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Life history studies on Urospora and Codiolum from southern British Columbia Hanic, Louis Anthony 1965

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The University of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of LOUIS ANTHONY HANIC . B.A., The University of British'Columbia, 1950 THURSDAY, JANUARY 14th, 1965 AT 2:00 P.M. IN ROOM 3.332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: I. McT. Cowan V. C. Brink R. F. Scagel Kathleen Cole Janet Stein G. L. Pickard G. H.' N. Towers External Examiner: Richard Norris Department of Botany University of Washington Seattle, Washington LIFE HISTORY STUDIES ON UROSPORA AND CODIOLUM. FROM SOUTHERN BRITISH COLUMBIA ABSTRACT L i f e h i s t o r y studies were conducted on Urospora and Codioluri). from a number of l o c a l i t i e s i n the S t r a i t of Georgia and Juan de Fuca S t r a i t . Urospora wormskioldii (Mertens) Rosenvinge (n = 12) is a dioecious species having a heteromorphic l i f e h i s t o r y with filament, dwarf and Codiolum stages» Asexual reproduction i s by means of acuminate quadri-f l a g e l l a t e zoospores» The Codiolum stage i s produced from zygotes, female and probably male gametes, The range i n v a r i a b i l i t y of morphological features of the vegetative filament, i s very great and encompasses a l l species now recognized from the P a c i f i c Coast of North America. Because of t h i s v a r i a b i l i t y . , the taxonomy of UTo_sp_ora should be based on l i f e h i s t o r y features as well as morphologi.cal ones. Mat i rigs were successfully made involving plants from six'widely-separate local!"' t i e s . Sexuality occurred i n cultures at 10°C.S through several s e r i a l subcultures i n the female but. only in the f i r s t c ulture in the male. A method employing a long th.errooper.iod was successful i n inducing sexuality in several vegetative clones of Ur ospora but f a i l e d i n others. Temperature,, filament: s i z e and nutrition, appear to be factors involved in the sexual response. F e r t i l i t y of c u l t u r a l Codiolum was low and attempts to increase it; met. with only p a r t i a l success. Urospora vancouverlana (Tilden) Setchell and Gardner (n = 9) i s an asexual species, producing a filamentous and dwarf stage via q u a d r i f l a g e l l a t e zoospores at: low temperatures and a Codioluro stage v i a b i f l a g e l l a t e zoo-spores at high temperatures. C u l t u r a l filaments are i n d i s t i n g u i s h a b l e from those of U. wormskioldii. Ur ospora speciosa (Carm.) Leblond ex Uamel (n = ?) i s recorded for the. f i r s t time, i n North America and was discovered to be uninucleate. It contains a filamentous and dwarf stage which reproduce via q u a d r i f l a g e l l a t e zoospores. Other features of i t s l i f e h i s t o r y are un-known . Codiolum gregarium A. Braun and _C. pusilium (Lyngbye) Kjellman, in the areas studied, are considered to be merely form variants belonging to the l i f e h i s t o r y of Urospora wormskioldii. F e r t i l i t y in natural Codiolum was i n h i b i t e d by short d a i l y warm thermoperiods and induced by cold treatment. Nuclear d i v i s i o n was studied but; meiosis was not demonstrated = Codiolum p e t r o c e l i d i s Kuckuck, found as an endophyte i n P e t r o c e l i s i s i n f e r r e d to belong to the l i f e h i s t o r y of Spongomorpha c o a l i t a (Ruprecht) C o l l i n s i n the areas investigated„ It i s considered to be u n i c e l l u l a r , uni-nucleate and capable of reversing i t s d i r e c t i o n of growth. The d i r e c t i o n of growth i s suggested to be governed by light, i n t e n s i t y . Cyt.ochero.ical' stij.di.es were done on the walls of U£pjp_ora Spongomorpha and t h e i r j^ojltjjljjm. stages. The inner walls of Urospgra and Spongomorpha are composed of c e l l u l o s e and pectic materials, while those of the Codiolum types are e n t i r e l y pectic* The pectic component appears to d i f f e r • from, that of higher plants* Urospora, Spongomorpha and t h e i r Codiolum. stages have an outer sheath of unknown composition. Three types of sheaths are represented, of which those of the two Codiolum types are the same, To be of f u l l taxonomic value-, future wall studies should i n -clude a l l stages where algae with heteromorphic l i f e h i s t o r i e s are concerned, Spongomorpha c o a l i t a was discovered to have operculate gametangia. This species should therefore be transferred to the genus Acrosiphonia, The Implications of t h i s transfer and the recognition of a u n i c e l l u l a r condition f ° r Codiolum p e t r o c e l i d i s are discussed in r e l a t i o n to l i f e h istory and taxonomic. problems ex i s t i n g i n the Acrosiphonia-Spongomorpha complex. GRADUATE STUDIES F i e l d of Studys-Genetics K, M. Cole, H, J . Muller & R. P„ Wagner Cytogenetics K.0 M„ Cole Cytology R. Eo CI. e l and Phycology R„ F. Scagel, R. C. Starr & J . R. Stein SCHOLARSHIPS 1960 - B. C. Sugar Refining Co. Ltd..Scholarship 1960 - Edith Ashton Memorial Scholarship 1961 - Research Assistantship, Indiana University (Nat. Health Found, grant to Dr. Starr, I.U.) 1962 - Indiana University grant to attend Woods Hole Summer Session 1963 - Graduate Fellowships and Scholarships, U. B. C. LIFE HISTORY STUDIES ON UROSPORA AND CODIOLUM FROM SOUTHERN BRITISH COLUMBIA LOUIS ANTHONY HANIC B.A., U n i v e r s i t y of B r i t i s h Columbia, 1950 A THESIS SUBMITTED TN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy i n the Department of ' Biology and Botany We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1965 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study* I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t ; c o p y i n g or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission® Department of Biology and Botany The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8? Canada Date J a n u a r y 17.1965  i ABSTRACT Life history studies, emphasizing cultural and cytological ap-proaches, were conducted on Urospora and Codiolum from a number of local-ities in the Strait of Georgia and Juan de Fuca Strait. Urospora wormskioldii (Mertens) Rosenvinge (n = 12) is a dioe-cious species having a heteromorphic l i f e cycle with a filamentous, dwarf and Codiolum stage. The Codiolum stage is produced "by syngamy of aniso-gametes or by parthenogenetic development of female and probably male gametes. A l l three stages produce asexual quadriflagellate zoospores which develop into filament or dwarf plants. The range in variability of morphological features of the vegetative filament is very great and en-compasses a l l species recorded from the Pacific Coast of North America. Crosses involving plants from six widely separate localities snowed no incompatability at the mating level. Sexuality occurred spontaneously at 10°C in cultures of both sexes through several serial subcultures in the female but only in the f i r s t culture in the male. Temperature changes, filament size and nutrition appear to be factors involved in the sexual response. Several vegetative clones, Including a two-year-old isolate from Cape Cod, were sexually induced by a method involving a long thermo-period. However, the same method failed on other ...clones. Growth of Codiolum in culture was poor and i t is suggested that daily exposure may be a requirement for normal growth. The f e r t i l i t y of zygotic and parthenogenetic Codiolum was low in cultures grown under constant temper-ature conditions. Attempts to induce f e r t i l i t y by cold treatments met with success in some isolates but not in others. Urospora vancouveriana (Tilden) Setchell and Gardner (n =9) I s i i an asexual species, producing.filamentous and dwarf stages via quadriflag-ellate zoospores at low temperatures and a Codiolum stage via Mflagellate zoospores at high temperatures. Cultural filaments are indistinguishable from U. wormskioldii. Urospora speciosa (Carm;) Leblond ex Hamel (n = ?) is recorded for the f i r s t time in North America and was discovered to be uninucleate. It contains filamentous and dwarf stages which reproduce via quadriflag-ellate zoospores. Other features of its l i f e history are unknown. Codiolum gregarium A. Braun and C. pusillum (Lyngbye) Kjellman, in the areas studied, are considered to be merely form variants belonging to the l i f e history of Urospora wormskioldii. The living nucleus and its! staining characteristics are described.. Nuclear divisions were followed but meiosis was not demonstrated. Fertility in both types was^induced by cold and inhibited by short daily thermoperiods. Codiolum petrocelidis Kuckuck, found as an endophyte in Petro-celis franciscana Setchell and Gardner is a unicellular, uninucleate plant producing ovate quadriflagellate zoospores which give rise to branching filaments. C. petrocelidis, as found in the areas studied, is inferred to belong to the l i f e history of Spongomorpha coalita (Ruprecht) Collins. The Codiolum cell is capable of reversing its direction of growth and this reversal is suggested to be governed by light intensity. It is also pro-posed that the mode of stipe growth rules out a multicellular condition. Operculation was discovered in S. coalita. Cytochemical studies were done on the walls of Urospora, Spon-goraorpha and their Codiolum stages. The inner walls of Urospora and Spongomorpha are composed of cellulose and pectic materials, while those i i i of the Codiolum types are entirely pectic. The pectic component appears different from that of higher plants. Urospora, Spongomorpha and their Codiolum stages have an outer sheath of unknown composition which is singly refractive, gives a negative test for cellulose, chitin, pectin and fat. Three types of sheaths are represented, of which those of the two Codiolum types are the same. Several taxonomic implications result from these studies. To he meaningful, identification of Urospora species should he based on more than just purely vegetative features. To be of taxonomic value wall studies should include a l l stages where algae with heteromorphic l i f e histories are concerned. Because of its operculate condition, Spongo- morpha coalita should be transferred to the genuB Acrosiphonia. This transfer would provide further evidence for the occurrence of a heter-omorphic l i f e history in Acrosiphonia. It would also re-establish Wille's basis for recognition of the two genera on nuclear condition. Acceptance of a unicellular condition for Codiolum petrocelidis, and a heteromorphic l i f e history for Acrosiphonia would re-establish the basis for Jonsson's \ new family, the Acrosiphoniaciae and at the same time remove the main barriers to the inclusion of the Acrosiphonia-Spongomorpha complex in the Ulotrichales (sensu Kornmann). iy TABLE OF CONTENTS . TOPIC PAGE ABSTRACT i ACKNOWLEDGEMENTS ' ' • . . v i i I. INTRODUCTION 1 II. MATERIALS AND METHODS 7 III. RESULTS AND OBSERVATIONS 13 1. Urospora wormskioldii (Mertens) Rosenvinge 13 2. Urospora vancouveriana (Tilden) Setchell and Gardner 32 3. Urospora speciosa (Carm.) Leblond ex Hamel 36 Summary 37 k. Codiolum gregarium A. Braun and C. pusillum (Lynghye) Kjellman "". ~ 38 5. Codiolum petrocelidis Kuckuck and Spongomorpha coalita (Ruprecht) Collins . k6 IV. DISCUSSION AND CONCLUSIONS ^9 V. FINAL SUMMARY 72 BIBLIOGRAPHY 76 TABLES 1. Description of collection sites, clones established and other data. 81 2. Filament growth in U. wormskioldii. 82 3. Germination of single zoospores, and carry over of sex-uality in U. wormskioldii female clone F l l . 83 k. Germination of single zoospores of U. wormskioldii male clone M2A &k 5. Effect of temperature on f e r t i l i t y of natural Codiolum. Qk 6. Effect of thermoperiod on f e r t i l i t y of natural Codiolum. ; 85 7. Effect of cold shock on f e r t i l i t y of cultural Codiolum. ,85 8. Effect of temperature on f e r t i l i t y of cultural Codiolum. 86 9. Effect of temperature on sexuality in U. wormskioldii. 86 FIGURES Key to symbols in Figures 1 - kO 87 1. Life cycle of U. mirabilis (Diagram) 88 2. Life cycles in Urospora and Ulothrix (Diagram) 88 3. Map, Field trip stations June, 1963 and matings. ,89 k. Collection sites.- - 91 5. Vertical-distribution of Urospora and Codiolum. 92 6. Codiolum, variation in abundance and morphology. 93 7. Life cycle of U. wormskioldii (Diagram) 9^4-8-1^. Cytomorphological features of U. vormskioldii. 96 15. Life cycle of U. vancouveriana (Diagram) 109 16. Temperature effects on U. vancouveriana. . 109 17-20. Cytomorphological features of U. vancouveriana. I l l 21. Cytomorphological features of U. vormskioldii, U. van-couveriana and U. speciosa. 119 22. Comparison of gamete morphology of U. vormskioldii and biflagellate zoospores of U. vancouveriana with those of European species. 120 23. Variation and matings in U. wormskioldii. 121 2k. Codiolum types and morphology. • • . 123 25. C. gregariura and cultural products. 125 26. Cytology, of C. gregariura and U. wormskioldii zoospores. 127 27. U. wormskioldii from C. gregarium. 129 28. Codiolum f e r t i l i t y , in transplants and nature. 131 29. Codiolum products from cold shocked cultural plants. ,." 133 30. Effect of thermoperiod on sexuality in Urospora. 134 '31. Cytochemical tests on the cell walls of U. wormskioldii and C. gregarium. 135 32. Cytochemical tests on the cell walls of S. coalita and C. petrocelidis. 136 v i 33-3^. Morphological features of treated walls of U. worm-skioldii and C. gregarium. 137 35• Morphological features of treated walls of S. coalita and S. petrocelidis. 1^ 2 36. Stipe comparison in C. petrocelidis, from nature and culture after various authors. ±1^ 37. Stipe morphology in C. petrocelidis and C. gregarium. 38, 39. Developmental types in C. petrocelidis. 1^ .3 kO. Developmental types, in C. petrocelidis and opercu-lation in S. coalita. : ~~ 152 I v i i AOqiOWLEDGEMMTS The author would like to express sincere gratitude to his ad-visor, Dr. K. M. Cole, for suggesting this problem, for her active inter-est in this research and for the personal warmth and generosity that she has bestowed upon the author and his family. The author also wishes to thank Dr. R. L. Fernald, Director of the Friday Harbor Laboratories, San Juan Island, for making available re-search facilities at that station; Dr. R. C. Starr, University of Indiana, for making i t possible to continue these studies at the Woods Hole Marine Biological Station; Dr. M. Dube, Western Washington State College, for his invaluable advice on photography and for being instrumental in finding Urospora vancouveriana; A. C. Mathieson, University of British Columbia, for aid in obtaining tidal data from Glacier Point; and R. K» S» Lee, Memorial University, Newfoundland, for making available his collections of Urospora and Petrocelis. The author is especially indebted to the members of his comm-ittee, Dr. J. R. Stein, Dr. K. M. Cole, Dr. R. F. Scagel, and Dr. T. M. C. Taylor for the valuable suggestions given thro ughout the course of this work. Finally, the author gratefully wishes to acknowledge financial assistance from the Biology - Botany Department of U. B. C , Dr. K. M. Cole's National Research Grant (N. R. C. -A-6V?), B. C. Sugar Refinery Scholarship (i960), Edith Ashton Memorial Scholarship (i960), the Univer-sity- of British Columbia Graduate Fellowship (I963) and Dr. R. C. Starr's National Health Foundation Grant (1962). Life History studies on Urospora and Codiolum  from southern British Columbia Introduction The genus Urospora, of the Cladophorales, was established by Areschoug in 1866. It comprises individuals which are filamentous, un-branched, having a rhizoidal holdfast formed by several basal cells. The ' vegetative ce l l has a netlike parietal chromatophore containing many pyrenoids, many nuclei and a large central vacuole. Asexual reproduction is by means of quadriflagellate posteriorly pointed zoospores and sexual reproduction by means of biflagellate iso- or heterogametes. About f i f -teen species are described inhabiting the cooler waters of the northern hemisphere. Setchell and Gardner (1920) recognized seven species from the west coast of North America of which six are recorded from British Columbia and northern Washington (Scagel, 1957). The genus Codiolum, formerly contained in the Chlorococcales, was established by A. Braun (1855)• It is comprised of small unicellular plants, both.free-living and endophytic. The cell has a basal stalk, a parietal chromatophore with several to many pyrenoids, and one nucleus. Reproduction is by means-of quadriflagellate zoospores. About nine species are described, of which a l l are marine except Codiolum lacustre Printz, which is freshwater. The three species described from the west coast of North America (Setchell and Gardner, 1920) are also recorded from British Columbia and northern Washington (Scagel, 1957)• In 1933 Jorde brought these two genera together in her report that Codiolum gregarium A. Braun (a free living form) belongs to the l i f e history of Urospora mirabilis Areschoug and thereby demonstrated the 2 . occurrence of a heteromorphic l i f e history in the green algae for the f i r s t time. Though Jorde's results remain unconfirmed in relation to the l i f e cycle of U. mirabilis, a similar Codiolum stage has been reported in other species of Urospora (Kornmann, l°/6l;b, c) and in other genera: . Ulothrix (Kornmann, 1963, 1964a) Spongomorpha (Hollenberg, 1957, I 9 5 8 ; Fan 1957, 1959; Jonsson 1959b, 1962 and Kornmann 1961a); Acrosiphonia (Jonsson 1951> 1958, 1959a, 1962, 1963, 1964a, b); Monostroma and Gomontia (for pertinent . literature see Kornmann, 1962, 1963, 1964b), and Cladophora (Archer and Burrows, i 9 6 0 ; Van de Hoek, 1964). However, these reports are not with-out conf l i c t . According to Kornmann ( I 9 6 2 ) , Acrosiphonia lacks a Codiolum stage and has an isomorphic l i f e history,whereas Jonsson claims the opposite. Kornmann (1961a) believes C. petrocelidis Kuckuck and Chloro-chyt'rum inclusum Kjellman to be merely endophytic modified forms of the zygote of S. lanosa, while Jonsson claims the former belongs to the l i f e history A. spinescens (= A. arcta, Kornmann 19&2) and the latt e r to S. lanosa. Most investigators e.g. Kuckuck ( 1894) , Zimmerman ( 1925) , Printz ( 1 9 2 6 ) , Hollenberg ( 1 9 5 8 ) , Fan ( 1959) , Jonsson (1958, I962) considered C. petrocelidis to be. unicellular, while Kornmann (1961a) suggests i t i s multicellular. Van de Hoek (1964) regards the Codiolum-like c e l l s in Cladophora as growth-suppressed germlings resulting from nutrient deple-tion. As a result of the recent findings mentioned above,attempts at taxonomic revisions have been made. Den Hartog (1959) proposed the new family Codiolaceae to include Urospora species. However, with the dis-covery of other Codiolum-containing genera the basis for this family i s removed. Jonsson (1959"b) created the family Acrosiphoniaceae to include Urospora, Spongomorpha and Acrosiphonia on the hasis of having in common a unicellular Codiolum sporophyte5, similar cell wall, chloroplast pyren-oid and vacuolar structure. Kornmann (19&2) rejected the Acrosiphoniaceae and redefined the old order, Ulotrichales, to include Ulothrix, Urospora, Monostroma and Gomontia. He excluded Spongomorpha and Acrosiphonia from this order mainly because they lacked a quadriflagellate acuminate zoo-spore .and had a different wall structure (Nicolai and Preston, 1952) and secondly because he felt Acrosiphonia had an isomorphic l i f e cycle and Spongomorpha, a multicellular Codiolum zygote. As a result of these con-flicting reports and different interpretations the taxonomy of the mar-ine green algae is, at present, in a fluid state. Cytological studies on Codiolum-containing genera are restricted to those of Hart (1928) and Jorde (1933) on Urospora and Jonsson (1962) on Acrosiphonia and Spongomorpha and have been concerned mainly with the filamentous stage. Hart described the nuclear condition of germlings and filaments of U. wormskioldii. Jorde studied nuclear division in U. mira-b i l i s and gave this species a chromosome number of four, but with some reservations since the chromosomes were very small. Jorde failed to ob-serve division in the Codiolum stage. Jonsson (1962) reported that nuclear division in the filament of Spongomorpha and Acrosiphonia during gametogenesis is mitotic, thereby providing indirect evidence for meiosis in the Codiolum stage. However, Moewus (1938) demonstrated meiosis in the Codiolum stage of Monostroma more directly by obtaining a 1:1 sex ratio from the Codiolum products. Our present knowledge of l i f e histories in Urospora is confined to European types. Little i s known about the genus from North America. Setchell and Gardner (1920) attempted to arrange the species reported from this coast, relying largely on herbarium material. Several species were placed into synonomy by them. The species were separated on the basis of differences in rhizoidal structure, cell proportions and dimen-sions, chloroplast structure and size of fertile sporangia. These authors pointed out the need for studies on living material. Frye and Zeller (1915) described U. tetraciliata, from San Juan Island, from sexual mater-i a l but did not follow zygote development. The unusual nature of gamete fusion, described as starting posteriorly> has been questioned by Collins (1918, p. 86) and Setchell and Gardner (1920, p. 195). Collins suggests that these may represent imperfectly separated microzoospores (= gametes). This species has not been recorded since, even though Frye attempted to relocate i t at later dates (Setchell and Gardner, 1920). Hart's (I928) cytological studies were done on U. wormskioldii from San Juan Island and included a minor amount of culturing. She was the f i r s t to discover dwarf plants and thick walled resting cells in culture and to note their hardy qualities. Fan (1959) attempted a cultural study of U. penicilli-formis from California but gave up after failure to obtain sexual mater-i a l . At this point a brief summary of the l i f e histories thus reported in Urospora is warranted. The l i f e history of Urospora mirabilis i3 shown in Fig. 1 and that of other Urospora species and Ulothrix, schem-atically in Fig. 2. U. mirabilis (Jorde, 1933) has three somatic stages: a filament, dwarf and Codiolum stage. It is dioecious and reproduces the filament and dwarf stage asexually via quadriflagellate zoospores. The Codiolum stage is produced from zygotes via sexual fusion.of biflagellate anisogametes or from female parthenogametes. Meiosis is suggested to 5. occur in the diploid Codiolum cell U. speciosa (Carra.) Leblond ex Hamel lo31> differs from U. mirabilis in being monoecious and isogamous (Kornmann, 1961c), (see Pig. 2, III). U. penicilliformis (Roth) Aresch. 1866, is similar to U. mirabilis dif-fering only in filament and gamete morphology (Kornmann, 1961c),. (see Fig. 2, V). . U. vormskioldii (Mertens) Rosenvinge 1893, has not been found sexual in nature and therefore its l i f e history is unknown U. wormskioldii var. biflagellatum Kornmann I961, lacks gametes. The filament and dwarf stage are produced from quadriflagellate zoo-spores and the Codiolum stage from biflagellate zoospores, (Kornmann, I96I b, c), (See Fig. 2, Via) '. U. wormskioldii var. caudatum Kornmann I96I, produces a Codiolum phase via the quadriflagellate zoospores but lacks a sexual phase, (Kornmann, I96I c), (See Fig. 2, VTb) U. bangioides (Harv.) Holm, et Batt. I89O, has no sexual phase, or Codio- lum stage, and reproduces only via quadriflagellate zoospores, (Kornmann, I961 c), (See Fig. 2,1) U. tetraciliata Frye and Zeller 1915, is reportedly isogamous but details on zygote development are lacking. The present study was undertaken to obtain information on the distribution, morphology and l i f e history of Urospora and Codiolum species from southern British Columbia. Endophytic Codiolum forms were also in-cluded since i t could not be assumed that only free-living forms give rise to Urospora. Special emphasis was placed on cytological and cultural 6. approaches. The main objectives were to establish the alternation of gen-erations on a cytological or genetical basis and.to determine the condi-tions required to bring about the complete l i f e cycle in culture. The latter involved studies on the sexual response in Urospora and the f e r t i l -ity response in Codiolum.. 7. II. MATERIALS AND METHODS  Collections Approximately thirty areas were investigated in the Strait of Georgia and Juan de Fuca Strait (see Fig. 3 for localities and Table 1 for their description). Three of these, Deadman's Bay (Fig. k G), Friday Harbor (Fig. 28 B) and Tsawwassen (Figs, ll- A and B) received monthly inspection for at least one year. Point No Point (officially known as Glacier Point)(Fig. h F), Ogden Point Breakwater (Fig. k C), Oak Bay (Fig. k D), Anacortes and Porlier Pass were visited two or more times during the summer and winter of at least one year. The remainder were visited infrequently. Nineteen of the areas (Fig. 3) were examined during a one week period in June, I963, to obtain comparative data on the dis-tribution, morphology and sexuality of Urospora. At each site samples of Urospora and Codiolum were taken throughout their vertical range and ex-amined on site with a dissecting microscope at magnifications of 25, 50 and 100. Where Urospora or Codiolum were not visible, scrapings were taken from rocks and logs throughout the intertidal area with the aid of a wire brush and syringe. Species of Urospora and Codiolum collected and later identified were; U. wormskioldii, U. vancouveriana and U. speciosa, C. gregarium and C. pusillum. C. petrocelidis Kuckuck (obtained from Petrocelis franciscana Setchell and Gardner) and Spongomorpha coalita were collected from Deadman's Bay and Porlier Pass during the summer of I96I and the summer and f a l l of 1963* Culture techniques Clones of Urospora were established representative of the upper, middle and lower range at each locality where a wide range occurred. The 8. morphology of the clones was compared with that of their field counterparts. Unless otherwise indicated cultures were grown and maintained in 20 x 180 mm test tubes containing 20 ml of UR1 medium (see below) at 10° ± 2°C, under 300 - kOOfjz. of illumination supplied by "cool-white" Sylvania fluorescent tubes on a lk hr/10 hr light/dark cycle. Single germlings ( 6 - 1 2 celled) derived from mass zoospore cultures were used as the in-oculum. The UR1 medium is equivalent to the SW1 medium of. Iwasaki (I96I) minus the TRIS, supplemented with 6 )\ g/l of biotin, 10 p. g/l.of thiamine and 0.02 jx g/l of vitamin B12. To minimize precipitation, the sterile medium was prepared by steaming one to two l i t r e quantities of filtered seawater on three consecutive days, following which, the minerals and vitamins were added from three pre-sterilized stock solutions, one con-taining the nitrate and phosphate, one the FeEDTA and one, the vitamins. The stock solutions were sterilized by autoclaving for three minutes at 15 psi. A 1.5$ agar preparation of UR1 was used to plate out zoospores and gametes. Other containers, as well as test tubes, used for experi-mental studies, were: 2 x 10 cm Petri plates (shallow) containing 50 ml of medium, 8 x 10 cm.Petri plates (deep) containing 125 ml of medium, and 125 ml Erlenmeyer flasks containing 100 ml of medium. Other media used to study Urospora growth were: Erdschreiber1s solution (Starr, 1956), Iwasaki's (I96I) SW1 and SW11 media and modifications thereof. Constant temperature of 5 ° , 10°, 15° and 20° were used for behavioral studies of Urospora, Codiolum and their motile reproductive structures. Methyl cellulose was used to slow down gametes for critical observation. The Urospora wormskioldii clone URX1 (derived from a large low tide form at Deadman's Bay) was used to study growth in different solu-tions (Table 2), different salinities ranging from 5 - 5 0 °/oo and different light intensities ranging from 25 - hOO f.c. Clone URX1 had produced Codiolum cells in great abundance on two occasions separated by over a year, yet evidence of gametes was never found at those times or in dozens of other subcultures. Infertile cells of Codiolum petrocelidi s.(Deadman's Bay) with the host, Petrocelis, were cultured in Erdschreiber1s solution in shallow petri dishes at 10° t 2°C and at various light intensities (25 - k-00 f . c ) . One lot was cultured in the liquid phase and one on moist f i l t e r paper supported at the surface of the medium with a glass triangle. The Petrocelis cultured in the latter manner underwent disintegration over a two week period leaving the Codiolum cells easily freed. This was done by passing moistened tissue several times through a narrow pipette. Freed cells were recultured in the liquid phase. Stain techniques Initial studies employed an acetocarmine technique in which the material was fixed in 3:1 (ethyl alcohol: glacial acetic acid), heated in glacial acetic acid at 6o°C for k hrs, stained in acetocarmine (Darlington and LaCour, i960), for 12 hrs at 6o°C in a screw cap vial and differentiated by applying a drop of 4$ ferric alum to the edge of a slide preparation and heating for a few seconds over an open flame. Though satisfactory as a nucleolar and chromosome stain, the chromatin in inter-phase nuclei of Urospora and Codiolum remained clear. Subsequently an intensive stain investigation was undertaken, which resulted in the adoption of three methods: a Newcomer-iron-propiocarmine method for Codiolum, a Feulgen-iron-propiocarmine method for Urospora and an aceto-orcein-iron-propiocarmine method for gametes. In the Newcomer-iron-propiocarmine technique, Codiolum was fixed in Newcomer's fixative ( 1 9 5 3 ) ; 10 heated in glacial acetic acid for k hrs at 60°C, washed, mordanted with 2.5$ ferric alum for 5 min, washed and stained on a slide with a drop of propiocarmine (prepared in the same manner as acetocarmine) and differ-entiated by brief heating (1 - 3 seconds) over an open, flame. This method also worked well on Urospora and C. petrocelidis. In the Feulgen-iron-propiocarmine method, Urospora was.cultured on slides, fixed in 3:1 for 12 hrs, stained with Feulgen (Darlington and LaCour, i960), using 8 min hydrolysis, 1 hr staining and 10 min in the sulfite bleach. On re-moval from the bleach, the slide preparation was stained with a drop or two of propiocarmine, mordanted a light purple blue with iron from an iron f i l e and differentiated with brief heating (1 - 3 sec) over an open flame. Over-mordanting and over-heating brought out nucleolar and cyto- , plasmic details. In the aceto-orcein-propiocarmine technique, gametes contained in a drop on a slide, were fixed over osmic acid vapours, stained with a drop of aceto-orcein, washed by drawing water under the coverslip, and then stained with a drop of propiocarmine mordanted lightly with iron from an iron f i l e , and differentiated with brief heating over an open flame. Crystal violet was used as a flagellar stain. For stain-ing germinating zoospores of Urospora, a 2$ colchicine in seawater solu-tion was applied for 9 hrs following the commencement of the dark cycle on the day when f i r s t c e l l divisions were observed (usually on the li-th day). Aniline blue in 10$ HC1 was used to stain C. petrocelidis in fresh or formalin fixed Petrocelis using mild heating to obtain separa-tion of the Petrocelis filaments. For nuclear studies, C. petrocelidis was fixed in 3:1 and stained with acetocarmine. The stained material was then drawn through a narrow pipette to bring about further separation of the Codiolum from the Petrocelis tissue. Wall Studies Cytochemical tests were done on fresh material and material fixed in 3:1 or % formalin. Ruthenium red (diluted to 1 part in 5,000 with water) was used to test for the presence of pectic compounds, Sudan III for fats and IKI - HgSO^  or ZnClIg (both prepared.according" to McLung, 1937) for cellulose. . The presence of chitin was tested according to Jensen (I962) on unsectioned material. In this test the material was autoclaved in 23 M KOH in a screw-cap vial for 15 min at 15 psi and subsequently treated with IKE and 1$ R^ SO^ . A violet-blue color indicated the presence of chitin. Schweitzer's reagent (Handbook of Chemistry and Physics, 1962 - 1963, Method 2, page 1650), HC1 - ZnClg (l.,pt cone. HC1: .1 pt ZnClg by weight) and slug cytase were used as cellulose solvents. The slug cytase was ob-tained from 3 - 6 In specimens of the slug Airiolimax columbianus Gould, starved for 12 hrs. The cytase was preserved with a few crystals of phen-ol according to Faberge (19^5) and used undiluted immediately after ex-traction. Except where heat or long periods were involved reactions were observed in progress at magnifications of 100 or ^ 50 times. Urospora matings o Field material to be used for mating was stored at 10 C, the temperature at which gamete formation was most prolonged. Mating of Urospora wormskioldii gametes was done by cooling filaments of both sexes on ice in the dark for about 30 min, transferring one or two f i l -aments of each sex to a small drop (5 - 7 mm diameter) on an ice-cooled slide, and illuminating with a bright light to stimulate gamete release. The slide was transferred to a cooled microscope stage and copulation was observed at magnifications of 100 or ^ 50 times. The slide preparation 12. was fixed over osmic acid vapours, stained with propiocarmine - lightly mordanted with iron from a f i l e , and made permanent in Euparal. De-hydration and embedding were done in a chamber saturated with ethyl a l -cohol. Several crosses were made of 8L female (Long Beach) with 18-3 male (Tsawwassen) for cultural purposes. In this case a dense male gamete suspension was prepared in a small drop about 0.5 nm diameter, and into this 50 to 100 female gametes were inserted. Induction of sexuality in Urospora and f e r t i l i t y in Codiolum The experiments on induction of sexuality in Urospora worm-skioldii were done mainly with female clone F l l , male clone M2A (both from Tsawwassen), clone URX1 (Deadman's Bay), and secondarily with asex-ual isolates derived from Codiolum or unknown Urospora types and with a two-year-old Urospora clone from Cape Cod, Massachusettes. The experi-ments on the induction of f e r t i l i t y in Codiolum were done with cultural Codiolum from clones F l l , M2A, URX1 (localities given above), female F8 (Long Beach), F 3 female (Oak Bay), and natural Codiolum from Deadman's Bay, Friday Harbor, Tsawwassen, Oak Bay, Point No Point, and Ogden Point Breakwater. The methods used are given together with the results. Tide level determination The tidal levels given are approximate and are based on inter-polations from tide table data (Anon, I962 - 3) with respect to measure-ments of water levels taken at specific times. Levels at Deadman's Bay and Friday Harbor were determined from the tide staff on the cantilever pier at the Friday Harbor Marine Laboratories, taking the zero mark and tidal amplitude as being roughly equal to that given for Fulford Harbour, B. C. 13. I I I . RESULTS AND OBSERVATIONS 1. Type I . Urospora vormskioldii (Mertens) Rosenvinge Ecology ( L i f e cycle, F i g . 7) Urospora vormskioldii vas the most common species encountered i n the areas studied. I t i s a mid-tide form ranging from the mean of the high-er high vaters down to the mean of the lover lov vaters ( F i g . 5)• Occasion a l l y i t i s found during the v i n t e r i n sparse quantities at high tide asso-ciated with f e r t i l e c e l l s of Codiolum gregarium A. Braun and i n the spring (March to early June) i n abundant quantities i n the low t i d e . In the spring and early summer U. wormskioldii i s associated i n the mid-tide with Porphyra lanceolata (Setchell and Hus) G. M. Smith, Bangia fuscopurpurea (Dillwyn)Lyngbye and U l o t h r i x species, mainly Ulothrix f l a c c a (Dillwyn) Thuret. In i t s smallest form (high tide) U. wormskioldii resembles U. p e n i c i l l i f o r m i s (Roth) Areschoug and i n i t s largest form (low t i d e ) , U. vancouveriana (Tilden) Setchell and Gardner. In i t s upper range i t f r e -quently suffers from fungal i n f e c t i o n and epiphytism. Sexual plants occur from early A p r i l to l a t e September arid are e n t i r e l y r e s t r i c t e d to the mid-tide. At Tsawwassen plants become sexual on the south side of the j e t t y about a month sooner than those on the north side. U. wormskioldii becomes more scarce and appears lower i n the i n t e r t i d a l as summer progresses and may disappear during June, J u l y , and August i n southerly exposures, i . e . southeast side of Tsawwassen j e t t y . In most areas the coverage of Urospora i s very spotty, often confined to patches on widely separate rocks. A continuous coverage i s found mainly on uniform substrates, i . e . the f l a t surfaces of the granite blocks forming the Ogden Point Breakwater ( F i g . k C), very large f l a t sur-faced boulders, beaches composed of small smooth rocks four to twelve i n -ches i n diameter as occur at Tsawwassen (Fi g . k A and B) or Oak Bay 11+. (Fig. k D), debarked sunken logs, and pilings. On one occasion i t was found on a newly painted Salmon Trol l e r at the bow near the waterline and in one instance (Departure Bay), on a tractor t i r e in the mid-tide. However, i t was never found as an epiphyte or in tide pools. Very large asexual forms (Fig. 23) were.found in the low tide re-gion ( 0 - 2 ft) in the same place in the spring and early summer (April -June) of two succeeding years at Tsawwassen, Deadman's Bay, Point No Point, and once at Kelsey Bay. Except at Kelsey Bay these were associated above with sexual plants of U. wormskioldii. At Tsawwassen and Deadman's Bay the two types were di s t i n c t l y separated, but at Point No Point they formed a continuous v e r t i c a l band, the size of the filaments gradually increasing with depth. Attempts were made to induce sexuality in several clones of large low tide forms but these f a i l e d . As a result, their assignment to U. wormskioldii cannot be certain. Urospora wormskioldii i s remarkably resistant to desiccation and heat. After prolonged exposure of up to six hours in the hot sun the f i l -aments can be rubbed to a powder between the fingers. Yet the plants swell up quickly on rewetting and, i f f e r t i l e , release masses of zoospores. The same behavior was observed for cultural plants. After fiv e days of drying at 20° - 23°C for four hours daily, active zoospores were released on rewetting. F i e l d plants can also withstand periodic heavy rains and freezing conditions. Dwarf plants survive cold much better than the filaments. When f i e l d filaments and cultural dwarf plants were kept in . o • the dark in frozen seawater (-5 C) the filaments died within three days, whereas the dwarf plants survived very well for more than three months. Also, dwarf plants survived at s a l i n i t y extremes of 10 °/oo and 50 °/oo for over a year. However, in spite of these hardy features of Urospora, . 15. its abundant spore production and its snort l i f e cycle (10 - 20 days) Urospara is not as abundant or widespread as might be expected. Morphology of field plants Vegetative plants vary considerably in dimensions and morphology from season to season or from locality to locality. The plant length varies from 0.5 - 24 cm and its width from 80 - 1200 }x. The holdfast is composed of 5 - 23 rhizoids, which generally are intramatrical in large plants (Fig. 10 A) but occasionally extramatrical in small ones. The number of rhizoids increases with the size of the plant. Usually, basal cells are shorter than wide (Fig. 10 A), subcentral cells isodiametric and slightly swollen, and more distal cells barrel shaped or spherical (Fig. 10 D). Generally the cylindrical cell is a feature of small plants and the barrel or spherical cell, of large plants. The chloroplast in small cells is parietal, in large cells cylindrical, and occasionally- is found withdrawn from the end walls in elongated basal cells (Fig. 8 A). The chloroplast varies from being coarsely reticulate in basal cells (Fig. 8 B), especially elongated ones, to finely reticulate in larger distal cells (Fig. 8 G and 10 B, E). Coarse reticulation, however, may occur in larger cells, especially at the end walls. This feature seems to be characteristic of rapidly growing filaments, for in culture the perforations become smaller with age of the filament. * Occasionally cells may be found in which the chloroplast con-sists of unconnected bands (Fig. 8 F, upper cell) arranged in a helical fashion. These are most often found next to dead cells or in cells grow-ing down through dead cells (Fig. 10 A). The latter type of cell was observed in stained material as well. In all cases the nuclei of the cell being perforated were degenerate. In several instances the nuclei in the cell adjacent to the one being perforated were also degenerate indicating that the cells were dying. Sexual plants, on the other hand, are much more uniform, vary-ing from 100 - 300 /u> in width and 2 - kcm in length. The vegetative cells and gametangia are most often isodiametric, or shorter than wide, and slightly swollen. A l l but the rhizoidal cells may become fertile to produce quadriflagellate zoospores, or sexual to produce biflagellate gametes. These are liberated through a pore (Figs. 12, D and G) formed by gelatinization of the inner walls (Figs. 9 A, B and 27 G). On liber-ation the outer membrane is ruptured and the spores are released in a vesicle (Figs. 9 D and E) or stream; the female gametes in a stream and the male gametes usually in a vesicle. Gametangia and small sporangia have a single discharge pore centrally located., but larger sporangia may have two to four pores located near the end walls. The zoospores (Fig. 10 H) range in size from 15 - 20 /u in length and 5-7 /u in width and are usually arranged parallel to the surface of the cell (Fig, 10 F). In large cells they may be;arranged in star-shaped clusters. The posterior tip of the zoospore may be long and thin (Fig. 10 H) or short and gradually tapered. In cross section the zoospores are quadrate (Fig. 27 K), but later round up (Fig. 27 L). The zoospore chloroplast is parietal and has four anterior projections (Fig. 25 B, 27 K and L). Four f i b r i l - l i k e structures (Figs. 11 E and 27 K, J) originate between the points of flagellar insertion and pass backwards along the four ridges of the wall. (The flagella are st i f f and gradually taper to the end. An area at their base appears very sensitive to external conditions, as in slide preparations, the flagellar sheath at 17. this point swells into a "balloon-like structure (Fig. 21 D) before the flagella are cast off. Detached flagella may exhibit flicking motions for several minutes and therefore show a degree of autonomy* Male and female gametangia are formed on separate plants with zoosporangia (Fig. 11 D). Similar to those in culture, they are usually isodiametric (Fig. 11 D) but may be longer than wide (Fig. 13 A and F). Female gametangia are of the same color as zoosporangia, a dark green, while male gametangia are yellow-orange. Unlike the vacuole of the zoo-sporangium, that of the male and female gametangium frequently contains several discrete semitransparent globular masses 10 - 20 ^u in diameter. Early-formed male gametes are spindle-shaped (Figs. 12 L and 13 K), and average 8 ^u in length and 2.5 ^u in width. Their spindle shape is readily detectable in gametangia with newly formed gametes. (Fig. 13 F and G). On aging, the male gametes become ovate or spherical (Fig. 11 D) and in this condition do not fuse with female gametes. The chloroplast is cupshaped and lacks an eyespot. Early formed female gametes are ovate (Fig. 13 A), but become quite asymmetrical within a few hours. The body develops two or three ridges and after becoming slightly curved and twisted (Fig. 12 K and 13 B, C) averages k in width and lk- /u in length. The chlor-oplast is cupshaped and contains a prominent posteriorly located eyespot. Both male and female gametes have two flagella, averaging 20 /\x in length, which become abruptly thinner at the tip. Occasionally both were found with four flagella of equal (Fig. 13 I) or unequal^size (Fig. 13 H). Both have a posteriorly located pyrenoid which in the male is only vis-ible on staining (Fig. 13 J)« Male gametes move rapidly through the water in more or less straight lines while the females move along a shallow spiral path. The anterior region of female gametes i s very 18. metabolic in that i t may stretch, shorten, twist, or rotate very much like Euglena. This feature is readily observable in gametes left behind in a newly discharged gametangium. Neither male nor female gametes showed any phototactic response over intervals of an hour when subjected to light intensities ranging from one to a thousand foot-candles. How-ever, gametes produced in culture, invariably settled in a very narrow band (0.1 mm) at the air/water/glass interface whether the cultures were illuminated from above or below. With side illumination the gametes gen-erally settled at the interface with greatest density nearest the light source. Gamete fusion is very rapid under ideal conditions, taking one to two minutes from the time of contact. Pairs fasten to each other by their anterior ends (Fig. lk A upper left) and rotate to a lateral position during fusion (Fig. lk A lower row). The quadriflagellate zygotes re-main active for some time (at least an hour) but show no phototactic response. Occasionally two females were observed copulating with one male (Fig. Ik A middle right). Male gametes are much more sensitive than females to external conditions. On warming the slide/; the males quickly round up and attach to the slide, while the females may exhibit normal motion for fifteen to twenty minutes longer. Nine successful matings were made between male and female gametes involving plants from six widely separate localities (Figs. 3 and 23). Zygote development was followed only for the cross between female 8L (Long Beach) and male 18-3 (Tsawwassen). ^ Culture Zoospores from field plants remain active at 10°C up to four days and, after attaching to the substrate, lose their flagella, germinate 19-to form either filaments (Figs. 11 A - D and 12), dwarf plants (Fig. 9 F), or both, depending on the culture conditions. Filament growth, though ini t i a l l y good in filtered seawater alone, declined through serial sub-culturing. Addition of nitrates and phosphates failed to improve growth. Of other media tested, growth was much better in Erdschreiber's solution but was inconsistent from culture to culture. Best and most consistent growth was obtained in Iwasaki.'s ( I 9 6 I ) SW1 and SW11 media (Table 2). The degree of.zoospore germination varied and appears to depend on the concentration of zoospores used, the quality of the seawater, and the quality of the spore itself. In i n i t i a l trials, 180 single zoospores, obtained from 18 plants (Tsawwassen, 1961) failed to germinate in Erdschreiber's solution. Similarly, zoospores from the same plants in sparse concentration or cultured singly failed to germinate in drop cul-tures down to 3 mm in diameter. On the other hand, germination was good when high concentrations of zoospores were used regardless of drop size. Curiously, single germlings of only a few cells (k - 9) originating from drop cultures with high zoospore concentration, grew very well in sea-water alone, indicating that the zoospores have a criti c a l germination period. At a later date, however, i t was found that single zoospores derived from a female filament F l l obtained from Tsawwassen (19^3) germ-inated in seawater alone but growth was much retarded (Table 3)• Sub-sequently i t was found that normal growth in the female cultures (Table 3) could be restored by addition of a small amount of SW1 medium which con-tained a supplement of nitrate, phosphate, and iron. On the other hand single zoospores from cultural filaments of male clone M2A (Tsawwassen) failed to germinate in seawater alone. However, these did germinate in media supplemented with FeEDTA (Table k). Zoospores from other cultures and from fertile Codiolum cells behaved in the same way, indicating that this critical germination period is a common characteristic of Uro spora and Codiolum zoospores. Cultural filaments show l i t t l e correlation to their fi e l d coun-terparts and lack distinction between isolates. Under crowded conditions they vary from 2 - k cm in length and 100 - 200 ^u in width and, in most cases, taper toward the base and apex (Figs. 11 A and 12). In uncrowded cultures the filaments may exceed 30 cm in length in which case they usually form rope-like strands. However, these long filaments are seldom wider than 200 y u . The rhizoids which number from 5 - 1 5 are, unlike their field counterparts, always extramatrical (Figs. 11 A and 12 A) and frequently ram-ified at their tips (Figs. 12 I and J). Rhizoids may also be formed at sharp bends in the filament as in Urospora vancouveriana (Fig. 17 F) or at the tip when i t reaches the air/water interface. Basal cells are generally shorter than wide, cylindrical or slightly swollen; central cells are mostly isodiametric and often swollen, while apical cells are frequently-very elongate (Fig. 12 N). Cell proportions, however, vary from culture to culture of the same clone and in some cases the cells, throughout the filament length, may be. up to ten times as long as wide. The chloroplast in cells of young rapidly growing filaments is usually coarsely reticulate and becomes finely reticulate.in larger or older cells. Chloroplasts re-tracted from the end walls were never found in culture and therefore appear to be an environmental feature. Cultural zoospores show the same variability in shape as fie l d "types but are generally smaller, averaging 10 yu in length and h ^u in width. However, in very large sportangia, the zoospores may reach 18 j\x in length. Cultural sporangia as well as game-tangia have a single pore. Both male and female gametangia in cultural 21 filaments are yellow-orange. The size, morphology and behavior of the gametes are the same as those from the field. Clones from a l l localities produced dwarf plants (Fig. 9 F) as well as filaments. The former consist of cell clusters attached to the. substrate by their own cells and/or by rhizoids developing out of several basal cells. Dwarf plants can be recognized at a very early stage in development by the random planes of cell division. . The cells have a central vacuole and a finely-reticulate parietal chloroplast with sev-eral pyrenoids in small cells or many in large ones. The proportion of dwarf plants and filaments depends on the cultural conditions. Dwarf plants increase with higher, or lower salinities and are predominant at 10 °/oo and 50 °/oo whereas filaments predominate at salinities from 20 - 30 °/oo. Over the range of light intensities used (25 - 1+00 f.c.) dwarf plants decreased and filaments increased with an increase in light intensity. Dwarf plants have many rhizoids (Fig. 9 F) at low salinities (10 °/oo) and none (Fig. 9 H and I) at high salinities e.g. 50 °/oo. At these salinities they frequently produce large thick-walled cells up to 200 in diameter which have persisted in unchanged media for over a year. Cells of this type frequently have no vacuole and look very much like stipeless Codiolum cells. These are very easily freed from the plant and on transfer to fresh medium of normal salinity, produce typical quadriflagellate zoospores which give rise to either dwarf plants or filaments. Dwarf plants were occasionally encountered during the winter months in the field but never contained such large cells. Male clone M2A and female clone F l l grew slowly at 5°C, best at 10° and 15°C, and only F l l grew at 20°C. It was not determined i f other male clones failed to grow at 20°C. 22. Of ten male and. twenty-four female cultures grown at 10°C, one male (M2A) and two female (Fll) cultures were found producing gametes after four months. Five of the males and fifteen of the females (includ-ing the sexual clones) contained typical Codiolum cells (Fig. lk B and C) which were most concentrated in a thin ring, less than 1 mm wide at the • air/water/glass interface (Fig. 8 E) and densest nearest the light. Codiolum cells derived from male and female cultures are termed male and female Codiolum respectively. Subsequently 10 clones of male (M2A) and 15 of female (Fll) were serially subcultured twice. None of the male clones became sexual or produced Codiolum cells; whereas most of the females did (Table 3). Over further subcultures sexuality was also lost in the female. Of the five male clones which produced Codiolum, three originated from Tsawwassen, one from Oak Bay and one from Long Beach. The origin of Codiolum in male cultures could not be determined. When cultured in liquid or agar media at various temperatures, zoospores from field and cultural plants gave rise to Urospora but never Codiolum. . and male gametes always died. Furthermore, though male M2A was made sexual later on a number of occasions, the cultures never produced Codiolum. Female gametes and zygotes developed into Codiolum gregariurn-like plants of about one-third to one-fifth the size of C. gregarium in nature.. These have a parietal, seldom-reticulate, chloroplast containing several pyrenoids in small cells and many in large cells. The stipe of cultural plants is frequently much reduced and the layers within are quite uniform (Fig. 37 A). Only one zygote was found fertile after 2k days of culture and this produced normal quadriflagellate zoospores, which germinated in the cell to form Urospora plants. Fertile haploid Codiolum cells from female clone F l l and F 3 in a l l instances contained abnormal products: irregular-sized masses without flagella; zoospores with 2 , k, or up to 6 flagellaj and in one cell two small Codiolum cells and a rounded up structure with an eyespot (Fig. 29 A). The latter c e l l was observed in cold shocked material. (Details p. 3 1 ) . In liquid and agar cultures the zoospores germinated within the Codiolum cells, the cell wall gelatinized and filaments or dwarf plants developed from the mass of germinating spores. Cytology The cells of filaments and dwarf plants of U. wormskioldii are multinucleate and nuclear division is synchronous. In the cells of rapid-ly growing filaments a concentration of nuclei is generally seen at either end wall and, occasionally, in longer cells in the mid-region as well (as in Fig, 19 G). Cell division is accomplished by a furrow (Figs, 33 D, F and as in Fig. 19 I).developing'in the middle of the cell protoplast, gradually extending inward to divide the cell into two equal halves. Cell division is independent of nuclear division. Additional wall material is formed about the cell which can be distinguished from the outer common daughter layers. The common layers appear to undergo some reorganization in the area of cell division, for in other non-dividing cells the layers appear continuous about each c e l l with only the outer filament sheath being common (Figs, 27 G and 33 C) 0 This reorganization, however>'may not be complete since the cellulose layers around adjacent cells appear connected or incomplete when viewed after autoclavlng in 23 M KOH (Fig. 3k A and C). When filaments from nature are allowed to stand in culture for several days, individual cells can be removed readily from the filament sheath with their inner walls intact. This can be done simply by squeezing the cells out of the sheath with a dissecting needle. This would indicate that the connection between cells is not strong. One of these cells is shown in Fig. 11 F. An additional layer with an inner ragged surface was frequently observed within empty sporangia and appears to be formed during zoospore formation or soon after (Fig. 27 G). This layer may be instrumental in building up turgor pressure in the cell by reducing its inner volume. Occasionally the cell division furrow starts obliquely, resulting in the two cells being coiled about each other (Fig. 8 D). In dwarf plants, the nuclei are frequently associated in random clusters. The large Cddiolum-like cells of dwarf plants were very dif-ficult to stain and were found to be multinucleate. Prior to spore formation in filaments, the nuclei divide rapidly, often remaining in clusters (Fig. 21 A). During this process the pyrenoids become very in-distinct (Fig. 8 H) and the chloroplast granular0 Initials of zoospores are f i r s t seen as spheres compressed at points of contact with other cells (Fig. 8 I) and later these elongate and develop flagella. Fertile cells which earlier had a coarsely-reticulate chloroplast (Fig. 11 G) reflect this reticulation in the arrangement of zoospores (Fig. 11 H). Nuclear clusters are characteristic of large cells and are reflected in the star-like arrangements of zoospores mentioned earlier. Zoospores (Fig. 27 I) and gametes (Figs. 12 K - M and 13 D, J) have a single nucleus located anteriorly, but occasionally both were found with two nuclei.1 However, nuclear number is not correllated with flagellar number for zoospores and gametes with one or two nuclei had either the normal or double number of flagella. The nuclei of filaments, dwarf plants, zoospores and gametes have a prominent often vacuolated nucleolus with about 2 - k small Feulgen-positive chromatic bodies (previously undescribed) in close contact with i t (Figs. 25 C, E and as in Fig. 20 A). The nuclei increase in size from about K ^u in small germlings to 10 /u in larger filaments. With increase in nuclear size the number of chromatic bodies increases up to ten and these become more distant from the nucleolus (as in Figs. 20 G and I). The chromatic bodies probably represent heterochromatic portions of inter-phase chromosomes. Nuclei of germlings and zoospores stained with Feulgen, but larger nuclei of mature filaments did not. A good nuclear stain was obtained when the Feulgen method was followed by staining in iron-pro piocarmine. The nuclear membrane and nucleolus disappear prior to meta-phase. The chromosomes become oriented at the metaphase plate with their long axis parallel to the spindle apparatus. The chromosomes are seldom more than 2 /u long and 1 /u wide and appear most frequently as small round dots. (Figs. 26 L - N, Q and R). The chromosome number of U. wormskioldii based on counts from metaphases in young germlings is twelve. Cultural Codiolum cells (zygotic and parthenogenetic) contain a single nucleus which, except in the very smallest young cells, fa i l s to stain either in Feulgen or Feulgen-iron-propiocarmine0 Since sectioned material behaves the same way, this lack of staining appears to be a property of the nucleus itse l f . The nucleus, however, stains well in Newcomer-iron-propiocarmine revealing the same type of vacuolated nucleolus and chromatic bodies as in Urospora. Nuclear division was not observed in cultural Codiolum. In the living state the pyrenoid of Urospora (Fig. 10 E) and Codiolum appears to consist of a central spherical hyaline mass surrounded by several starch plates. Whep stained with acetocarmine, Newcomer-iron-26. propiocarmine or Feulgen-iron-propiocarmine the central mass appears homo-genous (as in JJ. vormskioldii. Fig. 20 A), but occasionally contains sev-eral small dark staining bodies in the centre. When the third stain is used on sectioned material the central mass appears to be compound (Fig. 21 E and F). Hovever this may be an artifact of sectioning. Wall cytochemistry (see Figs. 31, 33, 3*0 The individual cells of Urospora vormskioldii are surrounded by . a thick vai l composed of several layers. A l l the cells" are bounded on the outside by a thin filament sheath. The layers and sheath are easily distinguished vhen treated with IKE - HgSO^  (Fig. 33 E) due to the result-ant svelling in the acid. The inner layers give a positive test for c e l l -ulose with IKI - R^ SO^  or ZnClI 2 "before and after heating in concentrated HC1, after autoclaving in 23 M KOH and after treatment with Schweitzer1s reagent, or slug cytase. They are also doubly refractive under polarized light before (Figs. 33 A and B) and after (Fig. 33 G) boiling in concen-trated HC1. The inner layers give a positive test with ruthenium red before but not after boiling in concentrated HC1 indicating the presence of acid soluble pectic compounds. The wall material left after acid treat-ment is readily soluble in Schweitzer's reagent or slug cytase. The sheath gives a negative test for cellulose, pectin, fat, or chitin; is insol uble in slug cytase, Schweitzer's reagent, and is singly refractive (Fig. 34 F ) . It is soluble in HG1 - ZnCl 2 (boiling) or 23 M KOH and in ZnClIg, Schweitzer's reagent and IKI - HgSO^  after brief-cheating in concentrated HC1. In the Codiolum cell, the protoplast is surrounded by layers, of material which only give a test for pectic compounds and which are soluble in hot concentrated HC1. The whole cell is bounded by a thin ' 27 membrane different to that of Urospora but also of unknown composition. Other details on the walls are given under C. gregarium (P..43 ) which gives the same tests and reactions as cultural Codiolum. Induction of sexuality- Temperature. After the loss of sexuality in the male clones and its reduc-tion in female clones when subcultured at 10°C, cultures of male M2A, female F l l and URX1 were cultured at 5°, 10°, 15°, and 20°C. Gamete pro-duction occurred in both sexes at 15°C and in the female at 20°C (Table 9) being highest in the female at 20°C, with about 50$ of the filaments becoming sexual. However, on subsequent cultures of both sexes at 15° the percentage of sexual filaments dropped to less than 1$. During this period 500 test tube Urospora clones were established from 50 fertile Codiolum. cells obtained from several areas at Tsawwassen and were cul-tured at 15°C, but in no case was sexuality evidenced over a two-month observation period. The medium in the cultures was then replaced.and the cultures were divided into two lots, one being placed at 15°C, the other at . l0°C. Again no sexuality was observed. These results clearly indica-ted that the sexual response can be restored or increased by culturing at warmer temperatures but not induced. ' -s Thermoperiod and desiccation Cultures of male M2A, female F l l and URX1 were grown in shallow Petri dishes at 10°C and after 10 days, when the filaments were 2.- 4 cm long and at their height of growth, one duplicate set received drying at 10°C and one at 20 - 23°C for 4 hrs on five consecutive days during the middle of the 14 hour daily photoperiod. After each treatment fresh medium was added and the cultures were examined for evidence of sexuality. 28. No sexuality was observed over the five day period in any culture. Death of cells and whole filaments occurred in a l l cultures,. and was higher in those receiving a thermoperiod and highest in the male cultures. After this period about 50$ of the male filaments and 10$ of the female f i l a -ments were dead. Under these conditions dwarf plants, small filamentous germlings and the rhizoids and basal cells of large filaments survived best.: The addition of fresh media each day resulted in fertile cells discharging zoospores which became active immediately on release. Three days following return to normal conditions (10°C) the two male and two female cultures which received a daily thermoperiod produced gametes in abundance. Gametangia occurred only in large filaments and comprised about 30$ of surviving cells. Under these conditions periods of desicca-tion and heat, but not desiccation alone,, increased sexuality in the fe-male and induced i t in the male. The effect of thermoperiod alone was then investigated. Thermoperiod , Male, (M2A), female (Fll) and URX1 clones were grown in test tubes at 10°C for 10 days and then duplicates'were transferred to thermo-period boxes (Fig. 30) where they received daily warming for four hours during the middle of a l4**hr photoperiod on 15 consecutive days. The. heat in each box was provided by wire wound resistors with various out-puts and was controlled by an air thermostat with 0.1°C sensitivity. Temperatures were measured in, and at the.bottom of the test tubes. After the k'hr thermoperiod cold air (5°C), forced into the boxes by means of a • pump,.was used to bring the cultures down to the basal temperature. The lag in reaching the maximum temperatures and the variation of temperature between tubes (± 2°C) makes precise interpretations difficult. Nevertheless the results have some value. The cultures at died in three days; those at 32°C,in lk days, while the rest survived. The male filaments showed considerable bubbling of the sheath at 21°, 2k°, and 29.5°C. On the fifteenth day a few gametes were observed in one of the two control female cultures and in one female at 29.5°C. A few Codiolum cells were o o observed in the female cultures at 10 to 32 C on the tenth day, which at the latter temperature were dead by the l^ +th day. On the l6th day the solutions were replenished and the cultures were returned to 10°C but- no sexuality was evident over a three-week observation period. Under these conditions, thermoperiods were not effective in increasing or inducing sexuality; nor was a nutrient change following fifteen cycles of thermo-periods. Since a thermoperiod failed to bring about sexuality here, but did in cultures subjected to daily thermoperiods and nutrient change, i t is suggested that nutritional factors may be involved as well. Prolonged thermoperiod and nutrient repletion' Because of the high rate of death in cultures subjected to thermo-period and exposures combined, and poorer growth of filaments in test tubes, i t was decided to see i f a long thermoperiod placed during maximum plant growth would induce sexuality. Male (M2A), female (Fll) and URX1 clones were cultured in deep Petri dishes at 10°C for ten days and then were placed at 20°C for six days. After this time the filaments of a l l clones showed yellowing and vacuolation. The nutrients of one lot were o replenished and the cultures returned to 10 C. After two to three days gametes were being produced in abundance in M2A and F l l , but not in URX1, in which the nutrients were changed. Gametangia were again confined to larger plants. Subsequently this same method was successful in inducing sexuality in two clones from Point No Point and one from Cape Cod, . 3 0 . Massachusettes but failed on one clone of" Urospora speciosa. The clone from Cape Cod had been maintained in culture asexually for two years pre-vious indicating that temperature is a prime factor involved in the sex-ual response. It is quite possible.that the success of this method was' in part due to the larger size of[the containers used, since filament production was much more abundant in these. > Induction "of f e r t i l i t y in cultural Codiolum The effect of temperature, was studied on the growth of Codiolum resulting from female gametes, of clone F"3; pn Codiolum plants from male clone M2A, female clone FU(both two months old), URX1 (four months old) and- one-day-old zygotes (8Lo. Xl8 -3 O* ). The F3 £ gametes were cultured . on agar, the M2A, F l l , URX1 Codiolum in shallow Petri plates and the zy-gotes on slides in Erlenmeyer flasks. The temperatures used were 5°> 10°, 1$^ throughout and also 20°C for. F3o. gametes. Only one zygotic Codiolum cell (8Lo_Xl8-3 O* ) became fertile over a twor-month culture period, this being on.the 2 4 t h day at 10°C. This one cell produced normal quadriflagellate spores which germinated within the cell. About 3$ of the F3£ Codiolum cells became fertile at 10° and 15°C o o and none at 5 and 20 C. A l l of these contained abnormal products, masses of varied size without or with many flagella and undivided zoospores in addition to normal spores. None of the No. 8Lo_ , M2Ad", Fllo_ or URX1 Codiolum cells became fertile. In a l l cases the growth was retarded at 5° and 20°C and was best at 10° and 15°C. A heavy bacterial growth occurred in agar cultures at 1 5 ° and 20°C and in such cultures Codiolum cells were very small lacking a stipe. Zygotes of (8L<j> X18-30* ) and Codiolum from Fllo_ were subjected to a daily 8-hr thermoperiod of 18 ± 2°C (coincident with an 8-hr light 31. period) for two weeks and then were returned to 10°C. None "became fertile. * - • o Plants of the same type were given a k-hr drying period at 22 ± 2 C for o one week and again none "became fertile on return to 10 C. Codiolum cells from No. 8L and URX1 were subjected to cold shocks .of one or six day duration at -2°C under dark conditions. On return to 10°C only the 8L Codiolum cells became fertile (Table 7).. Those cold-shocked for one day started to become fertile very much sooner, after three days, suggesting that longer periods of cold hasten f e r t i l i t y . In a l l cases these cells produced abnormal structures; (Fig. 29). Unfortun-. ately i t was not possible to try this method on zygotic material. 32. 2. Type II Urospora vancouveriana (Tilden) Setchell and Gardner (Life cycle, Fig. 15) Urospora vancouveriana * was found at Point No Point (Fig.'*)- F) during July and August, I 9 6 3 , where i t formed a dense covering on rocks in sandy areas in the low tide region (0 - 2 f t ) . The plants were largest at the lowest level, here frequently exceeding 30 cm in length and 2 mm in width, and decreased in size upwards, where they merged with a band of sexual U. wormskioldii. The level at which one ended and the other began could not be established without resorting to extensive culturing of samples from the suspected transition zone. However, i t appeared that this zone was between;, the two and three foot level. The following dis-cussion of the U. vancouveriana is based, on culture studies of six clones established from large filaments taken, from six areas along a hundred foot horizontal stretch of the beach between the zero and two foot tide level. The filaments of Urospora vancouveriana have a holdfast composed of 11 - 30 intramatrical rhizoids (Fig. 17 B), and taper gradually from 1 just above the holdfast to the end of the filament (Fig. 17 A). Indiv-idual cells at the base are isodiametric and slightly swollen, becoming more swollen sub-centrally and finally spherical in more distal regions (Fig. 17 A). In the largest filaments the cells may reach 3.mm in diameter. The chlproplast is frequently coarsely reticulate in basal cells and finely reticulate in mature cells (Fig. 17 C). The zoospores, which average 18 in length, 1 p in width, are arranged mostly parallel to the cell surface (Fig. 17 D) and are liberated through a central pore. .. * This form was brought to my attention by Dr. M. Dube of Western . Washington State College 33. Culture The six clones were cultured at different temperatures (5°, 10°, 15°, and 20°C) and light intensities (25 - ii-00 f.c.) As in i J . wormskioldii zoospores developed into filaments' (Figs. 17, F - I) and dwarf plants (Fig. 17 J). Cultural filaments are indistinguishable from those of U. wormskioldii. The chloroplasts in the cells in rapidly growing plants are, as in U. wormskioldii, coarsely reticulate (Fig. 17 G) and become finely reticulate with age. The dwarf plants contained one or two types of sporangia: one type producing quadriflagellate zoospores (Fig. 18 B) and the other type, biflagellate zoospores (Fig3 0 17 J and 18 A, C), The latter type were readily distinguished by their yellow-orange color. Filaments produced quadriflagellate zoospores at a l l temperatures. Cul-tural zoospores are generally about two-thirds the size of natural ones (Fig. 17 E). The number of dwarf plants increased with a decrease in light intensity and increase in.temperature, while the reverse was true of filaments. Yellow orange sporangia increased with an increase in temperature (Fig, 16) and decreased with a decrease in light intensity. The biflagellate zoospores are released in vesicles (Figs, 18 A, D, E and 19 F) and this release can be induced by cooling the culture on ice and subjecting i t to bright light. The zoospores, on release from the vesicle, are at f i r s t spherical to ovate (Figs, 18 C and 19 F), but over several hours become acuminate (Fig3, 18 F and 19 A) , At this time they average 8,6 /u in length and 3 /u in width. The flagella average 15.8 #u in length and become abruptly thinner at the tips (Fig. 18 G). They show no phototactic response, but in culture settle in greatest quantities at the air/water/glass interface nearest the light source. The contents of six vesicles were cultured separately on agar • 34. plates and in a l l cases the Mflagellate zoospores gave rise to typical Codiolum plants (Figs. 18 H, I and 19 C - E). The stipe of such cells contained very regular pectic layers which increased.in number with the age of the plant. On the other hand, quadriflagellate zoospores from dwarf plants or filaments never gave rise to Codiolum cells. WitMn thirty days of culture the Codiolum cells attained a length of 200 - 300 p and a width of 30 - 50 jx. About 3$ of these became fertile at 5°, 10°, and . o o 15 C, but none at 20 C. In a l l cases fertile cells produced normal quad-riflagellate zoospores which germinated within the cell. Out of this mass, filaments or dwarf plants emerged. A cold shock of one day at -5°C failed to increase f e r t i l i t y in vegetative Codiolum cells. At f i r s t i t was thought that the biflagellate zoospores were gametes and attempts were made to mate these with each other .and with gametes of U. wormskioldii. This was done on three separate occasions, but in a l l cases the biflagellate zoospores showed no copulation tendencies. Cytology and cytochemistry. Urospora vancouveriana nuclei stain similar to those of U. wormskioldii and show the same features. In small filaments and dwarf plants, the chromosomes at metaphase are very small and dot-like (Fig. 20 B). However, in early prophase the chromosome strands are readily visible (Fig. 20 C, upper left cell). In larger filaments the chromosomes at metaphase show some variation in size and shape (Fig. 20 E and H) where the largest ones are 2.5 p long and 1 wide. The chromosome number, based on twelve counts, appears to be nine (Fig. 20 D - F). The Codiolum cells were uninucleate except when undergoing f e r t i l i t y . Only two nuclear divisional stages were observed, both in f i r s t division anaphase. A spindle apparatus was clearly observed but chromosome counts could not 35. be made. The cell walls of U. vancouveriana gave the same color reactions and behaved in the same way to chemical treatments as those of U. worm-skioldii . 36. 3. Type III. Urospora speciosa (Carm.) Leblond ex Hamel In May of 1964, several small vegetative filament fragments (Fig. 21 G), less than 30 p. wide and 2 mm long, were found in a collec-tion of Urospora wormskioldii plants obtained from the low tide region (2 ft) at Deadman's Bay (Fig. 4 G - 1). Though these fragments appeared very similar to Ulothrix flacca (Dillwyn) Thuret, which was also present, one contained fertile cells which produced zoospores lacking eyespots. The fragments became fertile in culture and ten clones were established from the resultant germlings. Of these, eight gave rise to a small form of Urospora, one to U. wormskioldii, and one to UI. flacca. The cultural filaments of this Urospora species range from 1 - 2 cm in length and .30 -50 jx in width. The holdfast is composed of from one to four rhizoids. The cells are mostly isodiametric and slightly swollen. The chloroplast is cylindrical, irregular at the edges, and contains several pyrenoids. Fertile cells produce quadriflagellate acuminate zoospores ranging froirt 8 - 12 jx in length and 3 - 4 jx in width. The cells contain a single nucleus which is readily distinguished in the living state. When stained, the nucleus shows the same features. (Fig. 21 H) as those of the f i r s t two Urospora species. Other features of its l i f e history remain to be studied. One attempt was made to induce sexuality in this form but failed (Details P. 29). : -"9 37-Summary of the genus Urospora Three species of Urospora were found in this study. U. worm-sldLoldii (n = 12) is the most common. This species is dioecious, aniso-gamous, has three somatic stages: filament, dwarf and Codiolum. Codiolum plants are produced by zygotes,female gametes and probably-male gametes, and therefore may be diploid or haploid. The vegetative features of this species are extremely variable in the f i e l d and en-compass those of other Urospora species recorded from this coast; U. penicilliformis, U. tetraciliata, U. dolifera Setchell and Gardner, U. sphaerulifera Setchell and Gardner, and U. grandis Kylin. Urospora vancouveriana (n s 9) was found in only one locality. The filaments in nature are of a distinctive large form but in culture are indistinguishable from U. wormskioldii. This species is asexual, has three somatic stages: filament, dwarf and Codiolum. The latter stage is formed from biflagellate zoospores which are produced at higher temper-atures. Urospora speciosa (n s ?), found, once at one locality is a very small slender form and,unlike any other form,is uninucleate. This is the f i r s t record of i t s occurrence in North America. Other features of its l i f e history have not been studied. k. Codiolum. gregarium A. Braun and C. pusillum Lyngbye Kjellman Ecology Codiolum was generally found in a narrow band ( l - 1§- feet vertical depth) coinciding approximately with the higher of high tides from spring to early f a l l (April - September): below this level in sheltered areas (Friday Harbor, Tsawwassen) or above i t in more wave' beaten areas (Point No Point, Ogden Point Breakwater). (Fig. 5)• It occurs densest in protected areas, on long stretches of vertical faces (Ogden Point Breakwater) and is very sparsely scattered on beaches having rocky outcrops (Point No Point, Deadman's Bay). Codiolum is frequently asso-ciated with Prasiola meridionalis Setchell and Gardner (Tsawwassen, Ogden Point Breakwater, Deadman's Bay, Stanley Park,- Anacortes) where i t may be found intermingled with Prasiola or above or below i t . Codiolum is found in most abundance during f a l l and winter on flat faces of logs or rocks with a south, southwest or southeasterly exposure, above areas of maximum Urospora density. Juvenile forms appear in the spring at the upper limit of U. wormskioldii, soon after Urospora becomes.sexual. The Codiolum cells continue to grow and remain vegetative during the summer months. Gener-ally vegetative plants are most abundant and conspicuous during the f a l l and winter (Fig. 6). Fertility starts in the f a l l and reaches a peak during December and January.. By following the development of small iso-lated patches of Codiolum at Tsawwassen i t was noted that some plants may remain vegetative throughout the winter to become fertile in the next. Codiolum,as well as Urospora, may be killed off during hot summers partic-ularly in. areas with southern exposure. During the summer of I963 at Tsawwassen, Urospora disappeared completely from the southeast side of the jetty, whereas patches of Codiolum ( 0 . 1 - 0 .5 ur) remained, though with considerable reduction in numbers. Both survive much better on sunken logs or pilings. At Friday Harbor both plants were present on a sunken ; log (Fig. 28 B) throughout the summer, but absent from the rocks nearby. Codiolum is an extremely hardy plant; more so than Prasiola, Porphyra and Bangia which are killed in the summer in protected areas. Prolonged rainy spells or freezing conditions seem to have no effect.on Codiolum. In fact, in the winter of I 9 6 2 - 63 Codiolum was covered with ice for several weeks yet remained perfectly viable on thawing. Its ex-, treme hardiness is further demonstrated by the ability of very young plants to survive three months of dry storage at room temperature (20 - 23°C). Under this condition larger plants die within two weeks. Morphology and variability In the ten localities in which i t was found Codiolum shows a great variability (Fig. 6). The morphology, though constant for a part-icular locality in one year, may change in the next. It also differs according to the substrate, amount of crowding and exposure. Two ex-treme types are represented. The plants from Oak Bay, I963 (Fig. 2k D) are long and slender, tapering gradually from the clava (head of the cell) to the base. These were extremely crowded and formed a dense almost pure stand on rocks (Fig. k E). Mature plants range from 900 - 1500 p in length and 55 - 75 ^  in width with the clava constituting about one half of the total length of the ce l l . This type conforms very closely to Codiolum pusillum (Lyngbye) Kjellman. The plants from Tsawwassen (I963) (Fig. 2k E) are considerably shorter, clavate or ovoid above, with the stalk sharply delimited from the clava. These plants were not nearly as crowded as those at Oak Bay. Mature plants range from 50O - 700 p long, ho 80 - 100 p. wide at the clava, with a stipe 25 - 30 p. wide at the ciava tapering gradually to the "base, with the clava constituting from one quarter to one half the length of the ce l l . This type conforms closely to Codiolum gregarium A. Braun. The stipe of "both types is formed of periodic pectic layers continuous with similar layers in the clava (Fig. 2k A). In the winter of I96I plants at Deadman's Bay were scarce and resembled Codiolum gregarium (Fig. 6) . In I963 they were very abundant and resembled C. pusillum. The plants from Victoria Breakwater in I962 were very abundant, many resembling-C. pusillum; but in I963 they were reduced and conformed to C. gregarium. Plants on logs at Friday Harbor, •, Tsawwassen, and Point No Point were like C. gregarium but very reduced in- the latter two localities. The logs here were drift logs cast up on the shore during winter high tides. Plants on these logs persisted into mid-summer, but later perished. Plants at their upper tidal limit are generally smaller than those at lower levels, and as with Urospora are frequently infected by fungi, especially during winter months. Curiously, Codiolum was very scarce at Point No Point in the winter of 1963 on the rocks above the area where Urospora vancouveriana was so abundant in the summer. Less than 100 cells were found from scrapings taken from many rocks. Urospora appeared to be completely absent from this area during \ the winter, either in filament or dwarf form, yet very dense stands of extremely large Urospora plants were found again in the summer of 1964. Fertile Codiolum plants from seven localities a l l produced the typical Urospora quadriflagellate zoospore (Fig. 25 B). These are formed throughout the cell when no vacuole is present. Frequently the zoospores at the surface are arranged parallel to i t . Though many cells were examined, natural zoospore release vas never observed and only rarely were empty cells encountered. Such, cells always had a longitudinal split of variable length in the clava wall. A very small percentage of fertile cells could, however, be ruptured under laboratory conditions by drying at room temperature (20 - 23°C) for about eight hours and rewetting. In such cells the clava split to release the spores. Otherwise, under normal culture conditions the zoospores always germinated internally (Fig. 2k C). This occurs also in the field where, toward the end of the fertile period, one can distinguish very easily on logs :the many small oval patches of young Urospora germlings that have resulted from internal development of Codiolum spore masses. The Urospora filaments found at high tide in the spring are formed from such patches. The vegetative cell has a. parietal chloroplast containing sev-eral to many pyrenoids (depending on the cell size) and a central nucleus. One or two vacuoles were occasionally observed on either side of the nucleus but this is not an obligate feature. In crowded, rapidly-growing cells the chloroplast may be coarsely-reticulate, but in mature cells i t is dense and lobed inwardly. Culture Zoospores of fertile Codiolum plants were cultured from Deadman's Bay, Friday Harbor, Tsawwassen, Point No Point, Oak Bay and Ogden Point Breakwater. These always gave rise to dwarf plants or filaments which are indistinguishable from those of Urospora wormskioldii (Figs. 25 E - G and 27). The Codiolum plants obtained from Point No Point came from an area directly above where U i vancouveriana was found in the summer and although zoospores from these were cultured at 15°C, no biflagellate' zoospore-producing-sporangia were encountered as otherwise would be expected i f the Codiolum cells originated from U. vancouveriana. Sex-uality did not occur in any Codiolum-derived Urospora clones obtained from any of the localities'. However, of Ilk clones established from one fertile Codiolum cell obtained from Deadman's Bay, 8 produced Codiolum cells. Though these 8 clones were subsequently subcultured at 10° and 15°C none became sexual or produced Codiolum. Likewise, of 500 clones established from 50 Codiolum cells from Tsawwassen none became Bexual at 15°C (Details p. 27). Cytology and wall cytochemistry (Figs. 26, 31 B and 34) The nucleus of the vegetative cell of Codiolum increases with cell size, attaining a diameter of 30 ^u in larger plants. The living nucleus appears hyaline and contains a prominent vacuolated nucleolus (Fig. 26 P). Occasionally several smaller bodies (5 - 10) were observed in the nucleoplasm exhibiting a jiggling motion while they moved slowly and randomly in i t (Fig. 26 0). In material subjected to a combined four hour daily thermoperiod (23 - 2|j0C) and dessication, nuclei were fre-quently observed with five to ten nucleoli-like structures of variable size (Fig. 26 I and J). The nucleus of natural Codiolum, like that of cultural Codiolum, fails to stain with Feulgen or Feulgen-iron-propiocar-mine. When stained with Newcomer-iron-propiocarmine the chromatin is readily visible, appearing as a reticulum in the resting nucleus (Fig. 26 D). Though many thousands of Codiolum cells were examined, f i r s t div-isions were encountered in only four cells. These appeared to be mitotic (Fig. 26 A - C and E). Second and third divisions appeared normal (Fig. 26 G). However, occasional cells were found which had one or two large nuclei and two or more small degenerate nuclei indicative that nuclear division in some cells is abnormal. Nuclear divisions are synchronous and the nuclei decrease in size with further divisions. When y nuclear divisions are in progress the pyrenoids do not stain. This phen-omenon facilitates the search for divisions by enabling the use of lower magnification. A slight Feulgen stain-is obtained after about the fourth division (l6-nucleate stage) and this stain increases gradually with further divisions to zoospore formation. One cell was observed at ninth division metaphase in which 22^ of the 256 possible metaphases were lo-cated (Fig. 26 H), indicating that at least 512 zoospores may be formed by a single ce l l . The chromosome number of Codiolum obtained from Oak Bay, Deadman's Bay, Tsawwassen, and Ogden Point Breakwater appears to be twelve, based on the highest number found at metaphase. Similar to cultural material, natural Codiolum cells have an outer sheath of unknown composition and inner layers of pectic substances. The behavioral details of the wall components to stains and reagents are given in Fig. 31. The pectic layers give a positive test (red), with ruthenium red, and are insoluble in Schweitzer's reagent, slug cytase, or on autoclaving in 23 M KOH. They are doubly refractive (Fig. 3^ D) (a property generally not attributed to pectic compounds) and are readily soluble in boiling concentrated HCl. With the latter treatment the sheath becomesamuch distended (Fig. 3k E) or bursts. The sheath is singly refractive; gives a negative test for chitin, pectin, fat, or cellulose^ is insoluble in Schweitzer's reagent or slug cytase; is soluble in boiling HCl - ZnClg and on autoclaving in 23 M KOH but, unlike that of Urospora, after brief boiling in concentrated HCl, is insoluble in ZnClI,,, Schweitzer's reagent or IKI - HgSO^ . Both before and after boiling, in concentrated HCl a banding pattern was observed in the protoplast (Figs. 35 D and F). Induction and inhibition of f e r t i l i t y Infertile Codiolum cells from Deadman's Bay and Friday Harbor were cultured at various temperatures. A l l mature plants (larger ones) became fertile at 5°C and 10°C, a small percentage at 15°C and none at 20°C (Table 5). Codiolum cells from Point No Point, Ogden Point Break-water, Oak Bay and Tsawwassen behaved in a similar way. When plants were subjected to a daily thermoperiod of 8 hrs o 'at 18 i 2 C, f e r t i l i t y was inhibited (Table 6) and when returned to 10 C i t was induced. Subsequently exposure to a k hr thermoperiod every two days was found to be just as inhibitory. Intervals of three and four days resulted in a small percentage of plants becoming fertile and this increased with the length of interval. Plants subjected to a combined daily dessication period and thermoperiod survived much longer than those receiving a thermoperiod only. In such cultures epiphytism was markedly reduced, whereas in those receiving a thermoperiod only, the plants be-came overgrown with diatoms, fungi and other algae. Using the combined treatment, vegetative cells were kept in good condition on their natural substrates, wood or rocks, for four months. The finding of Codiolum in large quantities on a log at Friday Harbor made i t possible to study the effect of transplantation on. f e r t i l -ity. Small blocks containing Codiolum were removed from the log (Fig. 28) and placed on the back and front of ladder rungs positioned at 3 f t inter-vals from the +12 to the -9 f t ; level in the intertidal. Samples were removed on the 8th, l 6 t h , and 30th day and the percentage of f e r t i l i t y was determined from several hundreds of large mature looking cells. Samples were also removed from the log and from a rock at Deadman's Bay to serve as controls. *5. On the 8th day a small percentage of plants were fertile on the ladder rungs at levels from 0 - 9 f t while none were fertile on the log or rock (Fig. 28 E - G). On the l6 th day a greater percentage of plants was fertile on the rungs and f e r t i l i t y extended to the lowest level. None were fertile on the rock hut a small percentage was on the log. On the 30th day Codiolum was absent on several lower rungs due to their having become de-tached on f e r t i l i t y ; but where present, the percentage of fertile cells was much higher than before. . On the 30th day the percentage of f e r t i l i t y was also much higher on the log and rock but in general i t was lower than that on ladder rungs below the 6 f t level. No fertile plants were observed on the ladder rung at the 12 f t level over the thirty day period. Most of the larger plants on the south side at this level died over this period hut those on the north side survived. The plants at this level were about a foot above the daily high tides so would have remained dry over the en-tire period except when.high wave action coincided with high tides. Throughout the observation period the percentage of f e r t i l i t y was notice-ably reduced on rungs at the 6 and 9 f t level but was comparable to that found on the rock and log. Over the 30-day period the•south side of the rungs at the 6, 3 and 0 f t levels became increasingly covered with f i l a -mentous diatoms, Urospora and other algae, while the north sides remained free of epiphytes. By the 30th day the filaments of diatoms on the 6 f t level rungs attained a length up to 15 cm. It is suggestive that the lower percentage of f e r t i l i t y on these rungs was due to the epiphyte cov-erage. While the water temperatures remained fair l y constant over this period the aerial temperatures dropped considerably. The increase in the percentage of f e r t i l i t y of Codiolum on the rocks and log can be readily attributed to this atmospheric temperature drop. 46. 5. Codiolum petrocelidis Kuckuck and Spongomorpha coalita (Ruprecht) Collins Plants similar to Codiolum petrocelidis Kuckuck were found in abundance within the tissues of Petrocelis franciscana Setchell and ; Gardner in the mid-tide (2 - 7 ft) during the summer and f a l l of I96I and 1963 at Deadman»s Bay' and Porlier Pass where Spongomorpha coalita (Ruprecht) Collins occurred in dense patches below i t in the low tide (0 - 2 f t ) . Only vegetative cells were encountered during this period. At Deadman's Bay, C. petrocelidis was oriented stipe up (stipe directed towards the surface of the host) (Fig. 37 D) and at Porlier Pass, either up or down (Figs. 38 A, B and 39 A). On the other hand, fertile Petrocelis tissue collected from Point No Point in December, 1963 contained fertile plants of C. petrocelidis of which the majority were oriented with the stipe down (Figs. 39 E, F, and 40 E). At Porlier Pass about 50$ of the plants with the stipe directed down had a lateral appendage of variable length always directed upwards (Figs. 37 A, 39 B, and 40 C). A number of cells were found in which this lateral appendage was composed internally of periodic "V"-shaped layers identical to those of the stipe proper but pointing in the opposite direction (Fig. 37 B). A number of cells were also found which clearly showed the formation of a second stipe (Figs. 38 B - 3, and 4o A, D). In the f i r s t figure only the f i r s t two layers in the new stipe are visible. At Porlier Pass cells with stipes oriented down predominated in the summer and stipes oriented up predominated in the f a l l . Also, material collected from Porlier Pass showed plants oriented down to be more abundant in shaded areas than in sunny areas. Septa-like structures were occasionally observed in the stipes of C. petrocelidis from, Deadman's Bay (Fig. 37 C and E). However, in a l l three localities cells with a solid stipe composed of "V"-shaped layers were common. The protoplast of Codiolum petrocelidis contains a parietal chloroplast with several pyrenoids and, except at fe r t i l i t y , one central nucleus. In the culture studies i t was found that vegetative cells be-came fertile at low light intensities (25 - 100 f.c.) and only in the wet f i l t e r paper cultures. Fertile cells produced ovate quadriflagellate zoospores containing a prominent eyespot. The zoospores germinated in-ternally and later developed into branching multinucleate, multicellular filaments. Further development of these plants was not followed. Cytochemical tests done on Codiolum petrocelidis give identical results to those of C. gregarium and cultural Codiolum from Urospora  wormskioldii and U. vancouveriana (Fig. 32 B). The inner layers are pectic and the outer sheath is of an unknown composition. The septa-like struc-tures observed in some cells dissolved readily in boiling concentrated HCl (Fig. 35 0) indicating the absence of cellulose. Spongomorpha coalita was the only Spongomorpha species collected at Deadman's Bay and Porlier Pass. Mature sexual plants were collected at these localities from April to the end of August. A few unsuccessful attempts at mating were made in the summer of I961. The plant was re-investigated in the summer of I96U to obtain information on its wall structure for comparison with the walls of Urospora. . The inner walls of Spongomorpha cells appear to be composed of cellulose and small amounts of pectic materials. As in Urospora the cells of Spongomorpha are bounded on the outside by a common filament membrane of unknown composition. The cellulose layers appear thicker than in- • Urospora and give a positive test with IKI - HgSO^  or ZnCUg before or after heating in concentrated HCl or after autoclaving in 23 M KOH (Cytochem.details Figs. 32 A and 35) cellulose layers are also doubly . refractive under polarized light before and after removal of pectins. As in Urospora the cellulose layers are soluble in Schweitzer's reagent (Fig. 35 F) and slug cytase (Fig. 35 C and D), but only after boiling in concentrated HC1. The pectic material appears.to be less than in Urospora for only a slight swelling occurs on treatment with IKI - HgSOj^  and the filament remains more rigid after boiling in concentrated HC1. The ce l l wall gives a positive test for pectin with ruthenium red before but not after heating in concentrated HC1. The pectic materials are insoluble in Schweitzer's reagent, slug cytase or on autoclaving in 23 M KOH. The outer sheath gives a negative test for cellulose, pectin, chitin, or fat and is insoluble in Schweitzer's reagent or slug cytase. Unlike that of Urospora the sheath is insoluble in IKI - H2SO4, ZnClL^ or Schweitzer's reagent after brief boiling in concentrated HC1 and is s t i l l found present after autoclaving in 23 M KOH. However, after the latter treatment the . membrane dissolves with the addition of water. The • sheath of Spongo-morpha appears to be much thicker than that of Urospora and may also form part of the crosswalls. On treatment with Schweitzer's reagent or slug cytase after brief boiling in concentrated HC1, crosswalls are occasionally left (Fig. 35 D). These are generally found in cells in-volved in branching. When the filaments are boiled in HC1 - ZnCl^ the inner layers dissolve f i r s t , leaving the outer sheath with crosavail "shadows" (Fig. 35 F), but with further heating these disappear as the sheath becomes further dissolved. As a by-product of the wall studies i t was discovered that Spongomorpha coalita in this area of study has operculate gametangia (Fig. 39 C). Subsequently operculation was also found in a collection of 1+9. S . c o a l i t a f r o m M u s s e l B e a c h , i n t h e M o n t e r e y P e n i n s u l a , C a l i f o r n i a . * I V . DISCUSSION AMD CONCLUSIONS 1. G r o w t h a n d s e x u a l i t y o f U r o s p o r a T h e f r e q u e n t f a i l u r e o f s i n g l e z o o s p o r e s o f U r o s p o r a w o r m s k i o l d i i t o g e r m i n a t e i n c u l t u r e i s s u g g e s t e d t o h e due t o t h e r e q u i r e m e n t o f a d i f f u s i b l e endogenous f a c t o r r e q u i r e d d u r i n g a c r i t i c a l s t a g e i n g e r m i n a -t i o n . C o n c e n t r a t e d s p o r e s u s p e n s i o n s w o u l d t h e r e f o r e r e d u c e n e t o u t w a r d d i f f u s i o n o f t h e f a c t o r , w h i l e weak s u s p e n s i o n s w o u l d h a v e t h e o p p o s i t e e f f e c t , r e s u l t i n g i n i t s l o s s . The f a c t t h a t t h e s o u r c e o f t h e s p o r e s i s r e l a t e d t o t h e i r g e r m i n a t i o n a b i l i t y s u g g e s t s t h e amount o f t h e d i f f u s -i b l e f a c t o r p r e s e n t i n a s p o r e i s d e p e n d e n t on t h e p a s t e n v i r o n m e n t a l h i s t o r y o f t h e p a r e n t ( U r o s p o r a o r C o d i o l u m ) . S i n c e F e E D T A p r o m o t e s z o o -s p o r e g e r m i n a t i o n , i t i s p o s s i b l e t h a t t h e d i f f u s i b l e s u b s t a n c e i s i r o n i n a w e a k l y b o u n d f o r m . On t h e o t h e r h a n d , t h e b e n e f i c i a l e f f e c t o f F e E D T A may b e i n p r o m o t i n g r e t e n t i o n o f t h e f a c t o r . I f s i n g l e z o o s p o r e s a l s o h a v e a l o w g e r m i n a t i o n f r e q u e n c y i n t h e f i e l d i t w o u l d e x p l a i n why U . w o r m s k i o l d i i i s n o t a s a b u n d a n t o r more e v e n l y s p r e a d as m i g h t b e e x -p e c t e d . A s f a r a s I am a w a r e , a c r i t i c a l g e r m i n a t i o n p e r i o d has n o t b e e n r e p o r t e d f o r z o o s p o r e s i n o t h e r a l g a e . I n U r o s p o r a w o r m s k i o l d i i t h e male c l o n e M2A a p p e a r s t o b e f a r more s e n s i t i v e t o s t r e s s t h a n t h e f e m a l e F l l . I t does n o t grow a t 20°, d i e s s o o n e r w i t h d a i l y d e s s i c a t i o n p e r i o d s , t h e r m o p e r i o d s o r b o t h com-b i n e d , and e x p e r i e n c e s b u b b l i n g o f t h e o u t e r s h e a t h when s u b j e c t e d t o d a i l y t h e r m o p e r i o d s . H o w e v e r , i t was n o t d e t e r m i n e d i f t h i s i s a common * C o l l e c t e d b y A . C . M a t h i e s o n (U. B. C . ) i n J u n e , i960. 50. feature for a l l male plants, or in other words, sex linked. If i t is, the difference in heat tolerance of the two sexes could he U3ed to advan-tage in establishing the presence of meiosis in the Codiolum stage. It could also be used to determine the percentage of haploid and diploid Codiolum plants in nature and to. determine i f the haploid Codiolum plants can be of either sex. Though the gametes of Urospora wo imskioldii failed to show any phototactic response over short periods, the fact that they settled near-est the light in culture indicates they have a positive phototactic res-ponse. However, since they settled at the air/water/glass interface, even when illuminated from below, i t i s possible that selection of this site is determined by other factors as well. The spontaneous occurrence of sexuality in both sexes of Uros-pora wormskioldii, its gradual decrease in the female and abrupt loss in the male through serial subcultures suggests that internal as well as ex-ternal factors are involved in the sexual response. This carry-over of sex-uality probably occurs in the fie l d and therefore might be regarded as an adaptive feature, for i t would extend the sexual period and: thereby in-crease zygote production. However, the failure of clones derived from Codiolum • to became sexual indicates that, i f carry-over is due to some built-up stored factor, this factor is lost in passing through the Codiolum stage. The experiments on sexuality indicate that temperature change is the prime factor involved in the sexual response and that, in culture, nutrient factors are involved as well. Desiccation by itself does not appear to be important in promoting sexuality. Cyclic thermoperiods may not be a requirement for sex induction since a prolonged thermoperiod was found to be more effective. The proposal that temperature is the prime 5 1 . V sex-inducing factor agrees with the observation that sexual plants are found only in the summer and only in the mid-tide, and that plants with a southern exposure become sexual before plants with a northern exposure. The spontaneous occurrence of Codiolum in cultures of male Urospora wormskioldii from three widely different localities, and its absence in serial subcultures at the same temperature suggests that this is a natural and not a cultural, phenomenon. In order to account for the origin and production of Codiolum cells in original but not subsequent sexually induced cultures, i t is suggested that under ideal culture condi-tions male gametes can develop parthenogenetically. However, since male gametes from nature or culture die when cultured free of Urospora, and since the culture medium in original and subsequent cultures was the same, i t is suggested that the parthenogenic development of male gametes is de-pendent on a substance (or substances) produced by Urospora plants and that the substance ceases to be produced in subsequent subcultures. 2. Growth and f e r t i l i t y of Codiolum The poor growth of Codiolum in culture is hard to explain in view of its small size in nature and in view of the luxuriant growth of Urospora under the same cultural conditions. Reduced growth of cultural Codiolum was also obtained by Jorde (1933) and Kornmann (1961 b, c). The decrease in cell size with decrease in temperature implies that growth is dependent on temperature. The absence of f e r t i l i t y in a l l male and fe-male Codiolum cells of U. wormskioldii grown in liquid culture and its occurrence in female F3 Codiolum cells grown on agar medium suggests that the agar medium is a more favorable environment for Codiolum. However, this may not be true since cells on agar were invariably smaller than those in liquid culture. Since Codiolum is found only in the high tide, 52. i t is reasonable to suppose that daily periods of desiccation and heat may be required for normal growth. The studies of f e r t i l i t y in natural Codiolum show clearly that f e r t i l i t y is inhibited by daily warm thermoperiods and induced by cold conditions. That large cells become fertile f i r s t indicates a degree of maturity is required before f e r t i l i t y can be induced. The data also suggest that f e r t i l i t y commences about four to six days after the begin-ning of the cold treatment. This lag in f e r t i l i t y would be of survival value in areas where summer water temperatures approach 20°C for i t would ensure that plants become fertile only under relatively stable cool at-mospheric conditions as would occur with the approach of winter when water temperatures reach more tolerant levels for Urospora growth. Since fertile plants were never encountered in the field at lower levels, or at the lower limit of Codiolum, i t is proposed that the lower limit i s the lowest level at which gametes or zygotes settle, and that these settle only during high tide periods. ' The results from cultural studies are not as clear cut, making interpretations difficult. The absence of f e r t i l i t y at 20°C is probably due to inhibition by temperature, and its absence at 5°C to poor growth and immaturity. The failure of cold treatment following culturing at higher temperatures or following daily thermoperiods suggests that either the difference in temperatures was not great enough or that factors besides temperature are involved in the f e r t i l i t y response. The fonaer is probab-ly the case since a very high percentage of f e r t i l i t y was obtained in cold-shocked Codiolum cells of U. wormskioldii female clone F8. That other factors are involved is indicated by the observation that the same cold-shock treatments failed to induce f e r t i l i t y in Codiolum from isolate URX1. 53. If f e r t i l i t y is dependent on a temperature drop and on plant maturity, this would explain why some plants remain infertile throughout the winter months. Since sexuality.may occur on into September, Codiolum plants initiated at this time would develop under much colder conditions than those developing in the summer. These would experience a far smaller temperature drop which, during mild winters, might not be sufficient to induce f e r t i l i t y , and so the plants would, persist to the next winter. The abnormal products of fertile Codiolum cells from female clones F3 (not cold shocked) and F8 (cold shocked) appear, to be due to internal factors. These were probably not due. to cultural conditions since Codiolum from Urospora vancouveriana grown in liquid or agar media, always produced normal zoospores. These abnormalities would be under-standable i f meiosis was an obligate feature of haploid or diploid Codiolum cells in U. wormskioldii.; However, if. this were true, one would expect to find similar abnormalities in natural Codiolum and these were never encountered. 3. Cytology of Urospora and Codiolum The f i b r i l s in the walls of Urospora and Codiolum zoospores have not been described before. According to Areschoug (1874), Printz (1932), and Frye and Zeller (1915) the flagella in Urospora zoospores originate at the anterior end of the four zoospore ridges. However, in the present study, squash preparations of zoospores showed clearly that the flagella originate between the fi b r i l s of the ridges, and hence between the ridges themselves. Since the ridge f i b r i l s were found in zoospores of U. worm-skioldii, U. vancouveriana and their Codiolum stages, i t is possible that they are a general feature of Urospora. It would be of interest to determine i f this feature is also present in the acuminate quadriflagellate zoospores of other members in the Ulotrichales (sensu Kornmann). It would also be of interest to determine i f the f i b r i l s have any relation to flagellar roots as found in quadriflagellate zoospores of Ulothrix sp. (Manton, 1952) and other green filamentous algae (Manton et al, 1955; Manton, 1964). According to these authors, the flagellar roots arise be-tween the flagella as do the surface f i b r i l s in zoospores of Urospora. The morphology of the nucleus and nuclear division in Urospora are essentially the same as in closely related genera e.g. Ulothrix, Spongomorpha and Acrosiphonia. The chromosomes in U. wormskioldii and U. vancouveriana are comparable in size to those of A. spinescens (jonsson, 1962) but much smaller than those of Ulothrix (Sarma, I963) and Spongo-morpha (Jonsson, I 9 6 2 ) . In the latter two genera some chromosomes may measure 5 jx in length, whereas in Urospora they seldom are 2 /n. Chromatic bodies occurring in Urospora were not reported in the interphase nucleus of the other species. The decrease in Feulgen-stainability of larger nuclei of Urospora and Codiolum may be due to properties of the nucleus itself or to a spreading-out of the chromatin. The evidence for the latter possib-i l i t y is that, with increasing nuclear size the Feulgen-staining chromatin strands become finer and more distantly separated until they become in-visible. The evidence for the former possibility is that the chromatin appears quite dense when stained with Newcomer-iron-propiocarmine. It may be that some substance inhibits Feulgen staining in larger nuclei. If the chromatin does continue to increase in amount proportional to the increase in nuclear volume, i t is curious that the chromosomes in early divisions of Codiolum are so small. Though many cells were observed with nuclei of up to 30 jx in diameter, early-division chromosomes seldom 55. exceeded 3 p in length. By contrast, nuclei of Urospora, 10 p in dia-meter, produced chromosomes up to 2 p in length. The concentration of nuclei at the end •walls and in the middle of the cell in rapidly growing filaments or Urospora wormskioldii and U. vancouveriana suggests that nuclear migration takes place and that cell division is intimately related to nuclear concentration. A similar dis-tribution of nuclei was observed in U. mirabilis by Jorde (1933). Cell division, however, appears to be unrelated to nuclear division for the two occur independently, at least in multinucleate cells. This may. not be true in germlings where the cells are i n i t i a l l y uninucleate. Nuclear migration has also been reported in Acrosiphonia spinescens (Jonsson i960, I962). In this species some of the nuclei migrate to the plane of the future crosswall where they divide synchronously. More distant nuclei remain quiescent. The crosswall is formed directly after nuclear division by centripetal growth. In Spongomorpha lanosa, cytogamy and karyogamy are intimately linked (Jonsson I962). The mode of cell division in U. speciosa has not been studied.. h. Variability and Speciation in Urospotra The wide range in morphology exhibited by vegetative filaments of Urospora wormskioldii in nature places much doubt on the use of purely vegetative characteristics for dilineation of Urospora species, as has been done by Setchell and Gardner (1920) for species from this coast. This may apply as well to species from Europe which show similar variability (Printz 1932, Jorde 1933). In view of this,the taxonomy of the genus, to be meaningful, must be based on sexual, l i f e history and cytological features as well as morphological ones. At the present time, six taxa of Urospora may be recognized from 56. Europe; U. bangiodes, U. mirabilis, U. wormskioldii var wormskioldii, U. wormskioldii var biflagellatum, U. wormskioldii var caudatum and U. 3 p e c i o s a . The f i r s t , second, fourth and sixth taxa appear to be distinct entities; however, the remainder are questionable. U. wormskioldii var wormskioldii, a* low tide form, has not been found sexual in Europe. According to Jorde (1933) i t may be merely a growth form of U. mirabilis. According to Printz ( I 9 3 2 ) , U. bangiodes is synonomous with U. mirabilis, while Kornmann (196lc) considers i t to be a distinct species since i t differs from others in that cultural plants have a single richly branched rhizoid. Kornmann (1961c) considers U. bangiodes and U. wormskioldii var caudatum to be asexual since these were never found sexual in the fie l d or when cultured at various temperatures (3° - 15°C). Both reproduce via quadriflagellate zoospores which, in the latter taxa, are also capable of giving rise to a Codiolum stage.. His- evidence for the origin of Codiolum ' is, however, indirect and therefore questionable. To establish this point the development of zoospores would have to be followed. It is equally possible that the Codiolum cells arose from a small number of gametes which could have gone undetected in culture. Kornmann's conclusion that both species are asexual is also questionable since the same temperatures failed to induce sexuality in U. speciosa and U. penicilliformis which were found sexual in nature. In the present study several clones derived from sexual and vegetative filaments of U. wormskioldii behaved in a similar manner; some failed to produce Codiolum while others did in amounts ranging from one'to over,500 per test tube. Unless parthenogametic development of gametes is very low in culture- very few gametes would be required to produce these results. The appearance of Codiolum cells in 8 out of 11^ Urospora clones derived from a Codiolum cell from Deadman's Bay and none in the 500 clones derived from 50 Codiolum cells from Tsawwassen can he used as specific examples. In the first case, though the 8 clones were recultured at 5°/ 10° and 15°C, none became sexual or produced Codiolum. Since only U. wormskioldii was present at Tsawwassen and most abundant at Deadman's Bay, i t is very likely that the Codiolum cells used to establish these cultures were derived from sexual U. worm-skioldii . Therefore failure to obtain•sexuality in culture over the temperature ranges used cannot be taken as proof of asexuality. Of part-icular interest is the clone URX1 (a low tide form which I attribute to . U. wormskioldii). This clone has been recultured many times over the past three years and, in two instances, produced Codiolum cells in great abundance when cultured at 10°C; once after twelve months of culture and once after twenty months. 0n~ the last instance this occurred in a growth experiment involving various media. Of sixteen replicate cultures only one of these produced Codiolum, this occurring in medium k (Table 2). Sub-sequent cultures in the same medium failed to produce Codiolum. Though zoospores from this clone were cultured on liquid and agar, media at 5°> 10° and 15°C a l l developed into dwarf plants or filaments. The failure of URX1 to become sexual with any of the methods used in the present study might indicate i t to be a different species to U. wormskioldii - • perhaps an asexual one. However, further evidence i s needed to be sure of this point. Though i t is reasonable to expect that asexual species of Urospora exist in nature, means to obtain convincing evidence for proof of asexuality at present are not available. Transplantation studies might be usefully applied to this problem. Since Urospora is small and easily cultured in large quantities, cultural material could be 58 transplanted to areas in the intertidal where and when sexuality occurs in other species. Transplantation techniques might also he used to study the effect of habitat and seasons on growth and morphology of Urospora. In particular these would he useful in determining the nature of "asexual" low tide forms. To facilitate this type of work, an easily-handled sub-strate and labelling.technique would be required. Ceramics might be useful as a substrate since any shape or texture could be made. The ferric chloride-potassium ferrocyanide marking technique used by Astbury and Preston (19^0) on Cladophora or Calcofluor White in conjunction with fluorescent microscopy (Cole, 1964) might be useful for labelling and later, identification. The names applied to the three Urospora species found in this study are based on Setchell and Gardner's Key (1920) and are considered tentative. These species show close resemblance to European types but, because of incomplete or conflicting data, evaluation is not possible. Type I designated as U. wormskioldii conforms very closely in gametangium color and gamete size to U. penicilliformis from Helgoland (Kornmann, 196lc), but differs from i t in that the male gametes are spindle shaped, the female gametes are asymmetrical and the male as well as the female has a pyrenoid (Fig. 22). In U. penicilliformis both gametes are ovate and only the female has a pyrenoid. However, these distinctions may not be valid. In U. wormskioldii the small pyrenoid of the male gamete is difficult to distinguish unless stained and the asymmetry of the female gamete is not assumed until:some time after release. Kornmann did not stain his material nor did he observe gametes over long periods (personal communication). Future investigations of gamete morphology should take the time element into consideration. Since sexual material of U. 59. •Wormskioldii has not "been found in Europe, a comparison with i t is not possible. Type I appears to be different from U. mirabilis in gamete morphology and chromosome number. However, the description of the gametes of U. mirabilis given by Printz (1932) is at variance with Jorde's des-cription and drawings (Fig. 22). Furthermore, the chromosome number of four for U. mirabilis was given by Jorde with some reservation so that a mean-ingful comparison cannot be made. However, since both gametes of U. mirabilis have an eyespot which is present only in the male of Type I, i t would indicate that each is a distinct species. On the basis of information presently available on l i f e history and morphological features;in Urospora I am inclined to recognize five species from the Pacific Coast of North America; U. speciosa, U. vancouveriana, U. wormskioldii, U. tetraciliata and U. penicilliformis. However, the : last two are considered doubtful entities, since the un-usual nature of the gametes in U. tetraciliata has not been confirmed and since sexuality has, as yet, not been recorded in America in-U. penicilliformis*. U. dolifera, U. grandis and U. sphaerulifera are not regarded by me as distinct entities since they are known only from veg-etative material and they come within the range of U. wormskioldii. With these reservations the following key is proposed as a tentative one for Urospora species which I recognize from this coast. * Life history studies on Urospora penilliformis might be profitably done in the Monterey Peninsula since only this species is recorded from that area (Smith, l$kk). 6o. Key to Urospora species 1. Uninucleate; monoecious} isogamous; filaments under 60 p. wide: 1 - k extramatrical rhizoids ' U. speciosa 1. Multinucleate; rhizoids intra or extramatrical filaments up to 3 mm wide 2 2. Sexual • _ _ . 3 2. Asexual; Codiolum arising from biflag-ellate zoospores produced by dwarf plants at warm temperatures; filaments up to 3 mm wide U. vancouveriana 3. Monoecious; gametes quadriflagellate, isogamous; filaments up to 225 /» wide U. tetraciliata 3. Dioecious; gametes biflagellate, anisogamous, gam-etes ovate, male gametes smaller; filaments un-der 100 p. wide • U0 penicilliformis 3 . Dioecious; gametes biflagellate, anisogamous, male gametes smaller, spindle shaped; female gametes asymmetrical, slightly curved, twisted and having 2 or more blunt longitudinal ridges; filaments up to 1200 p or more in width U. wormskioldii Type II designated as Urospora vancouveriana is very similar to Kornmann^ (1961 b, c) U. wormskioldii var biflagellatura from Helgoland, with this similarity extending to the temperature requirements for the production of the biflagellate zoospores„ These are of similar size in both species but in U, wormskioldii var biflagellatum they are ovate 61. whereas In U. vancouveriana they are acuminate (Fig. 22). This difference may not he meaningful since Kornmann did not study the biflagellate zoo-spores over lengthy periods. Kornmann has observed zoospores with point- . ed ends which were not mentioned in his paper (personal communication), suggesting that a similar change may' take place in the biflagellate zoo-spores of this species over time. The main difference in the two species is that the fertile zoosporangia, producing biflagellate zoospores, in U. wormskioldii var bif lagellatum are. warty at the apex but smooth in U. vancouveriana. Comparison of the two on filament characteristics is not possible, since filaments of U. wormskioldii var biflagellatum have not been found by Kornmann in nature. . Type III is designated as Urospora speciosa, because of its close resemblance in vegetative features to that species at Helgoland as described by Kornmann (1961c) and because the Helgoland type is also uninucleate (Kornmann, personal communication). Further l i f e history information i s needed for the American type, however, before the two can be equated. The discovery of a uninucleate species of Urospora is further evidence for the evolution of. Urospora from Ulothrix. According to the precedent set by Wille (1900) for separating genera on the basis of nuclear condition, a new genus could be erected for uninucleate Urospora types as well. In view of the confusion that has resulted by creating new genera in the Acrosiphonia-Spongomorpha complex, i t should be avoided until cytological as well as l i f e history information i s available for other species. 5.. Variability and Speciation in Codiolum The. variability in the morphology of natural Codiolum appears 62. to be due to environmental conditions. Similar variability was observed for European forms by Printz (1932) and Jorde (1933), who considered these to be merely variants of C. gregarium A. Braun0 The two types encountered in the present study, C. gregarium and C. pusillum, are con-sidered to be synonymous and to belong to the l i f e history of U. worm- skioldii for the reasons that: they were found only in areas where U. wormskioldii was most abundant; they have the same chromosome number..' as wormskioldii;and Urospora clones derived from them,when cultured at 15°C,behaved like U. wormskioldii. However, It obviously cannot be con- ; eluded that free living Codiolum forms in other localities belong to this species. This would have to be determined from cultural and cytological studieso Codiolum petrocelidis is considered to belong to the l i f e history of S. coalita because of its close association with that species. According to Fan (1959), C. petrocelidis is multinucleate. In this study the plant was found uninucleate from April through to September, only be-coming multinucleate during the fertile period in the winter. Fan's observations of the nuclear condition seem to have been on cultural plants alone so that his results are not to be equated with conditions in the f i e l d . The different forms and orientations of Codiolum^petrocelidis cells observed in this study are best explained by assuming that the cells are capable of reversing their direction of growth. The direction of growth is suggested to be governed by light intensity, a high intensity promoting downward growth of the protoplast and low light intensity, upward growth. This would be in accord with the observation that plants at higher l a t i -tudes (Helgoland 54°N) grow mainly stipe down and plants at lower latitudes 6 3 . (Monterey, California 36°N) grow stipe up. This hypothesis could readily he tested with culture or transplantation studies. Growth reversal would also explain the significance of the lateral stipe appendage observed by other authors e.g. Kuckuck (189*0, Zimmerman (I925), Printz (1926), Kornmann (1961a). (Their drawings are brought together in Fig. 36 for comparison purposes). The appendage would merely represent the f i r s t -formed stipe. There are two facts favoring this proposal: the appendage is always directed upwards and its "V" shaped lamellations point in the opposite direction to those in the stipe proper. According to Zimmerman (I925) the lateral appendage i s a sec-ondary outgrowth resulting from the swelling of the wall where the wall is poorly attached to the c e l l . It is not clear whether he was referring to the outer sheath or inner walls, but the former seems the case. Jonsson (1962) concluded that in Brittany, France, C. petrocelidis changes its direction of growth, but in so doing reabsorbs the f i r s t formed stipe. In ihe latest stage the f i r s t stipe is represented by a thickened apical cap (Fig. 36 D - f ) . Jo'nsson's conclusion is based mainly on development of zygotes of Acrosiphonia spinescens in culture; the figures from nature shown here being arranged to conform with cultural observations. Jonsson,. however, did not suggest any reason for the change in growtho In order to obtain cells with appendages one would have to assume that in this area reverse growth occurs by the protoplast bulging out laterally and then bypassing the stipe. The fact that the lateral appendage can be found anywhere on the stipe indicates that growth of the outer membrane about the clava is diffuse. Otherwise, i f growth occurred only at the advancing end of the protoplast, the lateral appendage would eh. always "be located at the base of the main stipe. Though the descriptions of the morphology of C. petrocelidis are at variance in regards the stipe, to date a l l investigators, except Kornmann, regard the plant to be unicellular. According to Kornmann . (196la), the septa-like structures he observed in developing cultural zygotes of Spongomorpha lanosa (Fig. 36 G) and those observed by Fan (1959) in developing zygotes of S. coalita (Fig. 36 F) reflect an early multicellular condition. However, though Fan did not follow this devel-opment in detail, he clearly stated that the cells are unicellular. Hollenberg (1958), who worked on S. coalita from the same area as Fan, found developing zygotes to have an aseptate stipe (Fig. 36 H) indicating that septation is not an obligate feature. Jonsson (1958), who observed similar septa-like structures in zygotes of Acrosiphonia spinescens (Fig. 36 E), regards these as pseudosepta (personal communication). Kornmann did not follow development of C. petrocelidis in the field but agrees that in nature C. petrocelidis i s unicellular (personal communica-tion). The septa-like structures found in the present study are regarded to be merely the result of uneven deposition of pectic layers in the stipe. It is also suggested that the mode of stipe growth (deposition of pectic layers by the protoplast) rules out a multicellular condition. However, since different species are involved, Kornmann*a interpretations cannot be ruled out. A careful cytological study on the early development of zygotic material from Helgoland is therefore urgently needed. 6. Cytochemical studies The cytochemical studies have shown that there are qualitative and quantitative differences in the walls of Spongomorpha and Urospora, but not in the walls of their respective Codiolum stage. A l l four forms have 65. an outer membrane possessing the common characteristics of being singly refractive under polarized light, insoluble in slug cytase, concentrated <-HCl, or Schweitzer*s reagent, and of giving a negative test for cellulose, chitin, pectin, or fat. Three types of membranes appear to be involved since only that of Urospora dissolves in ZnClI 2 or Schweitzer's reagent after brief heating in concentrated HCl, and only that of Spongomorpha is insoluble on autoclaving in 23M KOH. The inner walls of Urospora and Spongomorpha differ in that pectic compounds seem predominant in the former and cellulose in the latter. The inner walls of the Codiolum types are .pectic and lack cellulose. The pectic component in a l l forms-appears to differ from that of higher plants, a feature also noted by Astbury and Preston ( I 9 I K ) ) for the pectic component in the walls of Cladophora. Some aspects of these results' are in conflict with those of Jonsson (1962) and earlier authors. Braun (1855) reported that the outer membrane of Codiolum gregarium gave a dark color with IKE. - BvjSO^ , which changed to green, but never blue, on addition of ZnClIg. I could not verify this color change. Zimmerman (I925) obtained a slight blue color in the anterior part of the cell with HCE - H^ SO^ , otherwise the outer membrane remained unstained. He found this membrane gave no stain with methylene blue or Congo red and was insoluble in the cellulose solvent, cupric-ferric-ammonium hydroxide and that the inner wall material dissolved readily In concentrated sulphuric acid. Zimmerman concluded that, in addition to a small amount of cellulose, the membrane is composed of some other material but not pectin. Jonsson (I962) failed to obtain a positive test for cellulose with ZnClIg in Spongomorpha, Urospora, Acrosiphonia, and also Cladophora, Rhizoclonium, and Chaetomorpha. In a l l of these he reported the cellulose fraction to be insoluble in Schweitzer1s reagent 66. and therefore different from that of higher plants. However, i t appears that he did not pretreat for removal of pectic compounds. It has been demonstrated in the present study and much earlier by Wurdack (lQ23) and Tiffany (1924) that pectic compounds act as a barrier to this cellulose solvent. According to Jonsson (I962) the outer membrane in Urospora, Spongomorpha and Acrosiphonia contains pectic materials, since he found i t to stain with ruthenium red. However, this membrane is so thin that coloring in adjacent layers might be,attributed to i t . If pectin is a component i t would have to be present in small quantities, since the membrane does not change noticeably in thickness after treatment for removal of pectins. A similar non cellulosic type of membrane has been described occurring in Cladophora (Brand 1901, Wurdack 1923), Bulbochaete (Tiffany 1924), and Oedegonium (Hirn 1900, Wurdack 1923). Cytochemical tests indicated the membrane to be chitin in Cladophora and Oedegoniurn (Wurdack 1923) and Bulbochaete (Tiffany 1924). Astbury and Preston (1940) confirmed the presence of chitin in Cladophora, using cytochemical and X-ray diffraction techniques, but did not localize i t . However, more recently the existence of chitin in Cladophora has been placed in doubt, Frey-Wyssling (1959), using X-ray techniques, and Jonsson (1962), using in addition cytochemical techniques, failed to demonstrate its presence in Cladophora. Though no conclusion can be made concerning the composi-tion of the membranes as reported, i t appears that a substance different to cellulose and chitin is involved. The banded appearance of the protoplast of Codiolum gregarium seen under polarized light, both before and after treatment with HC1, suggests the presence of oriented structures within the cel l . The presence of "microtubules" as found in root tip cells of several higher plants (Ledbetter and Porter, 1963) would provide an explanation for this banding. It has been suggested by these authors that the "microtubules" may be related to the deposition and orientation of cellulose in the cell wall, sisce a correlation has'been found in the orientation of cel l -ulose fibres and the "microtubules". This hypothesis would not apply here, however, for the sheath and inner walls of Codiolum appear to be non-cellulosic. Differences in wall composition as revealed by X-ray diffraction techniques have been used for taxonomic purposes. Jonsson (1959b) used his X-ray data as supporting evidence for the inclusion of Urospora, Spongomorpha and Acrosiphonia in his new family, the Acrosiphoniaceae, while Kornmann (I963) uses the data of Nicolai and Preston (1952) as supporting evidence for the inclusion of Urospora, Ulothrix, Gomontia and Monostroma in the Ulotrichales (sensu Kornmann) and the exclusion of ' Acrosiphonia and Spongomorpha from this order. However, interpretations of X-ray data obtained from studies on untreated bulk material, as done by these authors, have been questioned. . Frey and Preston (I96I) found that in a number of cases the ambiguous results obtained from some algae in the early work of Nicolai and Preston (1952) were due to contamination of clay minerals. Also, Cronshaw, Meyers and Preston (I958) have shown that X-ray diffraction patterns are influenced by other wall components. The results obtained in this present study indicate the walls of Urospora and Spongomorpha to be different but the walls of their respect-ive Codiolum stages to be the same. These results at once imply both a closer and more distant relationship of the two genera. Clearly, wall studies on algae having hetermorphic l i f e histories should include a l l 68. stages to be of f u l l taxonomic value. 7. The Acrosiphonia-Spongomorpha complex. The taxonomy of the Ac ro siphoni a-Spon gomor pha complex has been in a confused staite for some time. Part of this confusion i s due to Wille (1900), who proposed the retention of the two genera and the rele-gation of uninucleate types to. Spongomorpha and multinucleate types to Acrosiphonia. Setchell and Gardner (1920) preferred not to draw a dis-tinction for West Coast North American types until cytological informa-tion was available. Smith (19^6) rejected Wille's proposal completely, feeling that aside from nuclear condition the two genera agreed in a l l other respects. Jonsson (1957) was the f i r s t to discover operculation i n A c rpsiphonia and since then Kornmann(1962) has come to regard i t as a distinctive feature of Acrosiphonia. However, Jonsson also accepts its uninucleate condition as a distinctive feature. On the other hand, Kornmann rejects Wille»s proposal, pointing out that multinucleate as well as uninucleate, species exist in Acrosiphonia. He gives no.examples but i t appears he is referring to S. coalita from America (Fan 1959). 1° addition to operculation, Kornmann, on the basis of his studies, recog-nizes an isomorphic l i f e history as the distinctive feature of Acrosiphonia and a heteromorphic l i f e history as the distinctive feature of Spongomorpha. Kornmann would therefore claim S. coalita as a true member of the genus Spongomorpha. Jonsson (1957> 1959a, I962, I963), however, claims Acrosiphonia in Brittany, France to have a heteromorphic l i f e cycle and, more recently (19$+ a, b), has presented additional evidence to support this claim. Jonsson points out that Kornmann.'3 failure to obtain Codiolum from zygotes of A. arcta may be due to a complete failure of karyogamy since Jonsson reported that karyogamy in A. spinescens (= arcta, Kornmann I 9 6 2 ) is facultative. According to Jonsson, when karyogamy fai l s , ordinary Acrosiphonia plants are produced and when, i t occurs Codiolum is produced. Recently, Kornmann has come to consider A. arcta at Helgoland to he a diplont (personal communication) but gives no cytological evidence for this. The di scovery of operculation in Spongomorpha coalita now makes i t possible, at least for the present, to clear up this area of confusion. Since both Jonsson and Kornmann agree that operculation is a distinctive feature of Acrosiphonia then S. coalita should probably be relegated to the genus Acrosiphonia. Doing this would mean recognition of a hetero-morphic l i f e history for Acrosiphonia, and acceptance of Wille's'basis for distinguishing the two genera since S. coalita is multinucleate and has a heteromorphic l i f e history (Fan 1959)- Formal transfer, however, should await examination of the type specimen. Until more convincing evidence is brought forth, the sporophyte of Spongomorpha should be considered unicellular; Recognition cf this and a heteromorphic l i f e history in Acrosiphonia re-establishes the basis for Jonsson*s family, the Acrosiphoniaceae, and, at the same time, removes the main barriers to the inclusion of Acrosiphonia and Spongomorpha in the Ulotrichales (sensu Kornmann). The present author, however, is of the opinion that Urospora should not be placed in the Acrosiphoniaceae because of its asexual reproduction by means of acuminate quadriflagellate zoospores. Conflicting reports concerning the l i f e cycle of Acrosiphonia  arcta (= A. spinescens) at Helgoland (Kornmann) and Roscoff, France, (Jonsson) may indicate that a greater variability and fle x i b i l i t y in l i f e histories occur in this complex than has been suspected. It is clear that future l i f e history studies in this group should be very c r i t i c a l . The discovery of facultative karyogamy in Acrosiphonia by Jonsson (1962, 196k a, t>) emphasizes that cytology should form an integral part of such studies. Investigation of North American species in the Acrosiphonia-Spongomorpha complex should "be particularly rewarding, since ' six species of Spongomorpha are recorded from British Columbia, and northern Washington (Scagel, 1957) and four species from the northeastern coast of America (Taylor, 1957)* 8. Theoretical* considerations on the origin of Codiolum According to Jorde (1933)> the diploid Codiolum stage of Urospora can he considered as an advanced zygote. Though she gives no evidence to support this view, i t i s put forth, presumahly, on the basis that Codiolum exhibits growth over a prolonged period. These studies demonstrate that Codiolum may have a l i f e span of over six months, and that during this period growth may be continuous. Certainly, the Codiolum stage cannot be viewed as a resting zygote as is found in fresh water algae. Jonsson (1962), on the other hand, considers the Codiolum stage in Urospora, Spongomorpha and Acrosiphonia to be a much reduced sporophyte and suggests that in the latter two genera this reduction has come about as an adaptation to an endophytic habitat. However, as he states, this explanation loses some of i t s value when one considers that the sporophyte of Uro spora is never found endophytic, though i t i s just as reduced in form. In support of his hypothesis, Jonsson draws from information on l i f e histories in Stigeoclonium. In Stigeoclonium sub3pinosum the sporo-phyte consists of a few cells, whereas in Stigeoclonium amoenum i t is a single c e l l . (Juller, 1937)- Jonsson's reductional hypothesis is in keep-ing with the hypothesis put forth by Fritsch (19^2) that heteromorphic l i f e histories in the algae have arisen from isomorphic ones. 71. The hypothesis that the diploid state in algae, has arisen by prolongation and further development of a zygote (Smith, 1938) could be applied here as well. In this case competition for substrate light and . nutrition would be major factors affecting the survival of the zygote. This would be true, however, only on the assumption that a rich algal flora existed at the time when the green filamentous algae were under-going evolution from a haplontic to a haplodiplontic way of l i f e . In meeting this competition the newly formed zygote could take one of several steps to ensure its retention iri the l i f e cycle. The zygote could become fertile, soon after formation, to regenerate the haploid form. Nuclear division could take place to give rise to a coenocyte, or cell division could take place to give rise to a multicellular form different or iden-tical to the haploid generation. Lastly, the zygote stage could be pro-longed. In the last instance, competition would be very great unless a site were selected where competition could be avoided. Urospora, Aero- , siphonia, Spongomorpha, Gomontia and Monostroma a l l possess the unique feature that their Codiolum stages occupy habitats where competition is virtually absent. The extreme conditions of the high tide habitat of the Codiolum stage of Urospora is tolerated by few algae. The endophytic hab-itat of the Codiolum stage of Acrosiphonia and Spongomorpha,' in various red algae is protective. Similarly the burrows of the Codiolum stage of Gomontia and Monostroma in mollusc shells are protective. It is therefore equally reasonable to suggest that the heteromorphic l i f e cycle in these genera arose directly from haplont ancestors. VJhile either hypothesis may be true, adherence to the f i r s t , that a heteromorphic l i f e cycle arose from an isomorphic one by reduction, would require explanation as to why the diploid rather than the haploid 72 generation should have been reduced in these five closely related genera, and why this reduction went so far. If Kornmann is.correct,, that Acrosiphonia arcta at Helogland is a diplont and that the zygote of S. lanosa in culture is multicellular, this would, provide convincing evidence for Jonssons reductional hypothesis.' V. FINAL SUMMARY The distribution, morphology^ cytology and cultural behavior of Urospora and Codiolum.(free living and endophytic) have been studied from a number of localities within a radius of approximately 100 miles of Vancouver, British Columbia. Urospora wormskioldii (n : 12) is the most common species, rang-ing from low to high tide levels, being densest in the mid-tide. It is dioecious, anisogamous and has three somatic, stages, dwarf, filamentous and Codiolum. The vegetative characteristics of the filament stage are extremely variable and depend on the substrate, locality and season. Cultural plants show a similar variability, though not as great. The . Codiolum stage arises from zygotes, female gametes and probably male, gametes. C. gregarium and C. pusillum, as found in the areas studied,. are considered to be merely form variants and to belong to thellife history of U. wormskioldii. Fertility in natural Codiolum is inhibited by short daily thermoperiods and induced by cold conditions. On f e r t i l i t y , natural and cultural Codiolum produce quadriflagellate zoospores which give rise to the filamentous or dwarf stage. The last two stages re-produce asexually via similar zoospores. Sexuality occurred spontaneously in many Urospora clones cultured at 10°C, continuing through several serial cultures of female filaments but stopping after the f i r s t culture of the male filaments. Following loss of sexuality in culture, a long 73. thermoperiod during maximum filament growth followed by nutrient repletion successfully induced sexuality in.several Urospora clones. Nuclear div-ision was followed in natural Codiolum but meiosis was not demonstrated. Urospora vanco uveriana (n : 9) is a large low tide form and was found in only one locality. It is an asexual species having filamentous, dwarf and Codiolum stages. Cultural filaments are indistinguishable from cultural ones of U. wormskioldii. A l l three stages produce quadriflagellate zoospores which form either dwarf plants or filaments. In addition, dwarf plants when cultured at warmer temperatures, e.g. 15°C, produce biflagellate zoospores which give rise to the Codiolum stage. Urospora speciosa (n = ?) is a very slender form known from a fev filament fragments obtained from the low tide at one locality. F i l -amentous and dwarf stages are present, both of which reproduce via quadriflagellate zoospores. Other features of its l i f e history are un-known. U. speciosa was discovered to be uninucleate, this being a unique feature in the genus. Since Urospora dolifera, U. grandis and U., sphaerul- . ifera are known only from vegetative material and since these species come within the range of U. wormskioldii they should be regarded as doubtful entities. The discovery of a uninucleate condition in Urospora speciosa provides.further evidence for the hypothesis that Urospora evolved from Ulothrix. Rather than create a new genus, i t is suggested that this be avoided until cytological data for other species becomes available. Cytochemical tests were conducted on the walls of Urospora  wormskioldii, U. vancouveriana and their cultural Codiolum stages; C. gregarium, £. pusillum, C. petrocelidis and Spongomorpha coalita. The results indicate the inner c e l l walls of Urospora and Spongomorpha are composed of cellulose and pectic material, with pectic materials 7*. predominating in the former and cellulose in the latter. The inner walls of their respective Codiolum stages are pectic. The pectic material in a l l forms appears to he different to that of higher plants. The outer ensheathing membrane of Urospora, Spongomorpha and the Codiolum types is composed of an unknown substance which gives a-negative test for cellulose, pectin, chitin or fat. Three types of membranes appear to be involved, of which the Codiolum types are the same. The differences found in the cell walls of Urospora and Spon-gomorpha indicate a more distant relationship of the two genera, while the similarity of their Codiolum stages indicates a closer one. Obvious-ly, to be of taxonomic value, wall studies should include a l l stages in l i f e histories of algae having an alternation of heteromorphic generations. These studies also point to the need for re-examination of other green filamentous algae reported to have an outer membrane of different nature to cellulose. In the areas investigated, Codiolum petrocelidis, "found as an endophyte in Petrocelis franciscana, is suggested to belong to the l i f e history of Spongomorpha coalita. Morphological and developmental features of the cell are described. The protoplast of C. petrocelidis may reverse its direction of growth. The direction of growth is suggested to be governed by.light intensity. Accordingly, the mode of growth rules out a multicellular condition for C. petrocelidis. Spongomorpha coalita from this area and from the Monterey Peninsula, California where the major investigations on this species have been conducted, is reported for the f i r s t time to have operculate gametangia. This discovery supports a transfer of this species to the genus Acrosiphonia. However, formal transfer of the entity should await comparison with the type specimen. This transfer would validate a previous distinction based on nuclear condition. At the same time, i t would verify the occurrence of a heteromorphic l i f e history in the genus Acrosiphonia. Until more convincing evidence is presented, the zygote forms of Spongomorpha and Acrosiphonia must he considered unicellular. This removes the main objection to the inclusion of the two genera in the Ulotrichales (sensu Kornmann) and, at the same time, re-establishes the basis for Jonsson*s family, the Acrosiphoniaceae. Consideration is given to the possible origin of the Codiolum stage from a haplontic l i f e history as an alternative to Jonsson*s (1962) reductional hypothesis. BIBLIOGRAPHY Anon,(I962 - 3) . Pacific Coast Tide and Current Tables I963 - 4. Can. . Hydrographic Service Publ. Queens Printer, Ottawa. I962, vi 262 pp; I963, vi 280 pp. Archer, A. and E. M. Burrows (i960). Heteromorphic l i f e - history as a family criterion in the Cladophorales. Brit. Phyc. Bull. 2:31-33, i n c l . 1 table. Areschoug, J. E. (1874). Observations phycologicae. I. Nova Acta Reg. Soc. Sclent. Uppsaliensis VI: 1-26, 4 pis. add. Astbury, W. T. and R. D. Preston (I9A0).. Structure of the cell wall in some species of Cladophora. Proc. Roy. Soc. (London) B, 129? 55-76, i n c l . 3 tables, 16 figs; 1 pi. add. . Brand, F. (I90I). Ueber einige Verhaltnise des Baues und Wachsthums von Cladophora. Bei.'zumBot. Cent. 10:481-521. (Not seen) Braun, A. (l855)« Algarum unicellularum genera nova et minus cognita. Leipzig. Collins, F. S. (1918). The green algae of North America. Second supple-mentary paper. Tufts College Studies. Sci. Ser. 37, 4 (7): ' I-I06, 3 pis. ~~ Cole, K. (196^). Induced fluorescence in gametophytes of some Laminariales. Can. Jour. Bot. 42:1173-1181, 2 pis. add. Cronshaw, J., A. Myers and R. D. Preston ( I 9 5 8 ) . A chemical and physical investigation of the cell walls of some marine algae. Biochimica et Biophysica Acta 27:89-103, i n c l . 6 figs., 9 tables. . . . Darlington, C. D. and L. LaCour (i960). The handling of chromosomes. 3rd. ed., G. Allen, London. 2l+8 pp., illustrated. Faberge, A. C. (1945). Snail stomach cytase, a new reagent for plant cytology. Stain Tech. 20:1-4. Fan, K. C. (1957). Observations on the l i f e history of Codiolum  petrocelidis. Phyc. News Bull. 10:32. Fan, K. C. (1959). Studies on the l i f e histories of marine algae I. Codiolum petrocelidis and Spongomorpha coalita. Bull. Torrey Bot. Club 86:1-12, incl. 41 figs. Frei, E., and R. D. Preston (1961). Cell wall organization and wall growth in the filamentous green algae Cladophora and Chaetomorpha. 1. The basic structure and it s formation. Proc. Roy. Soc. (London) B, 154:70-94, incl. 11 pis. Frey-Wyssling, A. (1959). Die pflanzlich Zellwand (Springer, Verlag). (Not seen) -77 F r i t s c h , F. E. (1942). The a l g a l l i f e - c y c l e . Ann. Bot. 6:533-563. Frye, C , and S. M. Z e l l e r (1915) Hormiscia t e t r a c i l i a t a sp. nov. Puget Sound B i o l . Sta. Publ. 1:9-13, i n c l . 1 p i . Handbook of Chemistry and Physics (I962-I963). 44th ed., Chemical Rubber Publishing Co. 36o4 pp. Hartog, C. den. (1959). The e p i l i t h i c a l g a l communities occurring along the coast of the Netherlands. Wentia 1:1-241. (Not seen) Hart, H. T. (1928). Studies on Hormiscia v o r m s k i o l d i i . Puget Sound B i o l . . Sta. Publ. 5:355-359,- i n c l . 1 p i . Hirn, K. C. (1900). Monographie und Iconographie der Oedogoniaceen. Helsi n g f o r s . 383 pp. (Not seen) Hoek, Van den, C. (1964). Revision of the European species-of Cladophora. E. J . B r i l l , Leiden. 248 pp., i n c l . 39 tables, 18 maps; 55 p i s . add. Hollenberg, G.. J . (1957). C u l t u r e studies of Spongomorpha c o a l i t a . Phyc. News B u l l . 10:76. Hollenberg, G. J . (1958). Observations concerning the l i f e c ycle of Spongomorpha c o a l i t a (Ruprecht) C o l l i n s . Madrono 14:249-251, i n c l . 1 pi.. Iwasaki, H. ( I 9 6 I ) . The l i f e c y c l e of Porphyra tenera i n v i t r o . B i o l . B u l l . .121:173-187, i n c l . 5 tables, 16 f i g s . Jensen, W. A. (I962). B o t a n i c a l Histochemistry. W. H. Freeman and Co., San Francisco, 4o8 pp.,. i l l u s t r a t e d . Jonsson, S. (1957). Sur l a reproduction et l a s e x u a l i t e de 1'Acrosiphonia  spinescens (Kutz.) K j e l l m . C R. Acad. S c i . (Paris)~S**4:1251-55, i n c l . 15 f i g s . Jonsson, S. (I958). Sur l a structure c e l l u l a i r e et l a reproduction de Codiolum p e t r o c e l i d i s Kuck., Algue verte u n i c e l l u l a i r e endophyte. Cv R. Acad.. S c i . (Paris) 247:325-28, i n c l . l4 f i g s . a 1 Jonsson, S. (1959a). L'existence de l'alternance heteromorphe de generations entre 1'Acrosiphonia spinescens Kjellm. et l e Codiolum p e t r o c e l i d i s Kuck. C R. Acad. S c i . (Paris) 248:1565-67. i n c l . 1 f i g . Jonsson, S. (I959h). Le cycle de developpement du-Spongomorpha lanosa (Roth) Kutz. et l a nouvelle f a m i l l e des Acrosiphoniacees.. C R . Acad. S c i . (Paris) 248:1565-67, i n c l . 1 f i g . Jonsson, S. (i960) Nouveau type de cytodiere3e de l a cenocytique observe' • chez une Algue verte filamenteuse. C R . Acad. S c i . (Paris) 251:2390-2392, i n c l . 4 f i g s . 7 8 . Jonsson, S. (1962). Thesis. Recherches sur des Cladophoracees marines (structure, reproduction, cycles compares, consequences systematiques). Ann. Sc. Nat. Bot., 12 e serie, 3:25-230, incl. k tables, 4 8 figs; l 6 pis. add. . Jonsson, S. (1963). Sur quelques variations du cycle de development dans la famille des Acrosiphoniacees. C.R. Acad. Sci. (Paris) 256:5187-5189. . Jonsson, S. (1964a). Nouveau type de parthenogenese haplo'ids chez les Algues: la parthenogenese plasmogamique. C.R. Acad. Sci. (Paris) 258:2145-2148,- incl. 1 f i g . Jonsson, S. (1964b). Existence d'une caryogamie facultative chez 1'Acrosiphonia spinescens (Kutz.). C.R. Acad. Sc. (Paris) 258:6207-6209. Jorde, I. (1933). Untersuchungen Uber den Lebenszyklus von Urospora Aresch. und Codiolum A. Braun. Nyt. Mag. Naturvid. 73:1-19, incl. 5 figs., 1 pi. Juller, E. (1937);. Der Generations und Phasenwechsel bei Stigeoclonium  subspinosum. Arch. Protist. 89:53-93. (Not seen) : ~ Kornmann, P. (1961a). Uber Spongomorpha lanosa und ihre Sporophytenformen. Helgol. Wiss. Meeresunters. 7:195-205, incl. 6 figs. Kornmann, P. (1961b). Die Entwicklung von Codiolum gregarium. A. Braun, Helgol. Wis3. Meeresunters. 7 :252-259, incl. 5 figs. Kornmann, P. (1961c). Uber Codiolum und Urospora. Helgol. Wiss. Meeresunters. 8:42-57, incl. 9 figs. Kornmann, P. (1962). Eine Revision der Gattung Acrosiphonia. Helgol.. Wiss. Meeresunters. 8:219-242, incl. 11 figs. Kornmann, P. (1963); Die Ulotrichales, neu geordnet auf der Grundlage entvicklungsgeschichtlicher Befunde. Phycologia 3 :60-68, incl. 1 f i g . Kornmann, P. (1964a). Die Ulothrix-Arten von Helgoland.!. Helgol. Wiss. Meeresunters. 11:27-38 incl. 8 figs. Kornmann, P. (1964b). Uber Monostroma bullosum (Roth) Thuret und M. oxyspermum (Kutz.) Doty. Helgol.' Wiss. Meeresunters. 11:13-21, incl. 5 figs.. Kuckuck, P. (1894). Bemerkungen zur marinen Algenvegetation von Helgoland, Wiss. Meeresunters. Biolog. Anstalt. Helgol. N.F., 1:225-263, incl. 29 figs. Ledbetter, M. C. and K. R. Porter (I963). A "microtubule" in plant ce l l fine structure. Jour. Cell Biol. l£ : 2 3 9 - 2 5 0 , incl. 8 figs. 7 9 . Manton, I. ( 1952) . The fine structure of plant c i l i a . Symp. Soc. Exp. Biol. 6 :306-319, incl. 11 pis. Manton, I., Clarke, B. and Greenwood, A. D. ( 1955) . Observations with the electronmicroseope on biciliate and quadriciliate zoospores in green algae. Jour. Exp. Bot. 6:126-128, 10 figs. add. Manton, I. (1964) Observations on the fine structure of the zoospore and young germling of Stigeoclonium. Jour. Exp. Bot. 15:399-^-11, incl. 4 l figs. McLung, C. E. ( 1937) . Handbook of Microscopical Technique. 2nd edit. . Paul B. Hoeber Inc., New York, 698 pp., illustrated. Moewus, F. ( 1938) . Die sexualitat und der Generationswechsel der Ulvaceen und Untersuchungen uber die Parthenogenese der Gameten. Arch.. Protistenk. 91:357-441. (Not seen) Newcomer, E. H. ( 1953) . A new cytological and histological.fluid. Science I l 8 : l 6 l . ' Nicolai, E., and R. D..Preston (1952). . Cell-wall studies in the Chlorophyceae. I. A. general survey of submicroscopic structure in filamentous species. Proc. Roy. Soc. (London) B, l 4 0 : 244-274, incl. 4 tables, 21 figs. Printz, H. (I926). Die Algenvegetation des Trondhjemsfjordes. Norske ... Vidensk.-Akad. Oslo, Skrifter, I,.Mat.-naturw. Klasse, No. 5, 273 PP., incl. 8 tables, 29 figs., 10 pis., 1 map. Printz, H. (I932). Observations on the structure and reproduction in . . Urospora Aresch. Nyt. Mag. Naturvidensk. 70:273-287, 2 pis. add. Sarma, Y. S. R. K. (I963)• Contribution to the karyology of the Ulotrichales Phycologia 2:173-183, incl. 17 figs. Scagel, R. F. (1957)• An annotated l i s t of the marine algae of British Columbia and Northern Washington. (Including keys to genera)... Nat. Museum Can. Bull. No. 150, Biol. Ser. No. 5 2 , 289 pp. ' Setchell, W. A., and N. L. Gardner (1920). The marine algae of the Pacific coast.of North America. Part 2 , Chlorophyceae. Univ. Calif. Publ. Bot. 8:139-374, plates 9 - 3 3 add. Smith, G. M. (1938). Nuclear phases and alternation of generations in the Chlorophyceae. Bot. Rev. 4:132-139-Smith, G. M. ( 1944) . Marine algae of the Monterey Peninsula, California. Stanford Univ. Press, ix 622 pp., incl. 98 pis. Smith, G. M. ( 1 9 4 6 ) . On the structure and reproduction of Spongomorpha coalita (Rupr.) Collins. Jour, of the Indian Bot. Soc. Iyengar commemoration volume Pp. 201-208, incl, 1 table, 5 figs. 8o. Starr, R. C. ( I 9 5 6 ) . Culture collection of algae at Indiana University. Lloydia 19:129-149. Taylor, W. R. (1957). Marine algae of the Northeastern Coast of North • America. 2nd ed. Univ. Michigan Press. Ann Arbor. 509 pp. incl. 60 pis. Tiffany, L. H. (1924). A physiological study of growth and reproduction among certain green algae. Ohio Jour. Sci. 24:65-98, incl. 6 figs. Wille, N. (1900). Die Zellkerne bei Acrosiphonia (J. Ag.).Kjellm. Bot. Centralbl. 81:238-239. (Not seen). . Wurdack, M. E. (1923). Chemical composition of the walls of certain algae. Ohio Jour. Sci. 23:181-191, incl. 6 figs. Zimmerman, W. (1925). Helgolander Meeresalgen I-IV. Beitrage zur Morphologie, Physiologie und Okologie der Algen. Wiss. Meeresunt.ers, N.F. Abt. Helgoland 16:1-25, 1 pi. add. TABLE 1 COLLECTION SITES: CLONES ESTABLISHED AND FIELD TRIP DATA (June, 1963) W NO. OF June 1963 H - High mid-tide L - Low tide 5 UROSPORA H POSITION a CLONES o w NO. COLLECTION SITES IAT. N. LONG. W. DESCRIPTION OF AREA M E H SET UP SB3 o cn VANCOUVER ISLAND. B.C. 0 0 2 Sidney 48 39 123 23 Foot of Beacon Ave., 100 yds. south M 1 s -3 Oak Bay 48 25 123 !8 Below the Towers Apts, 1270 Beach Drive M 1*. 1 o* s c Ogden Pt. breakwater* 48 24 123 23 South side of breakwater and adjacent beach M 1*. 1 <? s c 5 Esquimalt 48 25 123 26 South of Yarrows Shipyard, 200 f t . (Duntze Head) M 1 s 8 Long Beach 48 59 125 37 South of Long Beach Store, 100 f t . j H M 1 1*. 1 d* s 9 Port Renfrew 48 31 124 27 Southwest area of Botany Beach j H M L 1 1? 1 s 10 Point No Point 48 23 123 55 About 125 yds. northeast of the point j H M L k 1,6 I d * !! s c 11 Kelsey Bay 50 23 125 58 Below Kelsey Bay Dock and north 100 yds. L 10 - -12 Campbell R. 50 01 125 13 South side of Discovery Crescent jetty M 1 ? -13 Qualicum Beach 49 21 124 27 Areas with boulder outcrops M 1 ? -14 15 Departure Bay SAN JUAN IS.WASH.U.S.A. Deadman's Bay 49 48 12 30 123 123 57 08 Southeast of ferry s l i p , 200 f t . A small bay 700 yds. south of Deadman's Bay M H M L 1 5 n 1*. l o * 5, 2,8!: s c 16 17 Friday Harbor FIDALGO IS.WASH. U.S .A. Anacortes 48 48 33 30 123 122 00 41 Below the new laboratory East of Shannon Point to the Gulf Island Ferry Terminal M M 1 1*. 1 0* s s c c 18 VANCOUVER. B.C. Tsawwassen Perry jetty 49 00 123 05 On both sides of the jetty past the ferry depot H M L 10 ,10,10 s c 19 20 Pt. Atkinson GALIANO ISLAND. B.C. Porlier Pass 49 49 20 01 123 123 16 35 Northwest of lighthouse, 50 - 100 yds. Below the Virago Point lighthouse M M 1 s * Also referred to as the Vic tor ia Harbour breakwater. ! Urospora s-peciosa. !! Urospora vancouveriana. cTUrospora wormskioldii 82. TABLE 2 FILAMENT GROWTH IN UROSPORA WORMSKIOLDII o ss 25 O M E H O CO E H Si CO O 2! CM 3 CD On 3 E H O U CO CO +> 0 nS >> si bD CO 1 o a a co E H C? On IN o W E H H •% CM O rH CM O OS M W rH CO 3 S3 CQ E H oq ml. mg. mg. mg. mg. mg. ml. mg. g. /Ag. /tg. >ug. S3 42 DAYS GROWTH Relative Density" Approx. Filament Length (cm.) 1 1000 (SEAWATER) 1000 ( ERDSCHREIBER ) 50 100 20 1000 72.2 8.8 0.5 500 10.5 (SWIl) 1000 72.2 8.8 0.5 500 10.5 6 10 0.02 ( SWII + BIOTIN + THIAMINE + B12 ) 1000 72.2 8.8 0.5 500 (3WI ) * Bar width represents approximate f r a c t i o n of flask-bottom covered with filaments. 83 TABLE 5 GERMINATION OP SINGLE ZOOSPORES AND CARRY-OVER OF SEXUALITY IN UROSPORA . WORMSKIOLDII FEMALE CLONE F11 GERMINATION and GROWTH of ZOOSPORES SEXUALITY ON o C°\ SUBCULTURE in W C*> vO vO vO vO C^l VO UR1 at 10°C. a § q cn REPLICA! June 20, July 2, Aug. 6, Sept.30, • > O Dec. 7, DEC. 11-31 JAN. 1-20 DAY 0 13 48 103 133 c s C S s 1 V — 500 (9 c elled) i i CO .H .aments look Dead ied 1 ml. .. 5 to each 1 t > T 2 -100 n >wth Vi Fil. 1 .copic .aments look Dead ied 1 ml. .. 5 to each t T T 1 3. </ ~ 40 II >wth Vi Fil. 1 .copic .aments look Dead ied 1 ml. .. 5 to each - 1 T t 4 u -o .aments look Dead ied 1 ml. .. 5 to each 5 1 only (5 mm) rH O O X! a -H i-i •H fx, O -a? 'CQ T T > T > 1 • > 100 1-2 T T > t 2 3 • >100 t T T T 4 V >100 t T t > T 4 5 >100 T T T > T 6 • >100 H t t T > T 7 Q 8 W E H 9 sa E H CO • > 100 R B I t 1 T > T LO CO • > 100 H i r T > T > T 1 g Hi 2 O • >100 IBB T T > t 5 3 • >100 T T 4 • >100 9B t T T > t 5 • >100 t T T T V Germination occurred ^ Sexual T Codiolum H H Abundant filament growth, filaments 2-3 cm long. Sol. 1 = Seawater Sol. 4 = SW1 + Biotin, Thiamine, B12 Sol. 5 = SW1 * UR1 = SW1 minus TRIS; supplemented with 6 Jig/l Biotin, 10 pg/l Thiamine, 0 . 0 2 pg/l B12 TABLE 4 GERMINATION OF SINGLE ZOOSPORES OF UROSPORA WORMSKIOLDII MALE CLONE M2A Qh. COMPOSITION OF MEDIA USED COMPOSITION 5-1 5-2 5-3 5-4 5-6 5-7 5-8 5-9 5-10 5-11 SEA WATER (32.7$) KNO3 KH2PO4 NaN03 NaaBPO/j. Fe EDTA TRI3 SOIL WATER Growth after 50 days culture ml. mg. mg. mg. mg. mg. mg. ml. 1000 72.2 8.8 0.5 1000 1000 72.2 8.8 0.5 500 1000 72.2 8.8 0.5 250 1000 72.2 8.8 0.5 0 1000 100 20 50 f-l o H I Sparse growth, filaments under 2 mm long. I Abundant growth, filaments 2-3 cm long. 1000 100 20 0.5 50 1000 72.2 8.8 1000 72.2 8.8 1000 72.2 8.8 1000 72.2 8.8 1000 500 250 TABLE 5 EFFECT OF TEMPERATURE ON FERTILITY OF NATURAL CODIOLUM Sept. 22 - Oct. 29. 1963 0/0 FERTILITY T°C 0 DAY 4 8 10 14 16 28 30 32 34 36 38 5° D 0 0 40 100 F 0 0 40 100 10° D 0 0 1 100 F 0 2 2 100 15° D 0 0 0 0 D I S C O N T I N U E D F 0 .5 0 10 0 Returned to 10°C 2 20° D 0 0 0 0 D I S C O N T I N U E D F 0 0 0 0 0 Returned to 10°C 0 D - Codiolum gregarium from a rock at Deadman's Bay. F - £. gregarium from a log at Friday Harbor 85 TABLE 6 EFFECT OF THERMOPERIOD ON FERTILITY OF NATURAL CODIOLUM Aug. 18 -Sept. 19 1963 O/O FERTILITY 0 1 DAY 4 5 6 7 8 9 10 11 12 13 17 18 19 CONTROL 0 60 97 TEST Thermoperiod OJ 19 13 in nuclear division 15 fertile 51 not fertile Thermoperiod - 8 hrs at 15 - 18 C coincident with an 8 hr photoperiodo Basal temperature - 10°C. . TABLE 7 EFFECT OF COLD SHOCK ON FERTILITY OF CULTURAL CODIOLUM Codiolum Isolate No. 8L°-UrXl 8L°-UrXl 8L°-UrXl 1 2 3 4 5 0/0 FERTILITY 9 10 11 18.8 8.1 12 13 C, Darkj CONTROL NOT FERTILE CONTROL NOT FERTILE TjT 16" 61J~ T C E 32 57 65 54 0 0 0 0 I T C E 0 100 100 84 86 • • • * 0 12 9.4 34.5 ^ ^ ^ • T C E 52 85 55 0 0 0 0 I ^ H B T C E 100 100 106 107 TCE - Total c e l l s examinedo 86. TABLE 8 EFFECT OF TEMPERATURE ON FERTILITY OF NATURAL CODIOLUM Origin Temp. Zygotes (8L x 18-3) M2A o* F l l o. URX1 F3 o. 5 C - - - - -10°C one c e l l - - - jfo 15°C - - - - jfo 20°C -fo - Percentage of Codiolum c e l l s f e r t i l e a f t e r 60 days growth. TABLE 9 EFFECT OF TEMPERATURE ON SEXUALITY IN UROSPORA WORMSKIOLDII Temp. Clone number 20°C 15°C 10°C 5°C F l l o_ S S _ M2A o* NG S - -URX1 NG - - -S - Sexual, NG - No growth. Key to symbols in Figures 1 - 40 c crosswall cb chromatic body ch chloroplast e eyespot f zoospore ridge f i b r i l f l flagellum g gametangium HHW higher high water 1 pectic lamella MHHW mean higher high water MLLW mean lower low.water n. . nucleus nu nucleolus o operculum P discharge pore py pyrenoid R reduction division s septa-like structure sh sheath z zoosporangium FIGURE 1 LIFE CYCLE OF UROSPORA MIRABILIS (After Jorde, 1933) FIGURE 2 LIFE CYCLES IN UROSPORA AND ULOTKRIX SPECIES (After Kornmaan, 1963) I II III I 9 t Y \ t Urotp. bang. Urotp. spea'osa UloHir. sp. (X) Ulafhnx sp. Ulothe tonata V Via Vib V f i f e i j | Urosp. penicilliformis V Urospora wormskioldii vatNfhfii- vetr. cai/rfat. FIGURE 3 FIELD TRIP STATIONS June 21 - 26, 1963 Scale of miles I 8 > S A W W A 8 3 E N ~ \ A N A C O R T E S «3 MATINGS OF UROSPORA WORMSKIOLDII June 29 - Jul;/ 3 1963 CO Tsawwassen Ferry Jetty, April 1, 1964 NW side, looking WE. • (l) Upper limit of Codiolum gregarium (13 f t . l e v e l ) . Close up of rectangular area in (A). (1) Large form of Urospora wormskioldii collected here April - May, I963 - 1964 at low tide (3 ft.) Refer to Fig. 24 for plant form. (2) U. wormskioldii most abundant on rocks in the mid-tide 15 - 8 ft.) but extending down to (1), (3 ft.) and up to (3), (11 ft.) Ogden Point Breakwater, June 21, I 9 6 3 . (1) Upper and lower limit of C. gregarium, (9.5 - 11.5 third t i e r from the top. ~" (2,3)U.. wormskioldii in the mid-tide, ( 4 - 9 f t ) . Water level 4 f t . Oak Bay, June 21, I 9 6 3 . (1) Position at which C. pusillum appeared in the f a l l . (2) U. wormskioldii in the mid-tide region. Rocks obtained at (1) in (D) with a dense covering of C. pusillum, October 9, I 9 6 3 . Point No Point, August 8, I963 looking NE. (1) U. vancouveriana, on rocks in the low tide area (3 -0 ftf. Water level 2.5 f t . U. wormskioldii from the water line up to the 9 f t . l e v e l . Deadman's Bay, August, I 9 6 1 . (1) Area where large forms of Urospora were collected in the spring of I 9 6 I , 1963 and 1964. (Refer to Fi g . 24 for plant form) and where U. speciosa was collected April 1, 1964.. Tidal level at (1) is 1 - 2 f t . (2) Rock on which a 3 m a l l patch (4 sq. in.) of f e r t i l e C. gregarium was found, January I 9 6 I . Tidal level at (2) i s 9 - 10 f t . (3) Rock referred to in Fig. 29G having C. gregarium on the upper surface and U. wormskioldii on the lower. FIGURE 5 92 VERTICAL DISTRIBUTION OF UROSPORA AND CODIOLUM H - i 13H . 12 m -p • r l O CD § CD fH CD «H 0 rl •3 9 • r l - P CD CD <H S • r l - P •a • r l CD •3 • r l E H OH s i O 7-1 o •3 . 4H 2H 1-0-- 1, 63 1963 CODIOLUM , UROSPORA o Q. O CD o rl PP ed z £? UJ ft SH CO o CO < < co { Q • d ,3 ft •H o 0 2 > rH O rH erf - P E D -p >» CD o P H 03 63,64 CC £ X Z < i s QC 111 L L Q IrH O •H CQ . fH } r S r ^ f-H O ft I I * I I o SH pq MEHW, MLLW, >— HEW from May 1 to Sept 30, 1963 HEW from May 1 to Sept 30, 1964 FIGURE 6 93, CODIOLUM - VARIATION IN ABUNDANCE AND MORPHOLOGY STATIONS NO LOCALITY Approx, Tide Level SEASONAL VARIATION IN ABUNDANCE DESCRIPTION TYPE P -H —I U O Cvj •H £*0 C^i CO O U O tiO O r-l •H -H Ti to ° £ 15 18 10 A 0 D F A J DEADMAN'S BAY FRIDAY HARBOR TSAWWASSEN JETTY OGDEN POINT BREAKWATER POINT NO POINT 9-10 9-10 9-10 11- 12 12- 13 13 9-11 60 62 63 1—1 I I I !_ 62 i i i i i i — 63 63 62 I 1 1 l _ 63 10-12 12.5 62 62 OAK BAY 10 61 63 64 63 64 64 63 64 63 63 64 62 63 One patch, 4 sq.in., on one rock. One patch, 4 so..ft., on one rock 0 On. east and west side • of sunken log i n a 6 i n . band,10 f t . long Patches, 1-2 sq.ft., on many rocks on the SW and SE side. Rocks on the SW side only One streak, 1 f t . x 6 i n 0 on a d r i f t log. Lower 2 ft.on the v e r t i c a l surface of the t h i r d t i e r blocks. Horizontal surface of fourth t i e r blocks. Few c e l l s from several rocks. Streaks, { x 12 i n . on NW side of d r i f t log-on rocks, 4-12 i n . ' diameter,upper sur-faces completely covered Scarce - A few c e l l s found from several rock scrapings. Common - Scattered patches, about 2 sq. i n . i n diameter, c e l l s widely separate. Abundant - Patches close together. Very abundant - Large surfaces covered, 2-6 sq. f t . , c e l l s closely appressed. Relative amounts of form variants. Worn away by rock abrasion during October gales. FIGURE 7 LIFE CYCLE OF UROSPORA WORMSKIOLDII (MERTENS) ROSENVINGE ( TYPE I ) CODIOLUM Urospora wormskioldii. • . (A - D, F - J, f i e l d . E, culture) Sections of filaments from Kelsey Bay, June 24, 1963. A. Basal c e l l with coarsely perforate chloroplast withdrawn from the end walls, x 800. B. Basal c e l l s of another filament with the chloroplast extending to the end walls, x 500. C. C e l l s further up the same filament as (B), with the chloro p l a s t perforations becoming f i n e r . . IKI. x 400. Filament showing abnormal c e l l d i v i s i o n * Two c e l l s are wound about each other r e s u l t i n g from oblique wall formation, x 500. Insert, x 50. Test tube culture of male clone M2A showing a r i n g of Codiolum (cr) at the o r i g i n a l air-water i n t e r f a c e . C e l l showing unconnected r i b b o n - l i k e c h l o r o p l a s t s . x 500. Developmental stages i n zoospore formation. G. F i n e l y r e t i c u l a t e chloroplast with conspicuous pyrenoids. IKI. x500.. H. Chloroplast becoming disorganized, pyrenoids incon-; spicuous. x 500. I. ' Zoospore d e l i m i t a t i o n , x 500. J. Beginning of zoospore elongation." x 500. Scale length, 50 p.-97. FIGURE 9 Urospora wormskioldii (A,B, D, E, G, field. C, F, H, I, culture) 'A, B. Formation of discharge pores (p) in sporangia. Safranin. x 1000. C. Ten-day-old germlings from zoospores. Acetocarmine. x 400. D, E. Zoospore release in vesicles, x 400. F. Dwarf plants after six months in 10 °/oo salinity seawater. Many rhizoids formed. IKI, eosine. x 500. G. Wall structure in empty sporangia, x 500. H. I. Dwarf plants after six months in 50 °/oo salinity seawater. Many large thick-walled cells produced. Rhizoids absent. H, x 400. I, x 80. Scale length, 50 yu FIGURE 9 9 8 . Urospora wormskioldii (Living field material from Deadman's Bay, I96I) Sections of one filament showing an increase in width apically. Rhizoids intramatrical. Note fourth cell from top growing through f i f t h c e l l . x 100. Sections of several filaments showing cells in different stages of development. x 50. Chloroplast of mature cell showing pyrenoids and perforate nature of the chloroplast. x 900. Surface of fertile sporangium showing star-like clusters of zoospores. x 500. Photograph of field plants in situ in the mid-tide region, x 2/3. Freshly liberated zoospores with four flage11a and long finely-drawn-out tails. x 500. Scale length, 50 /u. JTCKJRfi 1 0 100 Urospora vormskioldii (A - E , G - I, culture. F, field. Living material) Sections of filament from base to tip shoving extramatrical rhizoids and decreasing filament vidth. x 100. Cell of filament in (A) vith finely perforate chloroplast. x 500. .. .. • Cells of filament in (A) vith banded chloroplast. x 500* Male clone M2A, fertile zoosporangium (z) and fertile game-tangium (g). x 900. Zoospore squashed shoving the four "ridge" f i b r i l s (f) and four flagella ( f l ) . x 2000. End v a i l viev of mature cell of fi e l d filament .shoving chloroplast absent at both end vails.- x 100. Coarsely-perforate chloroplast showing prominent nuclei (n) and nucleoli (nu). x 1000. Fertile sporangium with zoospore arrangement reflecting a previous coarsely-reticulate chloroplast. x 1000. Cultural zoospores with short tails, x 500. Field zoospore. Acetocarmine. Phase, x 2000. Scale length A - D, F - I,. 50 / u ; E , 25 y u ; J , 20 /\x FI3URE 11 102. 103. FIGURE 12 Urospora vormskioldii (Culture, camera lucida drawings) A - H, N. Sections of one filament. A. Basal section shoving extramatrical rhizoids, x 200. B. Basal cell shoving ah early stage in rhizoid formation, x 400. C. Basal ce l l shoving coarsely-reticulate chloroplast. x 400. E. Vesicle of zoospores liberated through poire (p) in cell (D). x 400. F. Germling developing vithin empty sporangium, x 400. G. H,N. Sections of filaments shoving progressive decrease in filament -width tovards filament apex, x 400. I, J. Divaricate rhizoidal ends of another filament, x 400. K. Female gamete shoving asymmetry of body. Composite drawing of living and-stained gametes, x 2000. L. Nevly formed spindle-shaped male gamete, x 2000. M. Gametes in fusion. Aceto-orcein-iron-propiocarmine. x 2000. Scale length A - J; 50 j\x, K - M, 20 /U 10U. FIGURE 12 105. FIGURE 13 Urospora wormskioldii' (Culture) A. Female filament with gametangia. x 1000. B, C. Female gametes 12 hours after liberation. Note body asymmetry. B, x 2000. C, x 1000. D. Female gamete. Aceto-orcein-iron-propiocarmine. x 2000. E. Female gamete. Flagellar tips much finer. Crystal violet, x 1000. F. Male filament; gametangium in upper cell;:vesicle of male gametes liberated from middle c e l l . Methylene blue. . x 500. G. Male gametangium with spindle shape of male gametes evident. Methylene blue, x 1000, H. Abnormal male gamete with two normal flagella and two short . flagella. Iron-propiocarmine. x 1000. I. Abnormal male gamete with four flagella. Flagellar tips much finer. Crystal violet, x 1000. J. Male gamete with- small anterior projection. These are of common occurrence. Aceto-orcein-iron-propiocarmine. x 2000. . . • K. Newly liberated male gametes with characteristic spindle shape. Phase, x 1000. Scale length, 25 / U 1 0 6 . FiaURS 13 107. FIGURE 14 ' Urospora wormskioldii (A, f i e l d . B, C, culture) A. Stages i n gamete fusion. Middle r i g h t , two female gametes fusing with one male. Acetocarmine. Phase, x 2000. B. Codiolum c e l l s from female clone F l l ; t h i r t y days o l d . x 100. , C. Codiolum c e l l s from male clone M2A; s i x months.old. Phase, x 200. Scale length, 25 FIGURE 14 108. ,•» •» 'iff ' * i » — - ' ' i i r § ~ f 1 _^6s^ ^^^^ FIGURE 15 LIFE CYCLE OF UROSPORA VANCOUVERIANA (TILDEN) S.„ & G. ( TYPE II ) CODIOLUM FIGURE 16 EFFECT OF TEMPERATURE ON FILAMENT AND DWARF PLANT PRODUCTION IN UROSPORA VANCOUVERIANA I I Filaments.. •I Dwarf plants producing quadriflagellate zoospores. 1-551 Dwarf plants producing b i f l a g e l l a t e zoospores. 110. FIGURE 17 Urospora vancouveriana (A - E n f i e l d . F - J, culture) A. Dry mount of lower h a l f of one filament., C e l l s of upper h a l f were equal in" s i z e to those at the upper end of t h i s filament. B. Lower part of filament showing holdfast with several i n t r a m a t r i c a l rhizoids.. x 100. C. Chloroplast of mature c e l l showing f i n e perforations, x 500. D. Surface of f e r t i l e sporangium containing q u a d r i f l a g e l l a t e zoospores, x 1000. E. Surface of f e r t i l e sporangium containing q u a d r i f l a g e l l a t e zoospores, x 2000. Insert, x 1000. F. Filament showing development of l a t e r a l r h i z o i d s . x 100. G. Vegetative c e l l s showing coarsely-perforate c h l o r o p l a s t s . x 500. H. Empty sporangia showing discharge apertures and ruptured outer sheath, x 300. I. Holdfast of filament showing several extramatrieular r h i z o i d s . x 100. J. . Dwarf plants with f e r t i l e sporangia, containing b i f l a g e l l a t e zoospores, x 100. Scale length, 25 Urospora vancouveriana (Culture) Dwarf plant showing several empty sporangia and one liberated vesicle, x 500. Dwarf plant of an older culture with two sporangia contain-ing quadriflagellate zoospores, x 1000. Dwarf plant with liberated vesicle and several biflagellate zoospores. Note their round appearance. Phase, x 500. Liberation of vesicle containing biflagellate zoospores, two stages. Phase, x 500. Freshly liberated vesicle with biflagellate zoospores, x 1000. Biflagellate spores after 12 hours in the dark. Phase, x 1000. Biflagellate 3pores stained with iron-propiocarmine. Note the pyrenoid at the basal end, and the fine flagellar tips, x 2000. Codiolum cells produced by biflagellate.. zoospores; twenty-four days old. H, x 100.,. I, x 500. Scale length, 25 /u 113. FIGURE 19 Urospora vancouveriana (Camera lucida drawings of cultural material) A. Biflagellate zoospores. Iron-propiocarmine. x 2000. B, C. Germination stages of 'biflagellate zoospores. Feulgen. x 2000. D. Codiolum cell; ten days old. Feulgen. x 2000. E. Codiolum cell; thirty days old. Newcomer-iron-propiocarmine. x 2000. F. Vesicle of biflagellate zoospores, x 2000. G. Portion of filament showing concentration of nuclei at end walls and cell middle. Feulgen. x 2000. H. Young dwarf plant derived from quadriflagellate zoospores. '• Feulgen. x 2000. I. Dwarf plant with one cell undergoing division. Feulgen. x 1000. FIGURE 19 116. FIGURE 20 Urospora vancouveriana (Culture material stained with Feulgen-iron-propiocarmine) A. Germlings from quadriflagellate zoospores. Nucleolus un-stained. Note feulgen positive chromatic bodies (cb). x 2000. B. Pre-sporangial cells of dwarf plant; upper cell with nuclei in metaphase. x 1000. C. Cells of dwarf plant; upper left cell with nuclei in pro-phase; remaining cells with nuclei in interphase, x 1000. D. Apical portion of filament; upper cell with nuclei in meta-phase; lower cell with nuclei in interphase, x 1000. E. Apical cell of filament much squashed; nuclei in metaphase. Inserts prepared with the aid of camera lucida drawings. x 1000. F. Metaphases in a filament cell. Figure on left prepared with the aid of camera lucida drawings. x 3000. G. Filament showing multinucleate condition of each cell. Nuclei (n) are lightly stained and contain several Feulgen-positive chromatic bodies (cb). x 500. H. Metaphase in a filament cell showing some chromosomes to be elongate, x 3000. I. Rhizoidal nuclei (n), showing conspicuous nucleoli (nu) and 2-3 Feulgen positive chromatic bodies, x 2000. Scale length A - F, 25 yu; G, 50 /u; H, I, 10 yu -i FIGURE 20 117. Urospora wormskioldii. A cell in early stage of zoospore formation showing clusters of nuclei. Feulgen-iron-propiocarraine. x 1000. U. wormskioldii. A fertile cell with immature zoospores. Feulgen-iron-propiocarmine. x 1000. U. vancouveriana. A fertile cell with mature zoospores. Note the anterior projections of the chloroplast (ch). Feulgen-iron-propiocarmine. x 1000. U. wormskioldii. Plasmolized zoospores showing swellings (s) at the base of flagella. x 2000. U. wormskioldii. A germling with pyrenoids (py) appearing dissected. Feulgen-iron-propiocarmine. 10/u section, x 2000. ' Codiolum gregarium. A cell with pyrenoids (py) appearing dissected. Feulgen-iron-propiocarmine. 10 .u section, x 1000. I U. speciosa. Fertile field filament showing liberation of zoospores in a vesicle, x 500. U. speciosa. Cultural filaments showing their uninucleate condition. Newcomer-iron-propiocarmine. x 1000. Scale length, 25 yu FIGURE 21 119. FIGURE 22 120. U. PENICILLIFORMIS GAMETE COMPARISON OF TYPE I WITH U. MIRABILIS a U. PENICILLIFORMIS 20 10 0 10 20 KORNMANN (1961) TYPE I U. MIRABILIS PRINTZ (1932) JORDE (1933) AFTER DRAWINGS AFTER ^/MEASUREMENTS V GIVEN O f W0RMSK10LDI KORNMANN (I960 and TYPE II 1 1 1 . BIFLAGELLATA J O ~1 20 10 10 20 FIGURE 23 VARIATION AND MATINGS IN UROSPORA WORMSKIOLDII Codiolum gregarium from Deadman's Bay, February I96I. Nearly fertile cell showing pectic lamellations of the stipe. Com-posite photograph. Acetocarmine. x 300. C. gregarium. A fertile cell with quadriflagellate zoospores, x 800. C. gregarium zoospores germinating within the cell. IKI and eosine. x hOO. C. pusillum from Oak Bay, October 9, I963. Clava elongate with cell gradually tapering towards the base. Living, x 100. C. gregarium from Friday Harbor, August 30, I963. Clava" swollen with transition between clava and stipe abrupt. Living, x 100. Scale length, A - C, 50 /u; D, E, 500 /u Codiolum gregarium and cultural products Codiolum gregarium from Deadman's Bay, February I96I. A vegetative cell showing uninucleate - condition. Only the nucleolus is evident. Acetocarmine. x 1000. Quadriflagellate zoospores from C. gregarium. Note anterior projections of the chloroplast. Acetocarmine. x 2000. Developmental stages of germinating C. gregarium,zoospores. C. ~ Attached zoospore,, flagella lost. Acetocarmine. x 2000. D. Two-cell stage with zoospore t a i l s t i l l present. Acetocarmine. x 2000. E. Three-cell stage with lower cell developing into a rhizoid. Acetocarmine. x 2000. Germling showing filament and dwarf characteristics; upper five cells are filament-like; lower five cells are dwarf-plant-like. Acetocarmine. x 1000. Young'dwarf plant showing multinucleate condition and irregular arrangement of cells. Acetocarmine. x 1000. Scale length, 25 .u Codiolum gregarium in f i r s t division metaphase showing ten (?) chromosome pairs. Acetocarmine.. A, x 600. B, camera lucida. x 3000. C, x 3000. C. gregarium interphase nucleus showing coarse reticulation and nucleolus with large central vacuole. . Newcomer-iron-propiocarmine. x 1000. C. pusillum in f i r s t division metaphase showing thirteen chromosomes or twelve chromosomes and one nucleolus. Newcomer-iron-propiocarmine. x 2000. C. gregarium at the two-nucleate stage, with only the nucleoli evident. Acetocarmine. x 500. C. gregarium in third division metaphase. Insert is a higher magnification of rectangle showing eleven chromosomes. Aceto-carmine. G, x 500. Insert, x 3000. C. gregarium in ninth division metaphase. One optical plane "showing about 25 of the 226 nuclei seen; others were obscured by foreign material. Acetocarmine. x 500. C. pusillum subjected to daily thermoperiod. Optical sections of one nucleus showing five nucleoli-like structures and sev-eral smaller chromosome-like bodies (arrows). Living.x 1000.: One metaphase plate from cell (H) showing ten chromosomes. Camera lucida. Acetocarmine. x 3000. Zoospores of Urospora wormskioldii in f i r s t nuclear division showing eleven chromosomes. Colchicine arrested. N, camera lucida drawing of (M). Acetocarmine. L, x 1000. M, N, x 2700. C. pusillum in the four-nucleate stage showing a nucleus with a prominent nucleolus and several chromosome-like bodies (arrows). Living, x 1000. C. pusillum, interphase nucleus with prominent vacuolated nucleolus. Living, x 1000. U. wormskioldii germling from a_C. gregarium zoospore, colchicine arrested, ten chromosomes visible. R, is a higher magnification of rectangle in (Q). Acetocarmine. Q, x 1000. R, x 3000. Scale length, 10 /u 1 2 8 . F I G U R E 27. Urospora vormskioldii cultural filaments derived from Codiolum gregarium zoospores (Camera lucida dravings) A - D. Sections of one filament. A. Basal section shoving intramatrical rhizoids and divaricate rhizoidal ends, x 200. B-D. Distal sections of the same filament showing decrease in filament width, x 200. E, F. Cells of filament shoving multinucleate condition. Acetocarmine. x 800. G. Interpretive draving of vail structure during spore release. An additional inner wall appears to he formed during spore development. x 60O (approx.) H. Rhizoidal growth, showing middle wall.formed last, x 1500. I. Zoospore showing nuclear area unstained and prominent nucleolus. Acetocarmine. x 3000. J - L. Zoospores showing flagellar insertion between zoospore ridge f i b r i l s (f) and the four anterior projections of the chloroplast. x 3000. -Scale length, A - G, 50 yU; I - K, 25 yU Codiolum gregarium f e r t i l i t y i n nature and a f t e r transplantation Ladder on the o l d dock at the F r i d a y Harbor Marine Lab-o r a t o r i e s , lowered at (2) with weight (1) on the bottom. Rungs positioned every three feet with the lowest at the minus nine foot and the highest at the twelve foot t i d a l l e v e l . Log below the new laboratory b u i l d i n g from which the Codiolum samples were removed. Codiolum between arrows. Ladder rung with s i x samples stapled to the front and back surface of rung. Sampled area of log. Tabular r e s u l t s of transplant e f f e c t on Codiolum f e r t i l i t y . F e r t i l i t y of Codiolum on log i n (B). F e r t i l i t y of Codiolum on rock at Deadman's Bay. 131. FIGURE 28 TRANSPLANTATION EFFECT ON CODIOLUM FERTILITY Aug. - Sept. TRANSPLANTATION EFFECT E 30 29 f> Fertility or nuclear divisions FERTILITY IN NATURAL HABITAT DAY ft, 12.0 9.0 6.0 3.0 0.0 -3.0 -6.0 -9.0 3 s~ N "S _N_ 3 A 3 N_ "S N_ "S N_ "S N "3" o rH CO o 8 ND 1 r JL 2 1 3 l l 16 F ! FD i 2 i 2 i 5_ i 10 (XX 4 4 2 4' 1 3 8 9_, 7 • JO 15 ' 8 20 22 100 20 20 _ a _ a a a 100. a FD Log at Fri. Hartor f Approx. daily high tide; 1 Fertility on Log Date XXI 0 0 0 0 Aug.30 0 0 0 0 S*pt.7 0.3 - 6 Sept.15 20 1.5 50 30 Sept.29. Rock at Deadman1s f t 1 10 Air water TEMPERATUR 27.7 (1 PM) 11.6 " 10.6 E OC  13 (12 Noon) 14. 11 11. 11 10. -3 Qp t» • , 7 15 29 • 0 --0 -o - - 1 --20 f t . 10 F e r t i l i t y mWM Urospora. EZ53 Codiolum. FD - filamentous diatoms, x - sparse, xxx - dense ND - nuclear divisions, a - Codiolum absent 1 3 2 . FIGURE 2 9 Codiolum cells from Urospora wormskioldii female clone 8L, f e r t i l i t y induced with cold shock. Living material. A. _ Cell with two Codiolum cells, rounded up zoospores, one cell with an eyespot. (e) and one large donut-shaped mass, x 2 2 5 0 . B. Cell with rounded-up structures and one zoospore with two flagella. x 2 2 5 0 . C - E. . Cells forming irregular masses. C, x .2250. D, E, x 1000. FIGURE 29 133 FIGURE 30 EFFECT OF THERMOPERIOD OM iEQUALITY IU UROjPORA THERMOPERIOD BOX D a i l y 0 Culture 10 C Thermoperiod Culture 10 C 14 days F11 - Female clone of U. wormskioldii M2A - Male clone of U. wormskioldii URx1 - Unknown clone of U. wormskioldii (Produced Codiolum cells on two separate occasions) * Gametes in only one of the two tubes. ( ) Day of thermoperiod C - Codiolum G - Gametes BA3AL TEMP. 3-10°C 3 4 TIME IN HOURS FIGURE 51 CYTOCHEMICAL TESTS ON THE CELL WALLS OF U. WORMSKIOLDII AND C. GREGARIUM A. Urospora wormskioldii Sol ^ ?nCU 2 III . HCl-ZnCl (excest) ||g (cold 48 hrs) © n ^ Z n C l L ^ H C 1 _ Z n C 1 _ ^ Schweitzer^ || © s j I _ IKI-H2S0^ g j (excess) Soluble HCl-ZnCl2_ (Boil 2-3 min) _ Q . ZnClI S o l u b l e - 4 ^ I * * 0 9 - I K I - H ^ © ym /VZnClI © ^ •||: f ///+R. red © / « • ^^Schweitzer's ^ ^ m - H J S O ^ © (Heat 2-3 sec.) . ^ ,„....., , N HC1_ 0 „ S l u g — ^Schweitzer's. (8 min, 6ouC) cytase | ^ EH-H^O^ — S O 1 - f e l p 18^ HCl , I-II. KOH (Heat 2-3 sec)l II; >k (15 psi. \ 15 min) 1 vl-i-- • * 1 i Schweitzer's ZnClI 2 R. red IKI-H2S0, 1^* © © 2 R.rsd KI-H2S0^ © S o l u b l e ss) (t) (excess) ^ S o i u b l e B. Codiolum ereearium . , » ^ 15 min) ° " ' O O U ) ^ > „ K 1 - Z P C 1 2 Sol (Boil 2 -3-^ min) ^ ^ 5 s = E » Schweitzer's « r IKI-^SO^ © ^ Ar* ZnCH2 Q j V—H 2S0^,30^ n ?/ \ (warm) I j f. W 38i HCl * V / —•HCl-ZnCl,, * Soluble j \f (Boil 2-3 min) ZnClI2 © ^ 1 ^IKI-^SO^ © outer membrane, "»• inner cellulose walls, inv»«pectic material, ©positive test,©negative test Cellulose becomes blue with ZnClI2 or IKI-HgSO^  and pectin pink with ruthenium red FIGURE 32 CYTOCHEMICAL TESTS ON THE CELL WALLS OF S. COALITA AND C. PETROCELIDIS A. Spongomorpha coalita 0 ZnClI 2 — (excess) ZnClL. •^-Schweitzer's s IKI-H2S04 (excess) KOH 0 5 psi 15 min) / Sol H20- Soluble HCl-ZnClr (cold,48hrs"T HCl-ZnCl, (Heat 2-3 sec) HCl-ZnCl, KOH (15 psi 15+min) IKI-H2S0, © ZnClI 2 0 R. red © Schweitzer's 70*,H2S04—I | | (warm 2-3 I B S sec) „ N HC1 „ Slug (8 min,60°C) cytase 3Si HC1 I (Heat 1-3 sec) I Schweitzer's (excess) (Boil 2-3 min) © ZnClI2 (excess) R. red IKI-HoS0,, © ^1KI-H 2S0 4 © —-R. red © ^ Z n C l I 2 © 1 ^ -Schweitzer's -IKI-H2S0^ ^ IKI-H2S0^ © 0 (excess) >» Soluble B. Codiolum petrocelidis Sol Sol -1' H2S04 is* _KQH_ 15 mxn) HCl-ZnCl^ -(cold) - (Boil 2 - 3 ^ min) • •Schweitzer's C 72 hrs) IKI-H2S0^ © ZnClI2 R. red 30#, H2S0^ 38$, HC1 ZnCIL, © R. red © HCl-ZnCl, Soluble (Boil 2-3 min) •IKI-H2S04 © outer membrane, — inner cellulose walls, pectic material, ©positive test , © negative test Cellulose becomes blue with ZnClI2 or IKI-^SO^ and pectin pink with ruthenium red Urospora vormskioldii (A - H, field. I, J. culture. A l l fixed in 3:1) Filament under polarized light showing birefringence of walls. A, x 100. B, x 500. Wall layers of two adjacent cells. Ruthenium red. x 500. Walls stained with IKE - HgSO, showing separation of wall layers. D. Cells showing wall formation between dividing cells, x 500. E. Cells showing the sheath (sh) clearly separated from the inner wall layers, x 500. F. Note furrow (arrows) in cell•undergoing division, x 500. Filaments boiled in 38$ HC1 for 3 min showing birefringence, under polarized light, x 100. Basal section of filament after autoclaving in 23 M KOH at 15 psi for 15 min. Outer sheath dissolved, inner walls remained, IKE. H^ SO^ . x 500. Cultural filaments boiled for a few. seconds in 38$ HC1 followed by digestion with undiluted slug cytase for k hrs. Inner walls dissolved, outer sheath remained. .1. Outer sheath showing no birefringence under polarized light, x 300. J. Cells completely free in outer sheath.x 300. Scale length, 50 /u A, C - Urospora wormskioldii B, D - H - Codiolum gregarium ( A l l f i x e d i n 3:1) Autoclaved i n 23 M KOH at 15 p s i for 15 min and stained with ZnCll2. Inner layers remained, outer sheath dissolved on autoclaving. Phase, x 100. Untreated c e l l s , x 100. Untreated c e l l s under pol a r i z e d l i g h t showing birefringence i n walls, s t i p e and banded pattern i n protoplast, x 200. Boiled i n 38% HC1 for 2 min. C e l l s showing protoplast free i n outer sheath. Pectic layers of s t i p e dissolved. Phase, x 500. Boiled i n 38% HC1 for 2 min. C e l l s under p o l a r i z e d l i g h t showing absence of birefringence i n the outer sheath, and presence of a banding pattern i n the protoplast of one c e l l , x 200. Portion of s t i p e a f t e r autoclaving i n 23 M KOH at 15 p s i fo: 15 min. Outer sheath dissolved, inner p e c t i c layers re-main, x 500. Portion of s t i p e , untreated and stained with ruthenium red to show the p e c t i c l a m e l l a t i o n s . x 500. Scale length, 50 ,u FI3URB 34 H i O . A, C, D, F - Spongomorpha coalita B, E, G - Codiolum petrocelidis (A, C - G, fixed in 3:1. B, fixed in-formalin) -Untreated filaments, x 100. One cell of C. petrocelidis oriented stipe down in.Petrocelis  franciscana. Untreated. Aniline blue, x 300. Boiled in 38$ HCl for '2-3 min followed with digestion in undiluted slug cytase for k hours. C. Inner walls dissolved, outer sheath remained, x 100. D. Inner walls dissolved, outer sheath and part of cross-wall (c) lef t , x 100. Section of stipe. Untreated. Phase, x 500. Heated 2-3 sec. in HCl-ZnClg followed with an excess of Schweitzer's reagent, inner walls dissolved, crosswall shadows (c) left. Phase, x 100. Boiled in 38$ HCl for 2-3 sec, inner pectic layers dissolved, outer sheath remained. Phase. ' x 500. Scale length, 50 /u A f t e r Kuckuck, P. (1894, P. 259, F i g . 27). Codiolum p e t r o c e l i d i s n. sp. i n P e t r o c e l i s hennedyi. Helgoland, A normal c e l l , and c e l l s with l a t e r a l i n s e r t i o n s of the s t i p e on the c e l l . proper. Aft e r P r i n t z , H. (1926, F i g s . 64 - 72) Codiolum p e t r o c e l i s  Kuckuck i n Cruoria p e l l i t a showing c e l l s with l a t e r a l i n -sertions of the s t i p e and a l a t e r a l appendage on the s t i p e of one c e l l . A f t e r Kornmann, P. (1961a, p. 196, F i g . 1) Codiolum  p e t r o c e l i d i s plants from P e t r o c e l i s hennedyi, Helgoland. Several c e l l s having a l a t e r a l appendage on the s t i p e and i n one case on the c e l l proper.. Aft e r Jonsson, S. (1962, p. 74, F i g . 17, l a - f) Successive stages i n the development of Codiolum p e t r o c e l i d i s i n P e t r o c e l i d i s cruenta. Brittany, France. (a,b) i n d i v i d u a l s with t h e i r penetration tubes (stipes) oriented towards the e x t e r i o r of the host. (c,d) i n d i v i d u a l s with the f i r s t s t i p e reabsorbed. (e) i n d i v i d u a l which has changed i t s d i r e c t i o n of growth and i s producing a new s t i p e at the opposite end of the cell«> (f) a mature i n d i v i d u a l with a long second s t i p e and the f i r s t formed s t i p e remaining as an a p i c a l thickening. A f t e r Jonsson, S. (1958, p. 236, F i g . 11) A stage i n the development of Codiolum c e l l s reportedly derived from zoo-spores of Codiolum p e t r o c e l i d i s , Brittany, France, showing two membranes (septa) i n the s t i p e . A f t e r Fan, K. C. (1959, p. 6, F i g . 29, 34) (29) a stage i n the development of a zygote (C. p e t r o c e l i d i s ) from S. c o a l i t a . Moss Beach, C a l i f o r n i a . (34) Young c e l l s of C. p e t r o c e l i d i s which developed from zygotes of S_. c o a l i t a that were allowed to i n f e c t P e t r o c e l i s i n laboratory c u l t u r e s . The septa are shown here c l e a r l y . A f t e r Kornmann, P. (1961a, p. 196, F i g . 1). Codiolum  p e t r o c e l i d i s plants from P e t r o c e l i s hennedyi, Helgoland, showing the septate condition of the s t i p e . A f t e r Hollenberg, G. J . (1958, p. 250, F i g . 1 - 6) showing stages i n the germination and growth of zygotes of Spongomorpha c o a l i t a . Moss Beach, C a l i f o r n i a . FIGURE 36 Codiolum cell from Urospora clonal culture URX1 showing shallow""U" shaped pectic lamellations in the stipe. x 500. . . • Codiolum petrocelidis B. Cell from Porlier Pass, August 19^ 2, showing f i r s t and. second formed stipe. Note the opposite direction of the "V"-shaped lamellations taken in the f i r s t formed stipe ( 3 ) . Phase, x 1000. Whole cell, x 500. C-E. Cells from Deadman's Bay, June 1961.'" Note the "septa" like structures (s) distal.to the clava. Safranin. X 500. D. Cells growing with stipe directed up within Pj^ £°celis:'. franciscana. Safranin. x 500. Scale length, 50 /u FIGURE 38 \ C o d i o l u m p e t r o c e i L i d i s i n P e t r o c e l i s f r a n c i s c a n a A , B . C . p e t r o c e l i d i s f r o m P o r l i e r P a s s , A u g u s t 1962. 3 : 1 f i x . A n i l i n e b l u e . (1) C e l l w i t h s t i p e a t b o t h e n d s . (2) C e l l w i t h s t i p e o r i e n t e d down i n t h e h o s t . (3) C e l l w i t h s t i p e o r i e n t e d u p , and p r o t o p l a s t j u s t s t a r t i n g t o change i t s . d i r e c t i o n o f g r o w t h . L a m e l l a t i o n s (1) o f new s t i p e s e e n a t l o w e r e n d . A , x 100. B , x 600 . S c a l e l e n g t h , 100 , u FIGURE 38 1 4 9 . F. Cofllolum petrocelidis in Petrocelis franciscana • C. Spongomorpha coalita Two cells of Co petrocelidis from Porlier Pass, with stipes oriented up. 3:1 fix. Aniline blue. Phase. x 500*. A cell of C. petrocelidis from Porlier Pass, with change in direction of stipe growth. 3:1 fix. Aniline blue. Phase. x 500. Spongomorpha coalita from Mussel Point, Monterey, California, June- i960. A portion of a fertile filament showing an empty gametangiurn with an operculum (o). Dried herbarium -specimen. Phase, x 200. A cell of C. petrocelidis from Point No /Point, December 1963, showing a prominent apical cap. Formalin. Aniline . blue, x 200. Cells of C. petrocelidis from Point No Point, December , I963, oriented stipe down in fertile Petrocelis fran- ' ciscana. Formalin. Aniline blue, x 100. FIGURE 3 9 1 A, B, D -Scale length, 50 /U 151. FIGURE 40 Codiolum p e t r o c e l i d i s (A - D, f i x e d i n 3:1. E, f i x e d i n formalin) A - D. From P o r l i e r Pass. Stages i n formation of second s t i p e . C e l l A, B, D, are e a r l y stages and c e l l C, a l a t e r stage i n development. Phase, x 500. E. From Point No Point. Cell with first stipe broken off at arrow. Phase, x 200. Scale length equals 50 ,u 


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