i i appears to be r a t h e r patchy; however, I do not t h i n k t h i s i s a c t u a l l y the case. T h i s s p e c i e s has probably been overlooked because i t grows i n r a t h e r exposed areas where c o l l e c t o r s seldom go e a r l y i n the year. I t i s a l s o p o s s i b l e to mistake i t f o r £• n e r e o c y s t i s , P. m i n i a t a or P. p e r f o r a t a . The f a c t t h a t Porphyra t h u r e t i i grew cn the a r t i f i c i a l N e r e o c y s t i s suggests t h a t i t does not have a p h y s i o l o g i c a l dependance on the host p l a n t , but just r e q u i r e s the p h y s i c a l p o s i t i o n i n the environment which the host o f f e r s . T h i s c o n c l u s i o n i s f u r t h e r s u b s t a n t i a t e d by the f a c t t h a t i t was found on hosts as d i v e r s e as P h y l l o s p a d i x and N e r e o c y s t i s . I t would be d e s i r a b l e t o make a s e a s o n a l study of P. t h u r e t i i at the Monterey Peninsula i n order to see i f the d i f f e r e n c e s between these p l a n t s and study s i t e p l a n t s , i n the degree of marginal r u f f l i n g and t h a l l u s s i z e , are s i g n i f i c a n t . 3 8 Table I. Herbaria from which specimens were examined AHF: A l l a n Hancock Foundation, U n i v e r s i t y of Southern C a l i f o r n i a CSOH: C a l i f o r n i a S t a t e U n i v e r s i t y at Humboldt ES: Dudley Herbarium, Stanf o r d (specimens now i n UC) GJH: P r i v a t e Herbarium o f George J. Hollenberg GMS: G i l b e r t Morgan Smith Herbarium (Hopkins Marine S t a t i o n ) PS: P r i v a t e Herbarium of Robert B. Setzer UBC: U n i v e r s i t y of B r i t i s h Columbia UC: U n i v e r s i t y of C a l i f o r n i a at Berkeley UCSC: U n i v e r s i t y of C a l i f o r n i a at Santa Cruz T a b l e I I . *Host» p l a n t s of Porphyra g a r d n e r i Host Phaeophyceae A l a r i a marginata P o s t e l s et Ruprecht Egr e g i a l a e v i g a t a S e t c h e l l Egregia m e n z i e s i i (Turner) Areschoug E i s e n i a arborea Areschoug fljgdophyllutt s e s s i l e (C. Agardh) S e t c h e l l L aminaria q r o e n l a n d i c a Eosenvenqe L. s e t c h e l l i i S i l v a i* s i n c l a i r i i (Harvey) farlow L* yezoensis Mi ya be L e s s o n i o e s i s l i t t o r a l i s (Farlow e t S e t c h e l l J Reinke M a c r o c y s t i s p y r i f e r a (Linnaeus) C. Aqardh N e r e o c y s t i s luetkeana (Uertens) P o s t e l s e t Ruprecht IiSU£op_hycus g a r d n e r i S e t c h e l l e t Saunders P o s t e l s i a palmaef qrmis Ruprecht Fteryggphqra c a l i f p r n i c a Ruprecht Rhodophyceae Schizymenia p a c i f i c a K y l i n Acc, L MQJL L o c a t i o n on h^ost OBC 54894 midrib AHF OBC 77063 54889 t i p s o f l e a f l e t s t i p s o f l e a f l e t s OBC OBC 54890 54893 blade blade margin margin OBC 54129 blade margin OBC OBC 54 887 49214 blade blade margin margin OBC UBC 54202 54 883 blade blade margin margin GMS 13042 blade margin GMS OBC 103 55942 blade s t i p e margin UBC 54886 t o r n blade margin UBC 55751 s t i p e UBC 54885 s p o r o p h y l l margin Abbott 2516 t h a l l u s margin i n GMS 40 Table I I I . Biomass (grams dry weight) of Porphyra n e r e o c y s t i s per v e r t i c a l meter of n e r e o c y s t i s s t i p e . I g t e r s t u d y s i t e Queen C h a r l o t t e I s l a n a s 1 (top) 98 4 3 2 46 67 3 18 11 4 n e g l i g i b l e n e g l i g i b l e ( t o t a l of 3 ( t o t a l of 4 N e r e o c y s t i s N e r e o c y s t i s p l a n t s ) plants) 41 Table IV. Host p l a n t s of Porphyra t h u r e t i i Host Acc. no. L o c a t i o n on host A r t i f i c i a l host UBC 57199 upper s t i p e S a x i c o l o u s OBC 55302 upper s u b t i d a l Sea grasses f h y l l o s E a a i x sp. UBC 54447 on the l e a f Phaeophyceae Sgjcegia m e n z i e s i i (Turner) UBC 57198 on t i p s o f l e a f l e t s Areschoug E i s e n i a arborea Areschoug UBC 57202 blade margins Ne r e o c y s t i s l u etkeana DEC 57 200 s t i p e (Mertens) P o s t e l s et Euprecht Bhcdophyceae Gastroclonium c o u l t e r i GMS 1410 gen e r a l (Harvey) K y l i n G r a c i l a r i a s l o e s t e d t i i UC 791970 g e n e r a l K y l i n J i c r o c l a d i a c o u l t e r i Harvey UC 95607 gen e r a l ( t e n t a t i v e i d e n t i f i c a t i o n from fragment of host) t a b l e V. Annual v a r i a t i o n i n d u r a t i o n of d a y l i g h t (hr. and min.) on the 21st day of the month at 35°N and 48° 50.1'N (study s i t e ) . 35°N Study s i t e i M x l i S j l l i r a . j i l b J . January 10, 11 8, 58 February 11, 08 10, 32 March 12, 09 12, 12 A p r i l 13, 16 14, 00 May 14, 09 15, 28 June 14, 31 16, 07 J u l y 14, 10 15, 32 August 13, 18 14, 04 September 12, 12 12, 17 October 11, 07 10, 32 November 10, 11 8, 59 December s. 48 8, 17 43 F i g u r e 1a. Monosporic t h a l l i of Porphyra g a r d n e r i growing on the blade margins of Lag j. p a r i a s e t c h e l l i i . The type specimen (DS 306401 i n UC). F i g u r e 1b. A 2.7 m, f e r t i l e specimen of Pgrp_hjrr_a nereo.cxs.ii§ c o l l e c t e d a t the study s i t e (UBC 57180). F i g u r e 1c. The l e c t o t y p e of Porphyra t h u r e t i i (UC 791973). 44 45 F i g u r e 2a. The f i e l d work was c a r r i e d out i n B a r k l e y Sound on the west c o a s t of Vancouver I s l a n d ( i n s e t ) . The study s i t e was l o c a t e d o f f Diana I s l a n d ( c i r c l e d ) , near the B a o f i e l d Marine S t a t i o n (from Canadian Hydrographic S e r v i c e Chart #L-3001), F i g u r e 2b. A e r i a l photograph of Diana I s l a n d and the study s i t e . The d i r e c t i o n of the open P a c i f i c Ocean i s i n d i c a t e d by the arrow, (from B. C. Government a i r photograph #BC 7238-182). 46 47 F i g u r e 3a. C h a r a c t e r i s t i c heart-shaped new blade growth of Laminaria s e t c h e l l i i (OBC 54895) . F i g u r e 3b. E a r l y stage i n the s p l i t t i n g of the blade of L. s e t c h e l l i i (OBC 57204). F i g u r e 3c. Bature, l a c e r a t e d blade of L. s e t c h e l l i i with t h a l l i of Porphyra g a r d n e r i along i t s margins (OBC 54896). F i g u r e 3d. Honosporic t h a l l i of P. g a r d n e r i (UBC 54819). 49 Porphyra g a r d n e r i F i g u r e 4a. T h a l l u s of Porphyra g a r d n e r i which has spermatangia, carpogonia and young carposporangia along i t s l a t e r a l margins. Monospores are s t i l l being r e l e a s e d along the d i s t a l margin (OBC 54821}. F i g u r e 4b. O l d , l a c e r a t e d , and e p i p h y t i z e d t h a l l u s of Laminaria s e t c h e l l i i with a few t h a l l i of Porphyra g a r d n e r i on i t s margins (arrows). The new Laminaria blade i s j u s t beginning t c grow (OBC 57205). F i g u r e s 4c-e. M o r p h o l o g i c a l v a r i a t i o n i n t h a l l i o f P. g a r d n e r i which are spermatangial and c a r p o s p o r a n g i a l . Note the c h a r a c t e r i s t i c p a t t e r n formed by the spermatangia along the l a t e r a l margins. F i g u r e 4c: OBC 54138, from B o t a n i c a l Beach; F i g u r e s 4d and 4e: OBC 54891, from Cape Beale. F i g u r e 4f. T h a l l u s of P. g a r d n e r i from the type l o c a l i t y . T h i s p l a n t i s r e l e a s i n g monospores along i t s d i s t a l margin and spermatia and carpospores along i t s l a t e r a l margins (OBC 54818). S c a l e bar = 10 mm on F i g u r e s 4a, 4c, 4d, 4e, 4f 51 Porphyra n e r e o c y s t i s F i g u r e 5a. Young v e g e t a t i v e t h a l l i are c h a r a c t e r i s t i c a l l y long and s t r a p shaped, t a p e r i n g g r a d u a l l y to a p o i n t (UBC 57187) . F i g u r e 5b. The formation of spermatangia begins a t the l a t e r a l margins and proceeds b a s a l l y . These r e g i o n s of the t h a l l u s are e a s i l y r e c o g n i z e d by t h e i r pale yellow to white c o l o u r (OBC 57188). Fig u r e 5c. as a r e s u l t of spermatium l i b e r a t i o n , the l a t e r a l spermatangial margins begin to erode. T h i s s t a r t s a t the apex and proceeds b a s a l l y i , The remaining a p i c a l p o r t i o n of the t h a l l u s c o n s i s t s of v e g e t a t i v e c e l l s and carposporangia (OBC 57184). F i g u r e 5d., Spermatangia forming marginal and submarginal s t r e a k s which are o r i e n t e d i n an apex to base d i r e c t i o n (OBC 57180). F i g u r e 5e. The t i p s of o l d e r t h a l l i o f t e n have a s l i g h t l y mottled appearance caused by abundant carposporangia (OBC 57192) . 53 Porphyra t h u r e t i i F i g u r e 6a. M o r p h o l o g i c a l v a r i a t i o n i n t h a l l i which were growing on the s t i p e of N e r e o c y s t i s luetkeana i n Barkley Sound. Note the c h a r a c t e r i s t i c r u f f l e d margins (DBC 57200). F i g u r e 6b. The production of spermatangia begins i n the a p i c a l marginal r e g i o n of the t h a l l u s (UBC 52109). F i g u r e s 6c and 6d., On o l d e r t h a l l i s h o r t s t r e a k s of spermatangia form submarginally. Note th e c h a r a c t e r i s t i c p a t t e r n t h a t r e s u l t s (UBC 57201 and UC 95596 r e s p e c t i v e l y ) . F i g u r e 6e. Specimen from the Monterey Peninsula which resembles specimens from Barkley Sound i n s i z e spermatangial p a t t e r n . and 54 55 PART I I - SPERMATOGENESIS AND CARPOSPOBOGENES.IS I n t r o d u c t i o n Hus (1902) was the f i r s t to use the number of spermatia per spermatangium 1 and carpospores per carposporangium as a taxcnomic c r i t e r i o n f o r d e l i m i t i n g Porphyra s p e c i e s . He thought t h a t an u n d i f f e r e n t i a t e d v e g e t a t i v e c e l l f u n c t i o n e d as the carpogonium, and t h a t two a n t i c l i n a l d i v i s i o n s ( r e f e r r e d to by Hus as ' c r u c i a t e 1 d i v i s i o n ) c f a v e g e t a t i v e c e l l gave r i s e to a group of fou r spermatangia. Hus d e v i s e d a spore formula by c o n s i d e r i n g the spermatangium and carposporangium t o be cubes with *a* and *b' r e p r e s e n t i n g the h o r i z o n t a l c o o r d i n a t e s and 'c' the v e r t i c a l . A p o i n t which has been overlooked by a l l r e c e n t workers, with the e x c e p t i o n o f Krishnamurthy (1972), was t h a t Hus considered each carposporangium to be e g u i v a l e n t t o f o u r spermatangia. Hus' n o t a t i o n , a p p l i e d t o a cube 4x4x2, would be: 32 carpospores a/4, b/4, c/2 or 8 spermatia 1/2 a/2, 1/2 b/2, c/2 (Hus, 1902, p l a t e 22, f i g . 25) . As Krishnamurthy (1972) has pointed c u t , subseguent workers have dropped the 1/2 i n the spermatangium formula and a p p l i e d the formula e q u a l l y to both carposporangia and spermatangia. I have followed t h i s system because I c o u l d not f i n d any i n d i c a t i o n t h a t a carpogonium i s •-Hus (1902) and many subseguent workers used the t e r n Vpacket' i n place of spermatangium and carposporangium. See terminology s e c t i o n f o r f u r t h e r d i s c u s s i o n . 56 e q u i v a l e n t t o f o u r spermatangia. Judging by r e p o r t s i n the l i t e r a t u r e , summarized by Drew (1956), r e c o g n i t i o n of the carpogonium i n Porphyra has been an area o f c o n s i d e r a b l e c o n f u s i o n . According t o Drew, B e r t h o l d (1882) co n s i d e r e d the carpogonia of P. l e u c o s t i c t a Thuret i n Le J o l i s to be s l i g h t l y l a r g e r and p a l e r than v e g e t a t i v e c e l l s . The carpogonia B e r t h o l d (1882) f i g u r e d a l s o had s l i g h t b i p o l a r protuberances which d i d not push out the outer w a l l . Berthold a l s o i l l u s t r a t e d c e l l s with long b i p o l a r protuberances which caused a p r o t r u s i o n of the outer wall. He apparently c o n s i d e r e d these c e l l s t o be o l d u n f e r t i l i z e d carpogonia. Hus (1902) d i d not adequately c h a r a c t e r i z e the spermatangium or carpogonium of the Porphyra s p e c i e s he s t u d i e d . He thouqht they were formed by u n d i f f e r e n t i a t e d v e g e t a t i v e c e l l s and r e p o r t e d t h a t he c o u l d d i s t i n g u i s h v e g e t a t i v e d i v i s i o n from spermatangium d i v i s i o n by the wa l l t h i c k n e s s . Drew (1956) noted t h a t there was too much v a r i a t i o n i n w a l l t h i c k n e s s t o be ab l e t o d i s t i n g u i s h v e g e t a t i v e from r e p r o d u c t i v e d i v i s i o n s . Kunieda (1939) r e p o r t e d carpogonia with p r o t o t r i c h o g y n e s i n Porphyra t e n e r a . Dangeard (1927) i l l u s t r a t e d u n d i f f e r e n t i a t e d c a r p o g o n i a i n P. l i n e a r i s G r e v i l l e (as £• u m b i l i c a l i s f. l i n e a r i s ( G r e v i l l e ) Harvey) and carpogonia with p r o t o t r i c h o g y n e s i n P. u m b i l i c a l i s (Linnaeus) J , Agardh. Conway and Cole (1973) d e s c r i b e d ' c a r p o g o n i a - l i k e * c e l l s with ' t r i c h o g y n e - l i k e * p r o j e c t i o n s i n P. p a p e n f u s s i i Krishnamurthy. Based on t h i s d i s t r o m a t i c s p e c i e s , Conway et a l . (1975, f i g . 1) showed the carpogonium with one p r o t o t r i c h o g y n e . No i n d i c a t i o n 57 was given of the appearance of the carpogonium i n monostromatic s p e c i e s . Kurogi (1961), i n h i s e x c e l l e n t i n v e s t i g a t i o n of 4 monostromatic Porphyra s p e c i e s , r e p o r t e d t h a t the carpogonium had b i p o l a r p r o t o t r i c h o g y n e s . In a d d i t i o n to the d i f f e r i n g r e p o r t s about the appearance of the carpogonium, Drew (1S56) pointed out that there are c o n f l i c t i n g r e p o r t s of the o r i e n t a t i o n of the f i r s t d i v i s i o n o f the carposporangium. One group of workers b e l i e v e d the f i r s t two d i v i s i o n s were a n t i c l i n a l and a t r i g h t angles t o each other (often r e f e r r e d to as c r u c i a t e d i v i s i o n s ) (Hus, 1902; Krishnamurthy, 1959, 1969b, 1972; Conway, 1964b), whereas others have r e p o r t e d the f i r s t d i v i s i o n of the carposporangium as being p e r i c l i n a l ( in the plane of the t h a l l u s ) ( J a n c z e u s k i , 1873; B e r t h o l d , 1882; Ishikawa, 1921; Grubb, 1924; Dangeard, 1927; K u r o g i , 1961). Tanaka (1952) i n d i c a t e d t h a t the i n i t i a l d i v i s i o n c o u l d be e i t h e r a n t i c l i n a l or p e r i c l i n a l depending on the s p e c i e s . Conway e t a l . (197 5) d i d not i n d i c a t e the d i v i s i o n sequence of the carposporangium. Despite a c o n s i d e r a b l e amount of o b s e r v a t i o n of spermatangial development at the l i g h t microscope l e v e l (Janczewski, 1873; Thuret and Bornet, 1878; B e r t h o l d , 1882; Hus, 1902; Ishikawa, 1921; Grubb, 1924; Tanaka, 1952; Krishnamurthy 1959, 1972; Humford, 1973a; Conway et a l . 1975), the appearance and d i v i s i o n sequence o f the spermatangium of Por£hyra has not been w e l l documented. These s t u d i e s d i d l i t t l e more than i n d i c a t e t h a t the spermatia are formed by repeated d i v i s i o n of the spermatanqium. The data are c o n t r a d i c t o r y i n t h a t Janczewski (1873), Thuret and Bornet 58 (1878), Hus (1902), Ishikawa (1921) and Grubb (1924) maintained t h a t the f i r s t d i v i s i o n o f the spermatangium was p e r i c l i n a l , whereas Tanaka (1952) and Krishnamurthy (1959) thought i t was a n t i c l i n a l . as Drew (1956) pointed out, these v a r i o u s d i s c r e p a n c i e s are due perhaps t o the d i f f i c u l t y i n d i s t i n g u i s h i n g the spermatangium from a v e g e t a t i v e c e l l . I t was apparent to me t h a t Drew's (1956, p. 595) statement, " L i t t l e a t t e n t i o n has been paid to the method of d i s t i n g u i s h i n g normal c e l l - d i v i s i o n from spore f o r m a t i o n . . . " was s t i l l v a l i d . I t h e r e f o r e decided to t r y to c h a r a c t e r i z e the spermatangium and carpogonium and f o l l o w t h e i r d i v i s i o n seguence i n d e t a i l at the l i g h t microscope l e v e l f o r the three s p e c i e s I was s t u d y i n g . For the genus P o r p h y r e l l a Smith and H o l l e n b e r g (1943) repor t e d t h a t the carpogonium was formed by a c u r v i n g c e l l d i v i s i o n s i m i l a r t o t h a t of monospore d i v i s i o n i n E r y t h r o t r i c h i a . and t h a t t h e carpospores were r e l e a s e d s i n g l y . My p r e l i m i n a r y examination of specimens from the study s i t e i n d i c a t e d t hat the carpogonia and carpospores were not forming i n t h i s manner. These specimens f i t the d e s c r i p t i o n of £oj£hyra g a r d n e r i ( P o r p h y r e l l a g a r d n e r i at t h a t time) i n a l l e t h e r r e s p e c t s . A d e t a i l e d study of carpogonium and carpospore for m a t i o n i n p l a n t s a t the study s i t e and type l o c a l i t y was t h e r e f o r e e s s e n t i a l t o confirm t h a t the genus P o r p h y r e l l a was i n c o r r e c t l y d e s c r i b e d . 59 M a t e r i a l s And Methods Spermatangial and carposporangia1 t h a l l i were c o l l e c t e d a t the study s i t e and marginal and submarginal pieces (approx, 5mm2) were cut out and f i x e d i n a s o l u t i c n of glu t a r a l d e h y d e (50%), phosphate b u f f e r pH 7.2, and m i l l i p o r e f i l t e r e d seawater (1:4:4) (McBride and Cole, 1969) f o r 1.5 hours. Whenever p o s s i b l e m a t e r i a l was f i x e d immediately i n the f i e l d . F i x a t i o n was f o l l o w e d by dehydration i n a graded e t h a n o l s e r i e s and i n f i l t r a t i o n with g l y c o l methacrylate ( P o l y s c i e n c e s JB-4). S e r i a l s e c t i o n s (Henry, 1977) 2-3 um t h i c k were cut cn g l a s s k n i ves using a S o r v a l JB-4 microtome. Sections were s t a i n e d with t c l u i d i n e blue-0 i n benzoate b u f f e r or d i s t i l l e d water (Feder and O'Brien, 1968) mounted i n Euparal (GBI Labs L t d . , Heaton S t , Denton, Manchester) and examined on an Olympus l i g h t microscope (Model FHA) with planapochromatic o b j e c t i v e s . Photographs were taken w i t h a Nikon M i c r o f l e x (Model AFM) automatic photographic attachment using Kodak Panatomic-X f i l m . 60 B e s u l t s A. Porphyra g a r d n e r i 1. Spermatogenesis The d i v i s i o n sequence l e a d i n g to the f o r m a t i o n of mature spermatangia was f o l l o w e d i n s u r f a c e view and t r a n s v e r s e s e c t i o n . V e g e t a t i v e c e l l s have a s i n g l e s t e l l a t e c h l o r o p l a s t , c e n t r a l pyrenoid, and p e r i p h e r a l l y l o c a t e d nucleus (Figure 7a). During l a t e A p r i l the spermatangia began forming along the t h a l l u s margin. The f i r s t stage that v e g e t a t i v e c e l l s went through i n the t r a n s i t i o n t o a spermatangium was the production of a new w a l l l a y e r (Figure 7b) , The f i r s t d i v i s i o n of the spermatangium was p e r i c l i n a l (a/1, b/1, c/2) (Figure 7 c ) . The second and t h i r d d i v i s i o n s were a n t i c l i n a l and at r i g h t angles to each other ( F i g u r e s 7d and 8a). These were followed by a n t i c l i n a l and p e r i c l i n a l d i v i s i o n s ( F i g u r e s 7e-f) u n t i l a mature spermatangium c o n t a i n i n g 64 spermatia (a/4, b/4, c/4) was formed (Figure 7g). An immature spermatangium i s shown i n s u r f a c e view i n F i g u r e 7h, and a mature spermatangium i n F i g u r e 7 i . T h i s d i v i s i o n sequence i s summarized i n F i g u r e 8a. 2. Carposporogenesis The spermatangia, carpogonia and carposporangia (Figure 9b) are r e a d i l y d i s t i n g u i s h e d from v e g e t a t i v e c e l l s i n s u r f a c e view (Figure 9a). By e a r l y May carpogonia began forming i n the submarginal 61 r e g i o n of the t h a l l u s . No c u r v i n g , E r y t h r o t r i c h i a - l i k e n a i l s were i n v o l v e d i n t h e i r f o r m a t i o n . In t r a n s v e r s e s e c t i o n the carpogonia were e a s i l y d i s t i n g u i s h e d from v e g e t a t i v e c e l l s by t h e i r c h a r a c t e r i s t i c s p i n d l e - or lemon-shape due to the presence of b i p o l a r p r o t o t r i c h o g y n e s (Figure 9 c ) . The carpogonium c o n t a i n e d a s i n g l e s t e l l a t e c h l o r o p l a s t , pyrenoid, and abundant f l o r i d e a n s t a r c h g r a i n s . In some cases two carpogonia were c l o s e l y appressed (Figure 9d). T h i s appeared to r e s u l t from carpogonium d i f f e r e n t i a t i o n immediately f o l l o w i n g a v e g e t a t i v e c e l l d i v i s i o n before a t h i c k w a l l had formed between the daughter c e l l s . In determining the number of carpospores per carposporangium i t was necessary t o a s c e r t a i n t h a t the carpospores being observed were a c t u a l l y d e r i v e d from a s i n g l e carpogonium and not two c l o s e l y appressed ones. F o l l o w i n g f e r t i l i z a t i o n of the carpogonium (or perhaps without i t i n some cases; t o be discussed i n P a r t I V ) , the f i r s t d i v i s i o n of the carposporangium was p e r i c l i n a l (a/1, b/1, c/2) (Figure 9e). , Many of the carposporangia d i d not d i v i d e beyond t h i s stage, t u t some underwent second and t h i r d a n t i c l i n a l d i v i s i o n s to form 4 carpospores (Figures 9 f , 9g, and 9h). In some carposporangia these two d i v i s i o n s appeared to occur i n the same plane (a/1, b/2, c/2. F i g u r e 8b), but i n most cases they were o r i e n t e d a t r i g h t angles t o each other (Figure 8b). The l a t t e r carposporangia were e a s i l y seen i n s u r f a c e view ( F i g u r e s 9g and 9h).. O c c a s i o n a l l y f u r t h e r d i v i s i o n to produce 8 carpospores (a/2, b/2, c/2) occu r r e d (Figures 9 i and 9 j ) , The d i v i s i o n sequence of 62 c a r p c s p o r o g e n e s i s i s summarized i n Figure 8b. 3. Examination Of Type L o c a l i t y Plants In view o f the mode of carpogonium formation and d i v i s i o n sequence of the carposporangium observed i n study s i t e p l a n t s i t was apparent to me t h a t they belonged to the genus Porphyra, not P o r p h y r e l l a . Examination of carpogonia and carposporangia of herbarium specimens from- Alaska t o northern C a l i f o r n i a r e v e a l e d that they were i d e n t i c a l to study s i t e p l a n t s . T h e r e f o r e i t was necessary t o determine whether the p l a n t s a t the type l o c a l i t y on the Monterey P e n i n s u l a , C a l i f o r n i a , were the same taxon. From the o r i g i n a l d e s c r i p t i o n , herbarium specimens, and thorough notes made by Dr. Hollenberg on specimens i n h i s herbarium, i t was p o s s i b l e t o co n f i r m t h a t both taxa had the f o l l o w i n g c h a r a c t e r i s t i c s i n common: s i m i l a r host p l a n t s , a marginal d i s t r i b u t i o n on Lami n a r i a . a monospore c y c l e , 64 spermatia/spermatangium, s i m i l a r b a s a l region morphology and attachment, v e g e t a t i v e felade t h i c k n e s s 25-35 an, and ve g e t a t i v e t h a l l i minutely wrinkled. The only d i f f e r e n c e between type l o c a l i t y p l a n t s and those from the r e s t of the c o a s t was the report e d mode of carpogonium fo r m a t i o n and carposporangium d i v i s i o n . In order to sol v e t h i s problem, specimens were c o l l e c t e d i n J u l y 1976 at Point Joe (type l o c a l i t y ) , Pescadero P o i n t and Mi s s i o n P o i n t , C a l i f o r n i a . The number of carpogonia and carposporangia was low compared t o more northern p o p u l a t i o n s ; however, t h e i r mode o f formation and d i v i s i o n was i d e n t i c a l 63 with t h a t observed i n specimens from the study s i t e and other areas along the coast. T h i s o b s e r v a t i o n combined with f i e l d , c u l t u r e and chromosome s t u d i e s (see Part s I , I I I , IV) on the type l o c a l i t y p o p u l a t i o n c o n c l u s i v e l y demonstrated t h a t i t was the same taxon as was found at the study s i t e . I t seems probable t h a t the s m a l l number of carpogonia and carposporangia produced by t h a l l i at the type l o c a l i t y i s the reason t h a t t h e i r formation has been overlooked i n the pa s t . B. Porphyra n e r e o c y s t i s 1. Spermatogenesis Spermatogenesis was d i f f i c u l t t o f o l l o w i n Porphyra n e r e o c y s t i s because the spermatangia were not very d i s t i n c t from v e g e t a t i v e c e l l s . Unlike P. j a r d n e r i , no new wall was l a i d down around the spermatangium p r i o r t o i t s f i r s t d i v i s i o n . Furthermore, the spermatangia t h a t had undergone a s e r i e s of d i v i s i o n s were not w e l l d e f i n e d , making i t d i f f i c u l t to determine the number of spermatia i n mature spermatangia. In F i g u r e 9k a v e g e t a t i v e p o r t i o n of the t h a l l u s i s shown i n s u r f a c e view. The young spermatangia were d i f f i c u l t t o d i s t i n g u i s h from v e g e t a t i v e c e l l s u n t i l they had undergone s e v e r a l d i v i s i o n s (Figure 91). Spermatangia with 4 (a/1{2), b/2(1), c/2) or 8 (a/2, b/2, c/2) spermatia were s t a r t i n g t o l o s e pigmentation. Subseguent d i v i s i o n s o c c u r r e d ( F i g u r e 10a) u n t i l a mature spermatangium (a/4, b/4, c/8) c o n t a i n i n g 128 spermatia was reached ( F i g u r e s 10b and 10c). T h i s i s a maximum number; v a r i a t i o n s i n 64 the f i n a l number of spertaatia/spermatangium were observed. 2. Carposporogenesis at the study s i t e carpogonium formation began i n January i n the submarginal r e g i o n o f the t h a l l u s . In t r a n s v e r s e s e c t i o n the carpogonium was d i f f i c u l t t o r e c o g n i z e because i t d i d not form p r o t o t r i c h o g y n e s . The carpogonia were c h a r a c t e r i z e d as being r e c t a n g u l a r and elongate i n t r a n s v e r s e s e c t i o n (Figure 10e) compared to the square-shaped v e g e t a t i v e c e l l s (Figure 10d). They had a s i n g l e s t e l l a t e c h l o r o p l a s t , c e n t r a l pyrenoid and p e r i p h e r a l n ucleus. C a r p o g o n i a l r e g i o n s of the t h a l l u s c ould a l s o be recognized by the presence of spermatia forming f e r t i l i z a t i o n c a n a l s t o the carpogonia (to be di s c u s s e d i n Part IV) and by p e r i c l i n a l d i v i s i o n s of the carposporangia. F r e q u e n t l y 2 c l o s e l y appressed carpogonia were observed (Figure 1 0f), a phenomenon which may l e a d to m i s i n t e r p r e t a t i o n c f the number of carpospores per carposporangium. The f i r s t d i v i s i o n o f the carposporangium was p e r i c l i n a l (a/1, b/1, c/2) (Figure 10g) . I t i s f e l t t h a t t h i s d i v i s i o n n o r n a l l y f o l l o w s f e r t i l i z a t i o n (see Part IV). Both the second and t h i r d d i v i s i o n s were a n t i c l i n a l and a t r i g h t angles t c each other (Figure 10b). Carposporangia a t t h i s stage were e a s i l y d e t e c t a b l e i n s u r f a c e view (Figure 1 0 i ) . Subsequent d i v i s i o n occurs r a p i d l y t o produce 8 carpospores/carposporangium (a/2, b/2, c/2) (Fi g u r e s 10h and 1 0 j ) . Host carposporangia r e l e a s e d carpospores at t h i s stage (see P a r t I I I ) . F u r t h e r d i v i s i o n a lcng the »c ,-axis of the carposporangium (Figure 101) occurred 65 f r e q u e n t l y on o l d e r t h a l l i and r e s u l t e d i n a deeper pigmentation. Suspected v e g e t a t i v e c e l l s and i n c o m p l e t e l y d i v i d e d carposporangia which had l o s t t h e i r pigmentation were mixed i n with the mature carposporangia and gave the t h a l l u s a mottled appearance (Figure 5e). I was unable to f i n d carposporangia with 32 carpospores (a/4, fa/4, c/2) as has been t y p i c a l l y r eported f o r Porfhysa n e r e o c y s t i s (Has. 1902; Smith, 1944; Krishnamurthy, 1972; Mumford, 1973a; Abbott and Hollenberg, 1976; Conway e t a l . 1975). Groups of carpospores t h a t s u p e r f i c i a l l y appeared to have t h i s spore formula (Figures 10k and 11a) were too l a r g e to have been d e r i v e d from a s i n g l e carposporangium. Carposporangia a/4, b/2 appeared to form although I c o u l d not u n e g u i v o c a l l y determine t h i s . I suspect t h a t the carposporangia r e p o r t e d to be a/4, b/4 are a c t u a l l y made up of 2 c l o s e l y appressed a/4, b/2 carposporangia. C. Porphyra t h u r e t i i 1. Spermatogenesis Due to a l i m i t e d amount of m a t e r i a l of Porphyra t h u r e t i i i t was not p o s s i b l e t o f o l l o w spermatogenesis i n the same d e t a i l as was done f o r P. g a r d n e r i . The e a r l y d i v i s i o n sequence i s shown i n F i g u r e s 11b and 11c. The spermatangium i s w e l l d e f i n e d i n t r a n s v e r s e s e c t i o n , but I have not been a b l e t o determine i f t h i s i s due to the formation of a new w a l l l a y e r p r i o r t c d i v i s i o n . F i g u r e 11d shows an i n t e r m e d i a t e stage i n spermatangial 66 d i v i s o n . Mature spermatangia contained 64 spermatia (a/4, b/4, c/4) ( F i g u r e s 11e and 11f). 2. Carposporogenesis In t r a n s v e r s e s e c t i o n the carpogonia of Porphyra t h u r e t i i were seen to possess s l i g h t p r o t o t r i c h o g y n e s (Figure 11g). T h i s needs t o be re-examined because the m a t e r i a l I s t u d i e d was not f i x e d u n t i l 2 days a f t e r being c o l l e c t e d . My s t u d i e s o f i*« 9§£<Jng£i i n d i c a t e d that immediate f i x a t i o n i n the f i e l d was e s s e n t i a l f o r good carpogonium and spermatangium p r e s e r v a t i o n . The degree of p r o t o t r i c h o g y n e development i n P. t h u r e t i i may t h e r e f o r e be g r e a t e r than I have r e p o r t e d . The carpogonium c o n t a i n e d a s i n g l e s t e l l a t e c h l o r o p l a s t , c e n t r a l p y r e n o i d and p e r i p h e r a l nucleus. The f i r s t d i v i s i o n of the carposporangium ( f o l l o w i n g a suspected f e r t i l i z a t i o n ; see P a r t IV) was p e r i c l i n a l (a/1, b/1, c/2) (Figure 11h). The subseguent d i v i s i o n sequence (Figures 11i and 11j) was the same as t h a t observed i n P. g a r d n e r i and P. n e r e o c y s t i s f e a r l v s t a g e s ) . The mature carposporangium c o n t a i n e d 8 carpospores (a/2, b/2, c/2) (Figures 11k and 111), although carpospore r e l e a s e could occur any time a f t e r the f i r s t d i v i s i o n of the carposporangium. The d i s t a l region of older t h a l l i t y p i c a l l y had a mottled, deeply pigmented appearance due to the abundant mature carposporangia. 67 D i s c u s s i o n T h i s study o f spermatogenesis and c a r p c s p o r o g e n e s i s has y i e l d e d three major r e s u l t s : 1. The genus P o r p h y r e l l a i s not v a l i d . 2. An attempt has been made to c h a r a c t e r i z e the carpogonium and spermatangium of Porphyra. 3. The c l a s s i c a l * c r u c i a t e * d i v i s i o n seguence o f the carposporangia and spermatangia as proposed by Hus (1902) does not occur i n the Porphyra s p e c i e s examined. Examination of the mode of carpogonium formation and d i v i s i o n i n Porphyra g a r d n e r i demonstrated t h a t t h e carpogonia are not formed by unegual d i v i s i o n s and the carpospores are not r e l e a s e d s i n g l y . The c a r p o s p o r a n g i a l d i v i s i o n seguence i s t y p i c a l of the genus Porphyra. Furthermore, the s m a l l and v a r i a b l e number o f c a r p o s p o r a n g i a l d i v i s i o n s are not unique to S» g a r d n e r i . Kurogi (1961) has r e p o r t e d and f i g u r e d the same d i v i s i o n p a t t e r n f o r Porphyra k u n i e d a i Kurogi. I n view of t h i s , the genus P o r p h y r e l l a as d e s c r i b e d by Smith and H o l l e n b e r g (194 3) i s not v a l i d . T h e r e f o r e the f o l l o w i n g nomenclatural change was made (Hawkes, 1977b): Porphyra g a r d n e r i (Smith e t Hollenberg) comb. nov. Synonym: P o r p h y r e l l a g a r d n e r i Smith e t Hollenberg (1943), Am. J . Bot. , 30: 215-216, f i g s . 13-14. D e s c r i p t i o n : T h a l l i monostromatic 25-35 um t h i c k , one c h l o r o p l a s t per c e l l . T h a l l i up to 130 mm long 68 and 60 mm wide, but t y p i c a l l y 20-70 mm long and 10-30 mm wide. Reproducing a s e x u a l l y by monospores. Mature spermatangia c o n t a i n i n g 64 spermatia (a/4, b/4, c/4) maximum. Carpogonium formed by the t r a n s f o r m a t i o n of a v e g e t a t i v e c e l l and c h a r a c t e r i z e d by b i p o l a r p r o t o t r i c h o g y n e s . Carposporangium undergoing one t o three or r a r e l y seven d i v i s i o n s t o form two (a/1, b/1, c/2) , f o u r (a/1(2), b/2{1), c/2) or e i g h t (a/2, b/2, c/2) carpospores. The l i f e h i s t o r y i s heteromorphic, r e l e a s e d carpospores germinating i n t o a c o n c h o c e l i s phase. Conchocelis phase with p i t plugs as i s t y p i c a l of a l l Porphyra s p e c i e s t h a t have been examined (Lee and F u l t z , 1970; Bourne et a l . 1970; C o l e , 197 2b) . Spore fo r m a t i o n by unequal d i v i s i o n i s c h a r a c t e r i s t i c of the E r y t h r o p e l t i d a c e a e , whereas spore formation by repeated d i v i s i o n to produce s e v e r a l spermatia/spermatangium c r carpospores/carpospcrangium i s t y p i c a l of the Bangiaceae. On the b a s i s o f Smith and flollenberg's (1943) r e p o r t of the occurrence of both these modes of spore formation i n Porphyra a a r d n e r i . Drew (1956) suggested that t h i s s p e c i e s had a f f i n i t i e s with both f a m i l i e s . T h i s , however, i s not the case; P. g a r d n e r i i s t y p i c a l o f the Bangiaceae i n a l l asp e c t s of spore formation. Only one other s p e c i e s has been r e f e r r e d t o the genus £2I£hyxe.11 a, t h i s being P o r p h y r e l l a c a l i f o r n i c a H c l l e n b e r g , d e s c r i b e d by Hc l l e n b e r g (1945) from Santa Cruz I s l a n d and 69 s e v e r a l p l a c e s along the mainland c o a s t , southern C a l i f o r n i a . He separated i t from Porphyra garjinjarj. on the b a s i s of i t s s a x i c o l o u s h a b i t and shape of t h a l l u s . No mention was made of uneven c r o s s w a l l s i n carpogonium formation i n t h i s s p e c i e s . The holotype of P. c a l i f o r n i c a (GJH 1353) c o n s i s t s of one t h a l l u s permanently mounted on a g l a s s s l i d e . An examination o f i t r e v e a l e d abundant spermatangia along the t h a l l u s margin. Also i n the marginal r e g i o n were l a r g e r pigmented c e l l s , which are probably carpogonia and carpospores. No evidence o f uneven c r o s s w a l l s was seen. In view of t h i s i t i s suspected t h a t P or p hy r e 1 l a c a 1 i f o r n i c a should be t r a n s f e r r e d t o Porphyra; however, u n t i l f u r t h e r i n v e s t i g a t i o n of i t i s made, nothing c o n c l u s i v e can be s a i d about i t s taxonomic s t a t u s . There a r e a t l e a s t three d i f f e r e n t c a r p o g o n i a l morphologies i n the genus Porphyra. Hawkes (1977b) pointed out tha t i n those s p e c i e s f o r which p r o t o t r i c h o g y n e s have been re p o r t e d , t h e r e are two c a r p o g o n i a l types. Moncstrcoiatic s p e c i e s have carpogonia with 2 p r o t o t r i c h o g y n e s l o c a t e d on the s i d e s o f the carpogonium next t o the t h a l l u s s u r f a c e , whereas i n d i s t r o m a t i c s p e c i e s the carpogonium has o n l y one p r o t o t r i c h o g y n e , l o c a t e d next t o the t h a l l u s s u r f a c e . Judging by r e p o r t s i n the l i t e r a t u r e (Table V I ) , most Porphyra s p e c i e s have carpogonia with p r o t o t r i c h o g y n e s , The p r o t o t r i c h o g y n e s are produced by the developing carpogonia and f u n c t i o n as s p e c i a l i z e d r e c e p t i v e s i t e s f o r spermatium attachment and f e r t i l i z a t i o n (see P a r t I V ) , They give the carpogonia a c h a r a c t e r i s t i c elongate lemon-shape and cause the c u t e r c e l l w a l l of the t h a l l u s t o protrude t o v a r y i n g degrees depending on 70 how w e l l developed they are. The degree of p r o t r u s i o n appears to vary depending on the s p e c i e s . Porphyra p a p e n f u s s i i and £• tJLSsi (Yendo) Ueda are unique amongst the s p e c i e s l i s t e d i n Table VI because of t h e i r extremely long p r o t o t r i c h o g y n e s . Examination of these p r o t o t r i c h o g y n e s a t the E. M. l e v e l i s needed. The long protuberances reported by B e r t h o l d (1882) and J o f f e ' (1896) t o be formed by o l d u n f e r t i l i z e d carpogonia were not seen i n Porphyra g a r d n e r i . ft t h i r d type of c a r p o g o n i a l morphology i s d i s p l a y e d by Porchyra n e r e o c y s t i s i n which no p r o t o t r i c h o g y n e s are formed. The only f e a t u r e to d i f f e r e n t i a t e the carpogonia from v e g e t a t i v e c e l l s i s t h e i r s l i g h t l y more elongate, r e c t a n g u l a r shape. Only Dangeard (1927) and Tseng and Chang (1955) have s p e c i f i c a l l y noted t h a t a few Porphyra s p e c i e s l a c k p r o t o t r i c h o g y n e s (Table V I ) . P r e l i m i n a r y o b s e r v a t i o n of other s p e c i e s from the Northeast P a c i f i c Ocean suggests t h a t some of them lack p r o t o t r i c h o g y n e s . F u r t h e r i n v e s t i g a t i o n of t h i s i s needed. Dangeard (1927) was the f i r s t t o r e p o r t t h a t some Porphyra s p e c i e s possessed p r o t o t r i c h o g y n e s , whereas o t h e r s l a c k e d them (Table V I ) . Tseng and Chang (1955) noted t h a t there was v a r i a t i o n i n the occurrence of the p r o t o t r i c h o g y n e i n the three Porphyra s p e c i e s they s t u d i e d . They suggested t h a t a l l Porphyra s p e c i e s have the a b i l i t y t o form p r o t o t r i c h o g y n e s but t h a t t h i s may be suppressed i n those which possess a t h i c k , tough c e l l w a l l . Another e x p l a n a t i o n which I am proposing i s t h a t those s p e c i e s l a c k i n g p r o t o t r i c h o g y n e s have never had the a b i l i t y to produce these more s p e c i a l i z e d r e c e p t i v e areas and 71 may t h e r e f o r e by more p r i m i t i v e . T h i s needs t o be s t u d i e d i n other Porphyra s p e c i e s i n order t o completely c h a r a c t e r i z e the carpogonium of t h i s genus. Groups of 2 carpogonia were o c c a s i o n a l l y noted i n P. g a r d n e r i and P. n e r e o c y s t i s . but groups of 4 carpogonia r e s u l t i n g from a • c r u c i a t e 1 d i v i s i o n of a v e g e t a t i v e c e l l as re p o r t e d by Conway e t a l . 0975) were not seen. T h i s phenomemon appears to be l i m i t e d t o P. p a p e n f u s s i i and P. s c h i z o p h y l l a Hollenberg. The spermatangium i n Porphyra g a r d n e r i can be d i s t i n g u i s h e d from v e g e t a t i v e c e l l s by the new wa l l l a y e r t h a t i s l a i d down around i t p r i o r to d i v i s i o n . T h i s i s the f i r s t time t h i s f e a t u r e has been r e p o r t e d f o r Porphyra spermatangia, although Scott and Dixon (1973a) p r e v i o u s l y reported i t f o r the t e t r a s p o r a n g i a o f P t i l o t a hypnoides (Harvey) K y l i n . The new w a l l l a y e r g i v e s the spermatangium a well d e f i n e d appearance, making i t simple t o i d e n t i f y a l l the spermatia produced by one spermatangium. In c o n t r a s t , the spermatangium of Porphyra n e r e o c y s t i s l a c k e d the new w a l l l a y e r making i t d i f f i c u l t t o i d e n t i f y the products of a s i n g l e spermatangium. I t i s i n t e r e s t i n g to note t h a t t h i s l e s s s p e c i a l i z e d spermatangium i s a s s o c i a t e d with the simple carpogonium of P. n e r e o c y s t i s . adding f u r t h e r evidence i n support c f the theory that i t i s a more p r i m i t i v e member of the genus. In £• gairdneri the more advanced carpogonium i s a s s o c i a t e d with the more s p e c i a l i z e d spermatangium. Thus, t h i s p r e l i m i n a r y study suggests t h a t t h e r e may be a c o r r e l a t i o n between the type of carpogonium and spermatangium. / Study of other members of 72 the genus i s needed to confirm t h i s . The r e p o r t by Janczewski (1873) of spermatia and carpospores o c c u r r i n g i n the same sporangium remains an enigma. T h i s may have been a m i s i n t e r p r e t a t i o n by Janczewski. Such a phenomenon was not observed i n the 3 s p e c i e s I s t u d i e d . Once the carpogonium and spermatangium have been r e c o g n i z e d , the subseguent d i v i s i o n seguence can be f o l l o w e d . In a l l 3 s p e c i e s and f o r both the spermatangia and carpcsporangia the i n i t i a l d i v i s i o n s were i d e n t i c a l . The f i r s t d i v i s i o n i s p e r i c l i n a l and no • c r u c i a t e * d i v i s i o n s as f i r s t r e p o r t e d by Hus (1902) and l a t e r by Krishnamurthy (1959) occurred. I t i s p o s s i b l e t h a t the d i v i s i o n seguence i s d i f f e r e n t f o r other Porphyra s p e c i e s : however, t h i s should be r e - i n v e s t i g a t e d , p a r t i c u l a r i l y i n those cases where c r u c i a t e d i v i s i o n s have been reported. Hus (1902), Krishnamurthy (1972), and Conway e t a l . ; (1975) r e p o r t e d mature carposporangia of Porphyra n e r e o c y s t i s c o n t a i n e d up t o 32 carpospores (a/4, b/4, c/2). I was unable to i d e n t i f y c a r p o s p o r a n g i a o f t h i s s i z e . On o l d e r t h a l l i I observed carposporangia a/4, b/2, c/2-4, but they were never l a r g e r than t h i s . T h i s i s the f i r s t r e p o r t of carpcsporangia l a r g e r than c/2 i n Porphyra n e r e o c y s t i s . In the Japanese Porphyra s p e c i e s c a r p o s p o r a n g i a have s u r f a c e dimensions a/2, b/2 or a/4, b/2; there are no r e p o r t s of a/4, b/4 (Kurogi, 1972). Kurogi (1972) noted that the c l o s e contact of 2 carposporangia o f t e n occurs and s u p e r f i c i a l l y resembles a s i n g l e carposporangium of a/4, b/4. Kurogi detected t h i s phenomenon by comparing the dimensions of these 73 l a r g e 'carposporangia* with those of s m a l l e r c a r p o s p o r a n g i a and v e g e t a t i v e c e l l s . They should a l l have s i m i l a r dimensions; however, the suspected a/4, b/4 •carposporangia* were too l a r g e to be d e r i v e d from a s i n g l e carpogonium. I agree with Kurogi*s (1972) view and t h i n k t h a t the phenomenon of 2 c l o s e l y appressed carposporangia has been r e s p o n s i b l e f o r the r e p o r t s of carposporangia a/4, b/4. Other s p e c i e s from the Northeast P a c i f i c Ocean have been rep o r t e d to have carposporangia of these dimensions, f o r example, Porphyra MaBlJSggagis Mumford (Mumford, 1973b) and P. p e r f o r a t a <Hus, 1902; Conway et a l . 1975). Carposporogenesis should be re-examined i n these s p e c i e s t o see i f carposporangia as l a r g e as a/4, b/4 r e a l l y occur. In a l l 3 s p e c i e s I s t u d i e d there was some v a r i a t i o n i n t h e number of spermatia or carpospores per sporangium. Kurogi (1961) noted that the age of the t h a l l u s and e c o l o g i c a l c o n d i t i o n s c o u l d cause such v a r i a t i o n . Furthermore, he observed t h a t s p e c i e s with t h i c k t h a l l i g e n e r a l l y had l a r g e r d i v i s i o n numbers of spermatangia and carposporangia i n t r a n s v e r s e s e c t i o n than those with t h i n thalli.„ I n view of t h i s v a r i a t i o n , Krishnamurthy (1972) suggested t h a t l e s s emphasis should be put on the d i v i s i o n formulae of spermatangia and carposporangia as taxonomic c r i t e r i a , a t r e n d which i s g e n e r a l l y accepted (Mumford, 1973a; Conway et a l . 1975). In a d d i t i o n I would l i k e t o emphasize t h a t the d i v i s i o n formula i s meaningless u n l e s s the products of the d i v i s i o n of a s i n g l e spermatangium or carposporangium can be r e c o g n i z e d with c e r t a i n t y . Table VI. Summary of the s p e c i e s cf Porphyra which possess p r o t o t r i c h o g y n e s , and those which do not. Taxon J l t h o u t p r o t o t r i c h o g y n e s 1. Monostromatic Porphyra dentata Kjellman* 2* l i n e a r i s G r e v i l l e E* n e r e o c y s t i s Anderson Reference Tseng and Chang (1955) Dangeard (1927) - as P. u m b i l i c a l i s f» l i n e a r i s ( G r e v i l l e ) Harvey Hawkes ( t h i s t h e s i s ) With p r g t o t r i c h o j y j g e s 1. Honostromatic Porphyra angusta Ueda P. c a p e n s i s Ki i t z i n g emend. Agardh E* c r i s p a t a K j e l l man P. g a r d n e r i (Smith et Hcllenberg) Hawkes P. k a t a d a i Miura P. k u n i e d a i Kurogi P. l e u c o s t i c t a T b uret i n Le J o l i s £• p e r f o r a t a J . Agardh £• Sil i o i M S i i i a t a Kjellmanz P. taneqashimensjs Shinmora £. tenera Kjellman P. t e n u i p e d a l i s Miura £. u m b i l i c a l i s (Linnaeus) J . Agardh P. yezoensis Ueda P. sp. (amethystea Kiitzing?) D i s t r c m a t i c i2£2hl£§ bulbopes (Y en do) Okamura P. fiapenfjgssii Krishnamurthy Kurogi Graves (1961) (1969) Tanaka (1952) Hawkes (1977b, t h e s i s ) and t h i s Miura (1968) Kurogi (1961) Janczewski (1873) Be r t h o l d (1882) Bosenvinge (1909) Kornmann (1961a) Dr. T. F. Mumford (personal communication) Tanaka (1952) Shinmura (1974) Ishikawa (1921) Kunieda (1939) Kurogi (1961) Miura (1961) Dangeard (1927) Kurogi (1961) Yabu and T c k i d a (1963) Kornmann (1961b) Tanaka (1952) Krishnamurthy (1972) Conway and Cole (1973) Conway e t a l . (1975) 75 P. s c h i z o p h y l l a Hollenberg i n Smith and Hollenberg P. t a s a (Yendo) Ueda Krishnamurthy (1972) as J . n o r r i s i i , Krishnamurthy Conway e t a l . (1975) Nagai (1941) Tanaka (1952) »Tanaka (1952) f i g u r e s p r o t o t r i c h o g y n e s f o r t h i s s p e c i e s but does not mention them i n the d e s c r i p t i o n . 2 T s e n g and Chang (1955) reported they c o u l d not f i n d p r o t o t r i c h o g y n e s i n t h i s s p e c i e s . 76 PojEkXXI g a r d n e r i - spermatogenesis, l i g h t microscopy F i g u r e 7a. Vegetative c e l l with l a r g e c e n t r a l pyrenoid an s i n g l e s t e l l a t e c h l o r o p l a s t . The nucleus i s p e r i p h e r a l l y l o c a t e d . F i g u r e 7b. New w a l l l a y e r (arrow) l a i d down around spermatangium p r i o r to the f i r s t d i v i s i o n . F i g u r e 7c. The f i r s t d i v i s i o n o f the spermatangium i s p e r i c l i n a l . F i g u r e s 7d-f. Subseguent d i v i s i o n s of the spermatangium. Fig u r e 7g. Mature spermatangium 4 spermatia deep i n tr a n s v e r s e s e c t i o n . Figure 7h, Su r f a c e view of an immature spermatangium. Fig u r e 7 i . Surface view of a mature spermatangiua. 77 78 Porphyra g a r d n e r i F i g u r e 8a. Spermatogenesis. The d i v i s i o n seguence l e a d i n g t o a mature spermatangium c f 64 spermatia. Note the production o f a new w a l l l a y e r ( i n d i c a t e d by a t h i c k e r l i n e ) p r i o r to d i v i s i o n , and the absence of any * c r u c i a t e ' d i v i s i o n s . F i g u r e 8b. Carpcsporogenesis. D i v i s i o n seguence which produced 2, 4 or 8 carpospores per carposporangium. The upper carposporangium c o n t a i n i n g 4 carpospores i s most obvious i n s u r f a c e view and i s shown i n F i g u r e s 9g and 9h. The d i v i s i o n pathway i n d i c a t e d by s o l i d arrows i s most common. 79 4 z C T I b 80 Porphyra g a r d n e r i - carposporogenesis F i g u r e 9a. Surface view o f v e g e t a t i v e c e l l s . F i g u r e 9b. Surface view o f spermatangia (lower l e f t ) , carpogonia and carposporangia. F i g u r e 9c. T r a n s v e r s e s e c t i o n of tbe t h a l l u s showing two carpogonia. Note the b i p o l a r p r o t o t r i c h o g y n e s and attached spermatium (arrow). F i g u r e 9d. Transverse s e c t i o n showing two c l o s e l y appressed carpogonia. Note attached spermatium (arrow). F i g u r e 9e. Transverse s e c t i o n showing t h a t the f i r s t d i v i s i o n of the caposporangium i s p e r i c l i n a l . F i g u r e 9f. Transverse s e c t i o n showing the o r i e n t a t i o n of the t h i r d and f o u r t h d i v i s i o n s of the carposporangium. Shown i n s u r f a c e view i n F i g u r e s 9g and 9h. See a l s o F i g u r e 8a. F i g u r e 9g. Surface view o f carposporangium t h a t has undergone t h r e e d i v i s i o n s and c o n t a i n s 4 carpospores. See a l s o F i g u r e 8a. F i g u r e 9h. As i n F i g u r e 9g f but a d i f f e r e n t plane cf f o c u s . F i g u r e 9 i . Transverse s e c t i o n through a carposporangium c o n t a i n i n g 8 carpospores (only 4 v i s i b l e ) . F i g u r e 9 j . Surface view of a carposporangium c o n t a i n i n g 8 carpospores (only the top 4 are v i s i b l e ) . Porphyra n e r e o c y s t i s - spermatogenesis F i g u r e 9k. S u r f a c e view of the f o l i o s e phase showing v e g e t a t i v e c e l l s with a s i n g l e s t e l l a t e c h l o r o p l a s t . . F i g u r e 91. Surface view of spermatangia which have undergone a few d i v i s i o n s . 82 Porphyra n e r e o c y s t i s - spermatogenesis F i g u r e 10a, Transverse s e c t i o n through an immature spermatangium. F i g u r e 10b, Sur f a c e view of mature spermatangial r e g i o n of the t h a l l u s . I n d i v i d u a l spermatangia are not c l e a r l y d e l i m i t e d . F i g u r e 10c. Transverse s e c t i o n through a mature spermatangium which i s 8 spermatia deep. Porphyra n e r e o c y s t i s - carposporogenesis F i g u r e 10d. Transverse s e c t i o n through a v e g e t a t i v e c e l l r e g i o n of the t h a l l u s . Note the s i n g l e s t e l l a t e c h l o r o p l a s t , c e n t r a l pyrenoid and l a t e r a l n ucleus. F i g u r e 10e. Transverse s e c t i o n through a carpogonium. Note i t s s l i g h t l y more elongate shape compared t o the veg e t a t i v e c e l l s . F i g u r e 10f. Transverse s e c t i o n showing two c l o s e l y appressed carpogonia. Note the spermatium (arrow) and f e r t i l i z a t i o n c a n a l . F i g u r e 10g. Transverse s e c t i o n of two carposporangia showing th a t the f i r s t d i v i s i o n i s p e r i c l i n a l . F i g u r e 10h. Transverse s e c t i o n of two carposporangia. The one on the l e f t i s at the stage shown i n s u r f a c e view i n F i g u r e 1 0 i , whereas the one on the r i g h t i s at the stage shown i n F i g u r e 10j. T h i s d i v i s i o n seguence i s the same as that shown f o r Porphyra g a r d n e r i i n F i g u r e 8b. F i g u r e 10i. Surface view cf a carposporangium c o n t a i n i n g 4 carpospores. F i g u r e 10j. S u r f a c e view of a carposporangium c o n t a i n i n g 8 carpospores (only the top 4 are v i s i b l e ) . See a l s o F i g u r e 10h. F i g u r e 10k. Surface view of mature carposporangia. I t i s d i f f i c u l t t o determine the boundaries of a s i n g l e carposporangium. F i g u r e 101. Transverse s e c t i o n showing two c l o s e l y appressed carposporangia which have undergone more than one d i v i s i o n i n the plane of the t h a l l u s . 84 F i g u r e 11a. Porphyra n e r e o c y s t i s * t r a n s v e r s e s e c t i o n through a r e g i o n of mature carp o s p o r a n g i a . I t i s d i f f i c u l t t o determine the o r i g i n a l boundaries of a s i n g l e carposporangium. Porphyra t h u r e t i i - spermatogenesis F i g u r e s 11b and 11c. Surface view shewing e a r l y d i v i s i o n s of the spermatangia. F i g u r e 11d. Transverse s e c t i o n through an immature spermatangium. F i g u r e 11e. Mature spermatangium with s u r f a c e dimensions a/4, b/4 and c o n t a i n i n g a t o t a l of 64 spermatia. F i g u r e 11f. Transverse s e c t i o n through a mature spermatangium. Porphyra t h u r e t i i - carposporogenesis F i g u r e 11g. Transverse s e c t i o n through a carpogonium which appears to have very s l i g h t b i p o l a r p r o t o t r i c h o g y n e s . F i g u r e 11h. Transverse s e c t i o n showing t h a t the f i r s t d i v i s i o n o f the carposporangium i s p e r i c l i n a l . F i g u r e 11i. Transverse s e c t i o n of a carposporangium t h a t has undergone second and t h i r d a n t i c l i n a l d i v i s i o n s . A s u r f a c e view i s shown i n F i g u r e 11j. F i g u r e 11j. Surface view of a carposporangium which has undergone t h r e e d i v i s i o n s . F i g u r e 11k. Surface view of a mature carposporangium c o n t a i n i n g 8 carpospores {only the top 4 are v i s i b l e ) . See F i g u r e 111. F i g u r e 111. Transverse s e c t i o n of a mature carposporangium. 86 PART I I I - CULTURE STUDIES I n t r o d u c t i o n The f i r s t c u l t u r e s t u d i e s of Porphyra were made by Janczewski (1873) and, although crude by c u r r e n t standards, demonstrated t h a t the carpospores of Porjojiyra purpurea (Roth) C. Agardh (as P. l a c i n i a t a ( L i g h t f o o t ) J , Agardh) and £• l e u c p s t i c t a germinated i n t o a f i l a m e n t o u s phase. Thuret and Bornet (1878) and B e r t h o l d (1882) obtained s i m i l a r r e s u l t s ; however, B e r t h o l d thought t h a t the f i l a m e n t o u s growths were abnormal and concluded t h a t the carpospores remained unchanged u n t i l they germinated i n t o the f o l i o s e phase. Yendo (1919) germinated the carpospores of s e v e r a l Japanese Porphyra s p e c i e s and found they a l s o produced a filamentous phase. Grubb (1924), K y l i n (1922), Dangeard ( c i t e d from Drew, 1956, p. 574) and Rees (1940) obtained the same r e s u l t s with European Porphyra s p e c i e s . Kunieda (1939) thought t h a t the filamentous phase was an anomaly and suggested t h a t the e n t i r e carpogonium i s r e l e a s e d from the t h a l l u s and t h a t the carpospores pass the summer w i t h i n i t and are f i n a l l y r e l e a s e d i n the autumn when they germinate t o g i v e t h e f o l i o s e phase. Okamura e t a l . (1920) re p o r t e d t h a t immature carpospores germinated to produce r h i z o i d - l i k e f i l a m e n t s which f u n c t i o n e d to absorb n u t r i e n t s f o r the f o l i o s e t h a l l u s growth. They s t a t e d t h a t mature carpospores germinated d i r e c t l y i n t o f o l i o s e t h a l l i , Ueda (1929) suggested t h a t the carpospores germinated immediately 87 i n t o s m a l l p l a n t s which l a s t e d through the summer to produce mcncspores i n the autumn and thereby produce the f o l i o s e t h a l l i again. Kusakabe (1929) supported t h i s view. Drew (1949, 1954a, 1954b) was the f i r s t t o p o i n t out t h a t the f i l a m e n t o u s phase r e s u l t i n g from carpospore germination was a l r e a d y known to p h y c o l o g i s t s as C o n c h o c e l i s rjjsea B a t t e r s . T h i s s h e l l - b o r i n g a l g a was f i r s t d e s c r i b e d by B a t t e r s (1892) from m a t e r i a l c o l l e c t e d o f f the coast of S c o t l a n d . Drew*s f i n d i n g s s t i m u l a t e d a number of i n v e s t i g a t i o n s of v a r i o u s Porphyra s p e c i e s (Graves, 1955; H o l l e n b e r g , 1958; Krishnamurthy, 1959; Ccnway, 1964a, 1964b, 1964c}.; These c u l t u r e and c y t o l o g i c a l s t u d i e s d i d net determine the environmental parameters r e s p o n s i b l e f o r o p t i m a l growth and r e p r o d u c t i o n . Such i n f o r m a t i o n i s e s s e n t i a l to e f f i c i e n t Porphyra c u l t i v a t i o n and i t i s t h e r e f o r e no s u r p r i s e t h a t the Japanese are t h e l e a d e r s i n t h i s a r e a , having amassed a great amount of data on the b i o l o g y of the c o n c h o c e l i s phase. Kurogi (1953a, 1953b) was one of the f i r s t i n v e s t i g a t o r s to e l u c i d a t e the complete morphological l i f e h i s t o r y of Porphyra. He germinated the carpospores of 4 s p e c i e s , and o b t a i n e d the c o n c h o c e l i s phase. The c o n c h o c e l i s phase formed conchosporangial branches 1 which produced conchospores t h a t germinated i n t o the f o l i o s e phase. Kurogi f o l l o w e d t h i s p i o n e e r i n g work with a s e r i e s of e x c e l l e n t s t u d i e s on the e f f e c t s of p h o t o p e r i o d , l i g h t i n t e n s i t y , water temperature. *In some o f the Japanese l i t e r a t u r e the conchosporangial branch i s r e f e r r e d t o as a monosporangial branch and conchospores are c a l l e d monospores. 88 d e s i c c a t i o n and s a l i n i t y on growth and maturation of the c o n c h o c e l i s phase and cn conchospore l i b e r a t i o n (Kurogi and Hirano, 1955a, 1955b; Kurogi and Hirano, 1956a, 1956b; Kurogi, 1959; Kurogi, 1961; Kurogi e t a l . , 1962; Kurogi and Sato, 1962a, 1962b; Kurogi and Akiyama, 1965, 1966; Kurogi and Sato, 1967; Kurogi e t a l . , 1967). Other Japanese work has d e a l t with v a r i o u s aspects of the growth of the c o n c h o c e l i s phase (Yamasaki, 1954a; Ogata, 1955, 1961, 1S71; K u r o g i , 1956; M i g i t a , 1959a; Tanaka, 1959; Iwasaki, 1967; K i r i t a , 1970; Ogata and Schramm, 1971) or the f o l i o s e phase (Iwasaki and Matsudaira, 1958; Matsumoto, 1959^ Ogata, 1S63; Iwasaki* 1965; Imada et a l . , 1972). These s t u d i e s have enabled the Japanese to e f f e c t i v e l y manipulate t h e l i f e h i s t o r i e s of t h e i r Porphyra s p e c i e s to improve c u l t i v a t i o n and have enabled them to develop such novel technigues as g u i c k - f r e e z i n g s p o r e l i n g s on nets to -20°C and s t o r i n g them f o r l a t e r c u l t i v a t i o n ( M i g i t a , 1964, 1966, 1967a; M i g i t a e t a l . 1971; Miura, 1975). Another unigue approach t h a t has been taken t o the study of n u t r i e n t reguirements by the f o l i o s e t h a l l u s i s to c h a r a c t e r i z e the b a c t e r i a l f l o r a of the t h a l l i , then i n t r o d u c e i s o l a t e d s t r a i n s i n t o axenic c u l t u r e s of t h a l l i , and observe how the b a c t e r i a a f f e c t growth (Tsukidate, 1970, 1971, 1974). A few i n v e s t i g a t o r s o u t s i d e Japan have a l s o been i n t e r e s t e d i n the s p e c i f i c c o n d i t i o n s of spore formation and r e l e a s e by t h e c o n c h o c e l i s phase. Tseng e t ajL* (1963) s t u d i e d the e f f e c t of temperature and photoperiod on conchosporangial branch formation and conchospore r e l e a s e i n s i x s p e c i e s of 89 Porphyra, and Dring (1967) and Re n t s c h l e r (1967) have i n v e s t i g a t e d the e f f e c t of photoperiod on conchosporangial branch formation by Porphyra t e n e r a . Iwasaki (1961), working with Porphyra t e n e r a , was the f i r s t to f o l l o w the complete l i f e h i s t o r y i n v i t r o using w e l l d e f i n e d c o n d i t i o n s and growth medium. Koranann (1960, 1961a, 1961b) has done some e x c e l l e n t c u l t u r e work on the European PojEpJiyra s p e c i e s . Chen et a l . (1970) and B i r d et a l . (1972) have succeeded i n completing the l i f e h i s t o r i e s of Pophyra miniata and P. l i n e a r i s r e s p e c t i v e l y , through 5-6 g e n e r a t i o n s . Because of the d i f f i c u l t i e s of maintaining the f c l i o s e t h a l l u s i n c u l t u r e , there have been few s t u d i e s o f the environmental parameters r e s p o n s i b l e f o r spermatium, carpcspore and monospore* f o r m a t i o n . Iwasaki (1961) pointed out the importance of photoperiod i n i n d u c i n g carpospore formation. Kurogi (1961) suggested t h a t temperature p l a y s an important r o l e i n c o n t r o l l i n g monospore production. B i r d ej£ a l . (1972) and B i r d (1973) found t h a t P. l i n e a r i s s p o r u l a t e d under a v a r i e t y c f temperatures and photoperiods and suggested t h a t s p o r u l a t i o n was determined by the age of the t h a l l i r a t h e r than by an environmental s t i m u l u s . Hy c u l t u r e s t u d i e s of Porphyra g rardneri. P. n e r e o c y s t i s . and P. t h u r e t i i were intended t o complement my f i e l d s t u d i e s i n *In some of the Japanese l i t e r a t u r e these spores which are produced by the f o l i o s e t h a l l u s and which germinate t c produce more f c l i o s e t h a l l i , are r e f e r r e d to as n e u t r a l s p o r e s . The term monospore i n some o f the e a r l i e r Japanese l i t e r a t u r e r e f e r s to conchospores. 90 order to o b t a i n a d e t a i l e d p i c t u r e of the morphological l i f e h i s t o r i e s of these three algae. I had the f o l l o w i n g o b j e c t i v e s : to i d e n t i f y the r e p r o d u c t i v e bodies produced by the f o l i o s e t h a l l u s and see i f d i f f e r e n t photoperiods c o u l d a f f e c t t h e i r germination as has been reported f o r Bangja (fiichardson. 1970); to determine i f P. g a r d n e r i and P. t h u r e t i i have a c o n c h o c e l i s phase and i f so to c h a r a c t e r i z e the g e n e r a l f e a t u r e s of the c o n c h o c e l i s phase morphology and growth; and to determine the s p e c i f i c f a c t o r s r e s p o n s i b l e f o r conchosporangial branch formation and conchospore r e l e a s e . M a t e r i a l s And Methods Most c u l t u r e experiments were c a r r i e d out with m a t e r i a l from the study s i t e , although some spore germination s t u d i e s used m a t e r i a l from other s i t e s as a check f o r any population d i f f e r e n c e s i n the time or type of spore f o r m a t i o n . The other s i t e s i n c l u d e d : Porphyra g a r d n e r i : Second Beach and Execution Bock i n Barkley Sound; B o t a n i c a l Beach, Port Renfrew; P o i n t Joe, Monterey P e n i n s u l a . Porphyra n e r e o c y s t i s : A g u i l a r P o i n t , Seccnd Beach, Cable Beach and Cape Beale i n Barkley Sound; B o t a n i c a l Beach, Port Renfrew; F r i d a y Harbor, San Juan I s l a n d . Porphyra t h u r e t i i : Leach I s l e t , Barkley Sound. P r e l i m i n a r y monospore and carpospore germination s t u d i e s were c a r r i e d out under a photoperiod of 12:12 ( l i g h t : d a r k ) , a temperature o f 10°C and a l i g h t i n t e n s i t y of 25-35 f t - c . S y l v a n i a or Westinghouse F48T12 Cool White 40 W f l u o r e s c e n t tubes were used f o r i l l u m i n a t i o n . The s u i t a b i l i t y of these 91 c o n d i t i o n s f o r growing bangiophycidaen algae was determined i n previous c u l t u r e s t u d i e s (Hawkes, unpublished B.Sc. Honours t h e s i s ) . Small t h a l l u s p i e c e s were placed i n 100x15 am d i s p o s a b l e Nalgene P e t r i p l a t e s with 50 ml of P r o v a s o l i ' s e n r i c h e d seawater medium ( P r o v a s o l i , 1971) and GeOa (10 ug/ml) to c o n t r o l diatom growth (Lewin, 1966). Seawater used f o r a l l experiments was taken from the seawater system at the Bamfield Marine S t a t i o n and had a s a l i n i t y which v a r i e d from 30-32%.. C u l t u r e s were examined a f t e r 1-2 days and, i f spore r e l e a s e was s u f f i c i e n t , the t h a l l u s p i e c e s were removed and germination and development of the spores were observed. S u b - c u l t u r e s f o r f u r t h e r experiments were obtained by t r a n s f e r r i n g spores to f r e s h P e t r i p l a t e s and c u l t u r e medium. In a d d i t i o n to the standard c u l t u r e c o n d i t i o n s , a number cf other c o n d i t i o n s were used i n experiments on spore germination and c o n c h o c e l i s phase growth and r e p r o d u c t i o n . Pyrex c u l t u r e dishes 100x80 mm, 450 ml were used i n some of these experiments. Monospores and carpospores of Porphyra g a r d n e r i and carpcspores of P. n e r e o c y s t i s were germinated under photoperiods of 8:"l6, 12:12, 16:T and temperatures of 7 ° , 10° and 15°C. Both low (25-35 f t - c ) and high (150-200 f t - c ) l i g h t i n t e n s i t i e s were used. In experiments on the development o f monospores of Porphyra g a r d n e r i , c u l t u r e s were aerated or a g i t a t e d and p i e c e s of host p l a n t were added t o some to see i f growth c o u l d be enhanced. Conchospore c u l t u r e s of both P. g a r d n e r i and 92 f» t h u r e t i i were maintained at 10°C, 12:12 and a e r a t e d . A l i g h t i n t e n s i t y of 200-300 f t - c was used f o r the s e c u l t u r e s . The growth of the c o n c h o c e l i s phase of P. g a r d n e r i and P. n e r e o c y s t i s i n egg s h e l l , clam s h e l l (Saxidomus qiganteus) and o y s t e r s h e l l {Crassostrea giqas) was observed. For s t u d i e s o f the e f f e c t of temperature and photoperiod cn conchosporangial branch formation, the f o l l o w i n g c o n d i t i o n s were used: 7 ° , 10°, 15°C and 8:16, 12:12, 16:8", 25-35 f t - c . Only 10°C was used f o r P. t h u r e t i i . Temperature and photoperiod were a l s o the main f a c t o r s which were manipulated i n an attempt t o o b t a i n conchospore r e l e a s e . C u l t u r e s were maintained on a temperature g r a d i e n t t a b l e at temperatures from 7°-20°C. In another s e r i e s of experiments c u l t u r e s were t r a n s f e r r e d to d i f f e r e n t temperature and photoperiod regimes. These i n c l u d e d : Porphyra g a r d n e r i : from 10°C, 12:12 to 7°C, 12:TT and 7°C, 8:16; Porjghi£a n e r e o c y s t i s : from 15°C, 16:8 to 13°C, 11:'13~ and 10°C, 8:16"; from 10°C 12:12 to 10°C, 10: 14* and 7°C, 9:15; from 10°C, 16:~8 to 7°C ana 10°C, 8:16"; Porphyra t h u r e t i i : from 10°C, 12:T2 to 7°c, 9:15". Porphyra flgreocystis c o n c h o c e l i s phase was su b j e c t e d to s e v e r a l other c o n d i t i o n s i n an e f f o r t to induce conchospore r e l e a s e . These i n c l u d e d : pH over the range 7-10, s a l i n i t y from 20-40%» , d e s i c c a t i o n , a g i t a t i o n , and host p l a n t exudate. C u l t u r e d c o n c h o c e l i s phase was a l s o placed i n a s p e c i a l p l e x i g l a s s c u l t u r e tube and put out i n the f i e l d at the study s i t e i n the autumn at a depth of 7 m. The design of the c u l t u r e tube was based on that of the *Biomonitor* which i s 93 manufactured by the BioCo.nt.rcl Company, P o r t S a n i l i c , Michigan ( S c h l i c h t i n g , 1975a, 1S75b, 1976). B e s u l t s *• Porphyra g a r d n e r i 1. Monospores Soon a f t e r Porphyra g a r d n e r i f i r s t appeared i n the f i e l d i n February the c e l l s i n the marginal zone developed a f i n e granular appearance which p a r t i a l l y obscured the s t e l l a t e c h l o r o p l a s t . The g r a n u l a r appearance was best developed i n those c e l l s which were on the margin. Following the appearance of the g r a n u l a r c e l l c o n t e n t s , the e n t i r e d i s t a l margins began to r e l e a s e thousands of monospores which were 25-30 urn i n diameter (Figure 12a). In c u l t u r e the monospores germinated a f t e r 1-2 days by sending out a long r h i z o i d a i protuberance (Figure 12b). The f u n c t i o n o f t h i s r h i z o i d i s probably to ensure that the spore i s anchored i n the 'host* t i s s u e before the f o l i o s e t h a l l u s commences growth. Following r h i z o i d e l o n g a t i o n , a s e r i e s of p a r a l l e l d i v i s i o n s formed an u n i s e r i a t e t h a l l u s (Figures 12c and 12d). at the 4-5 c e l l stage d i v i s i o n s occurred a t r i g h t angles to the i n i t i a l ones t o form the f o l i o s e , monostromatic t h a l l u s ( F i g u r e s 12e and 1 2 f ) . S p o r e l i n g s grew w e l l i n c u l t u r e i n i t i a l l y , but when they reached the s i z e shown i n F i g u r e 12f growth ceased and they e v e n t u a l l y d i e d . 94 T h i s p r o l i f i c monspore production e x p l a i n s the g r e a t i n c r e a s e i n P. g a r d n e r i abundance i n the f i e l d throughout the s p r i n g months. During May, t h a l l i r e l e a s i n g monospores along t h e i r d i s t a l margin and spermatia and carpospores along t h e i r l a t e r a l margins were common. By June and J u l y , spore p r o d u c t i o n became almost e x c l u s i v e l y c a r p o s p o r i c , although the presence of a few b i p o l a r s p o r e l i n g s i n d i c a t e d t h a t some monospores were s t i l l being produced. In the autumn, moncspore production again became predominant, but not to the same extent as i t was i n the s p r i n g . In c u l t u r e a e r a t i o n was found to be e s s e n t i a l f o r good monospore germination. Under a l l photoperiods and temperatures t e s t e d , monospores underwent b i p o l a r germination to produce the f o l i o s e phase; however, at 16:8 the number of monospores germinating was c o n s i d e r a b l y lower than a t 12:12 or 8:16. Monospores were being produced by p l a n t s i n the other p o p u l a t i o n s examined. C u l t u r e work done a t Hopkins Marine S t a t i o n confirmed t h a t most of the t h a l l i c o l l e c t e d at Point Joe (type l o c a l i t y ) i n J u l y 1976, were e x c l u s i v e l y monospcric. The f a i l u r e of s p o r e l i n g s to reach maturity suggested they may have a p h y s i o l o g i c a l reguirement f o r the 'host* p l a n t . In view of the occurrence of Porphyra g a r d n e r i on 15 l a m i n a r i a l e a n algae and because one of the major photosynthates i n t h i s group i s mannitol, I t e s t e d the e f f e c t o f mannitol on s p o r e l i n g growth, Parker (1966) r e p o r t e d a mannitol c o n c e n t r a t i o n of 3,6% (v/v) f o r M i c r o c y s t i s p y r i f e r a (Linnaeus) C. Agardh. Using t h i s c o n c e n t r a t i o n as a g u i d e l i n e a 1,8$ and 0.9% s o l u t i o n of d(-) mannitol was t e s t e d ; however, no enhancement 95 of growth occurred. In another experiment 1 cm 2 p i e c e s of Laminaria t h a l l u s were added to the monospore c u l t u r e s . A c o n t r o l without Laminaria was a l s o r u n . , Monospores i n the c u l t u r e s c o n t a i n i n g host t i s s u e germinated before those i n the c o n t r o l , but the r e s u l t i n g s p o r e l i n g s d i d not grow t o maturity. 2. Spermatangium And Carpogonium Formation V a r i a t i o n i n the time c f spermatangium and carpogonium formation with l a t i t u d e (discussed i n P a r t I) suggested t h a t photoperiod was important i n i n d u c i n g t h e i r f o rmation. To determine i f t h i s i s a p h o t o p e r i o d i c or p h o t c s y n t h e t i c response, monosporic t h a l l i attached to the *host* p l a n t were kept i n 20 1 c o n t a i n e r s a t 10°C, 12:12 and 16:"8~, 300-400 f t - c . Spermatia formed under both photoperiods. U n f o r t u n a t e l y the t h a l l i died before carpospores sere formed. 3. Carpospore Germination - The C o n c h o c e l i s Phase At the study s i t e the f i r s t carpospores were produced by May. They were v i a b l e whether they had been formed by one or more d i v i s i o n s . Whether subseguent d i v i s i o n s occurred a f t e r the spores had been r e l e a s e d i s not known. Released carpospores were 14-20 um i n diameter ( F i g u r e 12g) and germinated i n 2-3 days to give c o n c h o c e l i s f i l a m e n t s t h a t were 4-7 um wide. The c e l l s of the c o n c h o c e l i s phase contained p a r i e t a l c h l o r o p l a s t s (Figure 12h). Under a l l temperatures and photoperiods t e s t e d , carpospores germinated i n t o the 96 c o n c h o c e l i s phase. Carpospores were produced by a l l c f the ether p o p u l a t i o n s which were examined. The number of carpospores produced by the p o p u l a t i o n a t the type l o c a l i t y was low compared t o more northern p o p u l a t i o n s , but they were v i a b l e and germinated i n t o the t y p i c a l c o n c h o c e l i s phase. The c o n c h o c e l i s phase grew p r o l i f i c a l l y and soon formed numerous t u f t - l i k e c o l o n i e s . Propagation of f i l a m e n t s by fragmentation o c c u r r e d r e a d i l y . S i n g l e s p o r e - l i k e s w e l l i n g s were formed i n some cases (Figure 1 2 i ) , but no evidence of them f u n c t i o n i n g as monosporangia was seen. A f t e r 10-12 weeks conchosporangial branches formed. They were i n i t i a l l y s h o r t and stubby (Figure 13a), but l a t e r became long and branched (Figure 1 2 j ) . By 24 weeks conchospore r e l e a s e occurred (Figure 13a). Released conchospores were 22-30 um i n diameter and a f t e r 2-3 days they underwent b i p o l a r germination to produce the f o l i o s e phase (Figure 13b). A e r a t i o n was found to be e s s e n t i a l f o r good conchospore germination. When grown i n s h e l l , the c o n c h o c e l i s phase had a c h a r a c t e r i s t i c p i n n a t e l y branched morphology (Figure 13c). Growth i n the e g g s h e l l was poor compared to the p r o l i f i c growth i n the clam and o y s t e r s h e l l s . In o l d e r p o r t i o n s of the c o n c h o c e l i s phase the branches became i r r e g u l a r l y l o b e d . The a b i l i t y of the c o n c h o c e l i s phase to s u r v i v e i n t o t a l darkness was t e s t e d . A f t e r 8 months of darkness the c o n c h o c e l i s f i l a m e n t s were pale pink, but were a l i v e and grew well when re t u r n e d to the l i g h t . 97 4. ; Ccnchosporangial Branch Formation The e f f e c t s o f photoperiod and temperature on c c n c h o s p o r a n g i a l branch formation were examined, Conchosporangial branches were i r r e g u l a r l y branched and v a r i e d i n s i z e , l e n g t h and branch d e n s i t y , making g u a n t i t a t i v e assessment of t h e i r abundance d i f f i c u l t , The technigue used by Dring (1967) on Porphyra tenera c o n c h o c e l i s phase was co n s i d e r e d i m p r a c t i c a l because, as Richardson (1970) has pointed o ut, i t does not determine the a c t u a l number o f conchosporangia. The t e c h n i c a l d i f f i c u l t i e s encountered i n t r y i n g t o o b t a i n t h i s data proved insurmountable. In each c u l t u r e the presence or absence of c o n c h o s p o r a n g i a l branches was noted and a g u a l i t a t i v e assessment of abundance made. at 10°C, c c n c h o s p o r a n g i a l tranches formed a t 8:16, and 12:12. One or two branches formed at 16:8, but most c u l t u r e s under lcng days remained v e g e t a t i v e . Conchosporangial branches formed at a l l 3 temperatures, but because of c u l t u r e chamber malfunctions i t was not p o s s i b l e to determine p r e c i s e l y the o p t i m a l temperature f o r conchosporangial branch f o r m a t i o n . The trend was f o r c c n c h o s p o r a n g i a l branches t o form sooner a t 10°C and 15°C than a t 7°C. 5. Conchospore Release Although i t was easy to induce c c n c h o s p o r a n g i a l branch form a t i o n , g e t t i n g them to r e l e a s e conchospores c o n s i s t e n t l y proved i m p o s s i b l e . Conchospore r e l e a s e occurred i n c u l t u r e s maintained at 7°C, 10°C and 8:16, 12:T2, with the most p r o l i f i c 98 r e l e a s e o c c u r r i n g at the l a t t e r photoperiod. A small ccnchcspore r e l e a s e was induced by t r a n s f e r r i n g a c u l t u r e from 10°C, 12:T2 to 7°C, 12:T2. £. Porphyra n e r e o c y s t i s 1. Carpospore Germination - The C o n c h o c e l i s Phase No evidence of mcncspore production by the f o l i o s e t h a l l u s was found. Carpospores were produced by a l l p o p u l a t i o n s of Porphyra n e r e o c y s t i s which were examined. Carpospores were r e l e a s e d from carposporangia which had undergone v a r y i n g degrees of d i v i s i o n (both c/2 and c/4, see P a r t I I ) . Released carpospores were 8.0-15 um i n diameter (Figure 13d). A f t e r 2-3 days they germinated to produce c o n c h o c e l i s f i l a m e n t s 4-6 um across (Figure 13e), the c e l l s of which c o n t a i n e d p a r i e t a l c h l o r o p l a s t s . Onder a l l temperatures and photoperiods t e s t e d , carpospores germinated i n t o the c o n c h o c e l i s phase. No p r o d u c t i o n of monospores by the c o n c h o c e l i s phase was observed, although i t r e a d i l y regenerated from fragments of v e g e t a t i v e f i l a m e n t s . A number of i r r e g u l a r s w e l l i n g s formed a t both the base and apex of f i l a m e n t s ( F i g u r e s 13f and 13g). Beaded branches 11-15 um across (Figure 13h) and s q u a r e - c e l l e d branches 13-17 um a c r o s s ( F i q u r e 13i) a l s o formed and were i n i t i a l l y mistaken f o r c c n c h o s p o r a n q i a l branches. These two branch types formed under a l l photoperiod reqimes t e s t e d . Whether they have any s p e c i f i c f u n c t i o n such as the production cf monospores i s 99 not known. They appear to form a t r a n s i t i o n from v e g e t a t i v e f i l a m e n t s t o c c n c h o s p o r a n g i a l branches (Figure 1 3 j ) . , Conchosporangial branches appeared a f t e r 10-15 weeks and were t y p i c a l l y candelabra-shaped ( F i g u r e s 13k and 14a). The branches were u n i s e r i a t e , 16-25 um across. Each c e l l of the conchosporangial branch had a s t e l l a t e c h l o r o p l a s t , t h i c k c e l l w a l l and p i t plugs between adjacent c e l l s (Figure 14b). I f a c l u s t e r of conchosporangial branches was removed from the v e g e t a t i v e f i l a m e n t s i t would continue t o grow, producing e i t h e r mere co n c h o s p o r a n g i a l branches or veg e t a t i v e f i l a m e n t s . The o l d e r p o r t i o n s o f some conchosporangial branches were two c e l l s t h i c k (Figure 14c). When grown i n s h e l l the v e g e t a t i v e c o n c h o c e l i s f i l a m e n t s developed pinnate branching (Figure 14d). Conchosporangial branches i n i t i a l l y formed i n the s h e l l and then grew cut cf i t to form s m a l l c o l o n i e s on the s h e l l s u r f a c e . , The v e r t i c a l o r i e n t a t i o n o f these branches suggested t h a t they were p h c t o t r o p i c . To t e s t t h i s , a c u l t u r e d i s h was covered so t h a t l i g h t was admitted at one end o n l y . Under t h i s treatment a l l c c n c h o s p o r a n g i a l branches grew toward the l i g h t , i n d i c a t i n g they are p o s i t i v e l y p h o t o t r o p i c . At the end of 8 months i n complete darkness, v e g e t a t i v e c o n c h o c e l i s f i l a m e n t s were as deeply pigmented as those grown i n the l i g h t . The c o n c h o c e l i s phase was observed i n the f i e l d growing i n b a r n a c l e s which were on the s t i p e of an o l d N e r e o c y s t i s p l a n t . I t was taken back to the l a b o r a t o r y and the s u r f a c e l a y e r s of ba r n a c l e d i s s o l v e d by p l a c i n g them i n a 1% s o l u t i o n of Na 2EDTA 1 0 0 f o r 12 hr (Prud'homme van Seine and van den Hoek, 1966). Small patches o f c o n c h o c e l i s f i l a m e n t s were removed and t r a n s f e r r e d to c u l t u r e d i s h e s . T h i s i s o l a t e grew w e l l and formed beaded, s q u a r e - c e l l e d and conchosporangial branches t y p i c a l o f Porphyra n e r e o c y s t i s . The g e n e r a l morphology of t h i s c o n c h o c e l i s phase and i t s f a i l u r e t o r e l e a s e co.nc.hospores suggests t h a t i t i s r e f e r r a b l e to Porphyra n e r e o c y s t i s . 2. Conchosporangial Branch Formation The e f f e c t s o f temperature and photoperiod on c o n c h o s p o r a n g i a l branch formation were examined. At 10°C, conchosporangial branches formed under a l l 3 photoperiods t e s t e d . , They f i r s t appeared i n c u l t u r e s a t 15°C and 16:8, or 12:12. At 8:16 most con c h o s p o r a n g i a l branches formed at 15°C. Conchosporangial branches which formed at 15°C were s h o r t and stubby. , 3. Conchospore Release None o f the attempts t o induce conchospore r e l e a s e succeeded. Because porphyra n e r e o c y s t i s appeared i n the l a t e autumn i t was suspected t h a t the decreasing daylength and seawater temperature were c r i t i c a l f a c t o r s ; however, ncne of the t r a n s f e r experiments, from higher temperatures and longer photoperiods t o c o o l e r temperatures and s h o r t e r photoperiods, r e s u l t e d i n conchospore r e l e a s e . The p o s s i b i l i t y t h a t some chemical cue from the aging N e r e o c y s t i s caused conchospore r e l e a s e was i n v e s t i g a t e d by 101 p l a c i n g p i e c e s of mature s t i p e , or s t i p e exudate, i n with the c o n c h o c e l i s phase. Ne i t h e r of these treatments was s u c c e s s f u l . The c o n c h o c e l i s phase of some Porph yra s p e c i e s has been found i n the i n t e r t i d a l zone (Drew and R i c h a r d s , 1953; Mumford, 19 73a) suggesting t h a t d e s i c c a t i o n c o u l d be a f a c t o r a f f e c t i n g conchospore r e l e a s e . No ccnchospores were r e l e a s e d by c c n c h o s p o r a n g i a l branches s u b j e c t e d t o 1, 2 and 3 hr of d r y i n g at 20°C. Because the c o n c h o c e l i s phase grows i n a c a l c a r e o u s s u b s t r a t e i t was suspected that there may be a s p e c i f i c pH r e g u i r e d f o r conchospore r e l e a s e . Six c u l t u r e s with a pH ranging from pH 7-10 were t e s t e d . At pH 10 the c u l t u r e d i e d . The other c u l t u r e s s u r v i v e d but the pH g u i c k l y changed t o pH 8-9. Changes i n s a l i n i t y had no e f f e c t i n t r i g g e r i n g conchospore r e l e a s e . The c o n c h o s p o r a n g i a l branches put out i n the f i e l d i n the c u l t u r e tube a l s o f a i l e d t o produce ccnchospores. C. Porphyra t h u r e t i i 1. Carpospore Germination - The C o n c h o c e l i s Phase No evidence of monospore production by the f o l i o s e t h a l l u s °f Porphyra t h u r e t i i w a s found. Released carpospores were 15-22 um i n diameter {Figure 14e) and germinated a f t e r 2-3 days to give a c o n c h o c e l i s phase {Figure 1 4 f ) . The f i l a m e n t s were 4-5 um a c r o s s and i n general morphology and c o l o u r they were d i s t i n c t 102 from those o f Porphyra g a r d n e r i and P. n e r e o c y s t i s . In c u l t u r e the v e g e t a t i v e f i l a m e n t s formed long, f i n e , h a i r - l i k e ' t u f t s * which were purple-gray. E a r l y i n my s t u d i e s I c o l l e c t e d t h a l l i which I c o u l d not i d e n t i f y . Herbarium specimens and c o n c h o c e l i s phase c u l t u r e s were made from t h i s m a t e r i a l f o r f u t u r e study. The c o n c h o c e l i s phase was so d i s t i n c t t h a t I was l a t e r able t o r e f e r i t t o Porphyra t h u r e t i i . The v e g e t a t i v e f i l a m e n t s developed i r r e g u l a r and beaded branches (Figure 14g) . A f t e r 8-14 weeks conchosporangial branches formed (Figure 14h). I n i t i a l l y they were s h o r t , but i n o l d e r c u l t u r e s they formed long candelabra-shaped c o l o n i e s (Figure 14i) which were p o s i t i v e l y p h o t o t r o p i c . In one experiment a t the end of 12 weeks, abundant conchospores formed w i t h i n the conchosporangial branches (Figure 14 j) f o l l o w e d by conchospore r e l e a s e (Figure 14k). Conchospores were not observed at the time o f t h e i r formation or r e l e a s e , so the d e t a i l s o f these processes are not known. F r e s h l y r e l e a s e d conchospores e x h i b i t e d pronounced amoeboid changes of shape and movement (F i g u r e s 15a-d). F o l l o w i n g t h i s the conchospores rounded up (14-17.5 um i n diameter) and w i t h i n a few days germination occurred. In the i n i t i a l stage of germination a l o n g r h i z o i d a l protuberance was formed ( F i g u r e 15e). The r h i z o i d d i d not develop t o the same extent as i t d i d i n Porphyra g a r d n e r i s p o r e l i n g s . , F o l l o w i n g the i n i t i a l r h i z o i d e l o n g a t i o n , a few s h o r t , broad r h i z o i d a l protuberances formed (Figure 15f) and p a r t i a l l y c o a l e s c e d t o produce a b a s a l pad ( F i g u r e s 15g, 1 5 i , 15j) . 103 Growth o f the f o l i o s e t h a l l u s began with a s e r i e s of p a r a l l e l d i v i s i o n s which r e s u l t e d i n an u n i s e r i a t e f i l a m e n t (Figure 15g). S p o r e l i n g s remained u n i s e r i a t e o n l y to about the 4-7 c e l l stage, at which time a s e r i e s of p e r p e n d i c u l a r d i v i s i o n s r e s u l t e d i n a b i s e r i a t e t h a l l u s (Figure 15h) and e v e n t u a l l y gave r i s e t o a parenchymatous, monostromatic t h a l l u s (Figures 15i and 1 5 j ) . In these young s p o r e l i n g s t h e r e was a d e f i n i t e a p i c a l growth zone. I n c u l t u r e , t h a l l i grew to about 1 am i n l e n g t h before growth ceased, 2. Ccnchosporangial Branch Formation The e f f e c t of photoperiod on c o n c h o s p o r a n g i a l branch formation was s t u d i e d , Conchosporangial branches formed f i r s t at 8:16 then at 12:12, C u l t u r e s a t 8:16 c o n s i s t e d almost e n t i r e l y of conchosporangial branches. In 1 out c f 3 experiments a couple of c o n c h o s p o r a n g i a l branches forned at 16:8, but most c u l t u r e s remained v e g e t a t i v e at t h i s photoperiod. 3. Conchospore Release Conchospore r e l e a s e occurred under two c o n d i t i o n s t e s t e d : at 10°C, 12:1~2, and by t r a n s f e r r i n g from 10°C, 12:12 to 7°C, 9:15. Subseguent exposure of other c u l t u r e s t o these c o n d i t i o n s f a i l e d t o induce conchospore r e l e a s e i n d i c a t i n g t h a t other f a c t o r s were a l s o i n v o l v e d . 1 0 4 D i s c u s s i o n 1. Monospores Porphyra g a r d n e r i i s the o n l y Porphyra s p e c i e s known from the Northeast P a c i f i c Ocean which has such a predominant monospore c y c l e . Although Conway e t a l . (1975) repo r t e d monospore production by s m a l l , high i n t e r t i d a l p l a n t s i n B r i t i s h Columbia they d i d not s p e c i f y which s p e c i e s t h i s was observed i n . Porphyra n e r e o c y s t i s and Porphyra t h u r e t i i do not form monospores, a s i t u a t i o n I t h i n k a p p l i e s t o most s p e c i e s i n t h i s a r e a . T h i s i s i n c o n t r a s t t o 10 Japanese s p e c i e s of Porphyra which are known t o produce monospores (Kurogi, 1972; Shirmura, 1974). In i t s p r o l i f i c monospore production P. g a r d n e r i i s s i m i l a r t c Porphyra k u n i e d a i and P. y e z c e n s i s which produce monospores on t h a l l i up to 70 mm i n l e n g t h (Kurogi, 1972), and to P. taneqashimensis Shinmura which l i b e r a t e s monospores through a l l seasons (Shinmura, 1S74). Why monospore production should be so p r e v a l e n t i n the Japanese s p e c i e s i s an i n t r i g u i n g g u e s t i o n . A study of the environmental c o n d i t i o n s under which the monospore-forming s p e c i e s grow and the f a c t o r s t r i g g e r i n g monospore production may provide i n s i g h t i n t o t h i s phenomenon., My s t u d i e s demonstrated t h a t monospore production by Porphyra g a r d n e r i was predominant i n the s p r i n g and autumn. T h i s i m p l i e s t h a t both s h o r t e r photoperiods and lower temperatures may play a r o l e i n i n d u c i n g monospore formation, K u r c g i (1961) s t a t e d t h a t water temperature seems to be c l o s e l y r e l a t e d to monospore l i b e r a t i o n by the f c l i o s e t h a l l i of 1.05 LPJphyya k u n i e d a i and P. t e n e r a . In c u l t u r e experiments with Eangia, Sommerfeld and N i c h o l s (1973) demonstrated t h a t water temperature a f f e c t e d monospore production. Kurogi (1961) a l s o i n d i c a t e d t h a t t h e r e i s a c o r r e l a t i o n between n u t r i e n t s and moDCspere production by I. Igzoejasig and £. tej$e.r_§. In some s p e c i e s , t h a l l u s s i z e determines whether monospores are produced. I n Porphyra angusta Deda f o r example, t h a l l i more than 2 mm high do not form monospores even when c o n d i t i o n s i n the f i e l d are s u i t a b l e (Kurogi, 1961) . The p r o d u c t i o n of monospores, spermatia and carpospores on the same t h a l l u s i n Porphyra g a r d n e r i i n the e a r l y summer i s not a unigue phenomenon. Kurogi (1961) r e p o r t e d t h i s f o r P. , kunjedai and P. y e z o e n s i s . Because of i t s monospore c y c l e , Porphyra g a r d n e r i could p o t e n t i a l l y be one o f the most v a l u a b l e s p e c i e s on our c o a s t f o r commercial c u l t i v a t i o n , assuming t h a t a method of growing i t on a s y n t h e t i c s u b s t r a t e can be developed. Japanese s p e c i e s of major economic importance have a monospore c y c l e (Kurogi, 1972). 2. Spermatangium And Carpogonium Formation My i n i t i a l f i e l d o b s e r v a t i o n s of spermatangium and carpogonium formation i n Por.phy.ra g a r d n e r i (see Part I) suggested t h a t photoperiod was r e s p o n s i b l e f o r t h e i r formation. The formation o f spermatangia a t a photoperiod o f 12:12 i n c u l t u r e ( l e s s than what appeared t o be the c r i t i c a l i n d u c i n g d a y l e n g t h ) , i n d i c a t e s t h a t spermatangium formation i s probably a p h o t o s y n t h e t i c e f f e c t r a t h e r than a t r u l y p h o t o p e r i o d i c 106 response as d e f i n e d by Bichardson (1970). C r i t i c a l e xperimental s t u d i e s are needed t o answer t h i s q u e s t i o n and to determine why some c e l l s produce spermatangia whereas others form carpogonia. Photoperiod c e r t a i n l y seems to be an important f a c t o r . Fujiyama and Suto {cited from Ogata, 1975) and Iwasaki (1965) r e p o r t e d t h a t when the daylenqth was g r e a t e r than 12 hr l i g h t , spermatangia and carpoqonia formed. Kuroqi (1961) t r i e d to c o r r e l a t e spermatanqiuni and carpogonium formation i n P. k u n i e d a i with water temperature; however, he concluded that t h i s was not the only f a c t o r i n d u c i n g s e x u a l r e p r o d u c t i o n . When experimental technigue i s s u f f i c i e n t l y r e f i n e d to permit manipulation of the r e p r o d u c t i v e s t a t u s of the f o l i o s e t h a l l i i n c u l t u r e , a new era i n Porphyra c u l t i v a t i o n w i l l begin., I t w i l l then be p o s s i b l e t o attempt h y b r i d i z a t i o n and s e l e c t i o n to improve the c u r r e n t l y c u l t i v a t e d P o r p h y r a s p e c i e s . Such s t u d i e s may already be going cn i n Japan (Dr. T. F. Mumford, personal communication) . 3. E f f e c t Of Photoperiod On Monospore And Carpospore Germination Germination of monospores of Porphyra g a r d n e r i - and carpospores o f P. g a r d n e r i , P. n e r e o c y s t i s and P. t h u r e t i i under d i f f e r e n t photoperiods showed t h a t once a p a r t i c u l a r spore type i s induced, i t s f a t e cannot be changed by photoperiod. T h i s i s i n agreement with the work of B i r d et a l . (1972) and Mumford (1973a) on Porphyra. and Cole (1972a) on Bangia. In c o n t r a s t , Bichardson (1970) r e p o r t e d t h a t i n Bangia 107 r e l e a s e d carpospores underwent b i p o l a r germination when c u l t u r e d under a photoperiod of l e s s than 12 hr l i g h t , and u n i p o l a r germination to give the c o n c h o c e l i s phase when c u l t u r e d under a photoperiod with more than 12 hr l i g h t . Conway (1964b, 1966) has suggested that under c e r t a i n c o n d i t i o n s carpospores of Porphyra can germinate i n f o b i p o l a r s p c r e l i n g s i n s t e a d o f the c o n c h o c e l i s phase. In such cases I t h i n k t h a t one should be very c a r e f u l t h a t a l l o f the spores being t e s t e d are o f the same type. Kurogi (1961) and Hawkes (1977b) have both noted that monospores and carpospores can be present on the same t h a l l u s . 4. The C o n c h o c e l i s Phase T h i s i s the f i r s t r e p o r t of a c o n c h o c e l i s phase i n the l i f e h i s t o r i e s of Porphyra g a r d n e r i and P. t h u r e t i i *. e s t a b l i s h i n g t h a t they have heteromorphic l i f e h i s t o r i e s . Cf the Porphyra s p e c i e s which have been c u l t u r e d , only Porphyra s a n j u a n e n s i s Krishnamurthy (Conway, 1973; Conway et a l . 1975) and Porphyra subtumens J . Agardh ex L a i n g 2 (Conway and Mylie, 1972) l a c k a c o n c h o c e l i s phase. I r r e g u l a r s w e l l i n g s o f v e g e t a t i v e c o n c h o c e l i s f i l a m e n t s and branch types which s u p e r f i c i a l l y resemble c o n c h o s p o r a n g i a l branches have been r e p o r t e d by s e v e r a l workers (Iwasaki, 1961; »The r e p o r t by Conway et a l . (1975) t h a t Porphyra t h u r e t i i has a c o n c h o c e l i s phase was based on my work. 2 I t should be noted t h a t i f the mode of spermatium fo r m a t i o n rep o r t e d by Conway and S y l i e (1972) i s c o r r e c t , then t h i s s p e c i e s does not belong i n the genus Porphyra. 108 Iwasaki and Matsudaira, 1963; Conway, 1964b; Conway and Cole, J977). Conway and Cole (1977) r e p o r t e d that these produced ncncspores. Iwasaki and Matsudaira (1963) found t h a t when Porphyra t e n e r a c o n c h o c e l i s phase was maintained under continuous i l l u m i n a t i o n i t formed s g u a r i s h branches which r e l e a s e d monospores. I cannot d e f i n i t e l y conclude t h a t the s w e l l i n g s I observed are monosporangia because they were never seen t o produce monospores. V e g e t a t i v e propagation of Porphyra n e r e o c y s t i s c o n c h o c e l i s f i l a m e n t s i n my c u l t u r e s o c c u r r e d by fragmentation r a t h e r than by monospore production. T h i s was a l s o observed by Mumford (1973a) i n h i s c u l t u r e s . C h y t r i d i n f e c t i o n of the c o n c h o c e l i s phase can a l s o produce s p o r e - l i k e s w e l l i n g s ( A r a s a k i , 1960, r e f e r r i n g to lendo's (1919) work; M i g i t a , 1973,) which c o u l d be mistaken f o r monosporangia. Under c e r t a i n c o n d i t i o n s , the beaded branches produced by Porphyra n e r e o c y s t i s and Porphyra t h u r e t i i may produce monospores, or they may have other f u n c t i o n s such as p l a y i n g some r o l e i n the s h e l l - b o r i n g p r o c e s s . Krishnamurthy (1969a) m i s i n t e r p r e t e d these beaded branches as being conchosporangial branches. Fukuhara (1968), i n h i s s t u d i e s of s e v e r a l Porphyra s p e c i e s of Hokkaido, noted t h a t the c o n c h o c e l i s phase of the s p e c i e s belonging t o the subgenus Porphyra c o u l d be d i v i d e d i n t o two types; one i n which the v e g e t a t i v e f i l a m e n t s and c c n c h o s p o r a n g i a l branches are a d i f f e r e n t c o l o u r and one i n which they are t h e same c o l o u r . In the 3 s p e c i e s I s t u d i e d the c c n c h o s p o r a n g i a l branches were more deeply pigmented than t h e 109 v e g e t a t i v e f i l a m e n t s but were not markedly d i f f e r e n t i n c c l o u r . The p i n n a t e l y branched morphology of the c o n c h o c e l i s f i l a m e n t s grown i n s h e l l was s t r i k i n g and pcses the g u e s t i o n of how the s h e l l matrix causes t h i s morphological change. Ogata (1959; 1961) presented some f a s c i n a t i n g photographs of the v e r t i c a l growth p a t t e r n o f the c o n c h o c e l i s phase i n s h e l l and noted t h a t the morphology was a f f e c t e d by the type of s h e l l . I found t h a t the degree and r e g u l a r i t y cf branching v a r i e d with the s h e l l type. S e v e r a l other Porphyra s p e c i e s develop pinnate branching i n s h e l l (Kurogi, 1953a, 1953b; Drew, 1954a; Kornmann, 1960; Kurogi and Sato, 1962a, 1962b, 1967; M i g i t a and Kim, 1970; Mumford, 1973a, 1975). M i g i t a and Kim (1970) found t h a t under d i f f u s e l i g h t c o n c h o c e l i s f i l a m e n t s developed the p i n n a t e l y branched morphology, whereas under u n i d i r e c t i o n a l l i g h t both the v e g e t a t i v e f i l a m e n t s and c o n c h o s p o r a n g i a l branches showed a d e f i n i t e p o s i t i v e phototropism. Mumford (1975) a l s o r e p o r t e d a p o s i t i v e phototropism f o r the conchosporangial branches of Pcrphyra brumalis Mumford. A p h y s i o l o g i c a l study i s needed t o e l u c i d a t e the mechanism of t h i s p h o t o t r o p i c response. Drew (1954a) r e f e r r e d to the branches she observed i n the s h e l l matrix as ' f e r t i l e - c e l l rows' and those on the s h e l l s u r f a c e as ' p l a n t l e t s * . M i g i t a (1961, 1962) has pointed out t h a t . they are a c t u a l l y one continuous s t r u c t u r e and should be r e f e r r e d to as the conchosporangial branches. My o b s e r v a t i o n s are i n agreement with M i g i t a ' s c o n c l u s i o n . M i g i t a (1962) a l s o r e p o r t e d t h a t the c o n c h o s p o r a n g i a l branches c o u l d r e v e r t t o v e g e t a t i v e f i l a m e n t growth which c o u l d r e - p e n e t r a t e the s h e l l . 110 I a l s o observed t h i s phenomenon i n P. n e r e o c y s t i s . Ogata (1961) used pH s e n s i t i v e s t a i n s to determine t h a t the i n t e r n a l pfi of c o n c h o c e l i s f i l a m e n t s was pH 5-6. He s p e c u l a t e d t h a t t h i s mild a c i d production was r e s p o n s i b l e f o r d i s s o l v i n g the s h e l l ; however, the a c t u a l p h y s i o l o g i c a l mechanism of t h i s process needs to be e l u c i d a t e d . The a b i l i t y o f the c o n c h o c e l i s phase t o s u r v i v e prolonged darkness i s another area i n which p h y s i o l o g i c a l study i s needed and should provide i n t e r e s t n g i n s i g h t i n t o a d a p t a t i o n s of the c o n c h o c e l i s . Sheath et a l . (1977) have r e c e n t l y r e p o r t e d the e f f e c t s of darkness and low l i g h t on c h l o r o p l a s t u l t r a s t r u c t u r e , pigmentation and p h o t o s y n t h e s i s i n the c o n c h o c e l i s phase of Porphyra l e u c o s t i c t a . Ogata (1961, 1971) observed t h a t c o n c h o c e l i s continued growing even a f t e r the s h e l l i t was i n was embedded i n l i g u i d p a r a f f i n . He concluded from t h i s t h a t the c o n c h o c e l i s was a b l e to use the s h e l l matrix as a carbon source. The c o n c h o c e l i s phase has been observed i n b a r n a c l e s or s h e l l s i n the f i e l d a number o f times ( B a t t e r s , 1892; Rosenvinge, 1931; Drew and R i c h a r d s , 19 53; van den Hoek, 1958; Miura and I t o , 1959; M i g i t a , 1959b; B i r d , 1973; Mumford, 1S73a, 1975). at the study s i t e the predominant c a l c a r e o u s s u b s t r a t e was provided by c r u s t o s e c o r a l l i n e algae. In view of Drew and Richards (1953) r e p o r t of Lithothamnion laevigatum F c s l i e ( r e p o r t e d as L i t hothamnion l a e v i g a t a ) being a s u i t a b l e c o n c h o c e l i s phase s u b s t r a t e , a thorough survey should be made before c r u s t o s e c o r a l l i n e s are r u l e d out as a p o t e n t i a l s u b s t r a t e f o r the c o n c h o c e l i s phase. 111 5. Conchosporangial Branches Conchosporangial branch f o r m a t i o n by Porphyra g a r d n e r i and l21Bfel£§ t h u g e t i i was most abundant under a photoperiod of 12 hr l i g h t or l e s s , whereas i n P. n e r e o c y s t i s no optimal photoperiod was observed. The s t u d i e s of most workers on ether Porphyra s p e c i e s i n d i c a t e t h a t photoperiod i s of major importance to conchosporangial branch i n d u c t i o n . Dring (1967) and B e n t s c h l e r (1967), both working with Porphvra t e n e r a . showed t h a t c o n c h o s p o r a n g i a l branch i n i t i a t i o n was a short-day, phytochrorae-mediated response. My r e s u l t s with P. g a r d n e r i and £• t h u r e t i i are s i m i l a r to those of Dring (1967) and Iwasaki and S a s a k i (1972), i n that conchosporangial branches formed under a l l photoperiods t e s t e d , but were r a r e under long days and abundant under s h o r t days. I t i s not known i f phytochrcme i s i n v o l v e d . Richardson (1970), however, r e p o r t e d that c o n c h o s p o r a n g i a l branch producion by pangja was an ' a l l or nothing' response. The data of Japanese i n v e s t i g a t o r s support the hypothesis t h a t photoperiod i s a major f a c t o r i n conchosporangial branch i n d u c t i o n . The main c o n c l u s i o n of s e v e r a l workers (Kurogi, 1959; Iwasaki, 1961; Kurogi and Sato, 1962a, 1962b; Kurogi et a l . 1962; Iwasaki, 1965; Iwasaki and S a s a k i , 1972; and Chihara, 1975) i s t h a t conchosporangial branch f o r m a t i o n i n the Japanese winter Porphyra s p e c i e s i s most abundant i n c u l t u r e s s u b j e c t e d to 12 hr or more dark per 24 hr. In the f i e l d such c o n d i t i o n s are encountered i n the autumn and winter when the f o l i o s e phase of most Japanese Porphyra s p e c i e s appears. In P. u m b i l i c a l i s . an autumn s p e c i e s , Kurogi and Sato (1967) found t h a t 112 c c n c h o s p o r a n g i a l branches were most abundant a t 16:8. Iwasaki and S a s a k i (1972), i n an experiment with Porphyra s u b g r b i c u l a t a Kjellman forma l a t i f o l i a , showed t h a t i t was photoperiod r a t h e r than the t o t a l amount of l i g h t r e c e i v e d ( p h o t o s y n t h e t i c e f f e c t ) which was r e s p o n s i b l e f o r c c n c h o s p o r a n g i a l branch f o r m a t i o n . The apparent l a c k o f an o p t i m a l photoperiod f o r c c n c h o s p o r a n g i a l branch i n d u c t i o n i n Porphyra n e r e o c y s t i s remains an enigma. I t must be po i n t e d out t h a t the branches formed were assumed to be c c n c h o s p o r a n g i a l branches, but because none of them r e l e a s e d ccnchospores t h i s i s perhaps a premature assumption. The o n l y r e p o r t i n the l i t e r a t u r e of p h c t c p e r i o d having no e f f e c t on conchosporangial branch formation i s t h a t o f B i r d e t a l . (1972) f o r Porphyra l i n e a r i s . Other f a c t o r s can have a modifying e f f e c t on the p h o t o p e r i o d i c i n d u c t i o n of conchosporangial branches. R e n t s c h l e r (1967) found that while temperature changes d i d not induce conchosporangial branches, they did i n f l u e n c e the d u r a t i o n from p h o t o p e r i o d i c i n d u c t i o n u n t i l the appearance of conchosporangial branches. In Porphyra n e r e o c y s t i s I observed t h a t c c n c h o s p o r a n g i a l branches f i r s t appeared i n those c u l t u r e s at a higher temperature. In many Porphyra s p e c i e s i t has been shown that conchosporangial branches formed over a range of temperatures, but t h a t there was u s u a l l y an o p t i m a l temperature (Kurogi and Hirano, 1956a, 1956b; Iwasaki and S a s a k i , 1972). Furthermore, Kurogi and Hirano (1956b) and Kurogi and Akiyaaa (1966) noted t h a t t h i s temperature was not always the optimal temperature f o r conchospore l i b e r a t i o n . These are general 113 tr e n d s and s h o u l d not be assumed t o be t r u e f o r a l l Porphyra s p e c i e s . For example, Kurogi and Sato (1967) r e p o r t e d t h a t P. u m b i l i c a l i s c o n c h o c e l i s formed conchosporangial tranches e g u a l l y as w e l l at 10°C as a t 20°C. L i g h t i n t e n s i f y has a l s o been shown to have a modifying e f f e c t on c c n c h o s p o r a n g i a l branch production (Kurogi and Hirano, 1955a, 1955b; Conway, 1964b). In Rhodochortcn, Knaggs (1966a, 1966b, 1967) demonstrated t h a t l i g h t i n t e n s i t y , or a combination of l i g h t i n t e n s i t y and n u t r i e n t l e v e l , was r e s p o n s i b l e f o r tetrasporangium i n i t i a t i o n ; however. West (personal communication) has pointed out that t h i s response was c n l y i n v e s t i g a t e d under i n d u c t i v e daylengths ( l e s s than 12:12). 6. Conchospore Release Only Porphyra t e n u i p e d a l i s Miura (Miura and I t o , 1959, as Poiphy.ra sp. ; Miura, 1961) and P. miniata (Krishnamurthy. 1969a, as P. cuneiform i s ( S e t c h e l l et Hus) Krishnamurthy) have been rep o r t e d t o produce the f o l i o s e phase d i r e c t l y from the c c n c h o s p o r a n g i a l branch without conchospore r e l e a s e . I t i s unfortunate t h a t the formation cf conchospores i n the conchosporangia was not observed i n e i t h e r Eorphyra g a r d n e r i o f P. t h u r e t i j . I t has been g e n e r a l l y assumed t h a t each c e l l of the c c n c h o s p o r a n g i a l branch f u n c t i o n e d as a conchosporangium by r e l e a s i n g i t s e n t i r e c o n t e n t s as a s i n g l e conchospore (Conway and C o l e , 1977); however, M i g i t a and Abe (1966), working with Porphyra tenera and P. y e z o e n s i s , observed that each conchosporangium produced 2-4 conchospores. M i g i t a (1967b, 1974) has subsequently 114 s t u d i e d t h i s i n f u r t h e r d e t a i l (see P a r t IV f o r more d i s c u s s i o n ) . Kornmann (1961b) i l l u s t r a t e d more than one conchospore per conchosporangium f o r Porphyra purpurea. T h i s o b s e r v a t i o n i s of c o n s i d e r a b l e importance because i t provides good c i r c u m s t a n t i a l evidence t h a t the d i v i s i o n of the conchosporangium to form conchospores i s the s i t e of meiosis and not the f i r s t d i v i s i o n t o form the conchosporangial branch as suggested by Conway et a l . (1S75). An i n t e r e s t i n g p o i n t i s t h a t s h e l l - l i v i n g c o n c h o c e l i s t y p i c a l l y produced two conchospores per conchosporangium whereas f r e e - l i v i n g c o n c h o c e l i s o f t e n produced four ( M i g i t a and Abe, 1966). In my experiments conchospore r e l e a s e proved to be i m p o s s i b l e to t r i g g e r c o n s i s t e n t l y . Conchospore r e l e a s e by Porphyra g a r d n e r i was o p t i m a l at 7-10°C, which c o r r e l a t e d reasonably w e l l with temperature c o n d i t i o n s i n the f i e l d when £• g a r d n e r i f i r s t appeared (Table V I I ) . , In T a b l e VII study s i t e s u r f a c e seawater temperatures are based on only a s i n g l e measurement each month, but the g e n e r a l trend i s the same as t h a t f o r Amphitrite P o i n t ( H o l l i s t e r , 1968), a nearby s i t e where d a i l y measurements are made. Although I was unable to demonstrate c o n c l u s i v e l y t h a t temperature was r e s p o n s i b l e f o r conchospore r e l e a s e by USEEh-Y-E 3 . 3SE^S®£i and P. t h u r e t i i , work which has been done on other Porphyra s p e c i e s p o i n t s t o temperature as the primary f a c t o r t r i g g e r i n g r e l e a s e (Tseng and Chang, 1956, Tseng e t a l . 1963; Kurogi and Hirano, 1956a, 1956b; M i g i t a and Abe, 1966; Kurogi and Akiyama, 1966; Kurogi and Sato, 1967; Kurogi et a l . 1967; B i r d e t a l . 1972; Shinmura, 1974; M i g i t a , 1974; 115 C h i h a r a , 1975). M i g i t a (1974) r e p o r t e d t h a t c o n c h o c e l i s phase c u l t u r e d without changing the medium f o r 3 months f a i l e d t o form conchospores even when i t was t r a n s f e r r e d t o an i n d u c i n g temperature. My f a i l u r e t o o b t a i n c o n s i s t e n t ccnchcspore r e l e a s e may be a t t r i b u t a b l e t o such a n u t r i e n t - r e l a t e d phenomenon. In c o n t r a s t t o most r e p o r t s , Iwasaki (1961) d i d not observe any d i r e c t c o r r e l a t i o n between temperature and the l i f e h i s t o r y phases cf Porphyra t e n e r a . He d i d note t h a t low l i g h t i n t e n s i t y or continuous i l l u m i n a t i o n i n h i b i t e d conchospore r e l e a s e . Other workers have a l s o r e p o r t e d t h a t l i g h t i n t e n s i t y has a modifying e f f e c t on ccnchospore r e l e a s e (Kurogi and Hirano, 1955a, 1955b; S a i t o , 1956; Kurogi and akiyama, 1965; Iwasaki and S a s a k i , 1972; Kurogi et a l . 1962). S e v e r a l workers have rep o r t e d t h a t ccnchospore l i b e r a t i o n i s highest i n the morning (Yamasaki, 1954b; Suto et a l . 1954; Kurogi and H i r a n o , 1955b; Tseng and Chang, 1956). Photoperiod i s a secondary f a c t o r mediating conchospore r e l e a s e . The work of Kurogi (1959), Iwasaki (1961), Kurogi and Sato (1962a, 1962b), Kurogi e t a l . (1962), and Kurogi ejt a l . (1967) has shown that t h e r e i s an optimal photoperiod f o r ccnchcspore r e l e a s e . In c o n t r a s t . B i r d e t a l . (1972) found t h a t photoperiod v a r i a t i o n d i d not a f f e c t conchospore l i b e r a t i o n by P. l i n e a r i s . Other f a c t o r s t hat have been r e p o r t e d to have an e f f e c t cn ccnchospore r e l e a s e are a g i t a t i o n (Tseng and Chang, 1956) and d e s i c c a t i o n (Graves, 1969). S a i t o (1955) c o r r e l a t e d v e r t i c a l 116 t u r b u l e n c e i n the water column with an i n c r e a s e of conchospores i n the s u r f a c e water. Kurogi (1953b) and Kurogi and Hirano (1956a) both reported a 1-2 week p e r i o d i c i t y i n conchospore l i b e r a t i o n but were unable to c o r r e l a t e i t with s p r i n g t i d e s as r e p o r t e d by Takeuchi et a l . (1954). although my attempts to induce conchospore r e l e a s e by Porphyra n e r e o c y s t i s f a i l e d , workers „• i n C a l i f o r n i a ( J . Woessner, p e r s o n a l communication August, 1977) have r e c e n t l y succeeded i n t r i g g e r i n g r e l e a s e by s u b j e c t i n g the c o n c h o c e l i s t o a temperature of 12°C. Judging from r e p o r t s i n the l i t e r a t u r e I t h i n k that the combination of s p e c i f i c temperature, photoperiod and l i g h t i n t e n s i t y are a l l important f o r conchospore r e l e a s e , with temperature being of primary importance. F u r t h e r c u l t u r e s t u d i e s under much more c r i t i c a l l y c o n t r o l l e d c o n d i t i o n s than I was able t o maintain must be done bef o r e we w i l l have a thorough understanding of the process of conchosporangial branch i n d u c t i o n and conchospore r e l e a s e i n the 3 s p e c i e s I s t u d i e d . From my ex p e r i e n c e , Porphyra t h u r e t i i would be the best one to s u b j e c t t o f u r t h e r i n v e s t i g a t i o n because i t responded f a s t e r and more d e f i n i t e l y to d i f f e r e n t photoperiod regimes than d i d the.other 2 s p e c i e s . 7. Conchospores Of p a r t i c u l a r i n t e r e s t was the photographic documentation of amoeboid change cf shape and movement by conchospores of j g r p h y r a t h u r e t i i . These two phenomenon are not unknown i n the Bhodcphyta, having p r e v i o u s l y been r e p o r t e d f o r : monospores of 117 Banjgia (Beinke, c i t e d from F r i t s c h , 1945, p. 431; B e r t h o l d , 1882; K y l i n , 1922; Sommerfeld and N i c h o l s , 1970), Porphyra (B e r t h o l d , 1882; Kunieda, 1939; Kuro g i , 1961; Graves, 1969), l O l i l o c i a d i a ( N i c h o l s and L i s s a n t , 1967), Helminthora ( S v e d e l i u s , 1917), Ljagpra (von Stosch, 1965) and Acjcochaetiuro (Rcsenvinge, 1909, as C h a n t r a n s i a ; Dangeard, 1929; Stegenga and B o r s j e , 1976; Stegenga and Vroman, 1976); f o r r e l e a s e d •carpogonia' o f Porphyra pa pent u s s i i (Conway and Cole, 1973); f o r Porphyra spermatia (Kunieda, 1939); f o r carpospores of SQI.2h.2LSi (Janczewski, 1873; Okamura e t a l . 1920; K y l i n , 1922; Graves, 1969), Bangia (Sommerfeld and N i c h o l s , 1970), Helminthora (Thuret and Bornet, 1867), Platoma (Kuckuck, c i t e d from F r i t s c h , 1945, p..,.602), S c i n a i a (Chemin, 1927), and Bonnemaispnia (Chemin, 1929); and f o r t e t r a s p o r e s of L i a g o r a (von S t o s c h # 1965), and Acrochaetium (Stegenga and Vroman, 1976} . Some rhodophycean spores e x h i b i t g l i d i n g movements with no amoeboid changes of spcre shape. Such locomotion has been re p o r t e d by Dangeard ( c i t e d from F r i t s c h , 1945, p. 602) f o r Acrochaetium c a r p o s p o r e s ; by Bosenvinge (1927) f o r monospores °f F r y t h r o t r i c h i a , t e t r a s p o r e s of Antithamnion. Ceramiuj, and Dumontia, and carpospores of Ceramium. and P o l y s i p h o n i a , and spermatia of Phyllophora; and by Chemin ( c i t e d from F r i t s c h , 1945, p. 602) f o r monospores of E r y t h r o t r i c h i a and Ggniotrichum. Recently L i n e t a l . (1975) made a d e t a i l e d study °f Porphyridium which e x h i b i t e d g l i d i n g movements with only s l i g h t changes of shape. They reported that t h i s phenomenon has a l s o been observed i n Chrpothece m o b i l i s Paseher and 118 Petrova and i n other Pcrphvridium s p e c i e s . Amoeboid changes o f shape and movement by conchospores have p r e v i o u s l y been r e p o r t e d f o r Porphyra ve z o e n s i s ( M i g i t a , 1972) P. angusta. P. k u n i e d a i ( K u r o g i , 1961), P. miniata (Chen et a l . 1970), P. s m i t h i i Hollenberg et Abbott (McDonald, 1972), and Bangia (Scmmerfeld and N i c h o l s , 1970) . The purpose of l i s t i n g a l l o f these examples i s to point out t h a t most are from the Bangiophycidae and Nemaliales. I s t h i s j u s t a c o i n c i d e n c e or i s there some e v o l u t i o n a r y l i n k between these two groups of Hhodophyta? Neither the f u n c t i o n nor mechanism of the amoeboid movements i s known, although B e r t h o l d (1882) p o i n t e d out that l i g h t had an i n f l u e n c e . A p o s s i b l e f u n c t i o n c o u l d be to i n c r e a s e the p r o b a b i l i t y o f the spore f i n d i n g a s u i t a b l e s u b s t r a t e f o r attachment and germination. The photcgraphs c f P. yezoensis conchospores p u b l i s h e d by M i g i t a (1972) suggest t h i s may be the case. The u l t r a s t r u c t u r a l study of L i n e t a l . (1975) on Porphyrjdium showed t h a t there was a production and polar e x p u l s i o n of mucilage from the moving c e l l s , although i t was not d e f i n i t e l y e s t a b l i s h e d t h a t t h i s was the cause of the movement. 119 Table VII. Annual t r e n d i n s u r f a c e water temperature (°C) a t the study s i t e (48° 50.11!*, 125° 11.1*W) and Amphitrite Point (48° 55.2*N, 1 2 5 ° 3 2 . 2 * W ) . Study s i t e Amphitri January 7.5 ——— February 7.0 8.5 March 7.0 8. 1 A p r i l 10.0 9.6 May 12.0 10.0 June 12.5 11.7 J u l y 12.8 12.3 August 13.8 12.2 September 13.5 12.2 October 11.3 11.2 November 9.0 9.9 December 8.0 9.2 120 Porphyra gardneri Figure 12a, Monospores being released along the margin of a th a l l u s c o l l e c t e d at the study s i t e . Figures 12b-f. Various stages in the development cf the f o l i o s e thallus from a monospore. Note the lon< r h i z o i d a l protuberances. Figure 12g. Released carpospores. Figure 12h. Carpospore germination to give the conchocelis phase. Figure 12i. Single spore-like swelling on the conchocelis filament. Figure 12jj. Conchosporangial branches. 121 122 Porphyra g a r d n e r i F i g u r e 13a. C o n c h o c e l i s f i l a m e n t s , c c n c h o s p o r a n g i a l branch (lower r i g h t ) and r e l e a s e d conchospore (arrow). F i g u r e 13b. B i p o l a r s p o r e l i n g r e s u l t i n g from conchospore germination. F i g u r e 13c. C h a r a c t e r i s t i c p i n n a t e l y branched morphology of the c o n c h o c e l i s phase growing i n o y s t e r s h e l l . Porphyra n e r e o c y s t i s F i g u r e 13d. Released c a r p o s p o r e s . F i g u r e 13e. Carpospore germination to g i v e the c o n c h o c e l i s phase. F i g u r e 13f. B a s a l s p o r e - l i k e s w e l l i n g of the c o n c h o c e l i s phase. F i g u r e 13g. A p i c a l s p o r e - l i k e s w e l l i n g of the c o n c h o c e l i s phase. F i g u r e 13h. I r r e g u l a r and beaded-branch types. F i g u r e 13i. S g u a r e - c e l l e d branch. F i g u r e 13j. Small c c n c h o s p o r a n g i a l branch connected t o the v e g e t a t i v e f i l a m e n t by a s q u a r e - c e l l e d branch. F i g u r e 13k. Ccnchosporangial branches. 123 124 Porphyra n e r e o c y s t i s F i g u r e 14a. Closeup of conchosporangial branches showing the s i n g l e s t e l l a t e c h l o r o p l a s t i n each conchosporangium. F i g u r e 14b, P i t plugs (arrows) between ccnchosporangia i n the conchosporangial branch ( t r e a t e d with chromosome f i x a t i v e and s t a i n ) . F i g u r e 14c. D i v i s i o n s w i t h i n the conchosporangial branch. F i g u r e 14d. C h a r a c t e r i s t i c pinnate branching of the c o n c h o c e l i s phase growing i n o y s t e r s h e l l . Note the i r r e g u l a r s w e l l i n g s . Porphyra t h u r e t i i F i g u r e 14e. Released carpospores. F i g u r e 14f. Carpospore germination to give the c o n c h o c e l i s phase. F i g u r e 14g. V e g e t a t i v e c o n c h o c e l i s f i l a m e n t s , beaded branches and young c c n c h o s p o r a n g i a l branch (arrow). F i g u r e 14h. C l u s t e r o f conchosporangial tranches showing s i n g l e s t e l l a t e c h l o r o p l a s t i n each conchosporangium. F i g u r e 14i. Candelabra-shaped c l u s t e r of conchosporangial branches showing t h e i r p o s i t i v e phototropism. F i g u r e 14j. Ccnchosporangial branch with conchospores. F i g u r e 14k. Released conchospores. 125 126 Porphyra t h u r e t i i F i g u r e 15a-d. Conchospores e x h i b i t i n g amoeboid change of shape and movement. F i g u r e s 15a and 15b taken 90 seconds apart. F i g u r e s 15c and 15d taken 45 seconds a p a r t . , F i g u r e 15e. Young b i p o l a r s p o r e l i n g r e s u l t i n g from conchospore germination. Note r h i z o i d a l protuberance. F i g u r e s 15f-15g. O l d e r , u n i s e r i a t e s p o r e l i n g s s t a r t i n g to form b a s a l pad. F i g u r e 15h. B i s e r i a t e stage of s p o r e l i n g growth.. F i g u r e s 1 5 i - 1 5 j . Older s p o r e l i n g s . Note the b a s a l r h i z o i d a l pad i n F i g u r e 15j. 127 128 PART IV - CYTOLOGICAL STUDIES General I n t r o d u c t i o n The f i r s t t h ree parts of t h i s t h e s i s presented a d e t a i l e d d i s c u s s i o n o f the morphological l i f e h i s t o r i e s cf Porphyra g a r d n e r i , P. n e r e o c y s t i s and P. t h u r g t j i . The primary purpose of P a r t IV i s to document the c y t o l o g i c a l l i f e h i s t o r i e s of these 3 s p e c i e s i n as much d e t a i l as p o s s i b l e , with emphasis on Porphyra gardneyi and the occurrence of sexual r e p r o d u c t i o n i n i t s l i f e h i s t o r y . I a l s o wanted t o examine the attachment of £orphyra g a r d n e r i and P. n e r e o c y s t i s to t h e i r host p l a n t s , and observe the u l t r a s t r u c t e r e of monosporogenesis i n P. g a r d n e r i . M a t e r i a l s And Methods C y t o l o g i c a l s t u d i e s were done on m a t e r i a l c o l l e c t e d at the study s i t e . The specimens of Porpfryra g a r d n e r i used i n the f e r t i l i z a t i o n study were c o l l e c t e d i n May, and those of £• n e r e o c y s t i s and P. t h u r e t i i i n January. For chromosome counts m a t e r i a l was. f i x e d i n 95% e t h a n o l ; g l a c i a l a c e t i c a c i d (3:1), and s t a i n e d with a c e t o - i r o n - h a e m a t o x y l i n - c h l o r a l hydrate (Wittmann, 1965). Spermatangial m a t e r i a l c o u l d be f i x e d a t any time to o b t a i n a chrcmoscffle count because o f the l a r g e number of d i v i s i o n s o c c u r r i n g and because the chromosomes i n the mature spermatia 129 are condensed. The best counts were obtained j u s t as the chromosomes were beginning to condense. The h i g h l y condensed chromosomes i n the mature spermatia did not g i v e r e l i a b l e counts because they tended to clump. Spermatial chromosome counts were made on type l o c a l i t y p l a n t s of Porphyra g a r d n e r i . In the case o f v e g e t a t i v e and c a r p o s p o r a n g i a l m a t e r i a l i t was necessary to f i x t h a l l i every hour f o r 24 hours to determine when m i t o s i s was o c c u r r i n g . T h i s was found to begin at sunset and continue f o r 1-2 hours. P r i n g l e and A u s t i n (1S70) obtained s i m i l a r r e s u l t s with another P o r p h y r a s p e c i e s . S e v e r a l workers have emphasized that m i t o s i s occurs most f r e g u e n t l y a t n i g h t ( K i t o , yabu and Tokida, 1967; M i g i t a , 1967b; Yabu, 1969a, 1970; K i t o , Ogata and Mclachlan, 1971). Methods of i s o l a t i o n and c o n d i t i o n s of growth of the c o n c h o c e l i s phase i n c u l t u r e have been d e s c r i b e d i n P a r t I I I . C u l t u r e d c o n c h o c e l i s was f i x e d at hourly i n t e r v a l s f o r 24 hours. D i v i d i n g c e l l s were i n f r e g u e n t i n the c o n c h o c e l i s phase compared to the f o l i o s e t h a l l u s . Most d i v i s i o n s were found i n m a t e r i a l f i x e d i n the dark p e r i o d , p r i m a r i l y i n the f i r s t few hours of darkness. Ramus (1969) i n work with Pseudogloiophloea and Sommerfeld and N i c h o l s (1970) i n work on B a n g i a c o n c h o c e l i s phase a l s o found t h a t the freguency o f c e l l d i v i s i o n was h i g h e s t d u r i n g the dark p e r i o d . In an attempt t o i n c r e a s e the number of c e l l s i n metaphase, c o n c h o c e l i s phase c u l t u r e s were t r e a t e d with 0.051?, 0.1% and 1$ c o l c h i c i n e f o r 24 hours. T h i s compound i s known to a r r e s t metaphase i n higher p l a n t s ( K l e i n and K l e i n , 1970); however, i t had no e f f e c t on the c o n c h o c e l i s phase. 130 M a t e r i a l f o r the attachment study and Feulgen s t a i n i n g was f i x e d and embedded i n methacrylate as d e s c r i b e d i n Part I I . For Eeulgen s t a i n i n g , s e r i a l s e c t i o n s (Henry, 1977) 3-6 um t h i c k were c u t . fin aldehyde blockade was c a r r i e d out using a s a t u r a t e d s o l u t i o n of 5,5-dimethylcyclohexane-1,3-dione (Dimedone) f o r 18 hours at rocm temperature. The s l i d e s were then r i n s e d i n running water f o r 30 minutes (Feder and O'Brien, 1968). H y d r o l y s i s was c a r r i e d out i n 1N HCl at 60°C f o r 20 minutes. S l i d e s were then t r a n s f e r r e d to S c h i f f " s reagent (made a c c o r d i n g t o the r e c i p e of Sharaa and Sharma, 1965) f o r 3 hours. T h i s was f o l l o w e d by 3 s u c c e s s i v e washes of 0.5% potassium m e t a b i s u l f i t e (2 minutes i n each), then a r i n s e i n running water f o r 10 minutes. S e c t i o n s were l i g h t l y c o u n t e r s t a i n e d f o r 1 minute i n a 1% aqueous s o l u t i o n of F a s t Green (Feder and O'Brien, 1968) . For e l e c t r o n microscopy m a t e r i a l was p o s t f i x e d i n 2% OsOy i n phosphate b u f f e r pH 7.2 (1:1) f o r 1 hour, followed by dehydration i n e t h a n o l and i n f i l t r a t i o n with Spurr's r e s i n . S e c t i o n s were cut on g l a s s knives using a fleichert Om 03 ultramicrotome. They were s t a i n e d with u r a n y l a c e t a t e i n 50$ e t h a n o l (Dawes, 1971) and l e a d c i t r a t e (Reynolds, 1963) and examined with a C a r l Z e i s s EM-10 e l e c t r o n microscope. M a t e r i a l f o r scanning e l e c t r o n microscopy was f i x e d and dehydrated using the above procedure. T h a l l u s p i eces were then run through a graded s e r i e s of ethanol:amyl a c e t a t e (3:1, 1:1, 1:3), 0.5 hr i n each and 0.5 hr i n 100% amyl a c e t a t e . Specimens were c r i t i c a l p o i n t d r i e d , mounted on s t u b s , gold coated and examined with a Cambridge Stereoscan Type 2k 131 scanning e l e c t r o n microscope, a. Epiphyt e - ' H o s t 1 Attachment I n t r o d u c t i o n The known e p i p h y t i c s p e c i e s of Porphyra have been summarized by Ohmi (1963) and Tckida (1960) . The nature o f the epiphyte-•host* r e l a t i o n s h i p i n these Porphyra s p e c i e s has r e c e i v e d l i t t l e i n v e s t i g a t i o n . Grubb (1923) d e s c r i b e d the attachment of Porphyra purpurea (as P. u m b i l i c a l i s var. l a c i n i a t a ) t o Fucus and concluded t h a t t h e b a s a l r h i z o i d s penetrated host c e l l s . K i t o (1966) reported t h a t the b a s a l r h i z o i d s of P^ k a t a d a i Hiura (as Porphyra sp.) penetrated deeply i n t o the t i s s u e of i t s host G r a t e l o u p i a f i l i c j , n a var. porracea (Mertens) Howe. Fukuhara (1968) found t h a t the basal r h i z o i d s of P. onoi Ueda penetrated the host t i s s u e whereas those of P. pseudocrassa Yamada et. Hikami d i d not. There have been no p h y s i o l o g i c a l s t u d i e s of the epiphyte-* h o s t 1 i n t e r a c t i o n i n the e p i p h y t i c Poiphyra s p e c i e s . The r e s t r i c t i o n of Porph vra n e r e o c v s t i s t o e s s e n t i a l l y one host s p e c i e s and the f a i l u r e of P. g a r d n e r i s p o r e l i n g s t o develop i n t o mature t h a l l i i n c u l t u r e (see Part I I I ) suggests t h a t they may have a p h y s i o l o g i c a l dependency on the »host t. As the most probable r e g i o n o f n u t r i e n t exchange i n such a r e l a t i o n s h i p would be the attachment zone, a l i g h t microscope study o f t h i s r e g i o n was made. 132 R e s u l t s 1. Porphyra g a r d n e r i Transverse s e c t i o n s were cut through the b a s a l p o r t i o n of £2ifihyra g a r d n e r i and the Lamina r i a blade margin. The r h i z o i d a l f i l a m e n t s which form t h e base c f the t h a l l u s of £• g a r d n e r i spread out s l i g h t l y on the Laminaria blade margin to form a basal pad {Figure 16a). The r h i z o i d a l f i l a m e n t s i n the c e n t r a l p o r t i o n o f t h i s pad penetrate deeply i n t o the medulla ( F i g u r e s 16a and 16b), and i n some cases they appear t o penet r a t e the medullary c e l l s , although an E. N. examination i s needed to v e r i f y t h i s . 2» Porphyra n e r e o c y s t i s S e c t i o n s were cut through the b a s a l r e g i o n of Porphyra n e r e o c y s t i s and the s t i p e of N e r e o c y s t i s i n a plane p e r p e n d i c u l a r to the s t i p e a x i s . &s i n P. g a r d n e r i . the b a s a l r h i z o i d a l f i l a m e n t s spread out to form a pad on the s t i p e s u r f a c e (Figure 16c)., T h i s pad i s much l a r g e r than t h a t formed fay £• 3§£^II€£i« I n the c e n t e r o f the pad the r h i z o i d a l f i l a m e n t s make a s l i g h t i n d e n t a t i o n i n t o the c o r t e x of the N e r e o c y s t i s s t i p e ( F i g u r e s 16c and 16d), but there i s no ex t e n s i v e r h i z o i d a l p e n e t r a t i o n of the s t i p e t i s s u e . 133 D i s c u s s i o n The i n t i m a t e attachment of Porjahjrra g a r d n e r i t o the Laminaria blade suggests t h a t t h e r e may be a t r a n s f e r of m e t a b o l i t e s from the Laminaria to the Porphyra t h a l l i . S t u d i e s by l i i n i n g et a l . (1972) have shown t h a t most Laminaria photosynthates are t r a n s p o r t e d i n the medulla t i s s u e . In c o n t r a s t to Porphyra g a r d n e r i . the attachment of E« n g r e o c y s t i s i s s u p e r f i c i a l , and the l a c k of s i g n i f i c a n t r h i z o i d a l p e n e t r a t i o n of s t i p e t i s s u e suggests there may not be exchange of m a t e r i a l s a t the attachment zone. I t should be noted though, t h a t H a r l i n (1973b) demonstrated t h a t a p e n e t r a t i o n of host t i s s u e i s not a p r e r e q u i s i t e f o r exchange of m a t e r i a l . The case may a l s o be that N e r e o c y s t i s i s exuding compounds i n t o the water where they are picked up by £• n e r g p c y s t i s . The mechanism determining the host s p e c i f i c i t y of P. n e r e o c y s t i s may be a c t i n g at an e a r l i e r stage i n development such as the time o f i n i t i a l conchospore settlement and germination. O l t r a s t r u c t u r a l and p h y s i o l o g i c a l s t u d i e s are needed to e l u c i d a t e the nature of the a s s o c i a t i o n between the epiphyte and "host* i n these o b l i g a t e e p i p h y t i c Porphyra s p e c i e s . 134 B. Monosporogenesis I n t r o d u c t i o n Sroithora naiadurn and Porphvropsis c ^ c c i n e a (Areschoug) Hosenvinge are the only members of the Bangiophycidae i n which mcncsporogenesis has been examined at an u l t r a s t r u c t u r a l l e v e l (HeBride and Cole, 1971; McDonald, 1972, r e s p e c t i v e l y ) . There has not been any u l t r a s t r u c t u r a l i n v e s t i g a t i o n of monospores i n Por.phyra, a s u r p r i s i n g f a c t i n view of the g r e a t importance of the monospore c y c l e i n commercially c u l t i v a t e d Porphyra s p e c i e s . , I t was t h e r e f o r e decided to make some p r e l i m i n a r y u l t r a s t r u c t u r a l o b s e r v a t i o n s of monosporogenesis i n Porpfeyra g a r d n e r i . R e s u l t s Monosporic t h a l l i were e a s i l y recognized i n the f i e l d by t h e i r g e l a t i n o u s margins r e l e a s i n g monospores (see P a r t I ) . Under the l i g h t microscope the monosporangia were s l i g h t l y l a r g e r than v e g e t a t i v e c e l l s and more s p h e r i c a l i n shape. They had f i n e granular c o n t e n t s and the s t e l l a t e c h l o r o p l a s t was somewhat obscured (see Part I I I ) . Tanaka (1952) a l s o noted t h a t the monospores of Porphyra okamurai Ueda had more gr a n u l a r contents than the v e g e t a t i v e c e l l s . The u l t r a s t r u c t u r e of the d i s t a l p o r t i o n of monosporangial t h a l l i was examined i n t r a n s v e r s e s e c t i o n . The f i r s t 135 i n d i c a t i o n t h a t the v e g e t a t i v e c e l l s were undergoing a t r a n s i t i o n to monosporangia was an i n c r e a s e i n dictyosome a c t i v i t y ( F i g u r e s 17 and 19a) which r e s u l t e d i n the production of s m a l l f i b r o u s v e s i c l e s . Mitochondria were found i n a s s o c i a t i o n with these dictyosomes ( F i g u r e 19a). Each monosporangium c o n t a i n e d a s i n g l e s t e l l a t e c h l o r o p l a s t with c e n t r a l pyrenoid and c e n t r a l t o l a t e r a l nucleus. F l o r i d e a n s t a r c h g r a n u l e s were abundant. The number of f i b r o u s v e s i c l e s i n c r e a s e d p r o l i f i c a l l y i n c i d e r monosporangia (those toward the r e l e a s i n g margin) ( F i g u r e 18). These abundant f i b r o u s v e s i c l e s appear to be the numerous granules observed i n the monosporangia at the l i g h t microscope l e v e l . The s m a l l f i b r o u s v e s i c l e s appeared to c o a l e s c e to form l a r g e r f i b r o u s v e s i c l e s ( F i g u r e s 18 and 19c) as w e l l as r e l e a s e t h e i r c o n t e n t s between the plasmamembrane and i n n e r c e l l w a l l , r e s u l t i n g i n a l a y e r of f i b r o u s m a t e r i a l (Figure 19b). In the more mature monosporangia the number of s m a l l and l a r g e f i b r o u s v e s i c l e s i n c r e a s e d (Figure 19c), and the l a r g e f i b r o u s v e s i c l e s became e x t r a c y t o p l a s m i c . T h i s phenomenon was a s s o c i a t e d with monosporangia that were near the r e l e a s i n g margin. U n f o r t u n a t e l y only the i n i t i a l s t a g e s of monospore r e l e a s e were observed, and were c h a r a c t e r i z e d by an apparent d i s s o l u t i o n of the c e l l w a l l l a y e r s beneath the outer c u t i c l e . 136 D i s c u s s i o n Monosporogenesis i n Porphyra g a r d n e r i i s c h a r a c t e r i z e d by the p roduction of s m a l l and l a r g e f i b r o u s v e s i c l e s which appear to be produced p r i m a r i l y by dictyosome a c t i v i t y . T h i s u l t r a s t r u c t u r a 1 study has demonstrated that monospores are more than u n d i f f e r e n t i a t e d v e g e t a t i v e c e l l s which have d i s s o c i a t e d frcm the t h a l l u s , as has been suggested by Dixon (1970) f o r monospores of Bangia and Porphyra. D l t r a s t r u c t u r a l l y the f i b r o u s v e s i c l e s appear the same as those r e p o r t e d i n Smithora monosporangia (McBride and C o l e , 1971) and i n other red a l g a l spermatia, carpospores and t e t r a s p o r e s (see Part IV, spermatogenesis, f o r a complete summary). McBride and Cole (1971) r e p o r t e d the p r o d u c t i o n of 2 d i f f e r e n t types of v e s i c l e s during monosporogenesis i n Smithora. In Porphyra g a r d n e r i I observed o n l y the one type, as d i d McDonald (1972) i n Porphyrppsis monosporogenesis. Released monospores l a c k a c e l l w a l l (observed, but p i c t u r e omitted) and are surrounded by a l a y e r of mucilaginous m a t e r i a l which no doubt has i t s o r i g i n i n the f i b r o u s v e s i c l e s . T h i s mucilage l a y e r may be a mucopolysaccharide ( P e y r i e r e , 1970) and appears to a i d i n monospore adhesion to the s u b s t r a t e . H istochemical study i s needed t o determine the chemical nature of the m a t e r i a l produced by the f i b r o u s v e s i c l e s . Another aspect which needs t o be examined i s whether c e l l w a l l breakdown a t the time of monospore r e l e a s e i s the r e s u l t o f enzymatic d i g e s t i o n . Such enzymes may be r e l e a s e d by the f i b r o u s v e s i c l e s . 1 3 7 C. Sexual Reproduction In Porphyra g a r d n e r i * I n t r o d u c t i o n The occurrence of s e x u a l r e p r o d u c t i o n i n the genus Porphyra has long been debated by p h y c o l o g i s t s . I t was f i r s t r e p o r t e d by B e r t h o l d (1882) f o r Porphyra l e u c o s t i c t a , and s e v e r a l subseguent i n v e s t i g a t o r s of other s p e c i e s concurred (Ishikawa, 1921; Grubb, 1924; Hamel, 1924; Dangeard, 1927; Kunieda, 1939; Hagne, 1952; Tseng and Chang, 1955; Kurogi, 1961, 1972; Yabu and Tokida, 1963; M i g i t a , 1967b; Giraud and Magne, 1968; and Yabu, 196 9b). However, Hus (1902) r e p o r t e d t h a t he could f i n d no evidence of s e x u a l r e p r o d u c t i o n , and other workers thought t h a t the evidence f o r i t was i n c o n c l u s i v e (Drew, 1954a; Krishnamurthy, 1959 ; Conway, 1964a, 1964b; Graves, 1969; Dixon, 1970, 1973; Conway and Cole, 1973, 1977; Conway e t a l . 1975). Conway (1964a) suggested t h a t because of the l a c k of c o n c l u s i v e evidence of sexual r e p r o d u c t i o n the two types of spore formed i n •packets' i n Porphyra should not be r e f e r r e d to as spermatia and carpospores. She proposed the terms -spore t o r e p l a c e spermatium and c^-spore to r e p l a c e carpospore. , I n a d d i t i o n to t h i s , the terms 'carpospore* mother c e l l (Dixon, 1973) and O^-spore mother c e l l (Conway et-a l . 1975; Hawkes, 1977b) have been used i n s t e a d of *This p o r t i o n of the t h e s i s has been accepted f o r p u b l i c a t i o n i n P hycologia and i s ' i n press* i n V. 17(3) . 138 carpogonium 1. The term J& -spore has met with c r i t i c i s m (McDonald, 1972) because the non-pigmented c e l l s t h a t i t r e f e r s to do not germinate and are t h e r e f o r e not spores. The m a j o r i t y o f c y t o l o g i c a l s t u d i e s have provided evidence of sexual r e p r o d u c t i o n . Magne (1952) reported a d i p l o i d chrcmoscme number f o r the carpospores of P. l i n e a r i s and subseguent workers have obtained d i p l o i d counts f o r the carpospores i n 22 other s p e c i e s (Table V I I I ) . D i p l o i d chromosome counts have a l s o been r e p o r t e d f o r the v e g e t a t i v e c e l l s of the c o n c h o c e l i s phase i n 9 Porphyra s p e c i e s (Table VIII) . There i s seme d i f f e r e n c e of o p i n i o n regarding the s i t e of meiosis. According t o Ishikawa (1921), Dangeard (1927) and Tseng and Chang (1955) the f i r s t d i v i s i o n of the f e r t i l i z e d carpogonium i s m e i o t i c and the carpospores are h a p l o i d , however, M i g i t a (1967b), Giraud and Magne (1968) and K i t o (1974) found t h a t t h e d i v i s i o n t c form the conchospores i n the cc n c h o s p o r a n g i a l branch i s the s i t e o f me i o s i s . The data which have been pointed to as evidence a g a i n s t the occurrence o f s e x u a l r e p r o d u c t i o n c o n s i s t s of K r i s h n a m u r t h y ^ (1959) r e p o r t that a l l l i f e h i s t o r y stages of !• purpurea (as P. u m b i l i c a l i s var. l a c i n i a t a ) are h a p l o i d * , and a r e p o r t by Conway and Cole (1973) t h a t the c o n c h o c e l i s lThe s t u d i e s which are being r e p o r t e d i n t h i s t h e s i s i n d i c a t e t h a t there i s no lon g e r any reason to continue to use the terminology proposed by Conway (1964a) f o r Porphyra g a r d n e r i . The terms spermatium, carpogonium, p r o t o t r i c h o g y n e and carpospores, which have p r e v i o u s l y been a p p l i e d t o Por Phyra. w i l l be used. See the terminology s e c t i o n f o r f u r t h e r comments. 2 I n c o n t r a s t K i t o e t a l . (1971) obtained a d i p l o i d count f o r the carpospores o f t h i s s p e c i e s . 139 phase of P. p a p e n f u s s i i i s h a p l o i d . The r e p o r t s of Janczewski (1873) that both carpospores and spermatia can o r i g i n a t e from the same carpogonium l e d Drew (1954a) and Conway e t a l . (1975) to express doubt t h a t they play a r o l e i n se x u a l r e p r o d u c t i o n . 1. The Spermatium as a f i r s t s tep i n attempting t o c l a r i f y t h i s problem, the nature c f the spermatium i n Porphyra would seem t o be of c o n s i d e r a b l e importance. Krishnamurthy (1959) commented cn the s i m i l a r i t y of mature spermatia of Porp.hy.ra with those i n the F l o r i d e o p h y c i d a e , and pointed out the need f o r f u r t h e r c y t o l o g i c a l i n v e s t i g a t i o n o f the spermatium. Spermatangial development has been s t u d i e d by s e v e r a l i n v e s t i g a t o r s u s i n g the l i g h t microscope (Janczewski, 1873; Thuret and Bornet, 1878; B e r t h o l d , 1882; Hus, 1902; Ishikawa, 1921; Tanaka, 1952; Krishnamurthy, 1959, 1972). These s t u d i e s d i d l i t t l e more than i n d i c a t e t h a t the spermatia a r e fornsed by repeated d i v i s i o n c f the spermatangium, and the data are c o n t r a d i c t o r y i n t h a t Janczewski (1873), Thuret and Bornet (1878) and Ishikawa (1921) maintained t h a t the f i r s t d i v i s i o n of the spermatangium was p e r i c l i n a l ( in the plane o f the t h a l l u s ) whereas Tanaka (1952) and Krishnamurthy (1959) thought t h a t i t was a n t i c l i n a l . Hus (1902) r e p o r t s t h a t t h e r e i s a • c r u c i a t e ^ d i v i s i o n to give f o u r spermatangia, and t h a t the f i r s t d i v i s i o n o f each spermatangium was p e r i c l i n a l . as Drew (1S56) pointed out, these v a r i o u s i n t e r p r e t a t i o n s are perhaps due to the d i f f i c u l t y i n d i s t i n g u i s h i n g the spermatangium from a v e g e t a t i v e c e l l . 140 L i t t l e i s known about the i n t e r n a l s t r u c t u r e c f the spermatiufii i n Porphyra. Hus (1902) was of the o p i n i o n t h a t , except f o r the l a r g e r number of d i v i s i o n s t h a t produce the spermatia, t h e r e i s l i t t l e t o d i f f e r e n t i a t e them from the carpospores. Krishnamurthy (1959) suggested t h a t the spermatia and carpospores were homologous based on the f a c t t h a t they a re formed by repeated d i v i s i o n s . Most workers mention the f a c t t h a t the spermatia tend to become l e s s pigmented as they develop. flagne (1952), Krishnamurthy (1959), Hawkes (1977b) and Mumford and Cole (1977) r e p o r t t h a t the nucleus o f the mature spermatium i s i n a condensed s t a t e . P e y r i e r e (1974) mentions that G, and J . Feldmann have seen abundant f i b r o u s v e s i c l e s i n the spermatia of Porphyra l e u c o s t i c t a . Sather s u r p r i s i n g l y only a few u l t r a s t r u c t u r a l s t u d i e s of Bangiophycidae spermatia have been made (McBride, 1972 Smithora; McDonald, 1972 - Bangia). In c o n t r a s t to t h i s , numerous u l t r a s t r u c t u r a l s t u d i e s have now been made of spermatium development i n s e v e r a l genera of the F l o r i d e o p h y c i d a e (Brown, 1969 - Batrachospermum: Kugrens and West* 1972 - L e v r i n q i e l l a and E r y t h r o c v s t i s : Kugrens, 1974 -Janczewskia j P e y r i e r e , 1971, 1974 - G r i f f 1th s i a, P cjLj sipjb_Q n i a, l a u r e n c i a , Polyneura and F u r c e l l a r i a : S c o t t and Dixon, 1973b -£tilota; Simon-Bichard-Breaud, 1971, 1972a, 1972b Eonnemaisonia: Young, 1977 - Bonnemaisonia). These s t u d i e s i n d i c a t e d t h a t the c h a r a c t e r i s t i c f e a t u r e s of spermatium formation and maturation are the production of f i b r o u s v e s i c l e s , and a g e n e r a l r e d u c t i o n i n c h l o r o p l a s t s i z e and complexity ( i n those which have c h l o r o p l a s t s ) . I n most cases 141 the nucleus of the nature spermatium i s i n a condensed s t a t e . S t a r c h i s u s u a l l y absent i n the mature spermatium {Young, 1977). I f the spermatium i n Porphyra i s i n f a c t f u n c t i o n i n g as a male gamete, as has been c l a s s i c a l l y s t a t e d ( B e r t h o l d , 1882), then one would expect i t would perhaps be u l t r a s t r u c t u r a l l y s i i i l a r t o the spermatium i n the F l o r i d e o p h y c i d a e . A study of spermatium development i n Porphyra g a r d n e r i was undertaken as the f i r s t s t e p i n attempting to determine i f s e x u a l r e p r o d u c t i o n occurs i n the l i f e h i s t o r y of t h i s a l g a . 2. Beports Of F e r t i l i z a t i o n The most v i t a l p i e c e o f i n f o r m a t i o n that has not been c o n c l u s i v e l y demonstrated, u n t i l my work with Porphyra g a r d n e r i , i s the a c t u a l occurrence of a sexual f u s i o n . Derbes and S o l i e r (1856) and Koschtsug ( a c c o r d i n g t o Drew, 1956) were of the o p i n i o n t h a t the spermatia and carpospores were gametes which underwent f u s i o n a f t e r they had been r e l e a s e d . Krishnamurthy (1959) obtained no evidence of such f u s i o n s , and n e i t h e r have subsequent workers. Drew (1956) summarized the l i t e r a t u r e d e a l i n g with sexual r e p r o d u c t i o n i n the Bangiophycidae. She noted t h a t most r e p o r t s on Porp.hyra i n d i c a t e d t hat a c e l l i n the f o l i o s e t h a l l u s i s transformed i n t o a carpogonium which i s then f e r t i l i z e d by a spermatium, and t h a t the carpospores are produced f o l l o w i n g t h i s f u s i o n . Drew noted t h a t there were two d i f f e r e n t mechanisms c f f e r t i l i z a t i o n d e s c r i b e d , based on the presence or absence o f a p r o t o t r i c h o g y n e produced by the 142 carpogonium. She i n d i c a t e d t h a t i n those s p e c i e s with p r o t o t r i c h o g y n e s the spermatium i s engulfed by i t ( J o f f e , 1896 and Kunieda, 1939 are c i t e d as examples), whereas i n those without p r o t o t r i c h o g y n e s a f i n e c a n a l - l i k e connection e x i s t s between the spermatium and carpogonium ( B e r t h o l d , 1882 and Dangeard, 1927 are c i t e d ) . I t should be noted t h a t the carpogonia B e r t h o l d (1882) f i g u r e d d i d have s l i g h t b i p o l a r protuberances (although he d i d not show the w a l l p r o t r u d i n g ) , and that Bosenvinge (1909) , working on the same s p e c i e s , d i d observe carpogonia with p r o t o t r i c h o g y n e s . S i m i l a r l y , Dacgeard (1927) a l s o f i g u r e d spermatia with f e r t i l i z a t i o n c a n a l s i n t o the p r o t o t r i c h o g y n e s i n one o f the two s p e c i e s he s t u d i e d . Hawkes (1977b) p o i n t e d out t h a t i n most monostromatic s p e c i e s there i s a pr o t o t r i c h o g y n e a t each pole o f the carpogonium, whereas i n d i s t r o m a t i c s p e c i e s , f o r which p r c t o t r i c h o g y n e s have been r e p o r t e d , t h e r e i s onl y one, l o c a t e d on the s i d e o f the carpogonium next t o the t h a l l u s s u r f a c e . S e v e r a l workers ( J o f f e , 1896; Bosenvinge, 1909; Dangeard, 1927; flagne, 1952; Tseng and Chang, 1955; Kurogi, 1961) have f i g u r e d spermatia attached t o carpogonia v i a f e r t i l i z a t i o n c a n a l s , Conway and Co l e (1973) reported suspected spermatia i n a s s o c i a t i o n with the p r o t o t r i c h o g y n e of P. p a p e n f u s s i i but they d i d not observe f e r t i l i z a t i o n c a n a l s . S i m i l a r l y , Krishnamurthy (1959) observed spermatia on the t h a l l u s s u r f a c e of P. purpurea (as P. u m b i l i c a l i s var. l a c i n i a t a ) but d i d not see f e r t i l i z a t i o n c a n a l s . He d i d r e p o r t s e e i n g c a n a l s but without spermatia over top of them. He s a i d t h at they ended b l i n d l y i n the mucilaginous sheath of the t h a l l u s or i n the i n t e r c e l l u l a r 143 l a y e r . Graves (1969) repo r t e d o c c a s i o n a l carpogonia with p r o t o t r i c h o g y n e s i n P. capensis K i i t z i n g emend. Agardh but f e l t t h a t the phenomenon was not common and never found spermatia a s s o c i a t e d with the p r o t o t r i c h o g y n e s . Drew (1956) summarized the c y t o l o g i c a l evidence f o r f e r t i l i z a t i o n up to that time. I t c o n s i s t e d of a r e p o r t by J o f f e (1896) o f two n u c l e i i n a c e l l , and Dangeard»s (1927) i l l u s t r a t i o n of two adjacent c e l l s , one of which has a spermatium attached to i t s c u t e r w a l l and a f e r t i l i z a t i o n c a n a l . In t h i s c e l l what Dangeard i n t e r p r e t e d as a male nucleus i s i n c o n t a c t with the c a r p o g o n i a l nucleus. In the neighbouring c e l l t h e r e i s a s i n g l e l a r g e nucleus which i s assumed to be a f u s i o n nucleus. Magne (1952) r e p o r t e d a nucleus i n prophase i n t h e f e r t i l i z a t i o n c a n a l c o n n e c t i n g the spermatium with the carpogonium, and was the f i r s t t o r e p o r t d i p l o i d carpospores. The o n l y r e c e n t work has been done by Yabu and Tokida (1963) on P. yezoensis,and Yabu (1969b) on P. t e n e r a . They f i g u r e a male and female nucleus i n c l o s e c o n t a c t with each other i n the carpogonium j u s t p r i o r t o f u s i o n . 3. Reports Of f u n g a l I n f e c t i o n Of Porphyra Dangeard's (19 27) r e p o r t of spermatia forming f e r t i l i z a t i o n c a n a l s l e d Kunieda (1939) to suggest t h a t what Dangeard had observed was not a s e x u a l f u s i o n but the p a r a s i t i z a t i o n cf the Porphyra t h a l l u s by an oomycetbus fungus. His o n l y b a s i s f o r making t h i s statement was t h a t he had observed w a l l p e n e t r a t i o n s i n the v e g e t a t i v e c e l l r e g i o n which 144 he thought were caused by a fungus, As has been pointed out by F r i t s c h (1945, p. 434), Kunieda d i d not provide any c o n v i n c i n g evidence f o r t h i s fungus theory. A p o i n t t h a t has been overlooked by Dixon (1970, 1973) i s t h a t Kunieda d i d b e l i e v e that a s e x u a l f u s i o n o c c u r r e d . Kunieda thought t h a t the p r c t o t r i c h o g y n e engulfed the e n t i r e spermatium and disagreed with Dangeard's concept t h a t the contents of the spermatium reached the carpogonium by a f e r t i l i z a t i o n c a n a l . Recently Krishnamurthy (1977) a l s o suggested t h a t a f u n g a l i n f e c t i o n c o u l d be mistaken f o r the; f e r t i l i z a t i o n of a carpogcniua; however no c o n v i n c i n g evidence was presented. Hus (1902) added to the s c e p t i c i s m surrounding the r e p o r t s of f e r t i l i z a t i o n . He commented t h a t b a c t e r i a i n f e s t i n g the walls of a l l r e g i o n s of t h a l l i he examined formed narrow l i n e s p e r p e n d i c u l a r t c the s u r f a c e of the f r o n d s , which he s a i d reminded him of the f e r t i l i z a t i o n c a n a l s f i g u r e d by B e r t h o l d (1882). No evidence was given. The only f u n g i t h a t have been .reported to i n f e c t the Porphyra t h a l l u s are s p e c i e s of Pythium (Ocmycetes) ( A r a s a k i , 1947, 1962; Tsuruga and N i t t a , 1960; F u l l e r et a l . 1966; Sasaki and Sato, 1969; Kazama and F u l l e r , 1970; S a s a k i and S a k u r a i , 1972; Sakurai e t a l . 1974; Takahashi e t a l . 1977) and a c h y t r i d ( A r a s a k i , 1960; A r a s a k i e t a l . 1960; fligita, 1969, 1973; Sparrow, 1969). In the case of the Pythium i n f e c t i o n (known as the 'red r o t ' d i s e a s e i n Japan), the f u n g a l hyphae penetrate the host c e l l s and cause a r a p i d p l a s m o l y s i s of them, r e s u l t i n g i n the formation of red pigment c r y s t a l s (Ogata, 1975). The i n f e c t i o n i s v i s i b l e t o the unaided eye and does 145 not bear any resemblance t o the r e p o r t s o f f e r t i l i z a t i o n . In the case o f the c h y t r i d , only s e t t l e d zoospores a t t h e i r i n i t i a l stage of i n f e c t i o n c o u l d be mistaken f o r a spermatium forming a f e r t i l i z a t i o n c a n a l . I t i s i n t e r e s t i n g to note that A r a s a k i {1960) s p e c u l a t e d t h a t Yendo*s {1919) r e p o r t of male and female swarming gametes from Porphyra c o n c h o c e l i s may be a t t r i b u t a b l e t o a c h y t r i d i n f e c t i o n , but that he d i d not make a s i i i i l a r statement r e g a r d i n g B e r t h o l d ' s (1882) and Dangeard*s (1927) r e p o r t s of f e r t i l i z a t i o n . Furthermore, Ogata (1975), i n a d i s c u s s i o n o f the pathology of Porphyra, made no mention t h a t the r e p o r t s of these workers are a t t r i b u t a b l e t o a f u n g a l i n f e c t i o n . In view o f the poor documentation t h a t the reported spermatia and t h e i r f e r t i l i z a t i o n c a n a l s were a c t u a l l y a fungal i n f e c t i o n , i t became apparent t o me that the method of f e r t i l i z a t i o n as proposed by B e r t h o l d (1882) merited c l o s e r examination. I t was t h e r e f o r e decided to t r y to f i n d f e r t i l i z a t i o n i n Porphyra g a r d n e r i and to examine i t using the l i g h t , t r a n s m i s s i o n and scanning e l e c t r o n microscopes i n an attempt t o r e s o l v e the problem. 146 R e s u l t s 1. Spermatogenesis - E l e c t r o n Microscopy Some general u l t r a s t r u c t u r a l f e a t u r e s of spermatogenesis were observed i n t r a n s v e r s e s e c t i o n . The v e g e t a t i v e c e l l (Figure 20a) con t a i n e d a s t e l l a t e c h l o r o p l a s t with a c e n t r a l l y l o c a t e d pyrenoid. The nucleus was l a t e r a l l y l o c a t e d . The new w a l l that was l a i d down around a vegetative c e l l undergoing the t r a n s i t i o n to a spermatangiua can be c l e a r l y seen i n Fi g u r e 20b. U l t r a s t r u c t u r a l l y the immature spermatia d i d not d i f f e r much from v e g e t a t i v e c e l l s ( F i g u r e s 20c and 20d)., The c h l o r o p l a s t was s t i l l w e l l developed and f l o r i d e a n s t a r c h g r a i n s were p r e s e n t . However, around the time of the f i n a l d i v i s i o n t o g i v e a mature spermatangium l a r g e f i b r o u s v e s i c l e s appeared (Figure 21a). At higher m a g n i f i c a t i o n (Figure 21b) s m a l l f i b r o u s v e s i c l e s were apparent as w e l l . These appeared to be r e l e a s i n g t h e i r contents t o the o u t s i d e o f , the spermatium, as w e l l as c o n t r i b u t i n g t o the formation of the l a r g e f i b r o u s v e s i c l e . Also at t h i s stage of maturation the c h l o r o p l a s t was c o n s i d e r a b l y reduced i n s i z e and s t r u c t u r a l complexity. I t had a degenerate appearance, t h e r e being only a few t h y l a k o i d s which were o f t e n d i s t o r t e d , and a few to s e v e r a l p l a s t o g l o b u l i . Mitochondria were a l s o present, and the nucleus possessed a nuclear membrane. P r i o r t o spermatium l i b e r a t i o n the l a r g e f i b r o u s v e s i c l e became e x t r a c y t c p l a s m i c ( F i g u r e 21c), and the nucl e a r membrane began t o di s a p p e a r . The mature spermatium (Figure 22) contained a nucleus which was i n a h i g h l y condensed s t a t e , and lac k e d a nucl e a r membrane. Small 147 f i b r o u s v e s i c l e s were abundant and a reduced c h l o r o p l a s t was present. Most spermatia d i d not possess f l o r i d e a n s t a r c h g r a i n s . At the time of r e l e a s e , there was no c e l l w a l l around the spermatium; i t was surrounded by a plasmamembrane and the contents of the l a r g e v e s i c l e s . The r e l e a s e d spermatia v a r i e d from 3-5 um i n diameter, most being 4 um. 2. F e r t i l i z a t i o n - L i g h t Microscopy While examining the e a r l y stages of carpogonium formation i n order to assess the v a l i d i t y of the genus P o r p h y r e l l a (Hawkes, 1977a, 1977b), numerous examples of suspected 'spermatia' a t t a c h i n g over the p r o t o t r i c h o g y n e s of the carpogonia were observed (Figure 23a). They had a very strong s i m i l a r i t y to B e r t h o l d ' s (1882) i l l u s t r a t i o n s of f e r t i l i z a t i o n i h Porphyra l e u c o s t i c t a . These 'spermatia* were only found a t t a c h e d to the p r o t o t r i c h o g y n e s , and they were i n t h e 3-5 um i n diameter s i z e range of the r e l e a s e d spermatia. In a d d i t i o n to t h i s , t here were f e r t i l i z a t i o n c a n a l s c o n n e c t i n g the •spermatia* with the i n s i d e of the carpogonium (Figures 23b and 23c). T h e i r s p e c i f i c attachment and p e n e t r a t i o n s t r o n g l y suggested t h a t they were spermatia; however, i t was impossible t c prove t h i s c o n c l u s i v e l y without using the e l e c t r o n microscope. 148 3. F e r t i l i z a t i o n - E l e c t r o n Microscopy Two scanning e l e c t r o n micrographs of the t h a l l u s s u r f a c e °f Porphyra g a r d n e r i are shown i n F i g u r e s 24a and 24b. The suspected spermatia are c l e a r l y v i s i b l e over the p r o t o t r i c h o g y n e s . Seme have a sunken and s h r i v e l l e d appearance, probably as a r e s u l t of having a l r e a d y r e l e a s e d t h e i r c o n t e n t s i n t o the carpogonium. Cyanophyta are a l s o present on the t h a l l u s s u r f a c e . A low m a g n i f i c a t i o n micrograph of a t r a n s v e r s e s e c t i o n through one of these spermatia and the carpogonium i s shewn i n F i g u r e 25a. Other examples a t a higher m a g n i f i c a t i o n to show the contents of the spermatia are shown i n F i g u r e s 25b-25d. In these, the presence cf a reduced, degenerate l o c k i n g c h l o r o p l a s t t y p i c a l of that found i n the mature spermatium demonstrates t h a t they are spermatia and not f u n g i . Mitochondria and non-membrane bound nuclear m a t e r i a l are present as w e l l . In F i g u r e s 25b and 25c a t h i n l a y e r of w a l l m a t e r i a l can be seen around the spermatium. The next p o i n t t o c l a r i f y i s whether anything i s t r a n s f e r r e d from the spermatium to the carpogonium. F i g u r e 26 shews two empty spermatia and t h e i r f e r t i l i z a t i o n c a n a l s . The s p e r m a t i a l n u c l e a r m a t e r i a l has been t r a n s f e r r e d to the carpogenium. The diameter of the f e r t i l i z a t i o n c a n a l was 0 . 6 - 0 . 9 um i n the s e c t i o n s examined. F i g u r e 27 shows another t r a n s v e r s e s e c t i o n cf a carpogonium i n which the p r o t o t r i c h o g y n e s and c e n t r a l pyrenoid are d i s t i n c t . The plane of s e c t i o n does not go through the f e r t i l i z a t i o n c a n a l ; however, the presence of a s l i g h t d e p o s i t of w a l l m a t e r i a l i n 149 the p r o t o t r i c h o g y n e below the spermatium i n d i c a t e s that a f e r t i l i z a t i o n c a n a l i s present. Some spermatia that have t r a n s f e r r e d t h e i r n u c l e a r m a t e r i a l r e t a i n what appears to be the remains of a c h l o r o p l a s t ( F i g u r e s 28a and 28b). These c h l o r o p l a s t s have a few d i s t o r t e d t h y l a k o i d s and some p l a s t o g l o b u l i . F o l l o w i n g the t r a n s f e r of the n u c l e a r m a t e r i a l , the f e r t i l i z a t i o n c a n a l i s r a p i d l y plugged by new w a l l m a t e r i a l d e p o s i t e d around i t (Figure 28b). T h i s appears to o r i g i n a t e from dictyosome a c t i v i t y i n the apex of the carpogonium. F i g u r e 28c i s a t r a n s v e r s e s e c t i o n through an empty spermatium and apex of the carpogonium. O n f o r t u n a t l y the plane of s e c t i o n does not go through the f e r t i l i z a t i o n c a n a l . In the apex of the carpogonium i s a s m a l l reduced c h l o r o p l a s t t y p i c a l of the c h l o r o p l a s t found i n the mature spermatium. Another example of t h i s i s shown i n F i g u r e 28d. I t would appear t h a t these c h l o r o p l a s t s have been t r a n s f e r r e d from the spermatium to the carpogonium along with the s p e r m a t i a l n u c l e a r m a t e r i a l . Carpogonia which co n t a i n e d what appeared to be h i g h l y condensed s p e r m a t i a l n u c l e a r m a t e r i a l were observed (Figure 29), but because of the apparent l a c k of a n u c l e a r membrane around the s p e r m a t i a l nucleus I was unable t o f o l l o w the d e t a i l s o f f u s i o n with the c a r p o g o n i a l nucleus a t an u l t r a s t r u c t u r a l l e v e l . The f i r s t d i v i s i o n of the presumed f u s i o n nucleus, and c y t o k i n e s i s to g i v e 2 carpospores, i s shown i n F i g u r e s 30a and 30b. The f e r t i l i z a t i o n c a n a l i s s t i l l v i s i b l e . 150 4. Chromosome Counts Evidence t h a t a f u s i o n o f s p e r m a t i a l n u c l e a r m a t e r i a l with the c a r p o g o n i a l nucleus does occur has been obtained from chromosome counts. Hawkes (1977b) repo r t e d t h a t the v e g e t a t i v e c e l l s of the f o l i o s e t h a l l u s and the spermatia have 4 chromosomes (n=4) (Fi g u r e s 31a and 31b) i In both the carpospores and v e g e t a t i v e c e l l s of the c o n c h o c e l i s phase a d i p l o i d chromosome number 2n=8 has been obtained ( F i g u r e s 31c,d and 3 1 e , f ) . 5. Feulgen S t a i n i n g In an attempt t o c l a r i f y t h e d e t a i l s of the t r a n s f e r of the s p e r m a t i a l n u c l e a r m a t e r i a l to the carpogonium, t r a n s v e r s e s e c t i o n s o f c a r p o g o n i a l r e g i o n s of the t h a l l u s were s t a i n e d u s i n g the Feulgen technique-. I n i t i a l l y the s t a i n was used on the spermatia. F i g u r e 32a shows a spermatangium which has undergone the maximum number of d i v i s i o n s , but the nucleus i n most spermatia i s s t i l l i n a d i f f u s e i n t e r p h a s e s t a t e . The condensing of the nucleus i s complete i n Fig u r e 32b. The r e l e a s e d spermatia l i k e w i s e have condensed n u c l e a r m a t e r i a l which s t a i n s a dense purple (Figure 32c). These o b s e r v a t i o n s agree with the 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 of spermatogenesis. In Figure 33a a spermatium c o n t a i n i n g condensed n u c l e a r m a t e r i a l can be seen attached to the p r o t o t r i c h o g y n e . No f e r t i l i z a t i o n c a n a l i s present. In F i g u r e 33b the s p e r m a t i a l n u c l e a r m a t e r i a l i s i n a d i f f u s e s t a t e and a f e r t i l i z a t i o n c a n a l i s present., F i g u r e s 3 3c and 33d show condensed 151 s p e r m a t i a l n u c l e a r m a t e r i a l and f e r t i l i z a t i o n c a n a l s are present. The c o r r e c t time sequence f o r Fig u r e s 33b-d i s not known. I t may be that the s p e r m a t i a l n u c l e a r m a t e r i a l deccndenses i n order t o code f o r an enzyme r e q u i r e d t c make the f e r t i l i z a t i o n c a n a l , and then re-condenses f o r the t r a n s f e r t o the carpogonium. Another e x p l a n a t i o n would be that the f e r t i l i z a t i o n c a n a l enzymes are present (or at l e a s t coded f o r ) i n the r e l e a s e d spermatium, and that the s p e r m a t i a l n u c l e a r m a t e r i a l remains condensed u n t i l the c a n a l i s complete, t h i s being f o l l o w e d by de-condensation and t r a n s f e r to the carpogonium. Once t r a n s f e r has o c c u r r e d the s p e r m a t i a l n u c l e a r m a t e r i a l shews up i n the carpogonium as a small densely s t a i n i n g purple mass (Figure 33e). I t i s not uncommon t o see from one t o three of these i n a d d i t i o n to the c a r p o g o n i a l nucleus (F i g u r e s 33f and 33g). O c c a s i o n a l l y up to 4 or 5 have been observed (Figures 33h and 3 3 i ) . On the b a s i s of the d i p l o i d chromosome number, and the f a c t t h at dense s p e r m a t i a l n u c l e a r masses are not found i n subseguent st a g e s , i t i s assumed t h a t one of them f u s e s with the c a r p o g o n i a l nucleus, and t h a t the r e s t degenerate. I t has not been p o s s i b l e t c observe t h i s f u s i o n ; however, carpogonia with l a r g e suspected f u s i o n n u c l e i have f r e q u e n t l y been seen (Figure 3 3 j ) . As has been shown at an u l t r a s t r u c t u r a l l e v e l , the f i r s t d i v i s i o n c f the f u s i o n nucleus i s p e r i c l i n a l ( F i g u r e 33k). The c h l o r o p l a s t and pyrenoid then d i v i d e f o l l o w e d by c y t o k i n e s i s (Figure 331) to g i v e 2 carpospores. Subseguent d i v i s o n s to g i v e 4 and 8 carpospores per 152 carposporangium are shown i n F i g u r e s 33m-o. A summary of the d i v i s i o n sequence i n v o l v e d i n carpospore fo r m a t i o n by Porghyra g a r d n e r i has been r e p o r t e d p r e v i o u s l y (Hawkes, 1977b) (see Part I I ) . D i s c u s s i o n 1. Spermatogenesis The main f e a t u r e s of spermatogenesis i n Porphyra g a r d n e r i are e s s e n t i a l l y the same as those r e p o r t e d by McDonald (1972) f o r Bangia fuscopurpurea (Dillwyn) Lyngbye, and s i m i l a r i n some r e s p e c t s t o the r e p o r t of McBride (1972) f o r t h e spermatia of Smithgra naiadum. U n f o r t u n a t l y n e i t h e r o f these workers i n d i c a t e d i f the n u c l e a r membrane remained i n t a c t i n the mature spermatium. There are some s t r i k i n g s i m i l a r i t i e s between spermatogenesis i n the F l o r i d e o p h y c i d a e and i n R2I£hlL\sk g a r d n e r i . The most c h a r a c t e r i s t i c f e a t u r e t h a t they have i n common i s the production of small and l a r g e f i b r o u s v e s i c l e s , and the r e l e a s e of the l a r g e f i b r o u s v e s i c l e p r i o r to spermatium l i b e r a t i o n . The r e d u c t i o n i n c h l o r o p l a s t s i z e and complexity i n the mature spermatium i s another f e a t u r e they have i n common., Kugrens (1974) notes t h a t t h i s i s a general phenomenon i n male gametes of algae i n s e v e r a l qroups. Another s i n i l a r i t y i s the q e n e r a l absence of s t a r c h q r a i n s i n the mature spermatia. Kuqrens (1974) has pointed out t h a t one p o s s i b l e e x p l a n a t i o n f o r the r e d u c t i o n i n c h l o r o p l a s t s t r u c t u r e 153 and storage product i s to prevent the male gamete from developing on i t s own, thereby bypassing sexual r e p r o d u c t i o n . Most workers r e p o r t that the nucleus of the mature spermatium i n the F l o r i d e o p h y c i d a e c o n t a i n s condensed chromatin (Grubb, 1925; F r i t s c h , 1945, p. 596; Kugrens and l e s t , 1972; P e y r i e r e , 1974; Young, 1977), as i s the case i n P. g a r d n e r i . S c c t t and Dixon (1973b) were u n c e r t a i n of the nu c l e a r c o n d i t i o n i n P t i l o t a s p ermatia, but judging from t h e i r micrographs, condensed chromatin i s present. The nucleus i n Janczewskia spermatia (Kugrens, 1974) does not c o n t a i n condensed chromatin. The disappearance of the nu c l e a r membrane i n the mature spermatium of Porphyra g a r d n e r i i s r a t h e r unexpected. T h i s has p r e v i o u s l y been r e p o r t e d at the l i g h t microscope l e v e l i n s e v e r a l F l o r i d e o p h y c i d a e (Grubb, 1925; F r i t s c h , 1945, p. 596) and at the u l t r a s t r u c t u r a l l e v e l by S c o t t and Dixon (1973b). Kugrens (1974) guestioned t h i s because he found i t d i f f i c u l t to e x p l a i n why the nuclear membrane would disappear i n the spermatium when there i s l i t t l e n u c l e a r membrane breakdown during m i t o s i s i n the Rhodophyta. The case may be that the nucle a r membrane i s p a r t i c u l a r l y s e n s i t i v e at t h i s stage of development and t h a t i t t h e r e f o r e was not preserved by the f i x a t i o n procedure used. A l t e r n a t i v e l y , i n those cases where an i n t a c t n u c l e a r membrane has been r e p o r t e d , perhaps c i d e r r e l e a s e d spermatia should have been examined to see i f the membrane does e v e n t u a l l y disappear. For the F l o r i d e o p h y c i d a e spermatia, many workers r e p o r t o n l y dictyosome involvement i n the formation of the f i b r o u s v e s i c l e s ( P e y r i e r e , 1971; Sc o t t and Dixon, 1973b; 154 Simon-Bichard-Breaud, 1972a); however, Kugrens and West (1972), Kugrens (1974) and Young (1977) have i n d i c a t e d t h a t the EB i s i n v o l v e d i n the e a r l y stages i n the s p e c i e s they s t u d i e d . The f i b r o u s v e s i c l e s i n the spermatia of P. g a r d n e r i may be mostly d e r i v e d from EB as few dictyosomes were observed during spermatogenesis. Reports on the chemical nature and f u n c t i o n of the f i b r o u s v e s i c l e s have been summarized by S c o t t and Dixon (1973b). They appear to c o n t a i n a mucopolysaccharide, although more c r i t i c a l a n a l y s i s i s r e g u i r e d to c o n f i r m t h i s . One suggested f u n c t i o n of the f i b r o u s v e s i c l e s i s that they may cause an i n c r e a s e i n the osmotic pressure r e s u l t i n g i n the r u p t u r i n g of the spermatangial w a l l and thereby a i d i n g i n spermatium r e l e a s e . L i k e w i s e , the mucopolysaccharide may a i d i n spermatium attachment to the t r i c h o g y n e . Recently, mucilage s t r a n d s have been shown t o be i n v o l v e d i n spermatium r e l e a s e and d i s p e r s a l i n T i f f a n i e l l a snyderae (Farlow) Abbott ( F e t t e r , 1977). The widespread occurrence of s i m i l a r f i b r o u s v e s i c l e s i n other red a l g a l spores such as the monospores of Smithora naiadum (McBride and C o l e , 1971), Porphyropsis c o c c i n e a (McDonald, 1972) and Porphyra g a r d n e r i see Part I V ) , carpospores of Porphyra n e r e o c y s t i s (Hawkes, unpublished o b s e r v a t i o n ) , L e v r i n g i e l l a and E r v t h r o c y s t i s (Kugrens and West, 1973, 1974) and P o l y s i p h o n i a novae-anqliae T a y l o r (Wetherbee and Wynne, 1973) and t e t r a s p o r e s of P t i l o t a hypnoides (Sc o t t and Dixon, 1973a) i n d i c a t e s t h a t they must have an important f u n c t i o n . 155 2. F e r t i l i z a t i o n T h i s i s the f i r s t c o n c l u s i v e documentation of the occurrence of a s e x u a l f u s i o n i n a s p e c i e s of Porphyra. and except f o r Hagne's (1960) e x c e l l e n t work on s e x u a l r e p r o d u c t i o n i n Bhodochaete parvula Thuret i t i s the only uneguivocal r e p o r t of f e r t i l i z a t i o n i n the Bangiophycidae. i t confirms t h a t the c l a s s i c a l i n t e r p r e t a t i o n by B e r t h o l d (1882) of sexual r e p r o d u c t i o n i n Porphyra l e u c o s t i c t a i s c o r r e c t . S i m i l a r l y , the o b s e r v a t i o n s by Bosenvinge (1909), Ishikawa (1921), Dangeard (1927), Magne (1952), Tseng and Chang (1955), Kurogi (1961), Yabu and T o k i d a (1963), M i g i t a (1967b) , and Yabu (1969b) of the f e r t i l i z a t i o n process i n other Porphyra s p e c i e s appear to be b a s i c a l l y c o r r e c t . The r e p o r t s o f Ishikawa (1921), Dangeard (1927) and Tseng and Chang (1955) of the f i r s t d i v i s o n o f the carpogonium being m e i o t i c are probably i n c o r r e c t . The mechanisms of f e r t i l i z a t i o n proposed f o r Porphyra by Derbes and S o l i e r (1856), Koschtsug ( c i t e d from Drew, 1956), Suto (1963) and Kunieda (1939) are probably i n c o r r e c t . Dixon*s (1973) suggestion t h a t f e r t i l i z a t i o n and the d i v i s i o n of the carpogonium t o produce carpospores are separated i n time by s e v e r a l or even many v e g e t a t i v e c e l l d i v i s i o n s i s l i k e w i s e not the case. The r e p o r t s by Krishnamurthy (1959), Conway and Cole (1973, and i n press) and Mumford and Cole (1977) of a h a p l o i d chromosome number i n the v e g e t a t i v e c e l l s o f the c o n c h o c e l i s phase of Porphyra are not n e c e s s a r i l y i n c o r r e c t . In view of the tremendous a d a p t a b i l i t y e x h i b i t e d by the Porphyra l i f e 156 h i s t o r y i t i s p o s s i b l e t h a t carpospore formation c o u l d occur p a r t h e n o g e n e t i c a l l y . S i m i l a r l y , the r e p o r t by Sommerfeld and N i c h o l s (1970) of a h a p l o i d chromosome number i n the c o n c h o c e l i s of Bangia may be due t o a parthenogenetic development of carpogonia; Rosenvinge (1909) observed the formation of carposporangia i n a po p u l a t i o n of p l a n t s which d i d not produce spermatia and i n which he co u l d not f i n d f e r t i l i z a t i o n c a n a l s . He suggested the carpospores developed apcgamously. Kurogi (1972) has pointed out there are some sp e c i e s f o r which he has never observed f e r t i l i z a t i o n c a n a l s . Drew (195tta), Krishnamurthy (1959) and Dixon (1973) have expressed doubt t h a t the number of carpospores which are produced are a l l the product of s e x u a l f u s i o n s . The case may be t h a t seme carpospores can form p a r t h e n o g e n e t i c a l l y whereas others are the r e s u l t of f e r t i l i z a t i o n . F u r t h e r i n v e s t i g a t i o n of t h i s point i s r e q u i r e d . C e r t a i n l y i n Porphyra g a r d n e r i the f u s i o n s are extremely abundant, t h e r e o f t e n beinq s e v e r a l f e r t i l i z a t i o n c a n a l s i n t o one carpoqonium. M u l t i - p e n e t r a t i o n s have a l s o been r e p o r t e d by Rosenvinge (1909) and Danqeard (1927) f o r Porphyra u m b i l i c a l i s . The other p o s s i b l e e x p l a n a t i o n f o r the occurrence o f a h a p l c i d chromosome number i n the v e g e t a t i v e c e l l s o f the c o n c h o c e l i s phase i s that meiosis occurs d u r i n g the f i r s t d i v i s i o n o f the f e r t i l i z e d carpogonium as has been r e p o r t e d by Tseng and Chang (1955); however, th e r e i s no evidence t h a t t h i s t akes p l a c e . Although the s i t e of meiosis i n Porphyra g a r d n e r i has not been i n v e s t i g a t e d yet i t probably occurs i n the conchospore 157 branch as has been reported f o r other Porphyra s p e c i e s by M i g i t a (1967b) Giraud and Magne (1968) and K i t o (1974). In view o f the l a r g e number cf Porphyra s p e c i e s f o r which f e r t i l i z a t i o n has been r e p o r t e d at the l i g h t microscope l e v e l , and i n which d i p l o i d chromosome numbers f o r the carpospores or v e g e t a t i v e c e l l s of the c o n c h o c e l i s phase have been r e p o r t e d , i t would seem reasonable to suggest t h a t sexual r e p r o d u c t i o n i n the genus i s a common occurrence. The a p p l i c a t i o n of technigues such as have been used i n t h i s study of Porphyra g a r d n e r i should be h e l p f u l i n demonstrating f e r t i l i z a t i o n i n other Porphyra s p e c i e s . I t i s expected that f u t u r e i n v e s t i g a t i o n s o f other Porphyra s p e c i e s w i l l show t h a t the t y p i c a l c y t o l o g i c a l l i f e h i s t o r y found i n the genus i s the same as t h a t o u t l i n e d by M i g i t a (1967b) i n h i s e x c e l l e n t work on Porphyra j e z c e j i s i s . The r e p o r t by Janczewski (1873) o f spermatia and carpospores o c c u r i n g i n the same sporangium remains an enigma. T h i s may have been a m i s i n t e r p r e t a t i o n by Janczewski. Such a phenomenon was not observed i n Porphyra g a r d n e r i . According to F r i t s c h (1945, p. 592) the carpogcnius and t r i c h o g y n e i n the F l c r i d e o p h y c i d a e (except c e r t a i n Hemaliales) l a c k c h l o r o p l a s t s . , F r i t s c h (1945, p. 597) a l s o notes t h a t t h e spermatium i n the F l o r i d e o p h y c i d a e t r a n s f e r s i t s c o n t e n t s t o the t r i c h o g y n e s as a r e s u l t of wall d i s s o l u t i o n i n the r e g i o n of contact. T h i s d i f f e r s from the s i t u a t i o n i n Porphyra g a r d n e r i i n which the carpogonium possesses a w e l l developed c h l o r o p l a s t and i n which the c o n t e n t s of the spermatium reach the p r o t o t r i c h o g y n e v i a a f e r t i l i z a t i o n c a n a l . 158 A d e t a i l e d examination and comparison of f e r t i l i z a t i o n at the u l t r a s t r u c t u r a l l e v e l i n suspected p r i m i t i v e members cf the F l c r i d e o p h y c i d a e ( f o r example; Acrochaetium and other Nemaliales) and other members of the Bangiophycidae may provide i n t e r e s t i n g i n s i g h t i n t o the e v o l u t i o n c f the female r e p r o d u c t i v e system i n these two groups of Bhodophyta. Although t h i s i n v e s t i g a t i o n has answered the major q u e s t i o n of the occurrence of s e x u a l r e p r o d u c t i o n i n the l i f e h i s t o r y of Porphyra g a r d n e r i , i t p o i n t s out s e v e r a l areas i n need of f u r t h e r study. What changes does the spermatium undergo from the time o f r e l e a s e to the time c f attachment to the p r o t o t r i c h o g y n e ? One change observed i n t h i s study of g a r d n e r i i s t h a t the attached spermatium possesses a t h i n w a l l which was not present at the time of l i b e r a t i o n . F r i t s c h (1945, p. 597) r e p o r t s t h a t i n Batrachospermum and Nemalion the s p e r m a t i a l nucleus d i v i d e s once i t c o n t a c t s the t r i c h c g y n e . Such a phenomenon was not observed i n P. g a r d n e r i but the p o s s i b i l i t y e x i s t s t h a t i t does d i v i d e once i t enters the carpogonium. The decondensing of the s p e r m a t i a l n u c l e a r m a t e r i a l a f t e r attachment to the p r o t o t r i c h o g y n e needs to be e x p l a i n e d because i t would seem t o argue a g a i n s t the nature s p e r m a t i a l nucleus being i n a prophase s t a t e as has been assumed by most workers (Grubb, 1925; F r i t s c h , 1945, p. 596; Drew, 1951; Magne, 1952; Krishnamurthy, 1959; Mumford and C o l e , 1977). H i s t o c h e m i s t r y may help to c l a r i f y changes t h a t the v e g e t a t i v e c e l l s undergo i n the t r a n s f o r m a t i o n to a carpogonium. The c u t i c l e of v e g e t a t i v e c e l l s of 159 Porphyra u m b i l i c a l i s has been s t u d i e d b i o c h e m i c a l l y (Hanic and C r a i g i e , 1969; Gunawardena and Williamson, 1974) and found t o c o n t a i n a high percentage o f p r o t e i n and g l y c o p r o t e i n . A s i m i l a r i n v e s t i g a t i o n of w a l l chemistry i n the p r o t o t r i c h o g y n e r e g i o n may p r o v i d e i n s i g h t i n t o mechanisms of the s p e c i f i c i t y cf spermatium attachment. An approach along the l i n e s c f the r e c e n t work c a r r i e d out by Evans et a l . (1977) on egg-sperm r e c o g n i t i o n i n Fucoids may be a p p r o p r i a t e here. With modified Feulgen procedures that can be used with e l e c t r o n microscopy (Jurand e t a l . 1958; Bryan and B r i n k l e y , 1963, 1964) i t s h o u l d be p o s s i b l e t o f o l l o w the s p e r a a t i a l n uclear m a t e r i a l i n the carpogonium at an u l t r a s t r u c t u r a l l e v e l . With such a technique i t may be p o s s i b l e to observe the formation of the f u s i o n nucleus. The known morphological and c y t o l o g i c a l l i f e h i s t o r y of Porphyra g a r d n e r i i s summarized i n F i g u r e 34. 160 D. Evidence Of Sexual Reproduction In Porphyra n e r e o c y s t i s and Porphyra t h u r e t i i I n t r o d u c t i o n F o l l o w i n g the d e t a i l e d i n v e s t i g a t i o n of sexual r e p r o d u c t i o n i n Porphyra g a r d n e r i I made a p r e l i m i n a r y survey of P. n e r e o c y s t i s and P. t h u r e t i i f o r evidence of sexual r e p r o d u c t i o n i n t h e i r l i f e h i s t o r i e s . R e s u l t s 1« Porphyra n e r e o c y s t i s The spermatium of Porphyra n e r e o c v s t j s i s u l t r a s t r u c t u r a l l y s i m i l a r to the spermatia of P. g a r d n e r i and the F l c r i d e o p h y c i d a e . Figure 35 shows a spermatium i n a spermatangium near the t h a l l u s margin., Note the reduced c h l o r o p l a s t with p l a s t c g l o b u l i , c e n t r a l n u c l e u s , and abundant sm a l l and l a r g e f i b r o u s v e s i c l e s . At the l i g h t microscope l e v e l spermatia were observed attached to the w a l l over the carpogonia and f e r t i l i z a t i o n c a n a l s were present (Figures 36a and 36b). The c e l l w a l l of £• n e r e o c y s t i s i s t h i c k e r than i n P. g a r d n e r i and as a r e s u l t the f e r t i l i z a t i o n c a n a l s are l o n g e r . Note a l s o t h a t the attachment of spermatia i s not as s p e c i f i c as i n P. g a r d n e r i . perhaps due to the absence of p r o t o t r i c h o g y n e s . An e l e c t r o n 161 micrograph of a t r a n s v e r s e s e c t i o n through a spermatium and a s s o c i a t e d f e r t i l i z a t i o n c a n a l i s shown i n Fi g u r e 36c. Evidence t h a t f e r t i l i z a t i o n occurs has been obtained from chromosome counts. The spermatia and v e g e t a t i v e c e l l s of the f o l i o s e t h a l l u s have 3 chromosomes, n=3 (Figures 37a and 37b), whereas the carposporangia and c o n c h o c e l i s phase have 6 chromosomes, 2n=6 ( F i g u r e s 37d,e and 37f,g). 2* Porphyra t h u r e t i i Spermatia attached to the s l i g h t p r o t o t r i c h o g y n e of the carpogonium and a s s o c i a t e d f e r t i l i z a t i o n c a n a l s were observed at the l i g h t microscope l e v e l ( F i g u r e s 36d and 36e). The spermatia have 2 chromosomes, n=2 (Figure 37c). U n f o r t u n a t l y I have not had s u f f i c i e n t m a t e r i a l or time to obt a i n counts f o r the carpospores or the c o n c h o c e l i s phase. 162 Discussion This preliminary investigation of Porphyra nereocystis and P. t h u r e t i i suggests that sexual reproduction i s a common occurrence i n t h e i r l i f e h i s t o r i e s . By evidence combined with that of other workers strongly suggests that sexual reproduction i n the genus Porphyra i s a widespread phenomenon. 163 Table V I I I . The s p e c i e s of Porphyra f o r which d i p l o i d chromosome numbers have been reported f o r the carpospores or v e g e t a t i v e c e l l s of the c o n c h o c e l i s phase. Taxcn -1. D i p l c i d Carpospores P°££il£a abbottae Krishnamurthy £• dentata Kjellman P. g a r d n e r i (Smith et H o l l e n t e r g ) Hawkes £• kanakaensis Mumford P. k a t a d a i Miura P. k i n o s i t a i (Yamada e t Tanaka) Fukuhara P. k u n i e d a i Kurogi P. l i n e a r i s G r e v i l l e P. miniata (C. Agardh) C. Agardh £• roo££ejttsis Oh mi P. n e r e o c y s t i s Anderson P. okamurai Oeda P. onoi Oeda P. £§£fgrata J . Agardh £• fisejii^linearis Ueda £ • £U££JJ£e§. (Both) C. Agardh P. s c h i z p p h y l l a Hollenberg i n Smith and Hollenberg P. s e r j a ta K j e l l man £• t e n e r a Kjellman £• u m b i l i c a l i s (Linnaeus) J . Agardh JE» yezoensis Oeda Reference Mumford and Cole (1977) Yabu (1971) Hawkes (1977a, 1977b, and t h i s t h e s i s ) Mumford and Cole (1977) K i t o (1966) - as P. sp. Yabu (1972) Yabu (1972) Yabu (1971) Magne (1952) K i t o e t a l . (1967) - as £• u m b i l i c a i i s f. l i n e a r i s ( G r e v i l l e ) Harvey K i t o et a l . (1967) - as P. amplissima (Kjellman) S e t c h e l l e t Hus Yabu (1970) - as P. amplissima K i t o e t a l . (1971) K i t o et a l . (1967) Hawkes ( t h i s t h e s i s ) Yabu (1969a) Yabu (1972) Mumford and C o l e (1977) Yabu (1969a) Giraud and Magne (1968) -as P. u m b i l i c a l i s var. l a c i n i a t a ( L i g h t f o o t ) Thuret K i t o et a l . (1971) Mumford (1975) Mumford and Cole (1977) a l . ( c i t e d 1975) Yabu (1969a) Fujiyama et from Yabu, K i t o (1968) Yabu (1969b) K i t o et al.„ (1971) Yabu and Tokida (1963) M i g i t a (1967b) Yabu (1969a) 16 P. spp. # 1 and 2 D i p l o i d C o n c h o c e l i s Porphyra g a r d n e r i E* Jsanakaensis P. m i n i a t a P. p e r f o r a t a P. p s e u d o l i n e a r i s !• purpurea P. s c h i z o p h y l l a P. tenera P. .  yezoensis Yabu (1971) Hasrkes (1 th e s i s ) Mumford a K i t o et a. Mumford a K i t o (197 Giraud an as P. um var. l a c Mumford a K i t o (197 K i t o (196 M i g i t a (1 K i t o (197 977a, and t h i s nd Cole (1977) 1. (1971) nd Cole (1977) <i) d Magne (1968) fcilicalis i n i a t a nd C o l e (1977) il) 7) 967b) <*) 165 F i q u r e 16a. Transverse s e c t i o n of the blade margin o f Lamj.naria s e t c h e l l i i and the base cf £2I£hyi.a g a r d n e r i . The r h i z o i d a l f i l a m e n t s can be seen p e n e t r a t i n q i n t o the medulla of the Laminaria blade. Fiqure 16b. Closeup o f the r h i z o i d a l p e n e t r a t i o n i n t o the medulla. F i g u r e 16c. Transverse s e c t i o n thrcuqh a N e r e o c y s t i s s t i p e and the base of a Porphyra n e r e o c y s t i s a t t a c h e d to i t . Fiqure 16d. Closeup of the c e n t r a l r e q i o n of the attachment d i s c . S c a l e bar = 100 um on a l l F i g u r e s . L i s t Of A b b r e v i a t i o n s Dsed On F i g u r e s For F a r t IV. C a r p o g o n i a l nucleus (cn) ; c h l o r o p l a s t (C,c)j c e l l w a l l (CW, cw); d i c t y o s c a e (D, d ) ; f l o r i d e a n s t a r c h (FS, f s ) ; l a r g e f i b r o u s v e s i c l e (LFV f I f v ) ; n e d u l l a (ME); mitochondrion (MI, J e f e o c y s J i s (NF) ; nucleus (N, n) ; Porphyra (PO) ; pyrenoid (P, p) ; s m a l l f i b r o u s v e s i c l e (SFV, sfv) ; s p e r a a t i a l n u c l e a r m a t e r i a l ( a ) ; spermatium (S). 166 167 F i g u r e 17. Tranverse s e c t i o n of Pprchyra g a r d n e r i t h a l l u s , through a young monosporangium. Note dictyosomes, s m a l l f i b r o u s v e s i c l e s and l a y e r of f i b r o u s m a t e r i a l being formed on the o u t s i d e of the plasma membrane. 168 169 F i g u r e 18. Transverse s e c t i o n of Eorphyra g a r d n e r i t h a l l u s through a monosporangium which has developed beyond the stage shown i n Fi g u r e 17., Small f i b r o u s v e s i c l e s are abundant and l a r g e f i b r o u s v e s i c l e s are beginning to form. 170 171 F i g u r e 19a. Dictyosome a c t i v i t y producing s m a l l f i b r o u s v e s i c l e s . F i g u r e 19b. Closeup of a monosporangial w a l l showing the depositon o f f i b r o u s m a t e r i a l along i t s i n n e r s u r f a c e (arrow) . F i g u r e 19c. Honosporangium i n the f i n a l stages of d i f f e r e n t i a t i o n p r i o r to monospore l i b e r a t i o n , Both sma l l and l a r g e f i b r o u s v e s i c l e s are abundant. 172 173 Z21£lJHS g a r d n e r i - spermatogenesis, e l e c t r o n microscopy (t r a n s v e r s e s e c t i o n s ) F i g u r e 20a. V e g e t a t i v e c e l l o f the f c l i o s e t h a l l u s . F i g u r e 20b. The f i r s t d i v i s i o n of the spermatangium i s n e a r l y complete. Mote the new w a l l l a y e r (arrow) that has been l a i d down i n the t r a n s i t i o n from a v e g e t a t i v e c e l l . F i g u r e s 20c-d. l a t e r d i v i s i o n stages of the spermatangium. S c a l e t a r = 5 um on a l l F i g u r e s . 174 175 Porphyra g a r d n e r i - spermatogenesis, e l e c t r o n microscopy (transverse s e c t i o n s ) F i g u r e 21a. F i n a l d i v i s i o n s i n the formation of a nature spermatangium. Note the p r o d u c t i o n of l a r g e f i b r o u s v e s i c l e s . F i g u r e 21b. Spermatium from a mature spermatangium. I t c o n t a i n s a nucleus, mitochondria, reduced c h l o r o p l a s t , s e v e r a l s m a l l and one l a r g e f i b r o u s v e s i c l e . Note s m a l l f i b r o u s v e s i c l e emptying i t s contents i n t o the l a r g e f i b r o u s v e s i c l e (arrow). F i g u r e 21c. Two spermatia i n a spermatangium near the r e l e a s i n g margin. The l a r g e f i b r o u s v e s i c l e has j u s t become e x t r a c y t o p l a s m i c . S c a l e bar = 1 um i n F i g u r e s 21b-c 176 177 F i g u r e 22. Mature spermatium j u s t p r i o r to r e l e a s e . Note the nucleus with h i g h l y condensed chromatin and no nu c l e a r membrane, reduced c h l o r o p l a s t with numerous p l a s t o g l o b u l i , mitochondrion and abundant s m a l l f i b r o u s v e s i c l e s . 178 179 Porphyra g a r d n e r i - f e r t i l i z a t i o n , l i g h t microscopy (t r a n s v e r s e s e c t i o n s ) F i g u r e 23a. Spermatium a t t a c h e d to the p r o t o t r i c h o g y n e of the carpogonium. F i g u r e 23b. Two spermatia with f e r t i l i z a t i o n c a n a l s going i n t c the carpogonium. F i g u r e 23c. Spermatia attached to both p r o t o t r i c h o g y n e s (note f e r t i l i z a t i o n c a n a l s ) . , S c a l e bar = 10 um on a l l F i g u r e s . 180 181 F i g u r e s 24a-b. Two scanning e l e c t r o n micrographs of the t h a l l u s s u r f a c e of Porphyra g a r d n e r i showing s e v e r a l spermatia attached t o the p r o t o t r i c h o g y n e s . 182 183 Porphv.ra g a r d n e r i - f e r t i l i z a t i o n , e l e c t r o n microscopy ( t r a n s v e r s e s e c t i o n s ) F i g u r e 25a. Low m a g n i f i c a t i o n view of a spermatium att a c h e d to the p r o t o t r i c h o g y n e . F i g u r e s 25b-d. Three examples cf spermatia a t t a c h e d t o the p r o t o t r i c h o g y n e p r i o r to the formation of the f e r t i l i z a t i o n c a n a l . They c o n t a i n a c h a r a c t e r i s t i c reduced c h l o r o p l a s t , mitochondria and non-membrane bound n u c l e a r m a t e r i a l . S c a l e bar = 2 um on F i g u r e s 25b-d 184 185 Porphyra g a r d n e r i - f e r t i l i z a t i o n F i g u r e 26. Transverse s e c t i o n thrcugh a carpogonium and two spermatia which have t r a n s f e r r e d t h e i r n u c l e a r m a t e r i a l dcwn the f e r t i l i z a t i o n c a n a l i n t o the carpogonium. 186 187 Porphyra g a r d n e r i - f e r t i l i z a t i o n F i g u r e 27. Transverse s e c t i o n through a carpogonium showing both p r o t o t r i c h o g y n e s , c e n t r a l pyrenoid and l a t e r a l nucleus. The presence of a s m a l l d e p o s i t of w a l l m a t e r i a l (arrow) i n the p r o t o t r i c h o g y n e below the spermatium i n d i c a t e s t h a t a f e r t i l i z a t i o n c a n a l has been formed, although i t i s not v i s i b l e i n the plane of s e c t i o n . Suspected Cyancphyta are a l s o v i s i b l e on the t h a l l u s s u r f a c e (double arrow). 188 189 Porphyra g a r d n e r i - f e r t i l i z a t i o n ( t r a n s v e r s e s e c t i o n s ) F i g u r e s 28a-fa. Two spermatia which have t r a n s f e r r e d t h e i r n u c l e a r m a t e r i a l down the f e r t i l i z a t i o n c a n a l i n t o the carpogonium, but which s t i l l have the remains of a c h l o r o p l a s t l e f t i n them. F i g u r e s 28c-d., P r o t o t r i c h o g y n e r e g i o n of the carpogonium with a reduced c h l o r o p l a s t which i s thought t o have come from the spermatium during the t r a n s f e r of nucle a r m a t e r i a l . S c a l e car = 1 um on a l l F i g u r e s 190 191 Porphyra g a r d n e r i - f e r t i l i z a t i o n F i g u r e 29. Transverse s e c t i o n through a carpogonium which has the remains of a spermatium attached t o the p r o t o t r i c h o g y n e , and what may be the h i g h l y condensed s p e r m a t i a l n u c l e a r m a t e r i a l (arrow). 192 Por phyra g a r d n e r i - carposporogenesis ( t r a n s v e r s e s e c t i o n s ) F i g u r e 30a. F e r t i l i z e d carpogonium showing the two daughter n u c l e i r e s u l t i n g frcm the f i r s t d i v i s i o n of the f u s i o n nucleus. The f e r t i l i z a t i o n c a n a l i s s t i l l present although the spermatium has d i s i n t e g r a t e d . F i g u r e 30b. The f i r s t d i v i s i o n of the carposporangium i s p e r i c l i n a l and produces 2 carpospores (note the f e r t i l i z a t i o n c a n a l s t i l l present i n the w a l l ) . S c a l e bar = 5 um on a l l F i g u r e s 194 195 Porphyra g a r d n e r i - chromosome counts F i g u r e 31a. Spermatium, n=4. Figu r e 31b. Vegetative c e l l of the f o l i o s e t h a l l u s , n=4. F i g u r e s 31c-d. Carpospore, 2n=8. F i g u r e s 31e-f. Vegetative c e l l of the c o n c h o c e l i s phase, 2n=8. Sca l e bar = 5 um on a l l F i g u r e s . 96T 1 9 7 Porphyra g a r d n e r i - Feulgen s t a i n i n g {transverse s e c t i o n s ) F i g u r e 32a. Mature spermatangium; most spermatia s t i l l have n u c l e i i n a d i f f u s e i n t e r p h a s e s t a t e (arrow). F i g u r e 32b. Mature spermatangium r i g h t on the r e l e a s i n g margin. The nuc l e a r m a t e r i a l i s h i g h l y condensed (arrow). F i g u r e 32c. Released spermatia with h i g h l y condensed nuc l e a r m a t e r i a l (dark d o t s ) . Scale bar = 10 um on a l l F i g u r e s . 198 % **** m 199 Porphyra g a r d n e r i - Feulgen s t a i n i n g ( t r a n s v e r s e s e c t i o n s ) F i g u r e 33a. Carpoqonium and attached spermatium which c o n t a i n s condensed n u c l e a r m a t e r i a l . No f e r t i l i z a t i o n c a n a l i s present. F i g u r e 33i>, Carpogonium and c a r p o g o n i a l nucleus l a t e r a l l y l o c a t e d . The attached spermatium c o n t a i n s n u c l e a r m a t e r i a l i n a d i f f u s e s t a t e , and a f e r t i l i z a t i o n c a n a l i s present. F i g u r e s 33c-d, Two examples of spermatia c o n t a i n i n g condensed nuclear m a t e r i a l i n c l o s e a s s o c i a t i o n with the f e r t i l i z a t i o n c a n a l . F i g u r e 33e. Carpogonium with attached empty spermatium and a densely s t a i n i n g mass of s p e r m a t i a l n u c l e a r m a t e r i a l i n the carpogonium c l o s e to the f e r t i l i z a t i o n c a n a l . F i g u r e s 3 3 f - i . . S e v e r a l examples of carpogonia with empty spermatia on t h e i r outer w a l l s and from 1-5 densely s t a i n i n g masses of s p e r m a t i a l n u c l e a r m a t e r i a l i n s i d e . F i g u r e 3 3 j . Carpogonium with l a r g e , suspected f u s i o n nucleus (note empty spermatium and f e r t i l i z a t i o n c a n a l ) . F i g u r e 33k. The f i r s t d i v i s i o n of the f u s i o n nucleus p r i o r t o c y t o k i n e s i s (see a l s o Figure 30a). F i g u r e 331. Two carpospores produced by the f i r s t d i v i s i o n of the carposporangium (see a l s o Figure 30b). F i g u r e s 33m-o. Subsequent stages of d i v i s i o n t o produce ** (Figure 33m) or 8 (Figure 33o) carpospores. S c a l e bar = 10 um on a l l F i g u r e s . 200 201 F i g u r e 34. Diagram summarizing the known morphological and c y t o l o g i c a l l i f e h i s t o r y of Porphyra g a r d n e r i . I t i s suspected t h a t meiosis occurs i n the conchosporangium a t the time of conchospore formation. 202 PORPHYRA GARDNERI L I F E HISTORY CARPOSPORANG IUM 203 F i g u r e 35. Porphyra n e r e o c y s t i s spermatium i n a spermatangium near the r e l e a s i n g margin. The spermatium c o n t a i n s abundant s m a l l and l a r g e f i b r o u s v e s i c l e s , a reduced c h l o r o p l a s t , mitochondria, and c e n t r a l nucleus., 204 205 Porphyra n e r e o c y s t i s F i g u r e s 36a-b. Transverse s e c t i o n cf the f o l i o s e t h a l l u s through carpogonia and suspected spermatia forming f e r t i l i z a t i o n c a n a l s . F i g u r e 36c. E l e c t r o n micrograph of a t r a n s v e r s e s e c t i o n through a suspected spermatium and f e r t i l i z a t i o n c a n a l . Porphyra t h u r e t i i F i g u r e 36d. Transverse s e c t i o n of the f o l i o s e t h a l l u s through a carpogonium and suspected spermatium. F i g u r e 36e. Transverse s e c t i o n o f the f o l i o s e t h a l l u s through a carposporangium t h a t has undergone the f i r s t d i v i s i o n . A suspected spermatium and f e r t i l i z a t i o n c a n a l are present. S c a l e bar = 10 um on F i g u r e s 36a-b, 36d-e. 206 207 F i g u r e 37a. Porphyra n e r e o c y s t i s spermatia. n=3. Figu r e 37b. Porphyra n e r e o c y s t i s v e g e t a t i v e c e l l s of the f o l i o s e t h a l l u s , n=3. F i g u r e 37c. Porphyra t h u r e t i i spermatium. n=2. F i g u r e 37d,e Porphyra n e r e o c y s t i s carpospore. 2n=6. (only 5 chromosomes v i s i b l e ) . F i g u r e 37f,g Porphyra n e r e o c y s t i s ccnchosporangial branch, 2n=6. Sc a l e bar ~ 5 um on a l l F i g u r e s 208 209 GENERAL SUMMARY F i e l d , c u l t u r e and c y t o l o g i c a l s t u d i e s of 3 e p i p h y t i c Porphvra s p e c i e s have y i e l d e d f o u r major r e s u l t s : 1. The genus P o r p h y r e l l a i s i n v a l i d and P o r p h y r e l l a g a r d n e r i has t h e r e f o r e been t r a n s f e r r e d to the genus Porphyra. 2. Porphyra g a r d n e r i . P. n e r e o c y s t i s and P. t h u r e t i i , 3 s u p e r f i c i a l l y s i m i l a r s p e c i e s , have been c l e a r l y c h a r a c t e r i z e d cn the b a s i s o f t h e i r seasonal occurrence, host s p e c i e s , v e g e t a t i v e and r e p r o d u c t i v e morphology, growth i n c u l t u r e , and chromosome numbers. 3. The spermatangium and carpogonium of these 3 s p e c i e s have been c h a r a c t e r i z e d , and t h e i r d i v i s i o n sequence f o l l o w e d i n d e t a i l . From these o b s e r v a t i o n s I have concluded t h a t there are two b a s i c types of c a r p o g c n i a l and spermatangial morphology i n the genus Porphyra. and th a t the c l a s s i c a l * c r u c i a t e d i v i s i o n * sequence as re p o r t e d by Hus (1902) may not be as common as p r e v i o u s l y thouqht. 4. The occurrence of sexual r e p r o d u c t i o n i n the qenus Porphyra has been u n e q u i v o c a l l y demonstrated f o r the f i r s t time. 210 Ss i s always the case r a i s e d mere questions than they r e q u i r i n g f u r t h e r i n v e s t i g a t i o n with r e s e a r c h , my s t u d i e s have have answered. Major t o p i c s i n c l u d e ; 1. The nature of the e p i p h y t e - 1 h o s t ' r e l a t i o n s h i p . P h y s i o l o g i c a l s t u d i e s are needed to e l u c i d a t e what determines host s p e c i f i c i t y . 2. Does meiosis occur i n t h e conchosporangium? C y t o l o g i c a l study of conchosporogenesis both at the l i g h t and e l e c t r o n microscope l e v e l i s needed to e s t a b l i s h t h a t t h i s i s where meiosis occurs. 3. What are the environmental c o n d i t i o n s and p h y s i o l o g i c a l mechanisms r e s p o n s i b l e f o r spermatangium and carpogonium i n d u c t i o n ? 4. Genetic s t u d i e s along the l i n e s of those which have been done on G r a c i l a r i a (van der Meer, 1977; van der fleer and Todd, 1977) may provide i n s i g h t i n t o what determines s e x u a l i t y and how the v a r i o u s p a t t e r n s of spermatangia and carposporangia on the t h a l l u s are e s t a b l i s h e d . Gf p a r t i c u l a r i n t e r e s t here are s p e c i e s l i k e Porphyra k a t a d a i (Kurogi, 197 2), P. brumalis-Kumford, and s e v e r a l other s p e c i e s l i s t e d by Mumford (1975), which have the spermatangia on one h a l f c f the t h a l l u s and the carposporangia on the other. 2 1 1 Can Pqrphyra hybrids be produced? Such r e s u l t s were reported by Sutc (1963), but h i s experiments were not w e l l documented. How widespread i s s e x u a l r e p r o d u c t i o n i n other members of the Bangiophycidae? Based on McDonald's (1972) 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 of the spermatia of l a n g i a fuscopurpurea. the r e p o r t s of Bosenvinge (1909), Dangeard (1927), and Cole (personal communication, 3 May, 1978) of f e r t i l i z a t i o n c a n a l s connecting spermatia with carpogonia, and Yabu's (1967) r e p o r t of f e r t i l i z a t i o n and a d i p l o i d chromosome number i n the c a r p o s p o r e s , s e x u a l r e p r o d u c t i o n would c e r t a i n l y seem to be o c c u r r i n g i n Bangja. In the E r y t h r o p e l t i d a c e a e , McBride (1972) has reported on the u l t r a s t r u c t u r e of the spermatia i n Smithora. They have some s i m i l a r i t i e s with the spermatia o f Porphyra g a r d n e r i and Eangia fuscopurpurea and may t h e r e f o r e be f u n c t i o n a l male gametes. HcBride a l s o r e p o r t e d the attachment of a spermatium o f E r y t h r o t r i c h i a boryana (Montagne) B e r t h o l d over a c a r p o s p o r e - l i k e c e l l i n the f i l a m e n t o u s t h a l l u s of t h i s a l g a . No f e r t i l i z a t i o n c a n a l was observed. i n view of t h i s evidence a r e - i n v e s t i g a t i o n of the l i f e h i s t o r i e s of these algae as w e l l as other members o f the Bangiophycidae would be worthwhile. 212 LITEEATUEE CITED Abbott, I. A., and G. J . H o l l e n t e r g . 1976. Marine Algae o f C a l i f o r n i a . S t a n f o r d U n i v e r s i t y Press, S t a n f o r d . x i i * 8 2 7 Pp. Abbott, I. A., and ». J , North. 1972. .Temperature i n f l u e n c e s on f l o r a l composition i n C a l i f o r n i a c o a s t a l waters, pp. 72-79 i n : K. Nisizawa, ed., P r o c . 7th Int. Seaweed Symp. , Sapporo, Japan 1971. Univ. Tokyo Press, Tokyo. Anderson, C. L. 1891. 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S. 1954./Vodorosli Okhotskogo Morya [ i n Russian, t r a n s l a t e d t i t l e 'Algae of the Okhotsk Sea']..Trudy Bet. I n s t . Akad. Hauk SSSB, s e r . 2, 9: 259-310. 234 APPENDIX I Porphyra C o l l e c t i n g S t a t i o n s And, Dates Of C o l l e c t i o n s J r i f i s h Columbia: A g u i l a r P o i n t , 48° 50.4*N, 125° 08.3'W, 31 MA? 1974, 2 APR 1977. Bolkus I s l a n d s , S k i n c u t t l e I n l e t , Queen C h a r l o t t e I s l a n d s , 52° 19.4»N, 131° 14.8*W, 27 MAR 1976. B o t a n i c a l Beach, 48° 32*N, 124° 27*W, 11 JUL 1975. Brooks P e n i n s u l a , 50° 06.7* N, 125° 54.5*13, 25 JUN 1S75. Cable Beach, 48° 49.6'N, 125° 09.1»fl, 8 AUG 1974. Cape Beale, 48° 48 *N, 125° 13*W, 28 JUL 1976. Execution Rock, 48° 48.8*N, 125° 10.6*8, 10 MAY 1974. Gordon I s l a n d s , Queen C h a r l o t t e I s l a n d s , 52° 06.1*N, 131° G8.7*W, 1 APR 1976. Grassy I s l a n d , Kyuguot Sound, 49° 55. 4* N, 127° 15.0*8, 14 MAY 1975.. Hope I s l a n d , 50° 54»N, 127° 57' H, 15 APR 1 975. Hot S p r i n g s Cove, 49° 21.5*N, 126° 15.8'«, 13 SAY 1975. Langara I s l a n d , Queen C h a r l o t t e I s l a n d s , 54° 13.54*N, 132° 57.7 'W, 29 MAR 1S76.. Leach I s l e t , B arkley Sound, 48° 49. 8* N, 125° 14.4*8, 2 APR 1977, 17 MAY 1977. Rennel Sound, Queen C h a r l o t t e I s l a n d s , 53° 22.65*N, 132° 30.7*8, 30 MAR 1S76. Ross I s l e t s , B a r k l e y Sound, 48° 52.2*N, 125° 09.5»W, 5 JUN 1974. Second Beach, Barkley Sound, 48° 48,9»N, 125° 10«H, 9 MAY 1974. South Pender I s l a n d , 48° 44,0*N, 123° 11.2«w, 4 SEP 1977. Study s i t e , B a r k l e y Sound, 48° 50.1*N, 125° 11.1»W, JUN 1974 through MAY 1977. Tree I s l e t s , Queen C h a r l o t t e Sound, 50° 58.9*N, 127° 42.6'W, 24 JUN 1975. 235 V o l c a n i c Cove, Kyuguot Channel, 49° 58.7*N, 127° 13.9*», 15 MAY 1975. H h i f f e n S p i t , Sooke, 48° 21.2»N, 123° 44.2»W, 15 OCT 1S73. Crejgcn: H a r r i s Beach, 42° 04»N, 124° 18*0, 23 SEP 1975. C a l i f o r n i a ; Ano Nuevo I s l a n d , 37° 06»N, 122° 20*W, 2 NOV 1975. Mission P o i n t , Carmel, 36° 32.5* N , 121° 56»H, 13 JUL 1976. Pebble Beach, Monterey P e n i n s u l a , 36° 34*N, 121° 57*8, 4 NOV 1975. Fescadero P o i n t , Monterey P e n i n s u l a , 36° 33.8*N, 121° 57»H, 12 JUL 1976., F e i n t Joe, Monterey P e n i n s u l a , 36° 36.6*N, 121° 57.4*8, 3 NOV 1975, 10 JUL 1976. 236 APPENDIX I I R e p r e s e n t a t i v e s p e c i m e n s o f p o r p h y r a g a r d n e r i * P. n e r e o c y s t i s * and P. t h u r e t i i P o r p h y r a g a r d n e r i M c n c s p o r i c A l a s k a : Murde r P o i n t , A t t u I s l a n d , 5 2 ° 4 8 ' N , 1 7 3 ° 11 « E , UBC 7 9 4 7 , 8 JUN 1 9 6 0 ; B i r d C a p e , A m c h i t k a I s l a n d , 5 1 ° 3 9 . 5 ' N , 1 7 8 ° 3 8 . 5 ' E , DBC 5 4 2 0 2 , 27 JUN 1 9 6 9 ; «Top C a m p ' , A a c h i t k a I s l a n d , 5 1 ° 3 4 . 7 » N , 178<> 5 0 . 5 « E , UBC 5 4 2 0 3 , 17 AUG 1 9 7 0 ; Cape A g a g d a k , Adak I s l a n d , 5 2 ° 0 0 » N , 1 7 6 ° 3 5 « H , UBC 7 7 8 3 , 12 JUN 1 9 6 0 ; H a r b o r P o i n t , L i t u j a B a y , S i t k a , 5 7 ° C 8 » N , 1 3 5 ° 1 5 » 8 , UBC 2 3 4 0 1 , 29 JUN 1 9 6 5 ; Cape H u z o n , D a l l I s l a n d , 5 4 ° 4 0 » N , 1 3 2 ° 41»H, UEC 2 1 4 6 9 , 13 JUN 1 9 6 5 . B r i t i s h C o l u m b i a : L a n g a r a I s l a n d , Queen C h a r l o t t e I s l a n d s , 5 4 ° 1 3 . 5 4 * N , 1 3 2 ° 57.7*W, UBC 5 4 8 8 3 , 29 MAR 1976 ; P l o v e r I s l a n d , 5 0 ° 5 5 ' N , 1 2 7 ° 5 8 « B , UBC 1 5 0 1 , 26 JUN 1 9 5 3 ; G a r d e n I s l a n d , K y u g u o t , 5 0 ° 0 1 « N , 1 2 7 ° 21»W , UBC 4 3 4 2 , 27 MAY 1 9 5 9 ; D i a n a I s l a n d , B a r k l e y S o u n d , 4 8 ° 50 . 1»N , 125 ° 1 1 . 1 * 8 , UBC 5 4 1 3 3 , 23 FEB 1 9 7 5 ; UBC 54127 and UBC 5 4 1 3 4 , 28 MAR 1975; UBC 5 4 8 1 9 , 16 APR 1S76 ; E x e c u t i o n R o c k , B a r k l e y S o u n d , 4 8 ° 4 8 . 8 ' N , 1 2 5 ° 1 0 . 6 » « , UEC 5 4 1 2 9 , 10 MAY 1 9 7 4 ; B o t a n i c a l B e a c h , 4 8 ° 3 2 ' N , 1 2 4 ° 27VH , UBC 5 4 8 8 4 , 14 MAY 1976. w a s h i n g t p n : H a a t c h P o i n t , Mukkaw B a y , 4 8 ° 2 0 » N , 1 2 4 ° 4 0 ' H , UBC 2 4 8 8 2 , 2 JUN 1966 . Or e ^ o n : I n d i a n B e a c h , C l a t s c p C o u n t y , 4 5 ° 5 6 » N , 1 2 3 ° 5 9 « « , UBC 4 7 3 2 8 , 14 HAY 1972 ; Chapman P o i n t , C l a t s o p C o u n t y , 4 5 ° 5 5 ' N , 1 2 3 ° 58'W, UBC 4 7 5 8 4 , 12 JUN 1972 ; S h o r t Sand B e a c h , 4 5 ° 4 5 * N , 1 2 3 ° 58'W, UBC 2 4 8 8 3 , 6 JUN 1966. C a l i f o r n i a : P i l l a r P o i n t , San Mateo C o u n t y , 3 7 ° 3 0 * N , 1 2 2 ° 3 0 ' 8 , RS 4 7 4 , 10 JUL 1968 ; P o i n t J o e , M o n t e r e y P e n i n s u l a , 3 6 ° 3 6 . 6 » N , 1 2 1 ° 57.4'W, GJB 2 6 7 2 , 18 JUN 1 9 3 9 ; UBC 54814 , 10 JUL 1 9 7 6 ; P e b b l e B e a c h , M o n t e r e y P e n i n s u l a , 3 6 ° 3 4 * N , 1 2 1 ° 5 7 ' I , GMS 1 2 4 6 1 , 7 MAY 1973 ; M i s s i o n P o i n t , C a r m e l , 3 6 ° 3 2 . 5 ' N , 1 2 1 ° 56*W, GMS 4 7 6 3 , 16 MAR 1S66 ; E s t e r o P o i n t , San L u i s O b i s p o C o u n t y , 3 5 ° 2 8 » N , 1 2 0 ° 58»W, AHF 8 0 4 3 7 , 6 AUG 1974. 237 Mexico : Punta Banda, Baja C a l i f o r n i a , 31° 44»N, 116° 44*8, AHP 77063, 25 JUL 1971. Spermatangial and c a r p o s p o r a n g i a l Alaska: Bingham I s l a n d , 60° 01*N, 144° 23»W, OBC 25459, 4 JUL 1966; Klokachef I s l a n d , 57° 25»N, 135° 53*8, OBC 23397, 30 JUN 1965; Loran S t a t i o n , B i o r k a I s l a n d , 56° 51* N, 135° 32*8, OBC 10291, 11 JUL 1960. B r i t i s h Columbia: Tasu Narrows, Tasu Sound, Queen C h a r l o t t e I s l a n d s , 52° 44,5»N, 132° 06«W, UBC 53651, 17 AUG 1970; Tree I s l e t s , 50* 58.9*N, 127° 42.6*W, UBC 54888, 24 JUN 1975; Cape S c o t t , 50° 47*N, 128° 25*8, UBC 35742, 11 AUG 1968; Solander I s l a n d , 50° 06. 5*N, 127° 56.3*8, UBC 53836, 25 JON 1975; Lynne Bock o f f Cautious P o i n t , 50° 04.3*N, 127° 33.4»W, BBC 36036, 16 AUG 1968; Diana I s l a n d , Barkley Sound, 48° 50.1»N, 125° 11.1*8, OBC 54821, 13 MAI 1976; UBC 54820, 16 JUN 1975; UBC 54136, 17 JUN 1975; UEC 54130, 19 JUL 1974; UBC 54137, 8 AUG 1975; UBC 54122, 14 SEP 1974; UBC 54132, 17 OCT 1974; Cape B e a l e , 48° 47*N, 125° 13*8, UBC 54891, 28 JUL 1976; B o t a n i c a l Beach, 48° 32'N, 124° 27»W, UBC 54133, 11 JUL 1975. Washington: Waadah I s l a n d , 48° 23*N, 124° 36*W, UBC 8767, 15 AUG 1958. Oregon: In d i a n Beach, C l a t s o p County, 45° 56*N, 123° 5 9*8, UBC 49873, 1 JUL 1973; Jockey Cap Bock, C l a t s o p County, 45° 51*N, 123° 58*8, UBC 48333, 8 JUL 1972. C a l i f o r n i a : T r i n i d a d Harbor, Humboldt County , 41° 03* N, 124° 09'8, CSUH 07353, 12 APB 1972; Bodega Head, Sonoma County, 38° 18*N, 123° 03*W, UBC 20010, 21 JON 1963; Tomales Bay, Marin County, 38° 14«N, 122° 55'W, AH J 55944, JON 1915; An"o Nuevo I s l a n d , 37° 06*N, 122° 20*8, Hansen 1631 i n OCSC { f i l e d under Lajdnarjjt s i n c l a i r i i ) , 8 AOG 1972; Davenport, 37° 00*N, 122° 11*W, GflS 2083, 6 J0L 1965; P o i n t Joe, Monterey P e n i n s u l a , 36° 36.6»N, 121° 57.4*8, OBC 54818 and OBC 54887, 10 JOL 1976; Pescadero P o i n t , 36° 33.8*N, 121° 57*8, OBC 54892, 12 JOL 1976; Mi s s i o n P o i n t , Carmel, 36° 32.5»N, 121° 56'W, OBC 54815, 13 JOL 1S76. 238 Porphvra n e r e o c y s t i s Alaska: Staraya Bay, Unalaska I s l a n d , A l e u t i a n I s l a n d s , 53° 37*N, 166° 32* W, UBC 2677 6, 16 JUL 1966; Kupreanof P o i n t , Kodiak I s l a n d , 57° 55* N, 153° 05*H, UBC 26949, 20 JUL 1966; Bousscle Head, 58° 23*N, 136° 55*1, UBC 21199, 29 JUN 1965. B r i t i s h Columbia: Langara I s l a n d , Queen C h a r l o t t e I s l a n d s , 54° 13.54*N, 132° 58.6*W, UBC 56677, 29 MAH 1S76; Masset, Graham I s l a n d , Queen C h a r l o t t e I s l a n d s , 54° 00 »N, 132° 09'8, UBC 50229, 10 MAS 1973; Off N. W. coast o f C a l v e r t I s l a n d , 51° 42»N, 128° 05«W, UBC 42946, 13 APB 1970; Tree I s l e t s , 50° 58.9'N, 127° 42.6'W, UBC 54105, 24 JUN 1975; Hope I s l a n d , 50° 54*N, 127° 57 »W, OBC 57182, 15 APB 1975; Amphi t r i t e P o i n t , Vancouver I s l a n d , 48° 54'N, 125° 33'W, UBC 39820, 8 MAY 1969; A g u i l a r P o i n t , Barkley Sound, 48° 50.4»N, 125° 08.3»W, UBC 57183 and 57184, 2 APB 1977; Cable Beach, Barkley Sound, 48° 49.6'N, 125° 09.1«H, UBC 57181, 8 AUG 1974; Second Beach, Barkley Sound, 48° 48.9*N, 125° 10'H, UBC 57186, 9 MAY 1974; Diana I s l a n d , B a r k l e y Sound, 48° 50.1»N, 125° 11.1 'SI, UBC 57185, 12 DEC 1976, UBC 57186 and 57189, 23 JAN 1977, UBC 57187 and 57190, 23 PEB 1975; Leach I s l e t , Barkley Sound, 48° 49.8*N, 125° 14.4'w, UBC 57191, 2 APB 1977; B o t a n i c a l Beach, 48° 32 *N, 124° 27'W, UBC 50652, 25 APB 1974, UEC 57193, 11 JUL 1975; Esguimalt, Vancouver I s l a n d , 48° 28'N, 123° 26'H, UC 96517, no date. B r i t i s h Columbia - S t r a i t of Georgia Maude I s l a n d , 49° 16*N, 124° 05* W, UBC 11583, 15 AUG 1959; Tugboat I s l a n d , 49° 09«N, 123° 41 »W, UBC 56603, 29 DEC 1976; Gowlland P o i n t , South Pender I s l a n d , 48° 44.0* N, 123° 11.2*8, UBC 57194, 4 SEP 1977; Sidney, Vancouver I s l a n d , 48° 39*N, 123° 24»B, UBC 1434, 26 SEP 1917. Washington: F r i d a y Harbor, San Juan I s l a n d , 48° 32*N, 123° 00*W, OBC 57195, 27 OCT 1S73; Whidbey I s l a n d , 48° 13»N, 122° 46'W, UEC 1244, no date. C a l i f o r n i a : Pebble Beach, Monterey P e n i n s u l a , 36° 34*N, 121° 57*W, UBC 57196, 4 NOV 1975. 239 Porphyra t h u r e t i i B r i t i s h Columbia: S h i e l d s Bay, R e n n e l l Sound, Queen C h a r l o t t e I s l a n d s , 530 22.65*8, 132° 30.7'W, UBC 55302, 30 MAR 1976; V o l c a n i c Cove, Kyuguot Channel, Vancouver I s l a n d , 49° 58.7*8, 127° 13.9*8, UBC 54446, 15 HAY 1975; Diana I s l a n d , Barkley Sound, 48° 50.1*N, 125° 11.1'W, UBC 57203, 23 JAN 1977; UBC 52019, 23 FEB 1975; UBC 57202, 29 APR 1975; Leach I s l e t , Barkley Sound, 48° 49.8* N, 125° 14.4*8, UBC 57200 and 57201, 2 APR 1977. C a l i f o r n i a : P a c i f i c Grove, Monterey County, 36° 37.6»N, 121° 55* K, UC 95610, 23 APR 1897; UC 95598 and 95596, JUN 1901; UC 95612, no date; Pebble Beach, Honterey P e n i n s u l a , 36° 34*N, 121° 57*8, GHS 1406, 24 JUN 1943; Carmel Bay 36° 33*N, 121° 56*8, 0C 791973 and 791970, 29 MAY 1900; GMS 6467, 23 JUN 1939. 

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