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A morphological and cytological study of Audouinella porphyrae and A. vaga (Rhodophyta) Tam, Carol Elizabeth 1985

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A MORPHOLOGICAL AND CYTOLOGICAL STUDY OF AUDOUINELLA PORPHYRAE AND A. VAGA (RHODOPHYTA) By CAROL ELIZABETH TAM B. SC., The U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE. in THE FACULTY OF GRADUATE STUDIES (Department of Botany) 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 August, 1985 © C a r o l E l i z a b e t h Tarn, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 ABSTRACT A comparative study was made between two s i m i l a r red a l g a l endophytes, Audouinella porphyrae (Drew) Garbary and Audouinella vaga (Drew) Garbary, Hansen et S c a g e l , (Acrochaetiaceae, A c r o c h a e t i a l e s ) and t h e i r red a l g a l hosts Porphyra spp. and Pt er os i phoni a bipinna:a, r e s p e c t i v e l y . Both endophytes have a x i a l , s t e l l a t e c h l o r o p l a s t s with a c e n t r a l p y r e n o i d and r e s i d e i n t h e i r host's c e l l w a l l s , producing e r e c t p o r t i o n s o u t s i d e the host that may bear monosporangia. The endophytes were c u l t u r e d " f r e e " from t h e i r hosts and m o r p h o l o g i c a l and c y t o l o g i c a l f e a t u r e s of the f r e e - l i v i n g forms were compared with f i e l d m a t e r i a l . Although the two endophytes d i f f e r s i g n i f i c a n t l y i n c e l l dimensions and branching p a t t e r n s , the f r e e - l i v i n g forms do not r e t a i n these d i f f e r e n c e s . C e l l dimensions, branching p a t t e r n s and developmental p a t t e r n s are i d e n t i c a l i n the two f r e e - l i v i n g forms. Asexual r e p r o d u c t i o n with r e g e n e r a t i o n by monospores was observed. Sexual r e p r o d u c t i o n was not observed i n e i t h e r endophytic or f r e e - l i v i n g forms of the endophytes. F r e e - l i v i n g forms were used f o r r e - i n f e c t i o n and c r o s s - i n f e c t i o n experiments. Under a l l experimental c o n d i t i o n s , the endophytes showed only e p i p h y t i c growth. The hosts seem to have some e f f e c t on both of the endophytes. Epiphytes, Audouinella porphyrae and A. vaga were not s e l e c t i v e and grew on both hosts, Porphyra tort a and Pterosiphonia bipinnata. Both epiphtyes growing on blades of Porphyra tended to branch and have more ex t e n s i v e p r o s t r a t e p o r t i o n s (3-5 c e l l s ) whereas both epiphytes - i i i -on Pt erosiphoni a bi pi nnat a tend not to branch and have only 1-2 c e l l s in the p r o s t r a t e p o r t i o n s . U l t r a s t r u c t u r a l s t u d i e s of both endophytes showed t y p i c a l f l o r i d e o p h y c e a n f e a t u r e s . U l t r a s t r u c t u r a l f e a t u r e s of f i e l d m a t e r i a l of the two endophytes were s i m i l a r and f r e e - l i v i n g , c u l t u r e d endophytes were s i m i l a r to f i e l d m a t e r i a l . A larg e vacuole was observed in s e c t i o n s of the f i e l d m a t e r i a l of both p r o s t r a t e and e r e c t p o r t i o n s whereas t h i s was not observed i n c u l t u r e d m a t e r i a l . Based on the r e s u l t s of t h i s study i t i s proposed that the two endophytes are c o n s p e c i f i c . Audouinella vaga i s r e f e r r e d to synonymy i n Audouinella porphyrae (Drew) Garbary, Hansen et S c a g e l . - i v-Table of Contents: Page A b s t r a c t i i L i s t of Ta b l e s v i L i s t of F i g u r e s v i i Acknowledgements ix D e d i c a t i o n x I n t r o d u c t i o n 1 M a t e r i a l s and Methods 8 F i e l d m a t e r i a l 8 C u l t u r e s 10 E s t a b l i s h i n g C u l t u r e s of F r e e - l i v i n g Endophytes.... 10 Developmental and L i f e H i s t o r y S t u d i e s 11 R e - i n f e c t i o n and C r o s s - i n f e c t i o n S t u d i e s 12 L i g h t and E l e c t r o n Microscopy S t u d i e s 13 R e s u l t s 16 F i e l d m a t e r i a l 16 Audouinella porphyrae 16 Audouinella vaga 17 Development of Endophytes: Audouinella porphyrae 20 Audouinella vaga 22 C u l t u r e d m a t e r i a l 23 Establishment of F r e e - l i v i n g Endophytes 23 I n f e c t i o n Experiments 26 L i g h t and E l e c t r o n Microscopy 28 D i s c u s s i o n 33 References 44 - V -F i g u r e s 52 - v i -L i s t of Tables Table Page I. S i m i l a r i t i e s and d i f f e r e n c e s between A u d o u i n e l l a porphyrae and Audouinella vaga a c c o r d i n g to Garbary et a l . (1982) 7 I I . Monthly f i e l d c o l l e c t i o n s of A. porphyrae i n v a r i o u s s p e c i e s of Porphyra 18 I I I . Dimensions of A. porphyrae growing i n s i t u , f i e l d c o l l e c t i o n s of Porphyra, and general host f e a t u r e s . . . 19 IV. Measurements of A. vaga i n i t s host Pi erosi phoni a b i p i n n a t a from f i e l d c o l l e c t i o n s 21 V. C e l l dimensions and branching p a t t e r n s of A. porphyrae and A. vaga from Point No P o i n t 24 VI. Summary of d i f f e r e n c e s and s i m i l a r i t i e s of mo r p h o l o g i c a l , r e p r o d u c t i v e and c y t o l o g i c a l c h a r a c t e r s of f i e l d and c u l t u r e d A u d o u i n e l l a porphyrae and A. vaga o b t a i n e d i n the c u r r e n t study 32 — v i i — L i s t of F i g u r e s F i g u r e s Page 1. Diagrammatic drawings of hosts 52 2-5. L i g h t micrographs of f i e l d m a t e r i a l 54 6-9. Komarski c o n t r a s t micrographs of f i e l d m a t e r i a l 56 10-14. Development of spores from c u l t u r e d m a t e r i a l 56 15-17. L i g h t micrographs of b a s a l and e r e c t f i l a m e n t s of c u l t u r e d m a t e r i a l 58 18-21. Nomarski c o n t r a s t micrographs of c u l t u r e d material..58 22-25. Host m a t e r i a l u n i n f e c t e d by endophytes 60 26-34. I n f e c t e d m a t e r i a l of Pt erosi phoni a bipinnata by Audouinella porphyrae 62 35-37. I n f e c t e d m a t e r i a l of Porphyra torta by Audouinella vaga 64 38-4-1. Me t h a c r y l a t e s e c t i o n s of A. porphyrae/Porphyra fuci col a 66 42-46. Met h a c r y l a t e s e c t i o n s of A. porphyrae/Porphyra fuci col a 68 47-50. Methacrylate s e c t i o n s of A. vaga/Pl erosiphonia bi pi nnat a 70 51-53. M e t h a c r y l a t e s e c t i o n s of A. vaga/Pt er os i phoni a bi pi nnat a 72 54-56. U l t r a s t r u c t u r e of A. porphyrae i n Porphyra fuci col a 74 57-60. U l t r a s t r u c t u r e of u n i n f e c t e d Porphyra fuci col a 76 - v i i i -61-64. U l t r a s t r u c t u r e of A. porphyrae i n Porphyra fuci col a 78 65-68. U l t r a s t r u c t u r e of A. vaga i n Pt erophoni a bi pi nnat a 80 69-71. U l t r a s t r u c t u r e of f r e e - l i v i n g A. porphyrae 82 72-75. U l t r a s t r u c t u r e of f r e e - l i v i n g A. vaga 84 - i X -Acknowledgements I am indebted to Dr. K. Cole f o r a l l of her wisdom, guidance and f i n a n c i a l support throughout t h i s t h e s i s . Her constant encouragement and enthusiasm were g r e a t l y a p p r e c i a t e d through my years as a graduate student. I would a l s o l i k e to thank Dr. D. Garbary f o r the i n i t i a t i o n of t h i s p r o j e c t , and f o r i n t r o d u c i n g me to the f i e l d of Phycology. E d i t o r i a l comments were g r a t e f u l l y accepted from Dr. K. Cole, Dr. D. Garbary and Dr. P. G a b r i e l s o n . Warm g r a t i t u d e i s expressed to Bev. Hymes fo r a l l of her h e l p and advi c e i n the l a b o r a t o r y . A p p r e c i a t i o n i s a l s o expressed to Kermit R i t l a n d f o r h i s support and computer knowledge throughout my t h e s i s . A s p e c i a l thanks i s given to Karen Morin f o r drawing f i g u r e one. Thanks a l s o go to Herb. Vandermeulen, Bev. Hymes, E l l e n Rosenberg, Dawn Renfrew, I r a Borgmann, Sandy Lindstrom and L a r r y Golden f o r t h e i r company d u r i n g many c o l l e c t i n g t r i p s to Point No P o i n t . I would l i k e to acknowledge Mr. L. Veto f o r t e c h n i c a l a s s i s t a n c e i n the E.M. l a b o r a t o r y . - X -Dedicated with love to my f a m i l y : Mom, Dad, S t e l l a and G l o r y . - 1 -INTRODUCTION Red algae may be found growing on other algae ( e p i p h y t i c ) , growing on the s u r f a c e s of animals ( e p i z o i c ) , and p a r t i a l l y i n s i d e the c e l l w a l l s of other algae (endophytic) as w e l l as on non-organic s u b s t r a t a . Epi/endophytic algae may grow p a r a s i t i c a l l y , or j u s t on or w i t h i n the host c e l l w a l l s without a f f e c t i n g the p h y s i o l o g y or morphology of the host c e l l s . Many symbiotic a s s o c i a t i o n s between p a r a s i t i c red algae and t h e i r h o sts have been w e l l s t u d i e d ( f o r review see Goff, 1982), but the r e l a t i o n s h i p between red a l g a l endophytes and t h e i r red a l g a l hosts has not r e c e i v e d much a t t e n t i o n (Goff, 1983). T r a d i t i o n a l l y , many red a l g a l endophytes have been c l a s s i f i e d as d i s t i n c t e n t i t i e s based on t h e i r a s s o c i a t i o n w i t h a p a r t i c u l a r host (White and Boney, 1969, 1970). These " o b l i g a t e " i n t e r a c t i o n s have been i n f e r r e d from o b s e r v a t i o n s of f i e l d - c o l l e c t e d m a t e r i a l s and have been only r e c e n t l y s u b j e c t e d to experimental a n a l y s i s . C u l t u r e s t u d i e s of some s p e c i e s of Audouinella (Bory) Dixon et I r v i n e , u sing r e - i n f e c t i o n and c r o s s - i n f e c t i o n s t u d i e s , have shown that they may not be as h o s t - s p e c i f i c as presumed (White and Boney, 1969, 1970; Garbary, 1979b). White and Boney ( 1969, 1970) s t u d i e d A. endophyt i ca ( B a t t e r s ) Dixon, and A. as paragopsis (Chemin) Dixon (both as Acrochaetium N a e g e l i ) , i n t h e i r hosts, Het erosiphonia plumosa ( E l l i s ) B a t t e r s , and Bonnemai soni a hamifera H a r i o t , r e s p e c t i v e l y . They measured c e l l dimensions of endophytes i n t h e i r hosts and compared these with c u l t u r e d , " f r e e - l i v i n g " -2-endophytes c o n c l u d i n g that the two taxa were d i s t i n c t s p e c i e s . They a l s o showed that the endophytes i n f e c t other hosts and do not show any s u b s t r a t e p r e f e r e n c e . Garbary (1979b) s t u d i e d s e v e r a l endophytic s p e c i e s of Audouinella, A. endophyt i ca, A. bonnemai soniae ( B a t t e r s ) Dixon, A. tetraspora Garbary et Rueness, from red a l g a l hosts as w e l l as an endozoic s p e c i e s from hydroids A. as par agopsis. He concluded that a l l i s o l a t e s e s t a b l i s h e d some degree of r e l a t i o n s h i p with a given host (both the o r i g i n a l and a l t e r n a t e h o s t s ) . Based on o b s e r v a t i o n s of c e l l s i z e and shape, Garbary (1979b) noted that the endophytic and f r e e - l i v i n g forms were m o r p h o l o g i c a l l y d i f f e r e n t s p e c i e s . The f r e e - l i v i n g forms tended to be l a r g e r i n c e l l l e n g t h and smaller i n c e l l diameter, and to branch l e s s f r e q u e n t l y . He found that only A. endophyt i ca grew e n d o p h y t i c a l l y i n t o given hosts and that when growing e n d o p h y t i c a l l y i t s morphology was a l t e r e d . He s p e c u l a t e d that the hosts caused m o d i f i c a t i o n s of the c e l l morphology of the endophyte and emphasised the importance of comparing c u l t u r e and f i e l d m a t e r i a l before d e l i m i t i n g s p e c i e s of endophytic and endozoic members of A c r o c h a e t i a c e a e . To c o n t r i b u t e to our knowledge of red a l g a l endophyte/host r e l a t i o n s h i p s , f i e l d and c u l t u r e d m a t e r i a l of two endophytic s p e c i e s of Audouinella, A. porphyrae (Drew) Garbary and A. vaga (Drew) Garbary, Hansen et Scagel (Rhodophyta, A c r o c h a e t i a l e s , A c rochaetiaceae) a s s o c i a t e d with Porphyra spp. and Pt erosi phoni a bipinnata ( P o s t e l s et Ruprecht) Falkenberg, r e s p e c t i v e l y , were s t u d i e d at one s i t e i n B r i t i s h Columbia. -3-Members of the Acro c h a e t i a c e a e are sma l l , u n i s e r i a t e branched f i l a m e n t s . The algae vary i n height from approximately 20 urn to 10 mm and t h e i r c o l o u r ranges from dark s t e e l blue to green and v i o l e t red to ro s e . The order A c r o c h a e t i a l e s i s d i s t i n g u i s h e d by the two l a y e r e d p i t plugs, absence of c a r p o g o n i a l branches, and a p i c a l growth of u n i s e r i a t e f i l a m e n t s t h a t are not e l a b o r a t e d i n t o pseudoparenchymatous t h a l l i . T h i s group of algae has e x p l o i t e d a wide range of environments and adapted to many d i f f e r e n t n i c h e s . D i f f e r e n t s p e c i e s occur i n f r e s h and marine h a b i t a t s ; however, they are p r i m a r i l y marine. Although some s p e c i e s are e p i l i t h i c , most taxa are symbiotic and e i t h e r e p i b i o t i c or e n d o b i o t i c . With regard t o ge n e r i c c l a s s i f i c a t i o n , at l e a s t 24 d i f f e r e n t schemes i n c o r p o r a t i n g one to e i g h t genera have been used s i n c e the e a r l y 20th century with over a dozen schemes employed s i n c e 1970 (Wo e l k e r l i n g , 1983). C r i t e r i a f o r generic segregation i n c l u d e f e a t u r e s of c h l o r o p l a s t s , l i f e h i s t o r i e s and c e l l morphology. A complete sexual l i f e h i s t o r y has been reported from only about 23 of the 390 d e s c r i b e d s p e c i e s of which 7 s p e c i e s are from f i e l d c o l l e c t e d p o p u l a t i o n s . Many s p e c i e s are known only to produce monosporangia (White and Boney, 1969) and/or t e t r a s p o r a n g i a (West, 1979; Garbary and Rueness, 1980). Four d i s t i n c t sexual h i s t o r i e s are known: 1) T r i p h a s i c , dimorphic -with s i m i l a r m o rphological gametophyte and te t r a s p o r o p h y t e ; 2) T r i p h a s i c , t r i m o r p h i c - with a d i m i n u t i v e gametophyte and a l a r g e t e t r a s p o r o p h y t e ; 3) D i p h a s i c , dimorphic - with a l a r g e t e t r a s p o r o p h y t e and no carposporopyte and 4) D i p h a s i c , dimorphic - with a d i m i n u t i v e c a r p o t e t r a s p o r o p h y t e and no te t r a s p o r o p h y t e , - 4 -( f o r more d e t a i l s , see Woelkering, 1983). Because of the few l i f e h i s t o r y s t u d i e s and inadequate d i s t i n c t m o r p h o l o g i c a l c h a r a c t e r s i t i s d i f f i c u l t to u t i l i z e these f e a t u r e s as generic c r i t e r i a . Drew (1928) proposed that a l l members of the A c r o c h a e t i c e a e be i n c l u d e d i n the genus Rhodochorton ( N a e g e l i ) . She suggested th a t c h l o r o p l a s t and sporangia f e a t u r e s were too v a r i a b l e to be used to d i s t i n g u i s h genera. Dixon and I r v i n e (1977) and Garbary (1979a) a l l concurred with Drew (1928) that a one genus system should be used. Dixon and I r v i n e , however, proposed the genus Audouinella s i n c e i t i s the o l d e r name. In t h i s t h e s i s I a l s o r e c o g n i z e only one genus Audouinella. Although many p h y c o l o g i s t s agree with t h i s c l a s s i f i c a t i o n (Garbary et a l . , 1982; W o e l k e r l i n g , 1983), the opposing schemes of Papenfuss (1945), Feldmann, (1962), Stegenga and van Wissen (1979), Bold and Wynne (1985) and t h e i r v a r i a n t s a l s o continue to be used. Within Audouinella a v a r i e t y of c h a r a c t e r s i s used to d i f f e r e n t i a t e s p e c i e s . These i n c l u d e : 1) b a s a l systems: u n i - o r m u l t i c e l l u l a r , 2) c h l o r o p l a s t s t r u c t u r e : p a r i e t a l vs s t e l l a t e , and one to many i n number, 3) mature c e l l : diameter and l e n g t h , 4) p y r e n o i d : present or absent, and one or more i n number, 5) branching p a t t e r n s : i r r e g u l a r , secund or o p p o s i t e , 6) h a b i t a t : e p i b i o t i c , e n d o b i o t i c or e p i l i t h i c , 7) morphology, s i z e , shape and c l u s t e r i n g of monosporangia and/or t e t r a s p o r a n g i a , 8) spore germination p a t t e r n , 9) morphology of gametangia and 10) post f e r t i l i z a t i o n development. For review of c h a r a c t e r s nine and ten, see Hansen and Garbary (1984). - 5 -The two s p e c i e s of endophytic Audouinella s t u d i e d i n t h i s t h e s i s have been p l a c e d i n s e v e r a l d i f f e r e n t genera. Smith (1944) t r a n s f e r r e d Rhodochort on porphyrae Drew from Rhodochorton i n t o Acrochaetiurn because the p l a n t s produced monosporangia. Simi l a r l y . , Jao (1937) t r a n s f e r r e d Rhodochort on vagum Drew from Rhodochorton to Acr ochaet i urn. Papenfuss (1945) t r a n s f e r r e d Rhodochor t on porphyrae to Chromastrum Papenfuss, and made the combination Chromastrum porphyrae (Drew) Papenfuss, based on the presence of a s t e l l a t e c h l o r o p l a s t . However, he r e t a i n e d Acrochaetiurn vagum Jao i n Acrochaeti urn based on the supposed presence of p a r i e t a l c h l o r o p l a s t i n the o r i g i n a l d e s c r i b e d m a t e r i a l (Drew, 1928). Papenfuss (1947) again r e v i s e d the c l a s s i f i c a t i o n of t h i s f a m i l y and r e p l a c e d Chromastrum with K y l i n i a Rosenvinge. Stegenga and Mulder (1979) t r a n s f e r r e d Acrochaet i urn vagum to Chromastrum based on the presence of a s t e l l a t e c h l o r o p l a s t and l i f e h i s t o r y presumed to be s i m i l a r to other s p e c i e s of Chromast rum. However, due to a l a c k of s t a b l e g e n e r i c c h a r a c t e r s Garbary (1979a) has chosen the one genus concept and both s p e c i e s are now r e f e r r e d to Audouinella (Garbary et a l . , 1982). Audouinella porphyrae, an endophyte which grows i n the c e l l w a l l s of a number of s p e c i e s of Porphyra (Bangiophyceae), i s found p r i m a r i l y i n the mid- to lower i n t e r t i d a l r e g i o n s . Endophytic Audouinella porphyrae i n s p e c i e s of Porphyra are widely d i s t r i b u t e d , i n western North America from Yakutat Bay, A l a s k a , U.S.A. (Hansen et a l . , 1981) to Baja, C a l i f o r n i a , Mexico (Garbary et a l . , 1982), New Zealand (South and Adams, 1976), A u s t r a l i a ( W o e l k e r l i n g , 1971) and T r i s t a n da Cuhna -6-(Baardseth, 1941). Audouinella vaga, an endophyte i n two f l o r i d e o p h y c e a n genera, Pt er osi phoni a, and Pol ysiphoni a, i s re p o r t e d from Alaska to C a l i f o r n i a but i s unknown elsewhere (Garbary et a l . , 1982). Audouinella porphyrae and A. vaga are m o r p h o l o g i c a l l y and o n t o g e n e t i c a l l y s i m i l a r (Garbary et a l . , 1982). Both have 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 that c o n t a i n s one pyr e n o i d , and s i m i l a r p a t t e r n s of spore germination. Based on d i f f e r e n c e s i n h o s t s , branching p a t t e r n s , presence or absence of h a i r c e l l s and t e t r a s p o r a n g i a , Garbary et a l . (1982) maintained them as d i f f e r e n t s p e c i e s (Table 1), but suggested that the two taxa might be c o n s p e c i f i c , the morphological d i f f e r e n c e s between them being a r e f l e c t i o n of the i n f l u e n c e of the hosts on t h e i r development. Using c u l t u r e s t u d i e s and l i g h t and e l e c t r o n microscopy, a comparative study was i n i t i a t e d on Audouinella porphyrae and Audouinella vaga. The f o l l o w i n g f e a t u r e s were i n v e s t i g a t e d : 1) mor p h o l o g i c a l and r e p r o d u c t i v e f e a t u r e s of both f i e l d and c u l t u r e d m a t e r i a l , 2) l i f e h i s t o r i e s of the endophytes i n u n i a l g a l c u l t u r e , 3) the r e l a t i o n s h i p between the endophytes and t h e i r h osts and 4) the q u e s t i o n of c o n s p e c i f i c i t y . Table I : S i m i l a r i t i e s and D i f f e r e n c e s between Audouinella porphyrae and Audoui net I a vaga a c c o r d i n g t o Garbary et a l . ( 1982). A. porphyrae vaga S i m i l a r i t i e s : Growth p a t t e r n C h l o r o p l a s t Pyrenoid Spore germination I n s i d e host c e l l w a l l S i n g l e , a x i a l , s t e l l a t e One I n t e r n a l d i v i s i o n I n s i d e host c e l l w a l l S i n g l e , a x i a l , s t e l l a t e One I n t e r n a l d i v i s i o n D i f f e r e n c e s : Host C e l l l e n g t h Tetrasporangia Hair c e l l s Branching p a t t e r n Porphyra Spp. 18-25pm Unknown Reported to be rare I r r e g u l a r Pi er ai phoni a sp. Less than 16pm Reported to be r a r e Unknown At r i g h t angles -8-MATERIALS AND METHODS F i e l d M a t e r i a l Monthly f i e l d t r i p s May 1984 - A p r i l 1985, were made to Poin t No Poi n t on the west coast of Vancouver I s l a n d , B r i t i s h Columbia (48° 23'N, 123° 59'W). Audoui nelI a porphyrae and Audouinella vaga var. vaga (henceforth A. vaga) were u s u a l l y c o l l e c t e d a t the same time. C o l l e c t i o n s were a l s o made at Eagle Cove (48° 28'N, 123° OTW) and C a t t l e P o i n t (48° 27'N 22° 58'W) on San Juan I s l a n d , Washington, U.S.A., i n May 1984, i n order to compare m a t e r i a l from d i f f e r e n t s i t e s . C o l l e c t i o n s i n the herbarium of the U n i v e r s i t y of B r i t i s h Columbia (UBC) c o n t a i n i n g A. porphyrae and A. vaga were examined t o determine p r e v i o u s c o l l e c t i o n s i t e s and dates of c o l l e c t i o n . Audouinella porphyrae tends to grow i n the h o l d f a s t r e g i o n of Porphyra spp. and i s de t e c t e d by red patches. Care was taken to remove h o l d f a s t s of Porphyra spp. when these were c o l l e c t e d . I n f e c t e d host specimens (Porphyra spp. and Pt erosiphonia bipinnata), which tended to be d e s i c c a t e d f o l l o w i n g exposure at low t i d e , were brought i n t o the l a b o r a t o r y on i c e d i r e c t l y from the f i e l d . Some were rehydrated i n l a r g e q u a n t i t i e s of seawater ov e r n i g h t f o r l i g h t and e l e c t r o n m i c r o s c o p i c s t u d i e s , and others were kept dry and c o l d i n the r e f r i g e r a t o r (4°C) f o r f u r t h e r examination of f i e l d c h a r a c t e r s . In c o n t r a s t , i t i s not p o s s i b l e to i d e n t i f y Pterosiphoni a bipi nnat a i n f e c t e d by Audouinella vaga with the unaided eye; t h i s r e q u i r e s m i c r o s c o p i c examination ( F i g . 4). Consequently, -9-s e v e r a l handfuls of P. bi pi nnat a were c o l l e c t e d from a number of s i t e s in the mid- to lower i n t e r t i d a l zone. There are other Audouinella s p e c i e s e p i p h y t i c on P. bipi nnata, but t h e i r c h l o r o p l a s t shapes and growth p a t t e r n s are d i f f e r e n t from those of A. vaga. Such taxa i n c l u d e : A. densa (Drew) Garbary, A. arcuata (Drew) Garbary, Hansen and Scagel and A. thuretii (Bornet) W o e l k e r l i n g (Garbary et a l . , 1982). Some i n f e c t e d specimens Porphyra s p e c i e s and of Pt er os i phoni a bipinnata were examined with d i s s e c t i n g and compound microscopes to determine the extent of i n f e c t i o n by the two Audouinella s p e c i e s . Measurements of the endophytes i n c l u d e d : ( c e l l dimensions) l e n g t h and diameter of c e l l s i n primary axes and of f u l l y e longated a p i c a l c e l l s of a f i l a m e n t ; maximal extent of i n f e c t i o n ( l e n g t h and width); and d i s t a n c e of the endophyte from the host's h o l d f a s t . The extent of growth o u t s i d e the host and the r e p r o d u c t i v e nature of both the host and the endophyte were a l s o noted. Three types of s t a t i s t i c a l a n a l y s e s were a p p l i e d to the data. A l i n e a r r e g r e s s i o n a n a l y s i s was used to r e l a t e the s i z e of Porphyra to the area of primary endophyte i n f e c t i o n (Sokal and R o h l f , 1973). Secondary i n f e c t i o n s were not c o n s i d e r e d i n t h i s a n a l y s i s . Another s t a t i s t i c a l a n a l y s i s was a 2X2 t e s t of independence and a Chi square t e s t t o v e r i f y i t s s i g n i f a n c e (Sokal and R o h l f , 1973). A " t - t e s t " was used to compare the s i g n i f i c a n c e of c e l l dimensions i n the two s p e c i e s , A. porphyrae and A. vaga from f i e l d m a t e r i a l (Sokal and Rohlf, 1973). For permanent c o l l e c t i o n s , i n f e c t e d Porphyra blades were d r i e d on herbarium sheets. P o r t i o n s of i n f e c t e d Pterosiphoni a bipi nnat a p l a n t s were mounted on s l i d e s i n 30% Karo. Other m a t e r i a l was preserved i n 5% formalin/seawater and where p o s s i b l e the Porphyra s p e c i e s were i d e n t i f i e d . Voucher specimens of f i e l d m a t e r i a l were d e p o s i t e d i n the U n i v e r s i t y of B r i t i s h Columbia p h y c o l o g i c a l herbarium (UBC). C u l t u r e s E s t a b l i s h i n g c u l t u r e s of f r e e - l i v i n g endophytic s p e c i e s . Some h e a v i l y i n f e c t e d host specimens were used to e s t a b l i s h f r e e - l i v i n g c u l t u r e s of the endophytes (Table I I ) . Ten m i l l i m e t e r d i s c s of non-reproductive, h e a v i l y i n f e c t e d Porphyra blades from c l o s e to the h o l d f a s t r e g i o n were p l a c e d i n 30-40 ml of PES medium ( P r a v o s o l i , 1968) with (5-10 mg/1) Ge0 2 to stop diatom growth on the hosts t h a l l u s (Lewin, 1966). D i s c s were screened to ensure no epiphytes were present. The m a t e r i a l was then kept at 6°C 16:8 h Light:Dark (L:D), photon f l u x d e n s i t y of 5-6 m i c r o e i n s t e i n m~2 s e c " 1 i n PES medium to induce s p o r u l a t i o n . C o n d i t i o n s were chosen to simulate f i e l d c o n d i t i o n s . Some stock c u l t u r e s were a l s o maintained at 10°C 8:16 h (L:D) photon f l u x d e n s i t y of 4-5 m i c r o e i n s t e i n s m"2 s e c " 1 i n PES medium. When spores were observed on the bottom of c u l t u r e d i s h e s , host m a t e r i a l was removed. Spores were then grown i n 60 X 20 mm p l a s t i c d i s h e s , with or without c o v e r s l i p s , and i n 125 ml f l a s k s c o n t a i n i n g 75 ml of medium. R e - i s o l a t i o n of s p o r e l i n g s of Audouinella porphyrae i n t o new v e s s e l s was necessary to achieve u n i a l g a l c u l t u r e s , and p e n i c i l l i n l e v e l s of about 150 mg/1 (Hoshaw and Rosowski, 1973) were sometimes used i n c u l t u r e s that - 1 1 -were o v e r l y i n f e c t e d by b a c t e r i a and f u n g i . The medium was changed every three weeks, and the c u l t u r e s were covered with c h e e s e c l o t h t o reduce the amount of l i g h t . I t was observed that a high photon f l u x d e n s i t y of 25 m i c r o e i n s t e i n s i r r 2 s e c " 1 tended to reduce pigmentation i f the medium was not r e p l a c e d weekly. Piec e s of i n f e c t e d Pt er os i phoni a bipi nnat a were t r e a t e d s i m i l a r l y t o e s t a b l i s h f r e e - l i v i n g c u l t u r e s of Audouinella vaga. F r e e - l i v i n g specimens of both endophytes were then used to compare t h e i r general morphologies, developmental p a t t e r n s , c r o s s - i n f e c t i o n a b i l i t i e s and l i f e h i s t o r i e s . Dimensions of the i n i t i a l spore, f i l a m e n t l e n g t h s , c e l l dimensions ( l e n g t h and width), and branching types were recorded. Developmental and L i f e H i s t o r y S t u d i e s . To study developmental p a t t e r n s , f i l a m e n t s of f r e e l i v i n g Audouinella porphyrae and A. vaga were grown on c o v e r s l i p s at 6°C 16:8 h (L:D), photon f l u x d e n s i t y of 4-5 m i c r o e i n s t e i n s m"2 s e c " 1 i n PES medium. V a r i o u s stages of the l i f e c y c l e , such as spore germination, f i l a m e n t i n i t i a t i o n branching p a t t e r n , monospore p r o d u c t i o n and spore r e l e a s e were photographed u s i n g b r i g h t f i e l d as w e l l as Nomarski modulation c o n t r a s t microscopy. Attempts were made to induce the sexual phase of the l i f e h i s t o r y . F i l a m e n t s of both f r e e - l i v i n g Audouinella s p e c i e s were grown on c o v e r s l i p s and p l a c e d under v a r i o u s temperature and daylength c o n d i t i o n s w i t h the same photon f l u x d e n s i t y (2 m i c r o e i n s t e i n m"2 s e c " 1 ) . The f o l l o w i n g c o n d i t i o n s were t r i e d : 6°C 8:16 h (L:D), 6°C 16:8 h (L:D), 15°C 8:16 h (L:D) and 15°C 16:8 h (L:D). 1 2 -R e - i n f e c t i o n and C r o s s - i n f e c t i o n S t u d i e s . T e t r a s p o r e s of Pi erosiphoni a bi pi nnat a ( c o l l e c t e d June, 1984) were grown i n the l a b o r a t o r y at 10°C 8:16 h (L:D) with PES medium i n 60 X 20 mm p l a s t i c d i s h e s . Filaments with r h i z o i d s were grown to approximately 2-3 cm i n le n g t h and then i n d i v i d u a l f i l a m e n t s were p l a c e d i n 250 ml f l a s k s c o n t a i n i n g 100 ml of PES medium at a photon f l u x d e n s i t y of 55 m i c r o e i n s t e i n rn"2 s e c " 1 with continuous a e r a t i o n . Clumps of m a t e r i a l approximately 4-5 cm long were used f o r subsequent s t u d i e s . Young u n i n f e c t e d blades of Porphyra torta Krishnamurthy and P. abbottae Krishnamurthy ( c o l l e c t e d Feb. 1985) were used i n the re - and c r o s s - i n f e c t i o n experiments. To ensure t h a t these blades were c l e a n , a method m o d i f i e d from P o l n e - F u l l e r and Gibor (1984) was employed. Young blades of Porphyra (3-8 cm i n length) were s o n i c a t e d g e n t l y i n s t e r i l e d i s t i l l e d water f o r 15 seconds i n an Artek Sonic Dismembrator Model 300 at the 30 p o s i t i o n , then r i n s e d i n s t e r i l e seawater. The procedure was repeated three more times using s t e r i l e seawater. To e l i m i n a t e protozoans and b a c t e r i a l e p i p h y t e s , blades were next soaked f o r 7 minutes i n a s o l u t i o n of 1 Betadine (TM): 100 seawater and r i n s e d i n s t e r i l e seawater u n t i l no t r a c e s of the brown Betadine were v i s i b l e . Blades were a l s o r i n s e d with a n t i b i o t i c s (Hoshaw and Rosowski, 1973). S e v e r a l i s o l a t e s of A. porphyrae (see Table II) were made to ensure that a l l i s o l a t e s were the same s p e c i e s . A n t i b i o t i c s (Hoshaw and Rosowoski, 1973) were a l s o used f o r one week when e s t a b l i s h i n g c u l t u r e s . I n f e c t i o n experiments i n v o l v e d three s e r i e s of f l a s k s : 1) c o n t r o l f l a s k s i n which p i e c e s of the host p l a n t s were c u l t u r e d -13-without any endophytes, 2) r e - i n f e c t i o n f l a s k s i n which Audouinella porphyrae grown on 18 X 18 mm2 c o v e r s l i p s was placed in the f l a s k s with cleaned p i e c e s of Porphyra and A.vaga was pl a c e d i n f l a s k s with Pt erosiphoni a bi pi nnat a and 3) c r o s s - i n f e c t i o n experiments i n which A. porphyrae was placed i n a 250ml f l a s k with P. bipi nnat a and A. vaga i n a 250ml f l a s k with Porphyra. A l l c o n d i t i o n s were e s t a b l i s h e d i n t r i p l i c a t e . F l a s k s were p l a c e d at photon f l u x d e n s i t y of 55 m i c r o e i n s t e i n s m"2 s e c " 1 at 10°C 8:16 h (L:D) and aera t e d using an a i r pump. Observations were made weekly f o r s i g n s of i n f e c t i o n . Since e p i p h y t i c growth only was observed i n the i n f e c t i o n experiments, an attempt was made to induce endophytic growth. Pt erosiphoni a bipi nnat a together with f r e e - l i v i n g endophytes were p l a c e d i n 250 ml erlenmeyer f l a s k s . The medium was poured out d a i l y and p l a n t s were allowed t o d e s i c c a t e from 2-48 hours with d e s i c c a t i o n times i n c r e a s i n g over a four week p e r i o d . Two r e p l i c a t e s of t h i s experiment were performed. C o n t r o l f l a s k s were not d e s i c c a t e d . The experimental c o n d i t i o n s were 15°C, 16:8 h (L:D), photon f l u x d e n s i t y of 55 m i c r o e i n s t e i n s m"2 sec" 1 . L i g h t and E l e c t r o n Microscopy S t u d i e s For l i g h t m i c r o s c o p i c s t u d i e s , rehydrated and i n f e c t e d Porphyra fucicola Krishnamurthy and Pt erosiphoni a bipi nnata were f i x e d with 2-3% glutaraldehyde/phosphate b u f f e r (0.067M; pH = 7.2) or 5% formalin/phosphate b u f f e r (0.067M; pH = 7.2). Specimens were dehydrated i n a methanol s e r i e s , f o l l o w e d by a propylene oxide s e r i e s , and then embedded i n g l y c o l methacrylate -14-p l a s t i c from P o l y s c i e n c e s , Warrington, Wa. (Feder and O'Brien, 1968). S e r i a l s e c t i o n s , approximately 2-2.5 urn t h i c k were cut usin g g l a s s knives on a S o r v a l l Porter-Blum microtome. S e r i a l s e c t i o n s were obtained by a p p l y i n g a small amount of rubber cement along the width of the blocks (Henry, 1977). S e c t i o n s were p i c k e d up, d r i e d on g l a s s s l i d e s , and p l a c e d i n v a r i o u s h i s t o c h e m i c a l s t a i n s : a l c i a n b l u e / p e r i o d i c a c i d s c h i f f (AB/PAS) f o r v a r i o u s p o l y s a c c h a r i d e s (Kiernan, 1981), f a s t green (FG) f o r p r o t e i n s (McCully et a l . , 1980), IKI f o r s t a r c h (O'Brien and McCully, 1981), p e r i o d i c a c i d / s c h i f f (PAS) f o r p o l y s a c c h a r i d e s and s t a r c h (O'Brien and McCully, 1981), sudan black (SB) f o r l i p i d s (O'Brien and McCully, 1981), C a l c o f l u o r White (CW) ST f o r B-1,3 or B-1,4 p o l y s a c c h a r i d e s (Hughes and McCully, 1975) and t o l u i d i n e blue-0 (0.05%, pH = 4.4) (TBO) was used as a general s t a i n (O'Brien and McCully, 1981). For t r a n s m i s s i o n e l e c t r o n micorscopy (T.E.M.) s t u d i e s of rehydrated f i e l d m a t e r i a l of Porphyra fuci cola/Audoui nelI a porphyrae and Pt erosiphoni a bi pi nnat a/Audoui nelI a vaga, as w e l l as f r e e - l i v i n g A. porphyrae and A. vaga were f i x e d i n 2-3% g l u t a r a l d e h y d e / phosophate b u f f e r (0.067 M; pH = 7.2)(Hymes and Cole, 1983a). S e v e r a l other f i x a t i v e s were found to be u n s a t i s f a c t o r y on f i e l d m a t e r i a l : phosphate-0.1M cacodylate-0.2M sucrose (pH = 7.2)/ 2-3% g l u t a r a l d e h y d e (Pueschel and Cole, 1985), seawater-NaCl/ 2-3% g l u t a r a l d e h y d e (Pueschel, p e r s . comm.). F r e e - l i v i n g specimens were f i x e d with PIPES/2-3% g l u t a r a l d e h y d e (Hayat, 1981), but the phosphate b u f f e r (0.067M; pH = 7.2)/ 2-3% gl u t a r a l d e h y d e (Hymes and Cole, 1983a) f i x a t i o n - 1 5 -proved most s a t i s f a c t o r y f o r f r e e - l i v i n g specimens. A l l m a t e r i a l s was p o s t f i x e d i n 2% 0s0«/phosphate b u f f e r (0.067M; pH = 7.2) at 4°C f o r 2 hours, except f r e e - l i v i n g A. porphyrae and A. vaga, which were p o s t f i x e d f o r 1 hour at 4°C to reduce dark c o n t r a s t i n the cytoplasm. Specimens were dehydrated i n a graded s e r i e s of methanol f o l l o w e d by graded s e r i e s of propylene oxide and embedded i n Spurr's p l a s t i c ( f r e s h p l a s t i c was r e p l a c e d every 24 hours f o r 7 days) that was hardened at 70°C for 7-8 hours (Spurr, 1969). S e c t i o n s of 60-90nm were cut using g l a s s knives on a R e i c h e r t 0mU3 ultramicrotome. S e c t i o n s were p i c k e d up on uncoated copper g r i d s , s t a i n e d f o r 45 mins with u r a n y l a c e t a t e (Watson, 1958) and r e s t a i n e d f o r 15 mins with le a d c i t r a t e (Reynold,1963). S e c t i o n s were viewed i n a Z e i s s EM-10 t r a n s m i s s i o n e l e c t r o n microscope. -16 -RESULTS F i e l d m a t e r i a l : Monthly c o l l e c t i o n s at P o i n t No Point showed that host p l a n t s , Porphyra spp. and Pi erosiphoni a bipinnala, were present from February to October. The endophytes Audouinella porphyrae and Audouinella vaga were observed from May to October, and t h e i r i n i t i a l appearance c o i n c i d e d with the maturation of the host p l a n t s i n l a t e s p r i n g . Endophytes and host p l a n t s were not p r e s e n t d u r i n g the winter. A study of the herbarium c o l l e c t i o n s i n UBC i n d i c a t e d that both endophytes o c c u r r e d i n t h e i r hosts from February to September and were most f r e q u e n t l y r e p o r t e d from June and J u l y . The abundance of herbarium c o l l e c t i o n s from these months may be a r e f l e c t i o n of c o l l e c t i o n e f f o r t , and c u r r e n t o b s e r v a t i o n s suggest t h a t p l a n t s are abundant throughout the p e r i o d s of May - June and September - October. The two endophytes were more d i f f i c u l t to c o l l e c t i n J u l y and in l a t e f a l l when host p l a n t s became l e s s common i n the i n t e r t i d a l zone. H o s t / endophytes c o l l e c t e d from San Juan I s l a n d (Washington) and T r i p l e I s l a n d , B.C. ( c o l l e c t e d by L. Golden) were m o r p h o l o g i c a l l y i d e n t i c a l t o those c o l l e c t e d from P o i n t No P o i n t . Audouinella porphyrae: Audouinella porphyrae i s a filamentous endophyte, growing i n the host c e l l w a l l , j u s t under the s u r f a c e . I t branches randomly and penetrates the w a l l between the c e l l s of the h o s t . -17-C e l l s of A. porphyrae tend to be c y l i n d r i c a l i n o l d e r p o r t i o n s of the f i l a m e n t s and to taper towards the apex. E r e c t f i l a m e n t s p r o j e c t outward from the host s u r f a c e where the asexual r e p r o d u c t i v e s t r u c t u r e s , monosporangia, are formed ( F i g . 2). Audouinella porphyrae i s not s p e c i e s s p e c i f i c . I t grows i n s e v e r a l Porphyra s p e c i e s when they are present (Table I I ) , most f r e q u e n t l y i n the h o l d f a s t regions (Table III and F i g . 1). Other p a r t s of the v e g e t a t i v e blade may a l s o be i n f e c t e d , but these i n f e c t i o n s are u s u a l l y l e s s e x t e n s i v e than the main i n f e c t i o n s i t e i n the h o l d f a s t a r e a . In A p r i l when the endophyte f i r s t appeared, i t was observed only i n the h o l d f a s t a r e a . By September many other areas of i n f e c t i o n were a l s o noted on more d i s t a l v e g e t a t i v e p a r t s of the b l a d e . I t was noted t h a t these l a t e r i n f e c t i o n s were g e n e r a l l y smaller than the o r i g i n a l i n f e c t i o n . No endophytes were seen i n the r e p r o d u c t i v e areas of the host b l a d e s . The area occupied by the primary i n f e c t i o n of A. porphyrae ranged from 0.1 cm 2 to 22.5 cm 2 (Table I I I ) . No c o r r e l a t i o n (b = 0.032; r 2 = 0.006) was found between the s i z e of the host blades and the s i z e of the primary i n f e c t i o n areas. The i n f e c t i o n i s r e s t r i c t e d to v e g e t a t i v e p o r t i o n s of Porphyra spp. Most of the i n f e c t e d blades were r e p r o d u c t i v e (Table I I I ) . The endophyte produces monosporangia ou t s i d e the host c e l l w a l l when the area of i n f e c t i o n i s g r e a t e r than 3.0 cm 2. AudouinelI a vaga Audouinella vaga i s an abundantly branching filamentous endophyte that grows j u s t beneath the s u r f a c e of the host, -18 -Table I I : Monthly f i e l d c o l l e c t i o n s of Audouinella porphyrae i n va r i o u s species of Porphyra. Date of Location Species of Presence of I s o l a t e s c o l l e c t i o n of c o l l e c t i o n Porphyra endophyte i n c u l t u r e May 16, 1 984 Eagle' S Cove , S . J . p. s c hi 2 o ph yl la May 16, 1984 Eagle' s Cove, s . J . p. I or l a + June 14, 19B4 T r i p l e I s . , B .c # * p. I or l a + June 29, 1 984 T r i p l e I s . , B .c * p. s c hi zophylI a + June 29, 1984 P.N.P. B.C. p. fucicola + June 29, 1984 P.N.P. p. S c hizophyl1ia + June 29, 1984 P.N.P. p. sanj uanes is + June 29, 1984 P.N.P. p. abbot t ae June 29, 1984 P.N.P. p. s c hizophylI a June 29, 1 984 P.N.P. p. t or i a + June 29, 1984 P.N.P. p. fuci col a + June 29, 1 984 P.N.P. p. fuci col a + June 29, 1984 P.N.P. p. fuci col a + June 29, 1984 P.N.P. p. fuci cola + June 29, 1984 P.N.P. p. fucicola + June 29, 1 984 P.N.P. p. fucicola + June 29, 1 964 P.N.P. p. fucicol a J u l y 15, 1984 P.N.P. very l i t t l e Porphyra _ Aug. 15, 1 984 P.N.P. p. I or l a Oct. 12, 1 984 P.N.P. p. kanakaensis + Oct. 12, 1 984 P.N.P. p. 1 or l a + Oct. 12, 1 964 P.N.P. p. t or l a + Oct. 12, 1 984 P.N.P. p. I or l a + Oct. 12, 1984 P.N.P. p. I or t a + Dec. 19, 1984 P.N.P. NO Porphyra -Feb. 4, 1984 P.N.P. Juven i l e -Mar. 4 , 1 964 P.N.P. J u v e n i l e -Apr . 10, 1 984 P.N.P. p. l or i a + S.J.= San Juan I s l a n d , Washington P.N.P.= Point No P o i n t , B r i t i s h Columbia * = c o l l e c t e d by L. Golden -19 -Table I I I : Dimensions of Audouinella porphyrae growing in situ, f i e l d c o l l e c t i o n s of Porphyra, and general host f e a t u r e s . Endophytes Host Length Width Area Reprod. Length Width Area Repi (cm) (cm) (cm 2) (cm) (cm) (cm 2) 7.5 3.0 22.5 12.5 (a) 13 162.5 + B 21 .5 20 + 14(a) 7 98 •+ 3.8 4 16.2 + 11.5(a) 8 92 + 3.5 3.5 12.25 + 11 (a) 7 77 2.5 2.3 5.75 + 6(a) 5.5 33 + 3.5 1.5 5.25 + 7(a) B 56 3.5 1.5 5.25 + 1.5(a) ' 3.5 5.25 + 2.0 2.4 4.6 + 6(a) 8 48 + 1.8 2.5 4.5 + 1.8(d) 2.5 4.5 + 2.5 1 .8 4.5 + 9.5(c) 7 .0 66.5 + 2.0 2.0 4 + 4.5(a) 5.5 24.75 -1 .6 1.8 3.24 + L B ( b ) 1 .8 3.24 + 1.8 1.8 3.24 + 11 (e) 6 66 + 1.5 1.5 2.25 - 4(a) 2.8 11.2 + 1.5 1.5 2.25 - 9.5(e) 7 66.5-1 1 1 - 10(a) 8 80 -0.5 0.5 0.25 - 4(a) 6 32 + 2 0.1 0.2 - 4.5(b) 5.5 24.75 + 0.1 0.1 0.1 - 15.5(c) 12.5 193.75 + • • reproductive s t r u c t u r e s were present - • reproductive structures were absent Note: a l l i n f e c t i o n s begin at the hold f a s t of the host and other subsequent smaller i n f e c t i o n s begin f u r t h e r up the host blade, a » Porphyra fuci col a b * Porphyra abbot Iae c « Porphyra s anj uanes i s d «= Par phyr a t or l a e • Por phyr a schi zophylI a -20-mainly i n the p e r i c e n t r a l c e l l w a l l s ( F i g . 4). The endophyte grows p a r a l l e l to the primary a x i s of the l o n g i t u n d i n a l a x i s of the host ( F i g . 5). C e l l s tend to be c y l i n d r i c a l i n the o l d e r p o r t i o n of the endophyte and taper toward the growing p o i n t . E r e c t f i l a m e n t s are e x t e r i o r t o the host w a l l and u s u a l l y abundant, a r i s i n g from almost any c e l l of the endophytic f i l a m e n t s . Monosporangia are formed from the e x t e r n a l , e r e c t f i l a m e n t s . Audouinella vaga grows most f r e q u e n t l y i n the w a l l of the main a x i s and i n l a r g e r branches of i t s host and only r a r e l y i n young a p i c a l t i p s and r h i z o i d s (Table IV and F i g . 1). I n f e c t i o n areas of A. vaga ranged from 180 urn2 to 7.5 mm2 (Table I V ) . Monospores u s u a l l y a t t a c h to the host w a l l i n the area between two adjacent p e r i c e n t r a l c e l l s . As f o r A. vaga, the s i z e of i n d i v i d u a l i n f e c t i o n areas i s p o s i t i v e l y c o r r e l a t e d ( x 2 = 3.03; p = 0.05) with the host being r e p r o d u c t i v e . Thus, the age of the host i s probably a determining f a c t o r f o r i n f e c t i o n by the endophyte. T h i s i s supported by the ob s e r v a t i o n t h a t o l d e r p o r t i o n s of the host tend to become h e a v i l y i n f e c t e d before secondary i n f e c t i o n s occur. The l a r g e r the i n f e c t i o n area ( g r e a t e r than 250 ;um2), the more l i k e l y the endophyte w i l l be r e p r o d u c t i v e (Table I V ) . Development of endophytes: Audouinella porphyrae I n f e c t i o n of both hosts by the two endophytes commences i n the same way. Monospores a t t a c h to outer w a l l s of t h e i r r e s p e c t i v e hosts and d i v i d e i n t e r n a l l y ( F i g . 6). A germination tube i s then produced from one of the two daughter c e l l s ( F i g . -2 1-Table IV: Measurements of Audouinella vaga i n i t s host Pterosiphonia bipi nnata from f i e l d c o l l e c t i o n s . Endophyte Length Width Area Reprod. Erect p o r t i o n s Reprod. Location outside of on host host host 1 2 3 (pm) (urn) (pm1) 151.5 50 7575 - - + / 138 42 5796 + + + / 165 28.5 4702.5 + + / 129 25.5 3289.5 + + / 49.5 60 2970 - + + 198 13.5 2673 + + + / 90 27 2430 - + / 75 21 1575 - - + / 77.1 IB 1387 .8 + - / 69 18 1242 - - + / 54 20 1080 - - + / 43 24 1032 + + + / 45 18 810 - - + / 42 15 630 - - + / 7.5 63 472.5 - - - / 30 15 450 - - + / 39 9 351 - - - / 52.5 4.5 236.25 - - / 37.5 6 225 - - + / 34.5 6 207 - - - / 13.5 9 121.5 - - - / 90 20 180 — T o t a l 2 / 13 6 + = reproductive s t r u c t u r e s (carpogonia, or spermatangia or tetrsporangia) are present - = reproductive s t r u c t u r e s are absent / = l o c a t i o n of endophyte on host according to a schematic drawing of Pterosiphonia bi pi nnat a ( F i g . 1) (see F i g . 1 for explanation of l o c a t i o n on host.) -2 2-7) and the r e s u l t i n g f i l a m e n t t a p e r s as i t pen e t r a t e s the host c e l l w a l l . The i n i t i a l spores remain o u t s i d e the hosts ' c e l l w a l l s throughout development. S e t t l e d monospores of Audouinella porphyrae measured 12.0 urn ±0.3 i n le n g t h and 9.0 urn ±0.3 i n width (n=50) a f t e r the i n i t i a l c e l l d i v i s i o n (Table V ) . C e l l s i n the primary f i l a m e n t were 13.5 pm ±2.3 i n le n g t h and 7.5 pm ±0.8 i n diameter (n=50). A l l 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 and one prominent pyrenoid ( F i g . 8 ) . As the endophyte grows w i t h i n the host w a l l , i t branches i r r e g u l a r l y , r a d i a t i n g out from the i n i t i a l p o i n t of i n f e c t i o n ; e v e n t u a l l y a somewhat c i r c u l a r r e d d i s h pink area i s formed. These f e a t u r e s c o u l d be seen e a s i l y u sing Hoffman microscopy ( F i g s . 6-9). As the endophyte grows areas of i n f e c t i o n become l a r g e r and l a r g e r , e v e n t u a l l y g i v i n g the blade a red b l o t c h y appearance. There were no i n d i c a t i o n s of the endophyte p e n e t r a t i n g host c e l l s . E r e c t f i l a m e n t s are formed e x t e r n a l t o the host w a l l . These developed p e r p e n d i c u l a r l y t o the host s u r f a c e , extended to about 25 pm, and had two to s i x c e l l s . In g e n e r a l , one to two monosporangia were present on each e r e c t a x i s . No t e t r a s p o r a n g i a or h a i r c e l l s were noted i n m a t e r i a l examined i n t h i s study. Development of endophytes: Audouinella vaga S e t t l e d monospores Audouinella vaga were 12 pm ±0.3 i n leng t h and 8 pm ±0.3 i n diameter (n = 50). The a p i c a l c e l l of growing f i l a m e n t s measured 21 pm ±0.4 i n l e n g t h and 3.0 pm ±0.3 in diameter (n = 50). The narrow diameter of a p i c a l c e l l s compared t o o l d e r v e g e t a t i v e c e l l s was a c o n s i s t e n t f e a t u r e of -23-both endophytes. There was a s i g n i f i c a n t d i f f e r e n c e i n the len g t h s of c e l l s on primary f i l a m e n t s of the two s p e c i e s and A , vaga had s i g n i f i c a n t l y s m a l l e r diameters (Table V ) . No d i f f e r e n c e s i n the c h l o r o p l a s t and pyren o i d were noted between the two s p e c i e s (Table V ) . Fi l a m e n t s of A. vaga tend t o grow p a r a l l e l to the main a x i s and to the l o n g i t u d i n a l dimension of p e r i c e n t r a l c e l l s of the hos t . In the p r o s t r a t e p o r t i o n s of the endophyte branching tends to occur at r i g h t angles ( F i g . 5). E r e c t p o r t i o n s a r i s e from the p r o s t r a t e f i l a m e n t s and protrude through the host c e l l w a l l s . E r e c t axes g e n e r a l l y c o n s i s t of one to two c e l l s and one to two monosporangia are produced on these e r e c t p o r t i o n s . The i n f e c t i o n d i d not show macroscopic p a t t e r n s or b l o t c h e s i n the host f i l a m e n t s , and i t was only by c l o s e examination with a compound microscope that the endophytic growth c o u l d be r e s o l v e d . M i c r o s c o p i c a l l y , the endophyte forms numerous p a r a l l e l f i l a m e n t s t h a t branch at r i g h t angles ( F i g . 5) . N e i t h e r t e t r a s p o r a n g i a nor h a i r c e l l s were observed i n f i e l d m a t e r i a l of A. vaga. C u l t u r e m a t e r i a l Establishment of f r e e - l i v i n g endophytes Audouinella porphyrae and A. vaga grew r e a d i l y as f r e e - l i v i n g endophytes i n c u l t u r e without the presence of host m a t e r i a l and t h e r e f o r e are not o b l i g a t e endophytes. Commencing with monospores r e l e a s e d from mature p l a n t s , the developmental p a t t e r n of both endophytes was s i m i l a r , and the f r e e - l i v i n g forms were m o r p h o l o g i c a l l y i d e n t i c a l . Upon -24 -Table V: C e l l dimensions and branching patterns of Audouinella porphyrae and Audouinella vaga in t h e i r r e spective hosts from Point No Point. (* p = <0.05; a l l cases n = 50) A. porphyrae A. vaga C e l l dimension: S e t t l e d spore**: 12pm ( 1 ) 12pm ( 1 ) 9pm (d)* Bpm (d)* Main filament: I3 .5jjm ( 1 ) * 15pm ( 1 ) * 7.5pm (d)* 6pm (d)* Mature a p i c a l c e l l : 15pm ( 1 ) * 21pm ( 1 ) * 3pm (d) 3pm (d) Host: Porphyra spp. Pt erosiphonia sp. Branching p a t t e r n : I r r e g u l a r At r i g h t angles ( 1 ) = length (d) = diameter * i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s at p = <0.05. ** measurements a f t e r i n t e r n a l d i v i s o n . -2 5-a t t a c h i n g to a v a r i e t y of s u b s t r a t a such as p l a s t i c , g l a s s or nylon thread, monospores underwent an i n t e r n a l d i v i s i o n ( F i g . 10). Attachment and i n t e r n a l d i v i s i o n occured w i t h i n 24-48 hours of spore r e l e a s e . T h i s was f o l l o w e d by the formation of a germination tube from one of the two c e l l s ( F i g s . 11, 12). Commonly, a second germination tube was formed l a t e r from the second c e l l of the o r i g i n a l spore. A small p r o s t r a t e f i l a m e n t o u s p l a n t was formed that gave r i s e to e r e c t f i l a m e n t s ( F i g . 13). At no time d i d e i t h e r of the two i n i t i a l c e l l s i n s i d e the spore w a l l g i v e r i s e t o e r e c t f i l a m e n t s . Basal c e l l s were 14.0 pm i n l e n g t h and 9.0 urn i n diameter, whereas the c e l l s i n the primary e r e c t f i l a m e n t were 16.5 urn i n l e n g t h and 9.0 urn i n diameter ( F i g . 14). Up to four e r e c t axes c o u l d be formed from a s i n g l e b a s a l system ( F i g s . 13). A p i c a l c e l l s were 18.0 um i n l e n g t h and 9.0 urn i n diameter. E r e c t f i l a m e n t s branch f r e q u e n t l y ; branching was p r i m a r i l y u n i l a t e r a l . C e l l s i n l a t e r a l branches were s m a l l e r than those of the primary a x i s ( F i g s . 15,18) and o c c a s i o n a l l y , the l a t e r a l branches, rebranched. These secondary branches always developed a d a x i a l l y ( F i g . 18). Most of the p l a n t i s comprised of e r e c t p o r t i o n s (50-100 c e l l s ) ; very few b a s a l c e l l s are formed (5-9 c e l l s ) . Growth i n the f l a s k s became q u i t e dense and p l a n t s grew on the s i d e s of the v e s s e l s . As the p l a n t s matured, monosporangia ( F i g s . 16,17) were produced. Monosporangia were e i t h e r t e r m i n a l or l a t e r a l on e r e c t branches, and e i t h e r s e s s i l e or on a o n e - c e l l e d p e d i c e l l ( F i g s . 16,17). In most cases only a s i n g l e monosporangium was formed on a supporting c e l l ; i n f r e q u e n t l y sporangia were formed -26-i n p a i r s ( F i g . 21). S i m i l a r to the formation of l a t e r a l branches, monosporangia were formed u n i l a t e r a l l y . Two to s i x sporangia may be formed i n s u c c e s s i o n w i t h i n the o r i g i n a l sporangium w a l l ( F i g s . 19,20). Remnants of empty sporangium w a l l s c o u l d be e a s i l y seen with Hoffman c o n t r a s t microscopy ( F i g . 20). Monosporangial p r o d u c t i o n was g r e a t e r i n long days 16:8 h (L:D) vs 8:16 h (L:D)), at a low temperature (6°C vs 15°C) and at low photon f l u x d e n s i t i e s (4-5 vs 22 vs 55 m i c r o e i n s t e i n s m " 2 s e c " 2 ) . Only a few monosporangia were produced under other experimental c o n d i t i o n s . Four months a f t e r i n o c u l a t i o n the bottom of a d i s h was covered with p l a n t s and monospores. The monosporangial c y c l e took three t o s i x weeks. Attempts to induce sexual r e p r o d u c t i v e s t r u c t u r e s by v a r y i n g c u l t u r e c o n d i t i o n s were u n s u c c e s s f u l . T h i s c o r r o b o r a t e s the absence of gametangia observed i n f i e l d m a t e r i a l . I n f e c t i o n experiments In the c o n t r o l s e r i e s , Pt erosiphonia bi pi nnat a p l a n t s grown from t e t r a s p o r e s were l a r g e enough t o be used f o r i n f e c t i o n experiments w i t h i n three weeks. Under the c u l t u r e c o n d i t i o n s p r o v i d e d , they branched and grew l a r g e r d u r i n g the e n t i r e time of the experiment ( F i g s . 22-24). Porphyra tort a, on the other hand, d i d not grow much l a r g e r than the o r i g i n a l f i e l d m a t e r i a l and there was no evidence of t h a l l u s d e t e r i o r a t i o n ( F i g . 25). In r e - i n f e c t i o n experiments, both endophytes showed e p i p h y t i c growth on Porphyra and Pt erosiphonia bipinnata but i n none of the r e - and c r o s s - i n f e c t i o n experiments was there evidence of endophytic growth. Monospores appeared to be p a r t i a l l y embedded -2 7-in the host c e l l w a l l ( F i g . 28). A l l p a r t s of Pterosiphonia bipi nnat a were i n f e c t e d ( F i g s . 29-33). The r e s u l t s of c r o s s - i n f e c t i o n experiments were i d e n t i c a l to those of r e - i n f e c t i o n experiments. The e p i p h y t i c growth was s i m i l a r to f r e e - l i v i n g p l a n t s i n that spores showed the same i n i t i a l i n t e r n a l d i v i s i o n ( F i g s . 26,27) and a s i m i l a r monosporangial r e p r o d u c t i v e c y c l e . However, s e v e r a l d i f f e r e n c e s were evident i n both Audouinella s p e c i e s between the f r e e - l i v i n g c u l t u r e s versus those growing e p i p h y t i c on Porphyra tort a or Pterosiphonia bipi nnat a. In the e p i p h y t i c p l a n t s there was l e s s development of the basal system (three to s i x v s . f i v e to nine c e l l s ) . T y p i c a l l y , one ( r a r e l y two to three) e r e c t a x i s was produced t h a t always arose from one of the two c e l l s i n the i n i t a l spore ( F i g s . 30, 32-33), and most e r e c t axes were unbranched except f o r the p r o d u c t i o n of l a t e r a l monosporangia ( F i g s . 31, 32-33). Monospores r e l e a s e d from the f r e e - l i v i n g c u l t u r e d Audouinella porphyrae and A. vaga a t t a c h e d to host c e l l w a l l s . Numerous p l a n t s were soon observed c o v e r i n g the host p l a n t s . Both endophytes completely covered the Pt er os i phoni a bipinnata p l a n t s w i t h i n 18 days except f o r the a c t i v e l y growing a p i c a l p o r t i o n s . Endophytes d i d not k i l l the Pt er osi phoni a bipinnata p l a n t s i n c u l t u r e , but r a t h e r slowed growth compared t o c o n t r o l c u l t u r e s . A f t e r 65 days, the host s t i l l grew and produced new shoots even though i t was t o t a l l y covered by ep i p h y t e s ( F i g . 34). When growing on Porphyra tort a, e p i p h y t i c p o p u l a t i o n s took much longer t o develop (two weeks t o three to f i v e days) than on Pt er os pi phoni a bipinnata. In a d d i t i o n , the p o p u l a t i o n was l e s s - 2 8 -dense on Porphyra t o r t a . Auduoinella porphyrae and A. vaga showed d i f f e r e n c e s i n t h e i r b a s a l p o r t i o n s and branching p a t t e r n s when growing on Porphyra torta and Pt e r o s i phoni a bipinnata. On Pt er os i phoni a bipi nnat a the bas a l system was l i m i t e d t o the i n i t i a l two c e l l s w i t h i n the spore and o c c a s i o n a l l y to one other c e l l . T h i s i s d i f f e r e n t from epiphytes on Porphyra tort a where up to f i v e b a s a l c e l l s might be produced ( F i g s . 28, 35, 37). In a d d i t i o n the e r e c t axes were more commonly branched when p l a n t s were growing on Porphyra torta (compare F i g s . 32 and 36). Va r i o u s c o n d i t i o n s i n c l u d i n g l a c k of a e r a t i o n , r e d u c t i o n i n l i g h t i n t e n s i t y and d e s i c c a t i o n of host were used i n an attempt to induce e p i p h y t i c p l a n t s to become endophytic i n Pt er osi phoni a bipinnata. The only e f f e c t of these experiments was a r e d u c t i o n in the r a t e of e p i p h y t i c growth which was noted when p l a n t s were d e s i c c a t e d . L i g h t and t r a n s m i s s i o n e l e c t r o n microscopy Using s e c t i o n s from methacrylate embedded f i e l d m a t e r i a l , v a r i o u s o b s e r v a t i o n s were made on the c y t o l o g i c a l r e l a t i o n s h i p s between the endophytes and t h e i r h o s t s . Many endophytic p r o s t r a t e f i l a m e n t s were seen i n the hosts' c e l l w a l l s . Audouinella porphyrae tended to branch i r r e g u l a r l y i n the Porphyra fucicola c e l l w a l l s ( F i g s . 38-39, 43), whereas the p r o s t r a t e p o r t i o n s of Audouinella vaga tended to grow p a r a l l e l to the l o n g i t u d i n a l a x i s of Pt er osi phoni a bipinnata. Thus, when the host w a l l curved, the endophyte a l s o appeared to grow i n an arc ( F i g s . 47, 49-50). I n t e r n a l l y d i v i d e d spores were observed - 2 9 -on top of the w a l l of both h o s t s . I t was noted that the germinating f i l a m e n t but not the spore penetrated the host c e l l w a l l . There were many f i l a m e n t s w i t h i n the outer c e l l w a l l l a y e r ( F i g s . 42,43) of Porphyra. Both endophytes produced e r e c t p o r t i o n s o u t s i d e t h e i r hosts, and monospores were formed from these e r e c t f i l a m e n t s ( F i g . 41). Filaments of A. porphyrae were not seen to pen e t r a t e the cytoplasm of the Porphyra c e l l s . However, on two oc c a s i o n s p e r i c e n t r a l c e l l s of Pterosiphoni a bipi nnata were penetrated by A. vaga ( F i g . 48). There were no d i f f e r e n c e s beween Audouinella porphyrae and Audouinella vaga i n t h e i r r e a c t i o n s to the h i s t o c h e m i c a l s t a i n s used i n t h i s study. S t a i n i n g with T o l u i d i n e blue-0 gave a red-purple metachromatic r e a c t i o n with the c e l l w a l l s and cytoplasm of the two hosts and the two endophytes. T h i s i s i n t e r p r e t e d to i n d i c a t e the presence of polyphosphates, p o l y s u l f a t e s and p o l y c a r b o x y l a c i d s (O'Brien and McCully, 1981). F a s t green showed a p o s i t i v e r e a c t i o n f o r p r o t e i n s i n the cytoplasm of a l l c e l l s as w e l l as i n the c u t i c l e of Porphyra fucicola ( F i g s . 44-46). When endophytic f i l a m e n t s were producing e r e c t axes, the host c e l l w a l l s adjacent to the emerging f i l a m e n t showed a p o s i t i v e r e a c t i o n with f a s t green ( F i g s . 45-46). In a d d i t i o n l o c a l i z e d areas of s t a i n i n g were observed immediately above c e r t a i n endophytic c e l l s ( F i g . 44-46, 52-53). T h i s suggests that some pro t e i n a c e o u s m a t e r i a l may be r e l e a s e d i n t o the host w a l l p r i o r to u p r i g h t a x i s i n i t i a t i o n . Audouinella vaga showed a s i m i l a r r e a c t i o n except there was no c u t i c l e present that s t a i n e d d a r k l y on the Pt er osi phoni a bipinnata host ( F i g s . 52,56). IKI and Sudan black d i d not show -30-any r e a c t i o n . No d i f f e r e n c e s between the c e l l w a l l s of the two endophytes were shown by the A l c i a n blue/PAS r e a c t i o n . Both showed p u r p l i s h c o l o r a t i o n i n d i c a t i n g a mixture of p o l y s a c c h a r i d e s (Kiernan, 1981). The two ho s t s , however, showed d i f f e r e n c e s i n t h e i r c e l l w a l l . Porphyra fucicola s t a i n e d p i n k - p i n k i s h red i n the cytoplasm and c u t i c l e whereas the outer w a l l s s t a i n e d b l u i s h , t h i s r e a c t i o n was a l s o observed i n Bangi a (Cole et a l . , 1985). The d i f f e r e n c e s i n d i c a t e d i f f e r e n t types of p o l y s a c c h a r i d e s . Pt erosiphonia bipinnata w a l l s showed a p u r p l i s h c o l o r a t i o n i n d i c a t i n g a mixture of p o l y s a c c h a r i d e s (Kiernan, 1981). Lack of f l u o r e s c e n c e with C a l c o f l u o r White ST t e s t i n d i c a t e d the absence of d e t e c t a b l e B-1,3 or B-1,4 p o l y s a c c h a r i d e s i n the w a l l s of the endophytes and t h e i r h o s t s . T r a n s m i s s i o n e l e c t r o n microscopy s t u d i e s showed that c e l l s of the two endophytes in vivo are u l t r a s t r u c t u r a l l y i d e n t i c a l ( F i g s . 69-71, 72-75). They have the "Nemalion" type c h l o r o p l a s t (Hara and Chihara, 1973) - a s i n g l e , s t e l l a t e , a x i l e c h l o r o p l a s t with one py r e n o i d that has an i r r e g u l a r p a t t e r n of t h y l a k o i d s w i t h i n the pyren o i d matrix ( F i g s . 54, 56, 62). P i t plugs have two cap l a y e r s and the nucleus i s surrounded with abundant s t a r c h ( F i g s . 69-71). Mi t o c h o n d r i a and dictyosomes are not abundant i n v e g e t a t i v e c e l l s ( F i g s . 69, 71-73); when present, they occur i n the p e r i p h e r a l cytoplasm of c e l l s . A l a r g e c e n t r a l v acuole i s o f t e n observed w i t h i n endophytic c e l l s i n f i e l d m a t e r i a l ( F i g s . 54, 56) which was not observed i n f r e e - l i v i n g m a t e r i a l . F u r t h e r u l t r a s t r u c t u r a l d e t a i l s w i l l be d i s c u s s e d l a t e r when comparing these taxa to other a c r o c h a e t i o i d a l g a e . -31 -P r e l i m i n a r y u l t r a s t r u c t u r a l s t u d i e s on endophytes growing in situ were c a r r i e d out. There were no major e f f e c t s on host p l a n t s ( F i g s . 57-60), however, some of the f i b e r s w i t h i n the w a l l s of Porphyra became d i s o r g a n i z e d d u r i n g i n f e c t i o n by Audouinella porphyrae ( F i g s . 61, 63-64). The s t r u c t u r a l o r g a n i z a t i o n of w a l l s of Pterosiphonia bi pi nnat a was u n a f f e c t e d by growth of A. vaga ( F i g s . 65-68). A summary of a l l r e s u l t s i s recorded i n Table VI. - 3 2 -Table VI: Summary of d i f f e r e n c e s and s i m i l a r i t i e s of morphological, reproductive and c y t o l o g i c a l characters of f i e l d and cultured Audouinella porphyrae and Audouinella vaga obtained in the current study. F i e l d material porphyrae A. vaga Cultured porphyrae Morphological features: C e l l dimensions: Hosts: Branching pattern: I n f e c t i o n c y c l e : Reproductive features: L i f e h i s t o r y : C y t o l o g i c a l features: R e - i n f e c t i o n exper iments: C r o s s - i n f e c t i o n exper iments: 12-15um(l) 3-7.5um(d) Porphyra spp. i r regular monospore asexual vi a monospore l2-2lum(l) 3-6pm(d) Pi er os iphonia bipinnat a r i g h t angular monospore asexual v i a monospore 14-l8um(l) 9pm(d) i r r e g u l a r monospore asexual v i a monospore ma t e r i a 1 A. vaga 14-lBjjm(l) 9um(d) i r regular monospore asexual v i a monospore s i m i l a r u l t r a s t r u c t u r a l features for f i e l d and cultured materials (see text) epiphyt i c growth ep i p h y t i c growth ep i p h y t i c growth epiphyt ic growth -3 3-DISCUSSION A study of f i e l d m a t e r i a l showed that there are s i m i l a r i t i e s and d i f f e r e n c e s between the two endophytes, Audouinella porphyrae and A. vaga. Many fe a t u r e s of the endophytes repo r t e d p r e v i o u s l y by Garbary et a l . (1982) have been c o r r o b o r a t e d i n t h i s i n v e s t i g a t i o n . Such f e a t u r e s i n c l u d e a s p e c t s of the s e a s o n a l i t y , i n f e c t i o n process, r e p r o d u c t i v e s t r u c t u r e s , morphology and spore germination i n the two t a x a . Some a d d i t i o n a l c h a r a c t e r s of the endophytes i n f i e l d m a t e r i a l were noted i n t h i s study. They r e l a t e p r i m a r i l y to the r e l a t i o n s h i p between the endophytes and t h e i r h o s t s . In n e i t h e r endophyte/host r e l a t i o n s h i p was t h e r e f a c o r r e l a t i o n between the s i z e of endophytic i n f e c t i o n areas and the s i z e of the h o s t s . A p o s i t i v e c o r r e l a t i o n between the s i z e of the endophyte and when the hosts were r e p r o d u c t i v e was noted. T h i s c o u l d i n d i c a t e t h a t the endophyte grows as the hosts mature. Both endophytes appear to i n f e c t the o l d e s t or o l d e r p o r t i o n s of t h e i r h o s t s . I n i t i a l l y , A. porphyrae f i r s t i n f e c t s the h o l d f a s t r e g i o n of Porphyra spp. whereas A. vaga i n f e c t s the o l d e r regions of the primary a x i s of Pt erosiphonia bipinnata, c l o s e s t to the r h i z o i d s . White and Boney (1970) a l s o noted t h i s when examining a s e r i e s of endophytic and endozoic Acrochaetiurn s p e c i e s , and suggested the b a s a l r e g i o n of a red a l g a w i l l u s u a l l y c o n t a i n any i n f e c t i o n s by endophytes. U n l i k e p a r a s i t e s which r e q u i r e n u t r i t i o n a l needs from t h e i r h o sts (Goff, 1982) and epiphytes which may r e q u i r e a c e r t a i n -3 4-c o l o n i z a b l e s u b s t r a t a ( H a r l i n , 1975), these endophytes do not appear to have any of these requirements. A p o s s i b l e reason f o r these symbionts to become endophytic i s t o "escape" environmental s t r e s s e s such as d e s i c c a t i o n , sand s c o u r i n g and wave a c t i o n . T h i s has yet to be s t u d i e d . The phenomenon of wounding by g r a z e r s plays an important r o l e i n the p e n e t r a t i o n of p a r a s i t e s (Goff, 1982). T h i s was not observed f o r the endophyte i n t h i s study and no obvious damage of host c e l l w a l l s was noted i n the area of settlement and p e n e t r a t i o n . Secondary i n f e c t i o n s occur i n v e g e t a t i v e r e g i o n s of both hosts probably from germination of monospores produced by the primary i n f e c t i o n . In support of t h i s argument, secondary i n f e c t i o n s are seen only when the primary i n f e c t i o n s are l a r g e .and r e p r o d u c t i v e . D i f f e r e n c e s which were p r e v i o u s l y noted and used t o separate the two species (Garbary et a l . , 1982 and T a b l e I) were a l s o observed i n t h i s study. For example, c e l l dimensions of the endophytes i n s i d e t h e i r h osts are s t a t i s t i c a l l y d i f f e r e n t , and c u r r e n t c e l l measurements f o r the two taxa o v e r l a p with those reported by Garbary et a l . (1982) (Table I and Table V ) . However, c e l l l e n gths were s l i g h t l y longer than those r e p o r t e d by Garbary et a l . (1982) thus extending the range of c e l l l e n g t h f o r Audouinella vaga. The branching p a t t e r n of the two endophytes i n the host m a t e r i a l d i f f e r s ; A. porphyrae branches i r r e g u l a r l y , whereas A. vaga branches at r i g h t a n g l e s t o the main growing f i l a m e n t j u s t i n s i d e the host w a l l . Although Garbary et a l . (1982) noted t e t r a s p o r a n g i a from one c o l l e c t i o n of A. porphyrae and h a i r c e l l s from one c o l l e c t i o n of A. vaga -35 -(Table I ) , n e i t h e r of these two c h a r a c t e r s was observed i n f i e l d or c u l t u r e m a t e r i a l from t h i s study. Since the presence of h a i r c e l l s and t e t r a s p o r a n g i a are so sporadic i n f i e l d m a t e r i a l , they are u n s u i t a b l e c h a r a c t e r s f o r d i s t i n g u i s h i n g these s p e c i e s . Some d i f f e r e n c e s between the endophytes i n a d d i t i o n to those i n d i c a t e d by Garbary et a l . (1982) were observed i n the c u r r e n t i n v e s t i g a t i o n . O c c a s i o n a l l y , f i l a m e n t s of Audouinella vaga were observed p e n e t r a t i n g c e l l s of Pt er os i phoni a bipinnata. T h i s i s the f i r s t r e p o r t of t h i s phenomenon. Drew (1928) noted the ra r e p e n e t r a t i o n of A. porphyrae i n t o Porphyra c e l l s , t h i s was not observed i n the present study. Since both endophytes have been observed to penetrate t h e i r hosts' c e l l s i n f r e q u e n t l y , i t appears that t h i s i s not a u s e f u l c h a r a c t e r to d i s t i n g u i s h between these two s p e c i e s (Garbary et a l . , 1982). The r a r i t y of t h i s phenomenon suggests that host c e l l p e n e t r a t i o n i s not a normal aspect of the growth s t r a t e g y of the two tax a . Thus, there are three major d i f f e r e n c e s that c o u l d be used in c h a r a c t e r i z i n g the two endophytes i n t h e i r h o s t s : 1) They r e s i d e i n separate h o s t s , Audouinella porphyrae occurs i n Porphyra whereas A. vaga i s present i n Pterosiphonia bipinnata, 2) A. porphyrae has s h o r t e r a p i c a l and main f i l m e n t o u s c e l l s than A. vaga (Table V ) , and 3) A. porphyrae branches i r r e g u l a r l y whereas A. vaga branches at r i g h t angles to the primary a x i s . Other than the d i f f e r e n c e s d i s c u s s e d above, the two endophytes are q u i t e s i m i l a r m o r p h o l o g i c a l l y and c y t o l o g i c a l l y . In a d d i t i o n , the monosporangial r e p r o d u c t i v e c y c l e s observed i n the f i e l d and the p a t t e r n of primary and secondary i n f e c t i o n s are the same. C h l o r o p l a s t s were c o n s i s t e n t with the "Nemalion" - 3 6 -- type as d e s c r i b e d by Hara and Chihara (1973). S i m i l a r c h l o r o p l a s t s have been shown i n other Audouinella s p e c i e s , such as Kyi i ni a sp. (Gibbs, 1970), Acrochaetium sp. (Hara and Chihara, 1974) and Rhodochorion sp. (Mitrakos, 1960). Other Audouinella spp. have d i f f e r e n t c h l o r o p l a s t types; t h i s was shown i n u l t r a s t r u c t u r a l s t u d i e s by Hymes and Cole, (1983a) and L i c h t l e , (1973) who r e p o r t e d a m o d i f i e d "Batrochospermum" - type of c h l o r o p l a s t . The two s p e c i e s of endophytic Audouinella have p i t plugs with two cap l a y e r s . T h i s i s c o n s i s t e n t with the o b s e r v a t i o n s of Pueschel and Cole (1982); Lee (1971); Hymes and Cole (1983a). Other than the l i m i t e d u l t r a s t r u c t u r a l s t u d i e s of c h l o r o p l a s t and p i t plugs (see above), the only papers d e s c r i b i n g the gen e r a l f i n e s t r u c t u r e of an a c r o c h a e t i o i d a l g a (Audouinella hermanni i (Roth) Duby) are by Hymes and Cole (I983a,b). L i k e A. hermannii, the f r e e - l i v i n g endophytes showed few mitochondria and dictyosomes; these were l o c a t e d at the p e r i p h e r y of the c e l l s . Furthermore, the c e l l w a l l s of the f r e e - l i v i n g endophytes were t h i c k , r e p r e s e n t i n g over 30% of the c e l l diameter. A l a r g e vacuole was observed i n f i e l d m a t e r i a l and i n o l d e r f i l a m e n t s of endophytes i n c u l t u r e , s i m i l a r to o l d e r c e l l s of A. hermanni i (Hymes and Cole, 1983a). U n l i k e Audouinella hermanni i, both endophytes showed a c e n t r a l prominent p y r e n o i d with four to f i v e t r a v e r s i n g t h y l a k o i d s . N u c l e i of both endophytes were commonly observed near the base of v e g e t a t i v e c e l l s i n e r e c t ( f r e e - l i v i n g ) axes ( F i g s . 72-73). T h i s has been observed i n one other s p e c i e s of Audouinella, A. parvula ( K y l i n ) Dixon (Garbary, 1978). In - 3 7 -c o n t r a s t , the nuclear p o s i t i o n i n A. hermanii i s more c e n t r a l l y l o c a t e d (Hymes and Cole, 1983a). C u l t u r e s t u d i e s were conducted to determine i f the endophytic c h a r a c t e r s i n f i e l d m a t e r i a l p e r s i s t e d i n the f r e e - l i v i n g s t a t e . F i e l d c h a r a c t e r s were not maintained i n the f r e e - l i v i n g endophytes. The data obtained r e v e a l e d c l e a r l y that the f r e e - l i v i n g forms of Audouinella porphyrae and A. vaga are s i m i l a r i n morphology, c e l l dimensions, branching and growth p a t t e r n s . They both demonstrate a monosporangial r e p r o d u c t i v e c y c l e i n t h e i r f r e e - l i v i n g forms, and c e l l l e n g t h appears to be w i t h i n the range encountered i n f i e l d m a t e r i a l . However, the c e l l diameters of the f r e e - l i v i n g forms i s gr e a t e r than t h a t of the endophytic forms (9 vs 3-7.5 urn). Garbary (1979b) a l s o noted some s i g n i f i c a n t d i f f e r e n c e s i n c e l l dimensions between the f r e e - l i v i n g and endophytic forms of A. endophyt i ca i s o l a t e d from Het er s i phoni a plumosa. In c o n t r a s t , White and Boney ( 1970) d i d not f i n d any s i g n i f i c a n t d i f f e r e n c e s i n c e l l dimensions and morphology when comparing the in vivo and in situ forms of A. endophytica. I t i s p o s s i b l e to conclude from the above o b s e r v a t i o n s that the d i f f e r e n c e s i n c e l l dimensions and branching p a t t e r n s i n f i e l d m a t e r i a l s are a consequence of the d i f f e r e n c e i n h a b i t a t s represented by the host c e l l w a l l s . Porphyra w a l l s are much t h i c k e r (50pm vs 20pm) than Pt er os i phoni a bipinnata w a l l s and t h i s c o u l d i n f l u e n c e the branching and growth p a t t e r n s of the endophytes. The o r i e n t a t i o n of m i c r o b i l s i n the c e l l w a l l matrix of f l o r i d e o p h y c e a e (Young, 1980) and the chemical c o n s t i t u e n t of the w a l l s of both h o s t s c o u l d i n f l u e n c e the -38-observed d i f f e r e n c e s i n the branching p a t t e r n of the endophytes. The w a l l s of m a c r o t h a l l i of Porphyra are composed of xylan and mannan ( F r e i and Prest o n , 1964) whereas members of Flo r i d e o p h y c e a e have s u l f a t e d p o l y s a c c h a r i d e and c e l l u l o s e i n the w a l l matrix (Mackie and Preston, 1974). Re- and c r o s s - i n f e c t i o n s t u d i e s demonstrated that both Audouinella porphyrae and A. vaga were not host s p e c i f i c . They both grew e p i p h y t i c a l l y on e i t h e r host under v a r i o u s c u l t u r e c o n d i t i o n s . Both White and Boney (1969) and Garbary (1979) s t u d i e d v a r i o u s endophytes, under f i e l d and c u l t u r e c o n d i t i o n s . They found that many endophytes such as Audouinella infestans and A. bonnemai soniae grew on v a r i o u s s u b s t r a t a i n c l u d i n g other red algae and h y d r o i d s . However, they both found that only A . endophytica grew e n d o p h y t i c a l l y i n v a r i o u s s u b s t r a t a such as s h e l l s and other red algae (not i t s normal h o s t ) . The other endophytes s t u d i e d e s t a b l i s h e d nonendophytic r e l a t i o n s h i p s ranging from simple entanglement t o some form of e p i p h y t i c growth on given s u b s t r a t a . White and Boney (1969, 1970) noted that only A. endophytica grew e n d o z o i c a l l y i n a hy d r o i d and showed d i f f e r e n t growth responses when given a v a r i e t y of ca l c a r e o u s s u b s t r a t a . D i f f e r e n c e s began s h o r t l y a f t e r spore germination and the germlings formed growth p a t t e r n s that were determined by the org a n i c matrix of the given substratum. For A. porphyrae and A. vaga, d i f f e r e n c e s i n responses when growing on the ho s t s , i n d i c a t e that the nature of the host c e l l w a l l s a l s o i n f l u e n c e s the development of the endophytes. The l a c k of endophytic growth c o u l d r e s u l t from s e v e r a l f a c t o r s . The c e l l w a l l s of the j u v e n i l e host p l a n t s may not be - 3 9 -" t h i c k " enough f o r the endophytes to penetrate and grow. T h i s argument i s presented by Garbary f o r the some of the Audouinella s p e c i e s he s t u d i e d (1979b). There c o u l d be some mechanism such as sand s c o u r i n g or wave a c t i o n which c o u l d prepare host c e l l w a l l s f o r p e n e t r a t i o n by the endophytes. Monospore p r o d u c t i o n i s the most common form of r e p r o d u c t i o n i n the genus Audouinella. Regeneration of monosporangia w i t h i n remnants of o l d monosporangial w a l l s was observed f o r both A. porphyrae and A. vaga. T h i s appears to be a t y p i c a l f e a t u r e of many Audouinella spp. (White and Boney, 1970; Boney, 1967; Hymes and Cole, 1983b). White and Boney, (1979) noted changes i n the abundance of monosporangial p r o d u c t i o n i n d i f f e r e n t c u l t u r e c o n d i t i o n s . D i f f e r e n c e s i n abundance of monosporangial p r o d u c t i o n were a l s o noted i n the c u r r e n t experiments c a r r i e d out i n c u l t u r e c o n d i t i o n s . Other m o r p h o l o g i c a l f e a t u r e s such as h a i r c e l l s and sexual r e p r o d u c t i v e s t r u c t u r e s were not seen under any c u l t u r e c o n d i t i o n s used i n the present study. White and Boney (1979) d i d not f i n d any sexual r e p r o d u c t i v e s t r u c t u r e s or h a i r c e l l s i n the endophytes, A. endophyt i ca, A. as par agops i s and A. infestans, even when experimenting with d i f f e r e n t daylengths and temperatures. In the asexual s p e c i e s , Audouinella proskaueri (West) Garbary Hansen et S c a g e l , West (1971) noted that h a i r c e l l p r o d u c t i o n was r e l a t e d p r i m a r i l y to l i g h t i n t e n s i t y r a t h e r than p h o t o p e r i o d or temperature. F i e l d m a t e r i a l of A. porphyrae has ' been r e p o r t e d with h a i r c e l l s (Garbary et a l . , 1982) but no h a i r s from f r e e - l i v i n g c u l t u r e s of the endophytes were observed - 40-under d i f f e r e n t photon f l u x d e n s i t i e s (4-5, 25, 55 m i c r o e i n s t e i n m"2 s e c " 1 ) . The l a c k of h a i r s i n c u l t u r e s of A. vaga and A. porphyrae may r e s u l t from an absence of s u i t a b l e c u l t u r e c o n d i t i o n s , or the f a c t that the e n t i t i e s do not have the g e n e t i c p o t e n t i a l to produce them. I t would appear that v a r y i n g i n d i v i d u a l environmental f a c t o r s i n the l a b o r a t o r y may be i n s u f f i c i e n t to induce c e r t a i n r e p r o d u c t i v e and morphological f e a t u r e s that were i n f r e q u e n t l y seen i n the f i e l d . Perhaps more p a r t i c u l a r combinations of c o n d i t i o n s of photoperiod, temperature and l i g h t i n t e n s i t y are needed to induce t e t r a s p o r a n g i a , h a i r c e l l s , and gametangia. I t i s a l s o p o s s i b l e that such s t r u c t u r e s are not formed by these e n t i t i e s . In t h e i r s t u d i e s on Audouinella arcuata (Drew) Garbary, Hansen et S c a g e l , Hansen and Garbary (1984) noted s i m i l a r i t i e s between two s p e c i e s of Audouinella: A. arcuata and A. vaga and suggested that they may represent t e t r a s p o r o p h y t i c and gametophytic phases of a s i n g l e sexual l i f e h i s t o r y . There are s e v e r a l examples where epiphytes with u n i c e l l u l a r bases and d i m i n u t i v e e r e c t f i l a m e n t s are the gametophytic stages i n t r i p h a s i c , t r i m o r p h i c l i f e h i s t o r i e s . In these l i f e h i s t o r i e s , t e t r a s p o r o p h y t e s are comprised of m u l t i c e l l u l a r bases and more ex t e n s i v e e r e c t f i l a m e n t s (eg. Stegenga and Mulder, 1979; Stegenga and Vroman, 1976; see W o e l k e r l i n g , 1983). Hansen and Garbary (1984) have observed A. arcuata with a few e r e c t f i l a m e n t s , and A. mi croscopi ca a l s o with a few e r e c t f i l a m e n t s (Naegeli) W o e l k e r l i n g growing with A. vaga on the same host, Pterosiphonia bipinnata. I have a l s o observed t h i s i n my f i e l d c o l l e c t i o n s . Audouinella arcuata and A. microscopica are - 4 1 -d i m i n u t i v e p l a n t s with u n i c e l l u l a r bases, one to s e v e r a l e r e c t axes and 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 s . Hansen and Garbary (1984) suggested that A. arcuata might be the gametophytic phase of A. vaga. Audouinella arcuata from San Juan I s l a n d was grown at 10°C 16:8 h (L:D) at a photon f l u x d e n s i t y of 4-5 m i c r o e i n s t e i n s m"2 s e c " 1 , and d i d not observe any sexual s t r u c t u r e s on t h i s p l a n t . Other c u l t u r e c o n d i t i o n s are needed to induce gametogenesis. S i m i l a r l y , A. mi croscopi ca (sensu Garbary et a l . , 1982 not Stegenga et a l . , 1979) maybe a p o s s i b l e gametophytic phase f o r A. porphyrae (Garbary et a l . , 1982). T h i s a l s o needs to be i n v e s t i g a t e d i n c u l t u r e . Thus, i f A . porphyrae and A. vaga are the same e n t i t y as shown from the c u l t u r e s t u d i e s of t h i s t h e s i s and i f they have the suggested gametophytes as pa r t of t h e i r sexual l i f e h i s t o r i e s , then at l e a s t three s p e c i e s of Audouinella c o u l d be synonymized when the l i f e h i s t o r i e s are r e s o l v e d . To summarize c u l t u r e o b s e r v a t i o n s , the endophytes are i d e n t i c a l i n t h e i r f r e e - l i v i n g forms, and both showed s i m i l a r r e s u l t s i n the r e - and c r o s s - i n f e c t i o n experiments. Based on f i e l d and c u l t u r e data (Table V I ) , I propose that the two endophytes are c o n s p e c i f i c . Audouinella porphyrae i s chosen over A. vaga because of l e s s taxonomical c o n f u s i o n i n i t s o r i g i n i a l d e s c r i p t i o n (Drew, 1928). I propose synonymizing the endophytes under Audouinella porphyrae. Another taxon A. vaga v a r . implicata (Drew) Garbary, Hansen et Scagel i s a s s o c i a t e d with t h i s complex. I t d i f f e r s from A. vaga v a r . vaga only i n i t s ' host (Halosaccion glandiforme (Gmelin) Ruprecht), and p r o v i s i o n a l l y may be synonymized with A. vaga (Garbary et a l . , -42 -1982). A complete l i s t i n g of the taxonomic and nomenclatural synonyms of Audouinella porphyrae i s as f o l l o w s : Audouinella porphyrae (Drew) Garbary, Hansen and Scagel, 1982 [1983]: 42 Basionym: Rhodochorton porphyrae Drew 1928: 188 Synonyms: Acrochaetium porphyrae (Drew) Smith 1944: 179 Chromastrum porphyrae (Drew) Papenfuss 1945: 325 Kylinia porphyrae (Drew) Papenfuss 1947: 438 Col aconema porphyrae (Drew) W o e l k e r l i n g 1971: 50 Audouinella porphyrae (Drew) Garbary, Hansen et Scagel 1982 [1983]: 42 Rhodochort on vagum Drew 1928: 188 Acrochaetium vagum (Drew) Jao, 1937: 111 Chromastrum vagum (Drew) Stegenga and Mulder 1979: 305 i n Tarn Rhodochor t on implicatum Drew 1928: 190 Chromastrum implicatum (Drew) Papenfuss 1945: 324 Kylinia implicata (Drew) Papenfuss 1 947: 438 Audouinella vaga v a r . implicata (Drew) Garbary Hansen et Scagel 1982 [1983]:63 In c o n c l u s i o n , f u t u r e s t u d i e s are needed t o pr o v i d e more i n f o r m a t i o n on Audouinella porphyrae ( i n c l u d i n g A. vaga) that would complement the c u r r e n t knowledge. For example, a complete l i f e h i s t o r y would be u s e f u l i n understanding the p o s s i b l e a l t e r n a t e l i f e h i s t o r y stages i n nature. More e x t e n s i v e e c o l o g i c a l s t u d i e s are needed to determine the l o c a t i o n of the endophytes, when the host p l a n t s are absent d u r i n g the winter as w e l l as the source of i n f e c t i o n f o r the young hosts d u r i n g e a r l y s p r i n g . There are s e v e r a l p o s s i b i l i t i e s which c o u l d be e x p l o r e d . The endophytes c o u l d be i n a c r y p t i c form i n the c r e v i c e s of rocks s i n c e they both i n f e c t the o l d e s t p o r t i o n s of the host p l a n t s . Another p o s s i b i l i t y i s that they c o u l d be r e s i d i n g i n an a l t e r n a t e h o s t . For example, A. vaga v a r . -43-implicata, i s d e s c r i b e d from Halos acci on glandiforme which i s abundant d u r i n g winter months, however, A. vaga v a r . implicata i s a p o o r l y known taxon. I t thus becomes important to c h a r a c t e r i z e adequately i t to r e s o l v e t h i s problem. Such s t u d i e s may add to our g e n e r a l knowledge of host-endophytic r e l a t i o n s h i p s and r e s o l v e taxonomic and e c o l o g i c a l problems in Audoui nel I a . - 4 4 -References: Baardseth, E. 1941. The marine algae of T r i s t a n da Cunha. Res. Norwegian Sci. Exp. to Tristan da Cunha 1937-1938 9: 1-174. Bold, H.C. and Wynne, M.J. 1985. Introduction to the Algae. 2ed. P r e n t i c e - H a l l , Inc., Englewood C l i f f s , N.J. pp.720. Boney, A.D. 1967. In vitro growth of the endophyte' Acrochaetium bonnemai soniae ( B a t t . ) J . et G. Feldmann Nova Hedw. Z. krypt ogamenkd. 23: 173-186. Cole, K.M., Park, CM., Reid, P.E. and Sheath, R.G. 1985. Comparative s t u d i e s on the c e l l w a l l s of sexual and asexual Bangi a at r opur pur ea (Rhodophyta). I. H i s t o c h e m i s t r y of p o l y s a c c a r i d e s . /. Phycol. 21 ( 4 ) : i n p r e s s . Dixon, P.S. and I r v i n e , L. M. 1977. Seaweeds of the British Isles. Vol. I Rhodophyta. Part I, Introduction, Nemaliales, Gi gart i nal es. B r i t i s h Museum ( N a t u r a l H i s t o r y ) , London. pp.252. Drew, K.M. 1928. A r e v i s i o n of the genera Chant ransi a, Rhodochorton, and Acrochaetium. Univ. Calif., Berkeley, Publ. Bot . 14: 139-224. Feder, N. and O'Brien, T.P. 1968. P l a n t microtechnique: some p r i n c i p l e s and new methods. Am. J. Bot. 55: 123-142. Feldmann, J , 1962. The Rhodophyta order A c r o c h a e t i a l e s and i t s -4 5-C l a s s i f i c a t i o n . Proc. 9th Pac. Sci. Congress 4: 219 221. F r e i , E. and Preston, R.D. 1964. N o n - c e l l u l o s i c s t r u c t u r a l p o l y s a c c h a r i d e s i n a l g a l c e l l w a l l s . I I . A s s o c i a t i o n of xy l a n and mannan i n Porphyra umbilicalis. Proc. Roy. Soc. (London) 160: 314-327. Garbary, D.J. 1978. Aspects of the taxonomy and b i o l o g y of the Acro c h a e t i a c e a e (Rhodophyta). Ph.D. T h e s i s , Univ. of L i v e r p o o l , England, U.K. pp.263. 1979a. Numerical taxonomy and g e n e r i c c i r c u m s c r i p t i o n i n the Acrochaeticaeae (Rhodophyta). Bot . Mar. 22: 477-492. 1979b. A r e v i s e d s p e c i e s concept f o r endophytic and endozoic members of the Acrochaetiaceae (Rhodophyta). Bot. Not. 132: 451-455. and Rueness, J . 1980. Audouinella l et raspora, a new member of the Acrochaetiaceae (Rhodophyta) from Norway. Norw. J. Bot. 27: 17-22. Hansen, G.I. and Scagel, R.F. 1982 [1983]. The marine alg a e of B r i t i s h Columbia and Northern Washington: D i v i s i o n Rhodophyta (Red A l g a e ) , C l a s s F l o r i d e o p h y c e a e , Orders A c r o c h a t i a l e s and N e m l i a l e s . Syesis: 15 (Suppl. 1) 1-102. Goff, L. 1982. The b i o l o g y of p a r a s i t i c red a l g a e . In: Progress in Phycological Research V o l . 1 . Eds. Round,F.E. and Chapman, D.J. E l s e v i e r Biomedical P r e s s , B.V. pp. 289-369. -46 -Goff, L. 1983. Marine a l g a l i n t e r a c t i o n s - e p i b i o s i s , e n d o b i o s i s , p a r a s i t i s m and d i s e a s e . Proc. Joint China - U.S. Phyc. Symp. Ed. Tseng, C K . Science P r e s s , B e i j i n g , c h i n a , pp.220-274. Gibbs, S.P. 1970. The comparative u l t r a s t r u c t u r e of the a l g a l c h l o r o p l a s t . N.Y. Acad. Sci. Ann. 175: 454-473. Hansen, G.I. and Garbary, D.J. 1984. Sexual r e p r o d u c t i o n i n Audouinella arcuata with comments on the A c r o c h a e t i a c e a e (Rhodophyta). Br. Phycol. J. 19:175-184. Hansen, G.I., Garbary, D.J., O l i v e i r a , J . C and S c a g e l , R.F. 1981. New records and range e x t e n s i o n s of marine algae from A l a s k a . Syesis 14: 115-123. Hara, Y. and Chihara, M. 1973. Comparative s t u d i e s on the 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 i n the Rhodophyta with s p e c i a l r e f e r e n c e to t h e i r taxonomic s i g n i f i c a n c e . Sc. Rep. T.K.D. 15: 209-235. H a r l i n , M.M. 1975. Epiphyte-host r e l a t i o n s h i p s i n seagrass communities. Aqua. Bot. 1: 125-131. . Hayat, M. 1981. Principles and Techniques of Electron Mi cr os copy. Biological Applications. V o l . 2nd ed. Univ. Park P r e s s . Baltimore Maryland, pp.505. Henry, E. 1977. A method of o b t a i n i n g r i bbons of s e r i a l s e c t i o n s of p l a s t i c embedded specimens. Stain Tech. 52: 79-60. - 4 7 -Hoshaw, R.W. and Rosowski, J.R. 1973. Methods f o r mic r o s c o p i c a l g a e . In: Handbook of Phycological Methods. Ed. S t e i n , J.R. Cambridge Univ. Press, Cambridge. pp. 53-68. Hughes, J . and McCully, M.E. 1975. The use of an o p t i c a l b r i g h t e n e r i n the study of p l a n t s t r u c t u r e . Stain Tech. 50: 319-379. Hymes, B.J. and Cole, K.M. 1983a. The c y t o l o g y of Audouinella hermanii (Rhodophyta, F l o r i d e o p h y c e a e ) . I. V e g e t a t i v e and h a i r c e l l s . Can. J. Bot. 61: 3366-3376. 1983b. The c y t o l o g y of Audouinella hermanii (Rhodophyta, F l o r i e o p h y c e a e ) . I I . Monosporogenesis. Can. J. Bot. 61: 3377-3385 Jao, C.C. 1937. New marine algae from Washington. Pap. Mich. Acad. S c i . Arts. Lett. 22: 99-115. Kiernan, J.A. 1981. Histological and Hi st ochemi cal Methods: Theory and Prat i c e . Pergamon P r e s s , O n t a r i o . pp.344 Lee, R.E. 1971. The p i t conne c t i o n s of some lower red al g a e : u l t r a s t r u c t u r e and phylo g e n e t i c s i g n i f i c a n c e . Br. Phycol J. 6: 29-38. Lewin, J.C. 1966. S i l i c o n metabolism i n diatoms. V. Germanium d i o x i d e a s p e c i f i c i n h i b i t o r of diatom growth. Phycol ogi a 6: 1-12. L i c h t l e , C. 1973. U l t r a s t r u c t u r e du p l a s t e de deux Rhodophycees: Rhodochort on purpureum ( L i g h t f . ) Rosenvinge - 4 8 -Rhodot hamni el I a floridula (Dillwyn) J . Feldmann. CR. Hebd. Seances Acad. Sci. Ser. D. , 277: 1865-1868. Mackie, M. and Preston, R.D. 1974. C e l l w a l l and i n t e r c e l l u l a r r e g i o n p o l y s a c c h a r i d e s . In: Algal Physiology and Biochemistry. Ed. Stewart, W.D.P. B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , Oxford, pp.989. McCully, M.E., Goff, L . J . and Adshead, P.C. 1980. P r e p a r a t i o n of algae f o r l i g h t microscopy In: Handbook of Phycological Met hods . Developmental and Cylol ogical Methods, Cambridge Univ. Press, Cambridge, pp. 263-283. Mit r a k o s , 1960. Feinbau and T e i l u n g b e i P l a s t i d e n e i n i g e r F l o r i d e e n - A r t e n . Protoplasma 52: 611-617. O'Brien, T.P. and McCully, M.E. 1981. The Study of Plant Structure: 'Principles and Selected Methods. Termarcarphi Pty L t d , Melbourne, pp.682. Papenfuss, G.F. 1945. Review of the Acrochaetiurn-Rhodochort on complex of the red al g a e . Univ. Calif. Berkeley, Publ . Bot. 18: 299-334. 1947. F u r t h e r c o n t r i b u t i o n s toward an understanding of the Acrochaetium-Rhodochorton complex of the a l g a e . Univ. C a l i f . , Berkeley, Publ. Bot. 18: 433-447. P o l n e - F u l l e r , M. and Gibor, A. 1984. Developmental s t u d i e s i n Porphyra. I. Bladed d i f f e r e n t i a t i o n i n Porphyra perforata as expressed by morphology, enzymatic d i g e s t i o n , and p r o t o p l a s t -49-r e g e n e r a t i o n . /. Phycol. 20: 609-616. P r o v a s o l i , L. 1968. Media and p r o s p e c t s f o r the c u l t i v a t i o n of marine a l g a e . In: Cultures and Collection of Algae. Eds. Watanabe, A. and H a t t o r i , A. Proc. U.S.- Japan Conf. Hakone, Sept. 1966. Jap. Soc. Plant Physiol, pp.63-75. Pueschel, CM. and Cole K.M. 1982. Rhodophycean p i t plugs: an u l t r a s t r u c t u r a l survey with taxonomic i m p l i c a t i o n s . Am. J. Bot. 69: 703-720. 1985. U l t r a s t r u c t u r e of germinating carpospores of Porphyra vari egat a ( K j e l l m . Hus B a n g i a l e s , Rhodophyta. J. Phycol. 21: 146-154. Reynold, E.S. 1963. The use of l e a d c i t r a t e at h i g h pH as an e l e c t r o n opaque s t a i n i n e l e c t r o n microscopy. J. Cell Biol. 17: 208-212. Smith, G.M. 1 944. Marine algae of the Monterey Peninsula., California. S t a n f o r d Univ. Press, S t a n f o r d , pp.622 S o k a l , R.R. and Rohlf, F. J . 1973. In: Introduction to Bi ost at i st i cs, Freeman and Co. San F r a n c i s c o , U.S.A. pp. 368. South,G.R. and Adams, N.M. 1976. The marine algae of the Kaikoura coast - a l i s t of s p e c i e s . Natl. Mus. New Zealand, Misc. Ser. No. 1:1-71. Stegenga, H. and Mulder, A.S. 1979. Remarks on the Audouinella mi cr oscopi ca (Naeg.) W o e l k e r l i n g complex with a -50-b r i e f survey of the genus Chromast rurn Papenfuss (Rhodophyta, N e m a l i a l e s ) . Acta Bot. Neerl. 28: 289-311. , and Van Wissen, M.J. 1979. Remarks on the l i f e h i s t o r i e s of three A c r o c h a e t i o i d algae (Rhodophyta; N e m a l i a l e s ) . Acta Bot. Neerl. 28: 97-115. , and Vroman, V. 1976. The morphology and l i f e h i s t o r y of Acrochaetium densum (Drew) Papenfuss (Rhodophyta, N e m a l i a l e s ) . Acta Bot. Neerl. 25: 257-280. Spurr, A.R. 1969. A l o w - v i s c o s i t y epoxy r e s i n embedding medium fo r e l e c t r o n microscopy. J. Ultrast. Res. 26:31-43. Watson, M.L. 1958. S t a i n i n g of t i s s u e s e c t i o n s f o r e l e c t r o n microscopy with heavy metals. /. Biophys. Biochem. Cyt ol . 4: 475-478. West, J.A. 1971. Environmental c o n t r o l of h a i r and s p o r a n g i a l formation i n the marine red a l g a Acrochaetium proskaueri sp. nov. Proc. Int. Seaweed Symp. 7: 377-384. 1979. The l i f e h i s t o r y of Rhodochort on membranaceum, an endozoic red a l g a . Bot. Mar. 2 2 : 111-115. White, E.B. and Boney, A.D. 1969. Experiments with some endophytic and endozoic Acrochaetium s p e c i e s . /. Exp. Mar. Biol. Ecol. 3: 246-274. 1970. In situ and in vitro s t u d i e s on c e r t a i n endophytic and endozoic Acrochaetium s p e c i e s . Nova Hedw. Z. Krypt ogamenkd. 19: 841-881. -51 -W o e l k e r l i n g , W.J. 1971. Morphology and taxonomy of the Audouinella complex (Rhodophyta) i n southern A u s t r a l i a . Austr. J. Bot. Suppl. 1: 1-91. 1983. The Audoui nell a (Acrochaet ium-Rhodocort on) complex (Rhodophyta): present p e r s p e c t i v e s . Phycol ogia 22: 59-92. Young, D.N. 1980. Unusual c e l l w a l l u l t r a s t r u c t u r e i n Antithamnion (Rhodophyta). Br. Phycol J. 15: 119-124. - 52-FIGURE LEGENDS 1 Diagrammatic r e p r e s e n t a t i o n s of Porphyra sp. and Pt erosiphoni a bi pi nnat a showing areas of i n f e c t i o n . 1a Porphyra blade: 1 = h o l d f a s t ; 2 = v e g e t a t i v e r e g i o n ; 3 = r e p r o d u c t i v e r e g i o n . 1b P. bi pi nnat a t h a l l i : 1 = h o l d f a s t ; 2 = main f i l a m e n t ; 3 = o l d e r branches; 4 = a p i c a l growth. - 5 3 -- 5 4 -F i g . 2 C r o s s - s e c t i o n of l i v i n g Porphyra torta (h), with the endophyte, Audouinella porphyrae. Note the e r e c t f i l a m e n t s (e) growing from the p r o s t r a t e p o r t i o n (p) of the endophyte. S c a l e bar = 18pm F i g . 3 P. torta h e a v i l y i n f e c t e d by A. porphyrae. Note the i r r e g u l a r branching p a t t e r n of the endophyte ( e ) . Scale bar = 19pm F i g . 4 Pt erosiphoni a bipinnata primary a x i s h e a v i l y i n f e c t e d by the endophyte A. vaga (en). Note growth p a t t e r n of the endophyte i n the w a l l s of the host p e r i c e n t r a l c e l l s . S c a l e bar = 75pm F i g . 5 Close up of A. vaga (arrow) as i t grows and branches i n the w a l l of i t s host. Note the branching and growth p a t t e r n of the endophyte. Scale bar = 25pm c - 5 6 -F i g . 6-9: Micrographs of l i v i n g f i e l d Audoinella porphyrae/ Porphyra fucicola taken with Nomarksi Modulation C o n t r a s t Microscopy. F i g . 6 A spore of A. porphyrae with an i n t e r n a l d i v i s i o n . Note germinating c e l l (arrow). S c a l e bar = 15pm F i g . 7 Note germinating c e l l (arrow) from s e t t l e d spore. S c a l e bar = 11 urn F i g . 8 P r o s t r a t e f i l a m e n t s i n the host c e l l w a l l s . Note prominent pyrenoid i n the c e l l (arrow). S c a l e bar = 14urn F i g . 9 E x t e n s i v e i n f e c t i o n by p r o s t r a t e f i l a m e n t s i n the h o s t . Note i r r e g u l a r branching p a t t e r n . Scale bar = 14pm F i g . 10 Monospore of f r e e - l i v i n g A . porphyrae s e t t l i n g and d i v i d i n g i n t e r n a l l y (arrow) on a p l a s t i c p e t r i p l a t e . S c ale bar = 12pm F i g . 11 Germinating c e l l from s e t t l e d spore (arrow). S c a l e bar = 8pm F i g . 12 Growing f i l a m e n t . Cytoplasm i s being "pushed" towards the growing t i p by an expanding vacuole (arrow). S c a l e bar = 8pm F i g . 13 P r o s t r a t e c e l l s (p) of A. porphyrae with two i n i t i a l e r e c t f i l m e n t s (e) (arrows). Scale bar = 20pm F i g . 14 E r e c t f i l a m e n t s of A. porphyrae showing a s t e l l a t e , a x i a l c h l o r o p l a s t (c) with a c e n t r a l p y r e n o i d (py) and a l a r g e vacuole ( v ) . S c a l e bar = 13pm -58-F i g s . 15-21: C u l t u r e m a t e r i a l of l i v i n g A. porphyrae ( s i n c e both f r e e - l i v i n g endophytes are i d e n t i c a l , only A. porphyrae i s shown). F i g . 15 E r e c t f i l a m e n t s with short l a t e r a l branches which form monosporangia. Scale bar = 14pm F i g . 16 E r e c t f i l a m e n t s ( c l e a r arrow) with d i s c h a r g e d monosporangia. Note p r o d u c t i o n of a new c e l l i n an empty monosporagium (dark arrow). Scale bar =15pm F i g . 17 Monosporangia can be l a t e r a l and/or t e r m i n a l on an e r e c t f i l a m e n t . Note p e d i c e l s below monosporangium (arrow). S c a l e bar = 15pm F i g s . 18-21: Nomarski C o n t r a s t Modulation Microscopy F i g . 18 E r e c t f i l a m e n t s with many l a t e r a l f i l a m e n t s . Monosporangia f i l a m e n t s can a l s o be formed on secondary b r a n c h l e t s (arrows). Scale bar = 8pm F i g . 19 A budding a p i c a l c e l l growing i n t o an empty sporangium. Note the vacuole (arrow). S c a l e bar = 17pm F i g . 20 Four to s i x monosporangia can be produced from one sporangium. Note o l d w a l l l a y e r s (arrow). S c a l e bar = 17pm F i g . 21 U s u a l l y one to two or o c c a s i o n a l l y three monosporangia are formed from one c e l l . S c a l e bar = 17pm -60 -F i g s . 22-25: U n i n f e c t e d l i v i n g Pt er os i phoni a bipinnata and Porphyra blades used f o r r e - i n f e c t i o n and c r o s s - i n f e c t i o n experiments. F i g . 22 A young branch on the main a x i s of P. bipinnata. Scale bar = 375pm F i g . 23 A p i c a l t i p s of P. bipinnata tend to curve a d a x i a l l y (arrow). S c a l e bar = 375pm F i g . 24 The main a x i s of c u l t u r e d P. bipinnata with a r h i z o i d (r) and a branch. Scale bar = 188pm F i g . 25 Cleaned Porphyra blade. S c a l e bar = 90pm - 6 1 --62-F i g s . 26-34: C r o s s - i n f e c t e d on Audouinella porphyrae on l i v i n g Pier osi phi na bipinnata. F i g . 26 Germlings of e p i p h y t i c A . porphyrae i n f e c t i n g a r h i z i o d of P. bipinnata. Note i n t e r n a l d i v i s o n (arrow). Scale bar = 10pm F i g . 27 Note e r e c t f i l a m e n t from s e t t l e d spore (arrow). Scale bar = 10pm F i g . 28 E r e c t f i l a m e n t growing from a monospore. Note the p a r t i a l p e n e t r a t i o n by the b a s a l c e l l (arrow). Scale bar = 12pm F i g . 29 E r e c t f i l a m e n t s growing on r h i z o i d (r) of P. bipinnata. Scale bar = 45pm F i g . 30 T u f t s of epiphytes growing on a r h i z o i d of i n f e c t e d host. Scale bar = 45pm F i g . 31 Older a p i c a l a x i s of host covered with e p i p h y t e s . Scale bar = 45pm F i g . 32 Epiphytes growing on a branching p o i n t of the host. Scale bar = 50pm F i g . 33 Heavy i n f e c t i o n by A. porphyrae o b s c u r i n g the host. Scale bar = 50pm F i g . 34 A young a p i c a l shoot of P. bipinnata growing from h e a v i l y i n f e c t e d host. The new shoot has a l r e a d y been i n f e c t e d (arrows). Scale bar = 40pm - 6 4 -F i g s . 35-37: Audouinella vaga c r o s s - i n f e c t i n g a l i v i n g Porphyra lorta blade. F i g . 35 I n f e c t i o n i s spar s e . Few e p i p h y t e s are seen on the v e g e t a t i v e a r e a of the blade. Note the long e r e c t f i l a m e n t s of the epiphytes (arrow). Scale bar = 30um F i g . 36 E r e c t f i l a m e n t s of A. vaga on h o s t . Note the ex t e n s i v e branching of the e p i p h y t e . Scale bar = 30um F i g . 37 Basal c e l l s (p) of epiphytes (e) growing on Porphyra blades (h) are more extensive (arrow) than e p i p h y t e s growing on P. bipinnata. S c a l e bar = 15um - 6 6 -F i g s . 38-41: M e t h a c r y l a t e s e c t i o n s of Audouinella porphyrae/ Porphyra fucicola (2.5um) s t a i n e d with T o l u i d i n e Blue-O. F i g . 38 E x t e n s i v e p r o s t r a t e f i l a m e n t s (p) i n host c e l l w a l l . Scale bar = 40pm F i g . 39 P r o s t r a t e f i l a m e n t growing between host c e l l s of P. f u c i c o l a . M a j o r i t y of endophytic growth i s w i t h i n the outer w a l l of the host. Scale bar = 36pm F i g . 40 Close-up of an e r e c t f i l a m e n t as i t grows through the wa l l of the host. Note the inner w a l l of the host (arrow). S c a l e bar = 11pm F i g . 41 An empty monosporangium on an e r e c t f i l a m e n t , o u t s i d e the host c e l l w a l l . Scale bar = 8pm - 6 7 -- 6 8 -F i g s 42-46: M e t h a c r y l a t e s e c t i o n s of A. porphyrae/Porphyra fucicola (2.5um t h i c k ) s t a i n e d with T o l u i d i n e Blue-0 ( F i g s . 42-43) and Fast Green ( F i g s . 44-46). F i g . 42 Close up of p r o s t r a t e p o r t i o n s growing e x t e n s i v e l y i n the outer w a l l of the host. Scale bar = 7pm F i g . 43 Close up of p r o s t r a t e p o r t i o n s growing between two host c e l l s (arrow) w i t h i n the inner w a l l of the host. E r e c t f i l a m e n t s (e) are a l s o p r e s e n t . Scale bar = 9pm F i g . 44 Suggestion of p r o t e i n o u s substance (dark s t a i n ) i s seen on the c u t i c l e (cu) and i n the cytoplasm of both the endophyte and the host. P i t plug (arrow) i s a l s o s t a i n e d . Scale bar = 14pm F i g . 45 Protuberance of an e r e c t i n i t i a l c e l l (arrow). Note dark s t a i n i n the host w a l l beside the protuberance. Scale bar = 6jum F i g . 46 Note the dark s t a i n j u s t above the endophyte c l o s e to the outer edge of the host (arrow). S c a l e bar = 3pm -7 0-F i g s 47-50: Met h a c r y l a t e s e c t i o n s of Audouinella vaga/ Pt er os i phoni a bipinnata (2.5um t h i c k ) s t a i n e d with T o l u d i n i e Blue-O. F i g . 47 Note the f i l a m e n t s of Audouinella growing along the wa l l of a p e r i c e n t r a l c e l l with a l a r g e vacuole ( v ) . Er e c t f i l a m e n t s are produced from p r o s t r a t e f i l a m e n t s (arrow). Scale bar = 8um F i g . 48 P e n e t r a t i o n of A. vaga i n t o the cytoplasm of the host (arrow). Note the nucleus (n) and l a r g e vacuoles (v) of the host c e l l . S c a l e bar = 6pm F i g . 49 Endophytic growth l i m i t e d by the host w a l l (dark arrow). Note the l a r g e vacuole (v) and cytoplasm a g a i n s t the p e r i p h e r y of the host c e l l ( c l e a r arrow). Scale bar = 8pm F i g . 50 F i l a m e n t s of the endophyte growing between two c e l l s of the host (arrow). S c a l e bar = 10^ im -7 2-F i g s . 51-53: M e t h a c r y l a t e s e c t i o n s of A. vaga/Pt er osi phoni a (2.5um t h i c k ) s t a i n e d with A l c i a n Blue/PAS ( F i g . 51) and Fast Green ( F i g s . 52-53). F i g . 51 Endophyte branching w i t h i n the host c e l l w a l l (arrow). Note the angular branching p a t t e r n of the endophyte. S c a l e bar = 10pm F i g . 52 Suggestion of proteinaceous substance l o c a t e d w i t h i n cytoplasm of both endophyte and h o s t . Note the l a r g e vacuoles of the endophyte (arrows). Note the darken area of the host w a l l j u s t above the endophyte. Scale bar = 10pm F i g . 53 A few e r e c t f i l a m e n t s (e) o u t s i d e of host (arrow). Note darkened areas i n host w a l l beside the endophyte. S c a l e bar = 8pm 81 - 7 4 -Figs 54-56: Ultrastructure of Audouinella porphyrae/ Porphyra fuci col a F i g . 54 Note the large vacuole (v) and the s t e l l a t e chloroplast (c) in the endophyte c e l l . The host c e l l wall appears to be affected by the endophyte. Note the irregular matrix of the host c e l l wall (hew). Scale bar = 2pm Fi g . 55 Section through a growing filament of A. porphyrae inside the host. Note the mitochrondria (m) and half p i t plug, (arrow). Scale bar = 1pm Fi g . 56 Note the nucleus (n) of the endophyte towards the end of the c e l l . Part of the s t e l l a t e chloroplast (c) i s seen within the vacuole (v). Scale bar = 2pm -76 -F i g s . 57-60: U l t r a s t r u c t u r e o f u n i n f e c t e d Porphyra f u c i c o l a . F i g . 57 A c e n t r a l p y r e n o i d (py) w i t h m i t o c h o n d r i a (arrow) i n t h e p e r i p h e r y of a P. fucicola c e l l . S c a l e b a r = 4pm F i g . 58 A n u c l e u s (n) w i t h m i t o c h r o n d r i a (m) a r o u n d i t . S c a l e b a r = 1pm F i g . 59 C l o s e - u p o f t h e c e l l w a l l o f P. fuci col a . Note t h e r e g u l a r f i b r o u s m a t r i x . Bar = 0.2pm F i g . 60 O v e r v i e w o f P. fucicola i n n e r w a l l . Note t h e r e g u l a r m a t r i x . S c a l e bar = 1.5pm -7 7--78 -Figs 61-64: Ultrastructure of Audouinella porphyrae/Porphyra fuel cola. F i g . 61 Section of endophyte growing outside of the host. Note the irregular matrix of the host c e l l wall (hew). A mitochondrion (m) and parts of the chloroplast (c) of the endophyte. Scale bar = 0.5pm Fi g . 62 Section through the chloroplast of the endophyte showing a central pyrenoid (py) with i r r e g u l a r thylakoids (arrow) traversing i t . Scale bar = 1pm Fig . 63 Note the irregular matrix of the host (hew) compared to the wall of the endophyte (ecw). Scale bar = 0.8_pm Fi g . 64 Enlargement of endophytic c e l l wall (ecw) and host c e l l wall (hew). Scale bar = 0.2pm -80-F i g s . 65-68: U l t r a s t r u c t u r e of Audouinella vaga/ Pt er os i phoni a bipinnata. F i g . 65 S e c t i o n of a part of u n i n f e c t e d host c e l l showing the re g u l a r matrix of the w a l l (hew) and p a r t of a vacuole (v) and mitochrondrion at the p e r i p h e r y of the c e l l . S c a l e bar = 0.8um F i g . 66 S e c t i o n of the endophyte p r o t r u d i n g o u t s i d e the host. Host c e l l w a l l (hew) does not appear t o be a f f e c t e d . S cale bar = 0.2um F i g . 67 S e c t i o n of endophyte i n s i d e the host c e l l w a l l (hew) showing o r g a n e l l e s of the host (h) which do not appear to be a f f e c t e d . Note l a r g e vacuole of endophyte (arrow). Scale bar = 2pm F i g . 68 Note how the growth p a t t e r n of the endophyte (e) f o l l o w s the l o n g i t u d i n a l growth of the host. S c a l e bar = 2pm - 8 2 -F i g s . 69-71: U l t r a s t r u c t u r e of f r e e - l i v i n g Audouinella porphyr ae. F i g . 69 Note the l a r g e c h l o r o p l a s t with a c e n t r a l p y r e n o i d (py) and the f l o r i d e o p h y c e a n s t a r c h (s) at the p e r i p h e r y of the c e l l . Note mitochondria (m) along the p e r i p h e r y of the c e l l and the t h i c k w a l l (double arrow). Scale bar = 1pm F i g . 70 Close up of a p i t p l u g . Note the two cap l a y e r s . S c ale bar = 0.5pm F i g . 71 A prominent nucleus (n) with a nu c l e o l o u s (nu) i s f r e q u e n t l y l o c a t e d at the base of the c e l l . Note the s t a r c h g r a i n s (s) surrounding the nucleus. S c a l e bar = 1pm -84 -F i g s . 72-75: U l t r a s t r u c t u r e of f r e e - l i v i n g Audouinella vaga. F i g . 72 Note the numerous l a y e r s (arrow) w i t h i n the c e l l w a l l of a growing f i l a m e n t . Note the nucleus (n) toward the base of the c e l l s . S c a le bar = 1pm F i g . 73 Close-up of a p i c a l t i p . Note the nucleus ( n ) , membranous bodies (mb) and p a r t s of a s t e l l a t e c h l o r o p l a s t (c) w i t h i n the cytoplasm of the c e l l . S c a l e bar = 0.8pm F i g . 74 Cross s e c t i o n of a f i l a m e n t . Note the nucleus (n) and s t a r c h g r a i n s (s) around i t . Scale bar = 0.8um F i g . 75 S e c t i o n through the p r o s t r a t e p o r t i o n of the endophyte. Note the c e n t r a l p y r e n o i d (py) and numerous s t a r c h g r a i n s ( s ) . S c a l e bar = 2pm 

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