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Cytological investigations of the genus Alaria greville : as it occurs on the West Coast of North America Robinson, Gordon George Christopher 1967

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The U n i v e r s i t y 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 GORDON G . C ROBINSON B.Sc, U n i v e r s i t y of Sfe Andrews, St Andrews, Scotland, 1964. MONDAY, OCTOBER 23, 1967, AT 3:30 P.M. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: B. N. Moyls T. • B i s a l p u t r a •. G. H.'N. Towers K. M. Cole E. B. tregunna R. F. Scagel V. C. Brink External Examiner: Alan P. Austin Department of Biology Un i v e r s i t y of V i c t o r i a Research Supervisor: K.'M. Cole CYTOLOGICAL INVESTIGATIONS OF THE GENUS ALARIA GREVILLE, AS IT OCCURS ON THE WEST COAST OF NORTH AMERICA, ABSTRACT Although the taxonomy of the-brown algal genus Alaria Greville of the order Laminariales'has recently been illucidated, neither the morphological nor the cytological aspects of the life-cycles of the species occurring on the west coast of North America has been investigated. Therefore a major part of this present project has been to establish the morphological and cytological phases of several species of Alaria.. Since the earliest investigations into the cytology of the Laminariales the concepts of the nuclear division processes have not changed as they have in higher organisms. A supplementary objective has therefore been the application of modern techniques and inter-pretations to nuclear divisions in the genus Alaria. During 1964-67 samples of Alaria marginata Postels et Ruprecht, Alaria nana Schrader, Alaria tenuifolia Setchell, Alaria taeniata Kjellman, Alarja fistulosa Postels e_t Ruprecht and Alaria grandjfolia J. Agardh were collected from the west coast of North America, from Cape St. Elias, Alaska to Pescadero Point, Ca l i -fornia. Cultures of these species were established from spore suspensions and maintained under controlled conditions until young sporophytes were produced. From these cultures i t has been demonstrated that the life-cycles of a l l six species show an alternation of heteromorphic generations: macroscopic sporophytic and microscopic dioecious gametophytic gene-rations. It has also been established that there is a corresponding chromosomal alternation of generations, the sporophyte being diploid and the gametophyte, haploid. The. development of male and femal gametophytes, the production of gametangia, f e r t i l i s a t i o n , and the early developmental stages of the young sporophytes have been examined. The similarities and differences between these phases in Alaria and other members of the Laminariales have been investigated and found to be similar except for the germination of zoospores of A. marginata, the formation of the egg cells in a l l the Alaria spp. and the occurrence of a possible " f e r t i l i s a t i o n pore" and "tube" in the eggs of A. tae.niata, The occurrence of parthenogenesis and the production of malformed haploid "parthenosporophytes" are reported. Meiosis in the immature zoosporangia and mitosis in gametophytes and young s.porophytes have been obr served, compared with these processes in other member of the Laminariales, and found to be similar in a number of cases.. However, the concepts of meiosis held by many earlier authors are not applicable to this process in Alaria. A haploid chromosome number of approximately 14 alternates with a diploid number of approximately 28 in A. marginata, A. nana, A. tenuifolia, A. fistulosa' and A. taeniata. The haploi number of A. grandifoila is approximately 24. Consequently only A. grandifolia can possibly be distinguished on the basis of chromosome numbers. The methods used in laminarian cytology and the d i f f i c u l t i e s involved in counting the extremely small chromosomes of the members of this order have been c r i t i c a l l y discussed. GRADUATE STUDIES Field of Study: Marine Phycology Dynamic Oceanography Synoptic Oceanography Chemical Oceanography Advanced Fresh Water Phycology Advanced Marine Phycology AWARDS 1964-1967 N-A.T.O. Studentship from S.R.C, London G.L. Pickard G.L. Pickard E. G r i l l J.R..Stein R.F. Scagel CYTOLOGICAL INVESTIGATIONS OF THE GENUS ALARIA GREVILLE, AS IT OCCURS ON THE WEST COAST OF NORTH AMERICA by GORDON G.C. ROBINSON B.Sc. U n i v e r s i t y of St Andrews, St Andrews, S c o t l a n d , 1964 A THESIS SUBMITTED IN. PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of BOTANY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, ,1967 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 o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y hi'S r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f B O T A K Y  T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a Date P^rd October. 1967 i i ABSTRACT Although the taxonomy of the brown a l g a l genus A l a r i a G r e v i l l e of the order Laminariales has recently been ifiJ.lucidated, neither the morphological nor the c y t o l o g i c a l aspects of the l i f e - c y c l e s of the species occurring on the west coast of North America has been investigated. Therefore a major part of this present project has been to e s t a b l i s h the morphological and c y t o l o g i c a l phases of several species of A l a r i a . Since the e a r l i e s t investigations into the cytology of the Laminariales the concepts of the nuclear d i v i s i o n processes have not changed as they have i n higher organisms. A supplementary objective has therefore been the application of modern techniques and interpretations to nuclear d i v i s i o n s i n the genus A l a r i a . During 1964-1967 samples of A l a r i a marginata Postels et Ruprecht, A l a r i a nana Schrader, A l a r i a t e n u i f o l i a S e t c h e l l , A l a r i a taeniata Kjellman, A l a r i a f i s t u l o s a Postels et Ruprecht and A l a r i a g r a n d i f o l i a J . Agardh were coll e c t e d from the west coast of North America, from Cape St. E l i a s , Alaska to lescadero Point, C a l i f o r n i a . Cultures of these species were established from spore suspensions and maintained under controlled conditions u n t i l young sporophytes were produced. From these cultures i t has been demonstrated that the l i f e - c y c l e s of a l l six species show an alternation of heteromorphic generations: macroscopic sporophytic and microscopic dioecious gametophytic generations. It has also been established that there i s a corresponding chromosomal alternation of generations, the sporophyte being d i p l o i d and the gametophyte, haploid. The development of male and female gametophytes, the production of gametangia, f e r t i l i s a t i o n , and the early developmental stages of the young sporophytes have been examined. The s i m i l a r i t i e s and differences between these phases i n A l a r i a and other members of the Laminariales have been investigated and found to be s i m i l a r except for the germination of zoospores of A. marginata, the formation of the egg c e l l s i n a l l the A l a r i a spp. and the occurrence of a possible " f e r t i l i s a t i o n pore" and "tube" i n the eggs of A. taeniata. The occurrence of parthenogenesis and the production of malformed haploid "parthenosporophytes" are reported. Meiosis i n the immature zoosporangia and mitosis i n gam-etophytes and young sporophytes have been observed, compared with these processes i n other members of the Laminariales, and found to be s i m i l a r i n a number of cases. However, the concepts of meiosis held by many e a r l i e r authors are not applicable to this process i n A l a r i a . A haploid chromosome number of approx-imately 14 alternates with a d i p l o i d number of approximately 28 i n A. marginata, A. nana, A. t e n u i f o l i a , A. f i s t u l o s a and A. taeniata. The haploid number of A. g r a n d i f o l i a i s approximately 24. Consequently only A. g r a n d i f o l i a can possibly be distinguished on the basis of chromosome numbers. The methods used i n laminarian cytology and the d i f f i c u l t i e s involved i n counting the extremely small chromosomes of the members of this order have been c r i t i c a l l y discussed. V TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . 3 a. General survey of the l i f e - c y c l e of the Laminar-i a l e s • • . . . a 3 b. The genus A l a r i a G r e v i l l e . . . '. . . !« . . '. . 16 MATERIALS AND METHODS . . . . 'V . . V . !i' v . 19 a1. Description of the macroscopic plants . :. . . 19 b. The i d e n t i f i c a t i o n of separate species. V !. ;. • v 19 c. The c o l l e c t i o n of material.: '. f. . \ <9 . 22 d. Culture methods • . '. . !. '. !. i* . . • !. v • 23 e. Fix a t i o n methods1. . !. !. . . '» . :. . . . . . . 26 f . Mordanting and staining methods ;. v i; y . . '• v 27 g. Pre treatments'; i«j . • • V • • > • . '•• . . • 30 h. Photographic methods. f. '. . . . . > !, ;. >. . 31 RESULTS : . V ! . v v V ! . .'• '.' '.' V »* W : i. :. 32 a!. Meiosis and the formation of meiospores !. . . • 32 b. Development of gametophytes and the formation of gametangia !. '. '. . y . !. \,< \m ; . v . . 36 c. F e r t i l i s a t i o n . . i. . . • >. v >. '. . > . . i . . v 39 di. Development of the young sporophyte . . B i . 40 e1. Mitosis i n gametophytes and young sporophytes '. '. 41 f . Chromosome numbers and s i z e . i . . . ^ . . . i. . 43 DISCUSSION1. ;. !.• ;. <»• !i- i . : >. !. >'i !. U! . !. '<.' 46 v i SUMMARY1, : : V '* !. V V I. !. f. . V '. *. * 60 BIBLIOGRAPHY . '.. !. V v . V . . j; . . r. . . i. j. t' . . r, 63 APPENDIX I Key to the genus A l a r i a . . 74 APPENDIX II S e m i - a r t i f i c i a l and a r t i f i c i a l culture media. 76 APPENDIX III Fixing, mordanting, softening and staining procedures 79 APPENDIX IV Ptetreatment procedures .82 APPENDIX V Tables I - VIII 84 Table I Members of the Laminariales i n which the l i f e -cycle has been investigated . . . . . . . . .85 Table II Chromosome numbers of the Laminariales . . . 89 Table III Frequency of male and female garnetophytes i n uniform cultures 92 Table IV Bivalent counts made during meiosis . . . . 94 Table V Haploid chromosome counts made at prometaphase of mitosis i n male and female gametophytes. . 95 Table VI Di p l o i d chromosome counts made at pro-metaphase of mitosis i n very young sporo-phytes. 96 Table VII Chromosome numbers of six species of the genus A l a r i a . 97 Table VIII Voucher specimens of A l a r i a .98 APPENDIX VI Plates 1 - 3 6 100 Plate 1 Habit drawing of A. marginata facing 101 Plate 2 Habit drawing of A. nana facing 102 Plate 3 Habit drawing of A. t e n u i f o l i a facing 103 Plate 4 Habit drawing of A. f i s t u l o s a facing 104 Plate 5 Habit drawing of A. taeniata . . . . . facing 105 Plate 6 Habit drawing of A. g r a n d i f o l i a . . . . facing 106 Plate 7 C o l l e c t i n g stations used i n i n v e s t i -gation facing 107 Plate 8 Meiosis i n A l a r i a spp facing 108 Plate 9 Meiosis i n A l a r i a spp.. . . . . . . . . facing 109 Plate 10 2nd d i v i s i o n of meiosis and the development of the sporangium . . . „ . facing 110 Plate 11 The germination of zoospores and the development of the female gameto-phyte/. . . <. '» . . . ! . ! . \ o . . !. !. facing 111 Plate 12 The development of the male gametophyte and the production of antheridia and antherozooids . !, ;. . • . . • ». . >. facing 112 Plate 13 Egg production and f e r t i l i s a t i o n . • • :. facing 113 Plate 14 F e r t i l i s a t i o n and the germination of the zygote. . . !. !, !. . . :. . j. <, . • !. facing 114 Plate 15 The development of the young sporo-phyte . « . . . [ . ! . V o . ! . . !. facing 115 Plate 16 The development of the young sporo-phyte facing 116 Plate 17 Mitosis i n A l a r i a spp . V . . . . . . . facing 117 Plate 18 Meiotic configurations i n A. marginata from which bivalent counts have been made. . . . . . . . . . . . . . o . . facing 118 Plate 19 Meiotic configurations i n A. nana from which bivalent counts have been made. . . . . . . . . . . . . . . . . facing 119 Plate 20 Meiotic configurations i n A. t e n u i f o l i a from which bivalent counts have been made facing 120 Plate 21 Meiotic configurations of A. f i s t u l o s a from which bivalent counts have been made facing 121 Plate 22 Meiotic configurations i n A. taeniata and A. g r a n d i f o l i a from which bivalent counts have been made . . . . . . . . facing 122 Plate 23 Meiotic configurations i n A. g r a n d i f o l i a from which bivalent counts have been made. . . . . . . . . . . . . . . . . facing 123 Plate 24 Mit o t i c configurations i n gametophytes of A»_ marginata and A. nana, from which haploid chromosome counts have been made facing 124 ix Plate 25 Mi t o t i c configurations { ± n gametophytes of A. nana and A. t e n u i f o l i a from which haploid chromosome counts have been made. . . . . . . . . . . . . . . . . . facing 125 Plate 26 Mi t o t i c configurations i n gametophytes of A. taeniata and A. f i s t u l o s a from which haploid chromosome counts have been made1. . '. :. . . „ . . !. . . !. . ^  '. facing 126 Plate 27 Mi t o t i c configurations i n a gametophyte of A. taeniata and a young sporophyte of A. marginata from which haploid and d i p l o i d chromosome counts have been made respectively. . . . . . facing 127 Plate 28 Mitotic configurations i n d i v i d i n g zygotes of A. marginata and A. nana from which d i p l o i d chromosome counts have been made1. . f. . !. v !. . . • facing 128 Plate 29 Mi t o t i c configurations i n di v i d i n g zygotes of A. nana and A. t e n u i f o l i a from which d i p l o i d chromosome counts have been made. . . . . . . . . . . . . facing 129 Plate 30 Mi t o t i c configurations i n young sporo-phytes of A. taeniata from which d i p l o i d chromosome counts have been made. . . . facing 130 X Plate 31 Mi t o t i c configurations i n a d i v i d i n g zygote of A. f i s t u l o s a and partheno-sporophyte of A. taeniata from which d i p l o i d and haploid chromosome counts have been made respectively facing 131 Plate 32 Graphic representation of chromosome counts made during meiosis i n A l a r i a spp.. . . . . . . . . . . . . . . facing 132 Plate 33 Graphic representation of chromosome counts made during mitosis i n male and female gametophytes of A l a r i a spp. . facing 133 Plate 34 Graphic representation of chromosome counts made during mitosis i n young cultured sporophytes of A l a r i a spp. . . facing 134 Plate 35 The e f f e c t of temperature on gametophytic growth i n A. g r a n d i f o l i a . . facing 135 Plate 36 The e f f e c t of temperature on gametophytic growth i n A. f i s t u l o s a . facing 136 x i ACKNOWLEDGEMENTS I sincerely wish to express my gratitude to Dr. K*iM. Cole for her d i r e c t i o n and advice during the course of t h i s study; to Dr. RiFi Scagel for making possible the c o l l e c t i o n of speci-mens from Alaska and for his continued interest and advice, and Dr. I£D. Druehl for financing the co l l e c t i o n s from Oregon and C a l i f o r n i a . I wish to record my indebtedness to Dr. E;Bv Tregunna and Dr. T. Bisalputra for t h e i r continued i n t e r e s t , and to the Department of Botany, University of B r i t i s h Columbia, as a whole, for continued help throughout the phases of thi s research. Further, I would l i k e to express my deep appreciation of the Science Research Council, London, U.Kfr, for providing me with a N.A.T.O. studentship for the years 1964-1967. F i n a l l y , I wish to thank Mrs Mary Robinson for her support and encourage-ment throughout t h i s study. INTRODUCTION Ideally a t o t a l understanding of the biology of any species and i t s r e l a t i o n s h i p with other s i m i l a r species demands a knowledge of a l l phases of i t s existence. However, the scope of the various aspects of biology i s so vast and b i o l o g i c a l research i s so compartmented that i n practice i t i s necessary deal with each s p e c i a l i s e d f i e l d i n d i v i d u a l l y . A r e l a t i v e l y large amount of information has been published concerning the Laminariales, an order of the Phaeophyta. This i s perhaps because of the economic importance of several genera of this order (Scagel, 1966). Taxonomic studies were obviously the f i r s t to be made, and i n i t i a l l y these were based almost completely upon the gross morphological c h a r a c t e r i s t i c s of the plants concerned. Subsequent taxonomic studies have unfortunately also been based upon incomplete information, which has resulted i n the weighting of ce r t a i n c h a r a c t e r i s t i c s over others. The l i f e h i s t o r i e s of many species of the Laminariales have been examined morphologically and i n almost a l l cases s i t has been shown that an heteromorphic alternation of generations e x i s t s , incorporating dioecious gametophytic generations. A few species have been investigated c y t o l o g i c a l l y . Others have not been studied at a l l . The taxonomy of the genus A l a r i a G r e v i l l e , a member of the Laminariales, has recently been investigated (Widdowson, 1964). -2-However, there have been no studies made of the l i f e - h i s t o r y or cytology of t h i s genus, as i t occurs on the west coast of North America. Since the time of K y l i n T s (1918) e a r l i e s t investigations into nuclear d i v i s i o n s i n the Laminariales there has been l i t t l e change i n the concepts of the d i v i s i o n processes. Although Naylor (1956) and Evans (1963b, 1965) have applied modern c y t o l o g i c a l techniques to nuclear d i v i s i o n s i n certa i n members of the order, there i s s t i l l an obvious need for the application of these to many more species. Consequently the present project was undertaken to esta b l i s h the l i f e h i s t o r i e s of several A l a r i a species morphologically and c y t o l o g i c a l l y , and to make a detailed comparative study of meiosis and mitosis within the genus. The species most thoroughly examined were those native to the shores of B r i t i s h Columbia. It was also proposed to determine chromosome numbers and to compare these numbers with others of the Alariaceae and the Laminariales as a whole. -3-LITERATURE REVIEf a. General survey of the l i f e - c y c l e of the Laminariales: In 1850 Thuret f i r s t observed the l i b e r a t i o n and germination of zoospores i n Sacchoriza bulbosa. Since that time a considerable amount of research has been conducted on the Laminariales, and a well defined picture of the l i f e - c y c l e t y p i c a l of t h i s order has emerged. The species i n which the l i f e - c y c l e has been investigated are l i s t e d i n Table 1. Thuret (1850) had c o r r e c t l y interpreted the product of the unilocular sporangia as zoospores. However, Drew (1910) regarded these sporangia i n Laminaria d i g i t a t a and Laminaria  saccharina as gametangia noting the "fusion" of non-motile "gametes". Sauvageau (1915a, 1915b, 1916a, 1916b, 1916c) and K y l i n (1916),, working independently on Laminaria f l e x i c a u l i s , Laminaria saccharina, Sacchoriza bulbosa and A l a r i a esculenta and Laminaria d i g i t a t a observed two morphological types of gametophyte, some of which had empty c e l l s . Although neither investigator was able to recognise antherozooids or f e r t i l i s a t i o n , they suspected that the two gametophytic types represented the d i f f e r e n t i a t i o n of male and female sexes and that f e r t i l i s a t i o n occurs i n t h i s generation. In 1918 Ky l i n established that reduction d i v i s i o n takes place i n the unilocular sporangia of the macroscopic plants of Chorda filum and Williams (1921) observed f e r t i l i s a t i o n i n Laminaria and Chorda. There followed Schreiber*s -4-(1930) observation that sex i s probably genetically determined i n Laminaria saceharina, since the male and female gametophytes are formed i n equal numbers from a single unilocular sporangium. Therefore, by the early 1930*s i t had been quite firmly established that the laminarian l i f e - c y c l e involves an alternation of heteromorphic generations, a conspicuous sporophyte and microscopic male and female gametophytes (Text F i g . 1). Text figure 1. Diagrammatic laminarian l i f e - c y c l e . SPOROPHYTE (macroscopic) A considerable amount of investigation has been conducted on the morphological phases of the laminarian l i f e - c y c l e . The development of the zoosporangium has been thoroughly examined i n Undaria undarioides, Eckloniopsis radicosa, Ecklonia cava (Nishibayashi & Inoh, 1960a ; Ohmori, 1967), A l a r i a valida, A l a r i a c r a s s i f o l i a , Laminaria yendoana, Laminaria cicherioides and Eisenia b i c y c l i s (Ohmori & Inoh, 1963; Ohmori, 1967), Chorda filum, Undaria p i n n a t i f i d a , Laminaria japonica -5-(Nishibayashi & Inoh, 1958; Ohmori, 1967), Laminaria  longipedalis, Laminaria angustata, Costaria costata, Ecklonia  s t o l o n i f e r a , Ecklonia kurome and A l a r i a angusta (Ohmori, 1967). Most workers have described the liberated zoospores as pyriform with two l a t e r a l l y inserted f l a g e l l a of unequal length. The longer flagellum projects ant e r i o r l y and the shorter, p o s t e r i o r l y . In Laminaria saccharina i t has been shown that the anterior flagellum i s t i n s e l l a t e d and the posterior whiplash (Manton & Clarke, 1951). After a short period of a c t i v i t y the zoospores come to rest, lose t h e i r f l a g e l l a , and secrete a r i g i d wall around themselves. Each spore then produces a germ tube and the i n i t i a l nuclear d i v i s i o n occurs. Both daughter nuclei migrate into the tube or one may remain i n the spore case as the germ tube i s cut off by a c e l l wall. Mitosis continues and a m u l t i - c e l l u l a r gametophyte develops. The antheridia produced on the male gametophyte may be either terminal or both terminal and i n t e r c a l a r y . A single b i f l a g e l l a t e antherozoid i s liberated from each antheridium. Each c e l l of the female gametophyte seems to be a pote n t i a l oogonium. It i s quite a common phenomenon for the female gametophyte to develop no further than one or two c e l l s before oogonium formation. A single egg i s extruded from each oogonium, and remains attached to the oogonium. The process of f e r t i l i s a t i o n has been observed i n a number of species, but very rarely has i t been investigated c y t o l o g i c a l l y . The nuclear cytology of t h i s process has been observed i n Pterygophora c a l i f o r n i c a (McKay, 1933), Eisenia  arborea (Hollenberg, 1939), Macrocystis i n t e g r i f o l i a (Cole, 1959) and Nereocystis luetkeana (Kemp & Cole, 1961). Both McKay (1933) and Hollenberg (1939) noted that nuclear fusion occurs while both nuclei are i n prophase. However, i n Nereocystis luetkeana f e r t i l i s a t i o n occurs while the nuclei of the eggsand the antherozooid are i n interphase, and involves a complete d i s s o l u t i o n of the nuclear membranes (Kemp & Cole, 1961). Following f e r t i l i s a t i o n the zygote divides m i t o t i c a l l y and develops into the young sporophyte. A uniseriate filament of four to twelve c e l l s i s f i r s t produced. D i v i s i o n i n a second plane then r e s u l t s i n the formation of a monostromatic blade. The basal c e l l s of t h i s blade give r i s e to rhizoids which i n i t i a l l y remain i n the empty oogonium. D i v i s i o n i n a t h i r d plane produces a distroma, within which i s l a i d down the p r e c o r t i c a l and premedullary t i s s u e . This sequence has been described for Costaria t u r n e r i , Undaria p i n n a t i f i d a and Laminaria (Yendo, 1911). A number of deviations from an alternation of d i p l o i d sporophyte and haploid male and female gametophytes have been recorded i n the Laminariales. In Chorda tomentosa (Sundene, 1963) i t i s reported that the gametophytic generation i s monoecious. The gametophytes of t h i s species give r i s e to both -7-oogonia and antheridia, although no antherozooids have actually been observed. It has also been demonstrated that i n this species sporophytes can be produced from uni-gametophytic cultures (Sundene, 1963). This observation does not, however, preclude the p o s s i b i l i t y of parthenogenesis, which i s often stated as taking place i n the Laminariales. In Laminaria d i g i t a t a and Laminaria hyperborea the formation of sporophytes without f e r t i l i s a t i o n of the egg has been reported by Shreiber (1930). This phenomenon has also been reported i n Laminaria r e l i g i o s a , Laminaria japonica, Laminaria angustata, Laminaria angustata var. longissima, Laminaria ochotensis, Laminaria d i a b o l i c a , A l a r i a c r a s s i f o l i a , Undaria p i n n a t i f i d a and Arthrothamnus bif i d u s by Yabu (1964a) and i n Nereocystis  luetkeana by Kemp and Cole (1961). Only i n Nereocystis  luetkeana wassit shown that these sporophytic plants possess an haploid chromosome complement. In the Laminariales i t appears that reproductive i s o l a t i o n i s not a sound c r i t e r i o n on which to base the d e f i n i t i o n of species, since r e l a t i v e l y free inter-crossing has been shown between a number of species. Yabu (1964a) demonstrated that crossing occurred between Laminaria r e l i g i o s a , Laminaria japonica and Laminaria angustata, and between Laminaria japonica and Laminaria d i a b o l i c a . P r i o r to t h i s Sundene (1958) had successfully crossed v a r i e t i e s of Laminaria d i g i t a t a , but not separate species. However, no chromosomal evidence has been -8-presented to substantiate any of the crossing experiments conducted. Investigations of the c y t o l o g i c a l aspects of the I.aminarian l i f e - c y c l e were i n i t i a t e d by K y l i n (19,18) . His account of meiosis as i t occurs i n zoosporangium of Chorda filum closely resembles modern day descriptions. In meiosis the interphase nucleus i s either r e t i c u l a t e or granular. A r e t i c u l a t e state i s described i n Laminaria angustata (Nishibayashi 8s Inoh, 1956; Ohmori, 1967), Chorda filum (Nishibayashi & Inoh, 1961a, 1961b; Ohmori, 1967), Costaria  costata (Nishibayashi & Inoh, 1957; Ohmori, 1967), Undaria  undarioides (Nishibayashi & Inoh, 1960b; Ohmori, 1967), Ecklonia s t o l o n i f e r a , Ecklonia cava (Ohmori, 1965, 1967), Undaria p i n n a t i f i d a (Inoh & Nishibayashi, 1955, 1960; Ohmori, 1967), Laminaria longipedalis, Laminaria yendoana, Eisenia  b i c y c l i s , Eckloniia kurome, Eckloniopsis radicosa (Ohmori, 1967), Pterygophora c a l i f o r n i c a (McKay, 1933), Egregia  menziesii (Myers, 1928) and Laminaria f l e x i c a u l i s and Laminaria  saccharina (Magne, 1953). Evans (1965) indicates a granulart interphase condition i n Laminaria saccharina, Laminaria d i g i t a t a , Laminaria hyperborea, Laminaria ochroleuca, A l a r i a esculenta, Chorda filum and Sacchoriza polyschides as do Kemp 8s Cole (196,1) i n Nereocystis luetkeana. There i s usually one d i s t i n c t nucleolus, although the occasional occurrence of two has been reported (Nishibayashi 8s Inoh, 1957, 1960b, 1960c, 1961a, 1961b; -9-Ohmori, 1965, 1967; Yabu, 1957, 1958, 1964b, 1965). At the onset of prophase I the nucleus enlarges. A reticulum either becomes more d i s t i n c t or, where a granular interphase has been observed, appears for the f i r s t time (Evans, 1965; Kemp & Cole, 1961). According to most workers the chromonemata then aggregate i n a mass of loops, which concentrates on one side of the nucleus. However, Evans (1965) states that t h i s aggregation occurs only very occasionally i n Laminaria saccharina, Laminaria d i g i t a t a , Laminaria hyperborea, Laminaria ochroleuca, Alaria.esculenta, Chorda filum and Sacchoriza polyschides. A description of the zygotene stage i s seldom presented. It appears that the congregation of chromonemata to one side of the nucleus and the synaptic stage are considered synonomous (Hollenberg, 1939; Inoh & Nishibayashi, 1954, 1955, 1960; McKay, 1933; Myers, 1928; Nishibayashi & Inoh, 1956, 1960b, 1960c, 1961a, 1961b; Ohmori, 1965, 1967; Yabu, 1957, 1958, 1964b, 1965; Yabu & Tokida, 1963). Evans (1965) describessthe appearance of "knots" at t h i s stage of d i v i s i o n . In Nereocystis luetkeana the .resuflt of this process i s • f a compared witb, " s y n i z e s i s " as i t occurs i n higher plants, although the actual process of pairing could not be observed (Kemp & Cole, 1961). Pachytene and diplotene of meiosis are most often described as a "spireme" stage. However, th i s concept i s questioned i n Nereocystis luetkeana due to the i m p r a c t i c a b i l i t y of observing -10-the continuous nature of the chromatin (Kemp 8s Cole, 1961). The spireme thickens towards the onset of diakinesis (Hollenberg 1939; Inoh 8s Nishibayashi, 1954, 1955, 1960; McKay, 1933; Myers, 1928; Nishibayashi 8s Inoh, 1956, 1960b, 1960c, 1961a, 1961b; Ohmori, 1965, 1967; yabu, 1957, 1958, 1964b, 1965; Yabu 8s Tokida, 1963), although Yabu and Tokida (1963) have reported a resting stage prior to d i a k i n e s i s , associated with a reduced s t a i n a b i l i t y of the chromatin. By diakinesis the nucleolus and nuclear membrane have generally disappeared, although i n Costaria costata (Nishibayashi 8s Inoh, 1957) , Laminaria  d i g i t a t a , Laminaria saccharina, Laminaria hyperborea, Laminaria  ochroleuca, A l a r i a esculenta, Chorda filum and Sacchoriza  polyschides (Evans, 1965) the nuclear membrance can persist u n t i l the chromosomes are aligned i n metaphase I. X-, Y-, 0-, V- and Il-shaped bivalent configurations are described i n diakinesis i n Laminaria angustata (Nishibayashi 8s Inoh, 1956; Ohmori, 1967), Costaria costata (Nishibayashi 8s Inoh, 1957; Ohmori, 1967), Undaria undarioides (Nishibayashi 8s Inoh, 1960b, 1960c; Ohmori, 1967), Chorda filum (Nishibayashi 8s Inoh, 1961a, 1961b; Evans, 1965; Ohmori, 1967), Ecklonia s t o l o n i f e r a and Ecklonia cava (Ohmori, 1965, 1967), Undaria p i n n a t i f i d a (Inoh 8s Nishibayashi, 1954, 1955, 1960; Ohmori, 1967), Pterygophora c a l i f o r n i c a (McKay, 1933), Nereocystis luetkeana (Kemp 8s Cole, 1961) Ecklonia radicosa, Laminaria longipedalis, Laminaria yendoana, Eisenia b i c y c l i s , Ecklonia kurome (Ohmori, .1967), Laminaria  d i g i t a t a , Laminaria saccharina, Laminaria hyperborea, Laminaria  ochroleuca, A l a r i a esculenta, Chorda filum and Sacchoriza  polyschides (Evans, 1965). Diakinesis marks the end of the meiotic prophase, and the chromosomes then amass to form the equatorial plate of metaphase I. Spindles appear at t h i s stage and centrosome-like bodies have beensreported i n a l l the species studied except Eisenia arborea (Hollenberg, 1939) Egregia menziesii (M^ers, 1928), Laminaria f l e x i c a u l i s and Laminaria  saccharina (Magne, 1953), Undaria undarioides (Nishibayashi 8s Inoh, 1960b, 1960c; Ohmori, 1967), Chorda filum (Nishibayashi 8s Inoh, 1961a, 1961b; Ohmori, 1967), Laminaria lon g i p ^ d a l i s, Ecklonia kurome (Ohmori, 1967) and the seven species studied by Evans (1965). Only one centrosome has been recorded i n the f i r s t d i v i s i o n of meiosis i n Laminaria japonica (Abe, 1939), Arthrothamnus b i f i d u s (Yabu 8s Tokida, 1963) and Nereocystis  luetkeana (Kemp 8s Cole, 1961) . During anaphase I the two t i g h t l y associated chromosome masses separate and move towards the poles. At t h i s stage i n Nereocystis luetkeana (Kemp 8s Cole, 1961) the presence of some bridges indicates a minor degree of chromosomal non-disjun c t i o n . In telophase I nuclear membrances and n u c l e o l i appear again. In Laminaria digitata,Laminaria saccharina, Laminaria hyperborea, Laminaria ochroleuca, Chorda filum, A l a r i a -12-esculenta and Sacchoriza polyschides prophase II appears to follow telophase I very quickly, since there i s a s c a r c i t y of binucleate c e l l s (Evans, 1965). The second d i v i s i o n i n the sporangium i s equational. Where chromosome coants have been possible at t h i s stage they have been used to confirm the fact that the i n i t i a l d i v i s i o n i s reductional. M i t o t i c d i v i s i o n s follow u n t i l 32 nuclei are produced. In Pterygophora c a l i f o r n i c a (McKay, 1933) and Eisenia  arborea (Hollemberg, 1939) 64 nuclei have been observed on occasion. In Chorda filum the f i n a l number of nuclei i s 16 (Nishibayashi & Inoh, 1961a, 1961b; Ohmori, 1967) and i n Sacchoriza bulbosa i t i s 128 (Sauvageau, 1915a). Mitosis has been most commonly described as i t occurs i n the gametophyte and developmental stages of the young sporophyte. In Laminaria f l e x i c a u l i s and Laminaria saccharina (Magne, 1953) and Chorda f ilum (Kylin, 1918) mitosis has been followed i n meristoderm c e l l s . McKay (1933) studied mitosis i n the inner c o r t i c a l c e l l s of Pterygophora c a l i f o r n i c a . Perhaps the most complete descriptions of mitosis are those of Naylor (1956) and Evans (1965) i n Laminaria d i g i t a t a , Laminaria saccharina, Laminaria hyperborea, Laminaria ochroleuca, A l a r i a esculenta, Chorda filum and Sacchoriza polyschides. The interphase nucleus i s granular with a d i s t i n c t nucleolus. Both Naylor (1956) and Evans (1965) have shown that the interphase nuclei -13-of the male and female gametophytes and the young sporophytes have d i f f e r e n t s t a i n a b i l i t i e s . The highest s t a i n a b i l i t y exists i n the male gametophyte and the lowest i n the female gametophyte. An intermediate s t a i n a b i l i t y was found i n the young sporophytes. At the Onset of the mitotic prophase the nucleus enlarges. This increase i n size i s most obvious i n the d i v i s i o n prior to egg formation i n the female gametophyte (Evans, 1965;Naylor, 1956). In prophase a reticulum of beaded threads appears. These threads contract during prophase u n t i l tiny spheres (Naylor, 1956) or rods (Evans, 1965) remain. By this prometaphase stage the nucleolus has disappeared and the nuclear membrane has broken down. At metaphase the chromosomes are t i g h t l y associated, forming a metaphase plate. Spindles have been observed (Naylor, 1956), and i t i s possible to distinguish chromatids at th i s stage only (Evans, 1965). In anaphase the chromosomes move apart i n t i g h t l y associated masses. Bridges and lagging chromosomes have been observed at this stage (Naylor, 1956). In telophase there i s a reversion to the granular state, accompanied by the reappearance of n u c l e o l i and nuclear membranes. Walker (1954), describing mitosis i n Laminaria d i g i t a t a gametophytes and young sporophytes, introduced a theory of endomitosis. He claimed that the nuclear membrane breaks down and the chromosomes multiply i n the cytoplasm. No other worker has reported any such phenomenon. Although -14-multinucleate c e l l s have been observed i n some young sporophytes, i t has been assumed that these abnormalities would not survive to maturity (Kemp & Cole, 1961). There are r e l a t i v e l y few accounts of the morphology of the chromosomes of the Laminariales, perhaps because of t h e i r exceedingly small s i z e . The r e l a t i v e sizes of the chromosomes of both the haploid and d i p l o i d complements are reported for Egregia menziesii (Myers, 1928). The eight chromosomes of the haploid complement were measured at mitotic metaphase i n the sporangium; f i v e are quite large, two are smaller and one i s smaller s t i l l . Four of the chromosomes of the d i p l o i d complement at anaphase i n somatic mitosis are very small and twelve are much larger. McKay (1933) reported the actual sizes of chromosomes, as measured at metaphase i n d i p l o i d somatic c e l l s , i n Pterygophora c a l i f o r n i c a . She observed three pairs of rod-shaped chromosomes, the largest of which measured 0.8 microns, nine pairs of ovoid chromosomes measuring 0.3 - 0.6 microns, and one pair of minute spheroid chromosomes, too close to the ultimate resolution of the l i g h t microscope to measure. In Laminaria  d i g i t a t a , Laminaria saccharina, Laminaria hyperborea, Laminaria  ochroleuca, A l a r i a esculenta and Chorda filum the chromosomes of the gametophytM generation, as measured at mitotic metaphase, range i n s i z e from 0.4 to 1.7 microns (Evans, 1963a, 1965). One chromosome i n the female gametophyte i s 1.3 to 1.7 microns long. -15-Such a chromosome could not be found i n the male gametophyte. The implication behind such an observation i s obviously that heteromorphic sex chromosomes exist i n these species. To e s t a b l i s h t h i s c l e a r l y requires a meiotic configuration showing the pairing of unequal chromosomes. No t r u l y conclusive configuration has been found (Evans, 1965). In Sacchoriza  polyschides, however, a much larger chromosome has been recorded. In this species the chromosomes of the female gametophyte can be grouped according to s i z e : three are 0.4 - 0.7 microns; the majority, 0.8 - 1.0 microns; three, 1.1 - 2.6 microns; and one, 4.0 - 6.0 microns (Evans, 1965). No chromosome has been accounted for i n the male gametophytes which are as large as 4.0 - §.0 microns. In diakinesis t h i s large chromosome has been observed paired to a smaller. From these observations Evans (1965) concluded that an X/Y sex mechanism exists i n Sacchoriza  polyschides. Chromosome numbers i n the Laminariales have shown a wide d i v e r s i t y since the i n i t i a l c y t o l o g i c a l investigation of K y l i n (1918). This d i v e r s i t y i s spread throughout a number of species and i n some cases even i n a single species. Chromosome counts which exist at the time of this research are presented i n Table I Kemp & Cole (1961) have tenta t i v e l y suggested that perhaps a polyploid series around the number ten might exi s t i n the Laminariales, but that much more information i s required before any conclusive statement can be made. -16-b. The genus A l a r i a G r e v i l l e The genus A l a r i a , characterised by "fronds membranaceous, furnished with a percurrent cartilaginous midrib, the stem pinnated with d i s t i n c t l e a f l e t s " , ( G r e v i l l e , 1830), has for some considerable time presented a major problem i n the taxonomy of the Laminariales. However, Widdowson (1964) has applied numerical methods to the taxonomy of the genus, and following extensive population studies was able to confirm the i d e n t i t y of ten species. There are four other species which Widdowson (1964) was SQt able to study to the same extent. The 14 species of the genus are as follows : A l a r i a angustata Kjellman A l a r i a c r a s s i f o l i a Kjellman A l a r i a c r i s p a Kjellman A l a r i a esculenta (L.) G r e v i l l e A l a r i a f i s t u l o s a Postels et Ruprecht A l a r i a marginata Postels et Ruprecht A l a r i a ;nana Schrader A l a r i a praelonga Kjellman A l a r i a taeniata Kjellman A l a r i a t e n u i f o l i a S e t c h e l l -17-A l a r i a g r a n d i f o l i a * J . Agardh A l a r i a ochotensis* Yendo A l a r i a paradisea* (Miyabe et Nagai) A l a r i a p y l a i i * (Bory) G r e v i l l e . (* studied less extensively). Except for taxonomic and d i s t r i b u t i o n a l records of this genus, very few investigations have been conducted. Chromosome numbers have been recorded for three species (Table I I ) . A t y p i c a l alternation of generations has been established i n A l a r i a  esculenta (Sauvageau, 1916b, 1916c; Evans, 1965), and A l a r i a  c r a s s i f o l i a (Yabu, 1964a). Yabu (1964a) has reported the formation of parthenosporophytes i n A l a r i a c r a s s i f o l i a , and the p o s s i b i l i t y of an X/Y sex mechanism has been proposed, but not substantiated, for A l a r i a esculenta (Evans, 1965). An " A l a r i a -type" ontogeny of the zoosporangia has been shown i n A l a r i a  c r a s s i f o l i a by Nishibayashi 8s Inoh (1958), Ohmori 8s Inoh (1963) and Ohmori (1967). According to these authors the meristoderm c e l l of the sporophyll divides to give r i s e to an outer c e l l and a basal c e l l . The outer c e l l produces a paraphysis, and the basal c e l l f i r s t produces another paraphysis and then the zoosporangial mother-cell. However, this i s not consistent throughout the genus. In A.valida (-A.marginata (Widdowson, 1964)) the basal c e l l gives r i s e d i r e c t l y to the zoosporangial mother-cell (Ohmori 8s Inoh, 1963; Ohmori, 1967). This i s a -18 -"Laminaria-type" ontogeny, and has been shown to occur i n A . c r a s s i f o l i a also (Ohmori 8s Inoh, 1963; Ohmori, 1967) . The formation of the zoosporangia has been described i n A . f i s t u l o s a Ofcibbe, 1915) , but not with the same accuracy. -19-MATERIALS AND METHODS a. Description of the macroscopic plants: (Text, F i g . 2) The A l a r i a plant i s characterised by a d i s t i l blade and midrib, and a proximal stipe attached to the substrate by haptera. At the junction of the blade and the sti p e there i s an i n t e r c a l a r y meristem. Below the in t e r c a l a r y meristem i s the rachis zone, marked by the attachment of sporophylls and remnants of sporophylls. The sporophyll t y p i c a l l y consists of a f e r t i l e sorus areas on both surfaces surrounded by ?i s t e r i l e areas. The s o r i , formed i n late spring or early summer, are generally r e s t r i c t e d to the sporophylls, although one population of A l a r i a  t e n u i f o l i a has been found i n which the basal area of the blade also possesses f e r t i l e t i s s u e . The growth of A l a r i a i s mainly r e s t r i c t e d to the in t e r c a l a r y meristem. However a ce r t a i n amount of d i f f u s e growth presumably occurs from the meristoderm layer of c e l l s over the surface of the whole plant. The genus A l a r i a i s extremely commonly represented i n the low i n t e r t i d a l and sub-tidal zones of the North East P a c i f i c . o There i s no evident northern l i m i t to the genus, while the 20 C isotherm of maximum sea temperature appears to be the southern l i m i t (Widdowson, 1964). b. The i d e n t i f i c a t i o n of separate species. In Widdowson's investi g a t i o n of the taxonomy of A l a r i a , Haptera -21-thirteen measurements were incorporated. However, from the results obtained, the author constructed a key based upon stipe length and shape, sporophyll width and shape, mode of attachment of sporophylls and shape of the rachis (Widdowson, 1964). This key (Appendix I) has been used throughout the current study for the i d e n t i f i c a t i o n of A l a r i a plants. When c o l l e c t i n g material for c y t o l o g i c a l investigation, at least t h i r t y plants were used from each population before an i d e n t i f i c a t i o n was made. Id e n t i f i c a t i o n s were always made on wet f e r t i l e plants. The species which have been i d e n t i f i e d and used i n this project are; A l a r i a marginata Postels et Ruprecht A l a r i a nana Schrader A l a r i a t e n u i f o l i a S e t c h e l l A l a r i a taeniata Kjellman A l a r i a f i s t u l o s a Posels et Ruprecht A l a r i a g r a n d i f o l i a J . Agardh Habit drawings of these s i x species are presented i n Figures 1 - 6 . A. marginata, A. nana and A. t e n u i f o l i a have been treated more extensively than A. taeniata, A. f i s t u l o s a and A. gr a n d i f o l i a , because of th e i r l o c a l abundance on the coast of B r i t i s h Columbia. -22-In almost a l l cases the location of these species has corresponded with documented records of t h e i r d i s t r i b u t i o n . The material i d e n t i f i e d as A l a r i a g r a n d i f o l i a , however, did not. This species has previously been recorded only i n Labrador, Greenland and Japan (Widdowson, 1964), and yet the plants named A l a r i a g r a n d i f o l i a i n thi s project were collected on Coronation Island, Alaska. Voucher specimens of the s i x species studied are recorded i n Table VIII. c. The c o l l e c t i o n of material (Figure 7.) In obtaining material for c y t o l o g i c a l investigation each species was co l l e c t e d from a variety of locations. In t h i s way a number of morphological forms of each species, except A l a r i a  g r a n d i f o l i a , was obtained. Collections were made at the following locations: A l a r i a marginata W i f f i n Spit, Sooke, Vancouver Island, B.C. Glacier Point, Vancouver Island, B.C. Jordan River, Vancouver Island, B.C. Beaver Point, Saltspring Island, B.C. Indian Beach, Ecola State Park, Oregon, USA. Short Sands Beach, Oregon, U.S.A. Volga Island, Alaska, U.S.A. A l a r i a nana Long Beach, Vancouver Island, B.C. Glacier Point, Vancouver Island, B.C. -23-Wigham Island, Alaska, U.S.A. Cape Muzon, Alaska, U.S.A. Pescadero Point, C a l i f o r n i a , U.S.A. A l a r i a t e n u i f o l i a Goose Island, Alaska, U.S.A. Brockton Point, Vancouver, B.C. A l a r i a f i s t u l o s a Cape Muzon, Alaska, U.S.A. Port Conclusion, Alaska, U.S.A. A l a r i a g r a n d i f o l i a Coronation Island, Alaska, U.S.A. A l a r i a taeniata Wigham Island, Alaska, U.S.A. Volga Island, Alaska, U.S.A. Cape Spencer, Cross Road, Alaska, U.S.A. Where possible, f i x a t i o n of material was carried out i n the f i e l d , and l i v i n g material transferred d i r e c t l y to the laboratory for c u l t u r i n g . The most e f f e c t i v e method of transporting l i v e material for short distances was by wrapping freshly c o l l e c t e d plants i n paper, dampened with sea water, and placing them i n a darkened refrigerated box. Over long periods of time, such as those involved i n transporting plants from Alaska, t h i s method was not used. Instead, plants were maintained i n large marine plywood aquaria boxes f i l l e d with sea water, which was changed regularly. d. Culturing Methods. Gametophytic cultures were established f o r a l l species -24-c o l l e c t e d . The method used for the i n i t i a t i o n of cultures was that of Hollenberg (1939). F e r t i l e sorus material was cut into small pieces. These were rinsed i n s t e r i l e sea water and blotted dry. After a short period of time the pieces were immersed i n a culture medium, i n 500 ml. c r y s t a l l i s i n g dishes. Once spore release had been established, the pieces of sorus were removed. Several c.c. of the remaining suspension of motile spores was them pipetted into culture containers. These culture containers were 12" x 6" x 4" "p l e x i g l a s s " boxes, i n which racks of either cover-glasses or microslides were set. The sl i d e s and cover-glasses provided a large s e t t l i n g area for the spores, and subsequently permitted very convenient study of the developing plants. Three d i f f e r e n t culture media were used during t h i s project; Erdschreiber medium (Starr, 1956), a controlled enrichment medium, and a modified ASP a r t i f i c i a l medium (West, personal communication) (Appendix I I ) . B a s i c a l l y , the Erdschreiber and controlled enrichment media are supplemented sea water. In this project the sea water was obtained from the v i c i n i t y of Glacier Point, Vancouver Island, to ensure a high s a l i n i t y . The Erdschreiber supplement consists i n part of a s o i l extract, whereas the controlled enrichment medium has a defined supplement. The a r t i f i c i a l medium of West (Personal communication) i s a modification of the ASP medium of Provasoli, -25-McLaughlin and Droop (1957) and allows absolute control of the constituents of cultures. Of the sea water solutions the controlled enrichment medium was preferred, since the gametophytes grown i n Erdschreiber showed a wide d i v e r s i t y i n development and morphology. This d i v e r s i t y might be accounted for by the unknown, and possibly variable, composition of the s o i l extract. Ultimately West Ts a r t i f i c i a l medium proved to be superior and was used extensively for a l l six species of A l a r i a examined. o Cultures were maintained at 10 C. i n a refrigerated culture chamber, under a l i g h t i n t e n s i t y of approximately 200 foot candles emitted from a single cool white fluorescent tube (Sylvania F48T12CW). The photo-period was altered throughout t h i s project to correspond approximately with seasonal conditions. o o Since two constant temperature culture chambers (5 C. and 10 C.) were available for the c u l t u r i n g of A l a r i a gametophytes, a preliminary experiment was conducted on A l a r i a g r a n d i f o l i a and A l a r i a f i s t u l o s a to ascertain which was the more suitable o temperature. One culture of each species was maintained at 5 C. o and the other at 10 C. A l l other environmental conditions were o i d e n t i c a l . The cultures at 10 C. showed s i g n i f i c a n t l y faster vegetative growth, which provided s u f f i c i e n t somatic d i v i s i o n s o for i n v e s t i g a t i o n (Figs. 256 8s 257). The lt.0 C. culture of A l a r i a f i s t u l o s a also produced young sporophytes within 30 days, -26-o whereas the 5 C. culture never did produce sporophytes. For the purposes of t h i s project a considerable amount of vegetative growth and the production of antheridia, oogonia and sporophytes have been used as the c r i t e r i a for determining the success of cultures. e. Fixation methods. An i d e a l f i x a t i v e k i l l s and preserves l i v i n g c e l l s without disturbing t h e i r i n t e r n a l structure or external arrangement. Such a sol u t i o n must act quickly and have rapid penetration properties. It was found that for the f i x a t i o n of the macroscopic sorus material and the microscopic gametophytes and young sporophytes of A l a r i a , Carnoy Ts 3:1 absolute ethanol: g l a c i a l acetic acid f i x i n g solution has a number of advantages. Not only does i t f i x material quickly, but bleaches out the chloroplastspigments. In a l g a l cytology this i s e s s e n t i a l , otherwise the chloroplasts tend to mask the nuclei completely. The 3:1 f i x i n g s o l u t i o n does, however, also have some disadvantages. It hardens material to the extent that a squash technique cannot be used without the i n c l u s i o n of a softening step. It also has a tendency to d i s t o r t the i n i t i a l f r a g i l e stages of the gametophytic generation and occasionally causes the interphase nucleus to collapse. For the f i x a t i o n of the f r a g i l e development stages i t became -27-necessary to employ a less violent f i x a t i v e . A medium chromic acid-acetic acid f i x a t i v e was used, v i z : 10% aqueous chromic acid 7.0 c.c. 10% aqueous acetic acid 10.0 c.c. F i l t e r e d sea water to 100 c.c. f. Mordanting and Staining methods; Some stains can be applied a f t e r c e r t a i n f i x a t i o n s , without the i n c l u s i o n of a mordanting procedure. For example c r y s t a l v i o l e t can be applied d i r e c t l y a f t e r f i x a t i o n i n a chromic acid s o l u t i o n . It was found i n A l a r i a , as i n other members of the Laminariales, that an intermediate mordanting step i s required a f t e r ethanol:acetic acid f i x a t i o n and p r i o r to an aceto-carmine or haemotoxylin s t a i n . Without th i s step nuclei stained very poorly and i n d i v i d u a l chromosomes could hardly be distinguished. For the s t a i n i n g of chromosomes i n A l a r i a a number of mordanting procedures were used with d i f f e r i n g success: 1. F e r r i c acetate. A saturated solution of f e r r i c acetate i n 45% acetic acid has been used as a mordant i n conjunction with an aceto-carmine s t a i n . It has been added to the f i x a t i v e (5-10 drops/100 c.c. f i x a t i v e ) , applied as a separate step for 10 to 20 minutes between f i x a t i o n and staining, or has been added to the s t a i n (5-10 drops/100 c.c. s t a i n ) . 2. F e r r i c chloride. A 4% solution of f e r r i c chloride i n 45% acetic acid has been used as a post f i x a t i o n step. This step was applied with some success prior to an aceto-carmine staining procedure. 3. F e r r i c ammonium sulphate. A 2% aqueous solution of f e r r i c ammonium sulphate has been used prior to an acet i c -haemotoxylin s t a i n i n a modification of Wittmann's staining procedure (1962). 4. F e r r i c chloride/EDTA. The most successful mordanting procedure was a modification of the technique used by McClaren (1967) for the staining of meiotic chromosomes i n Basidiomycetes. A 4% aqueous solut i o n of f e r r i c chloride was saturated with ethylenediaminetetraacetic acid (EDTA) . The re s u l t i n g s o l u t i o n was used to mordant fixed material for 24 hours p r i o r to an aceto-carmine staining procedure. An acetic-haemotoxylin s t a i n was also applied af t e r t h i s mordant, but with limited success. The s t a i n i n g of the microscopic stages and sorus material of A l a r i a has necessitated somewhat d i f f e r e n t techniques. For the examination of sorus material a squash method was used i n preference to the sectioning methods most often used i n the past. Since the sorus material has thick c e l l walls and had been hardened by the ethanol:acetic acid f i x a t i o n i t was necessary to-soften i t before squashing. Three softening techniques were used a f t e r the mordanting of material and pri o r to staining: 1. A 6% aqueous solut i o n of sodium bicarbonate (Naylor,1957) . 2. A solution of i o d i c acid, aluminum alum and chrome alum i n absolute ethanol and concentrated hydrochloric acid (Wittmann, 1962). 3. A one molar aqueous solution of lithium chloride as used by Evans (1963b). The t h i r d method proved to be very e f f e c t i v e and has been widely used throughout t h i s project. The mordanted, softened sorus material was then squashed i n a drop of Belling*s aceto-carmine (Darlington & LaCour, 1962) on a s l i d e . Squashing was ca r r i e d out under a cover-glass using a f l e x i b l e " p l e x i g l a s s " rod. Heat was applied gently during the squashing procedure i n order to speed up the d i f f e r e n t i a t i o n and penetration of the s t a i n . Such preparations were made permanent by f l o a t i n g off the cover-glass i n 95% ethanol. The squashed material generally remained attached to the cover-glass, which was t£en passed through several changes of absolute ethanol, and mounted on a s l i d e with Euparal. This procedure stained nuclei very c l e a r l y and allowed observation of d i v i s i o n f igures. Throughout this project i t has been used i n preference to a l l others, although the a c e t i c -haemotoxylin procedure of Wittmann (1962) did y i e l d r e s u l t s . The major disadvantage to the l a t t e r method was a serious tendency for the s t a i n to become progressively i n t e n s i f i e d to the extent that nuclear d e t a i l became ob l i t e r a t e d . It has also been found that haemotoxylin stains, such as those used by a l l Japanese workers on the Laminariales, are not necessarily n u c l e i - s p e c i f i c . -30-The gametophytie and microscopic sporophytic stages of A l a r i a were s i m i l a r l y stained with aceto-carmine, but without squashing. Squashing of t h i s material made i t extremely d i f f i c u l t to di s t i n g u i s h male and female gametophytes and young sporophytes. The complete procedures which were used predominantly i n this project are presented i n Appendix I I I . g. Pretreatments t In the i n i t i a l stages of this research i t was noted that gametophytie nuclear d i v i s i o n s appeared very infrequently i n the stained preparations. This suggested that either nuclear d i v i s i o n s were occurring extremely quickly thereby making i t d i f f i c u l t to f i x material i n the process of d i v i d i n g ; or i n fact they occurred very seldom, creating the same problem. This second p o s s i b i l i t y was d i f f i c u l t to accept since the number of c e l l s i n the cultured plants increased very r a p i d l y . One other* p o s s i b i l i t y i s that d i v i s i o n was p a r t i a l l y synchronous and occurred at a p a r t i c u l a r time during the day. Such a phenomenon, however, was not observed during several 24 hour periods during which material was fixed at thirty-minute i n t e r v a l s . Several pretreatments were applied i n an attempt to either increase the rate of d i v i s i o n (Bactojphytohemagglutinin M) or halt d i v i s i o n s at metaphase (colchicine and paradichlorobenzene). Combinations of these pretreatments were also applied (Appendix -31-IV). Regrettably these pretreatments seemingly had no affect on the d i v i s i o n rate. Colchicine pretreatments have previously been applied to members of the Phaeophyta by Levan & Levring (1942), Evans (1966) and Roberts (1966) with l i t t l e or no success, but t h i s represents the f i r s t application of Bactophytohemagglutinin M and paradichlorobenzene to the plants. h. Photographic methods: A l l photographs were taken with Kodak "Panotomic X" f i l m , developed i n Edwal "FG7" developer. Prints were made on I l f o r d bromide paper (Numbers 3, 4 and 5) and developed i n Kodak "D 11" developer. Photographs were taken using a Leica DBD camera with a Lietz Wetzlar x l extension tube mounted on a Nikon S-Ke research microscope. -32-RESULTS a. Meiosis and the formation of meiospores: The sorus of A l a r i a i s c h a r a c t e r i s t i c a l l y composed of unilocular sporangia and s t e r i l e paraphyses, both of which arise from the meristoderm layer of c e l l s . The f i r s t nuclear d i v i s i o n i n immature unilocular sporangial i s reductional. Meiosis has been studied i n the immature zoosporangia of A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a and A.grandifolia, and was found to be s i m i l a r i n a l l f i v e species. There was i n s u f f i c i e n t material available to examine th i s process thoroughly i n A.taeniata. The dimensions of the immature zoosporangia prior to the onset of meiosis are approximately 10 x 25 microns. The interphase nucleus appears granular (Figs 8 & 9) and measures 8-10 microns i n diameter. There i s no obvious reticulum connecting the heterochromatic granules (chromomeres) at th i s stage. A singl e nucleolus and d i s t i n c t nuclear membrane were v i s i b l e i n only a few preparations of a l l the species studied. At the onset of d i v i s i o n the chromomeres become more condensed and have an increased s t a i n a b i l i t y with aceto-carmine. At th i s stage there i s some evidence of thread-like connections between the chromomeres (Figs. 10 & 11). However, the resultant beaded threads cannot be discerned as d i s t i n c t chromosomes. This stage i s interpreted as leptotene. The following stage, zygotene, when homologous chromosomes pair, cannot be c l e a r l y delimited i n A l a r i a species. Although there i s some in d i c a t i o n of the pairing of chromomeres (Figs. 12, 13, 14 8s 15), there i s often only one instance of pairing v i s i b l e i n a zygotene nucleus. Added to t h i s d i f f i c u l t y i s the fact that the chromomeres at t h i s stage are only 0.2-0.5 microns i n diameter and cannot re a d i l y be distinguished under the l i g h t microscope. Since the centromeres cannot be distinguished at zygotene i t i s impossible to state at which region of the chromonemata synapsis commences. Pachytene i s marked by the increased size of the paired chromomeres and the o v e r a l l condensation of the chromosomes (Figs. 16, 17, 18, 19, 20, 21, 22, 23, 24 8s 25). The chromosomes s t i l l appear as beaded threads, but the beads are now fewer and much larger while the threads are much shorter. There i s seldom any i n d i c a t i o n of the double structure of the bivalents at t h i s stage. This i s the e a r l i e s t time at which chromosome counts can be obtained, although i t has been found to be more desirable to r e s t r i c t counting to a l a t e r stage of prophase or even metaphase I. Due to an abundance of pachytene configurations i n squash preparations i t seems that this stage may l a s t some considerable time. This i s e s p e c i a l l y so i n A . t e n u i f o l i a where pachytene may i n fact represent a resting stage i n the d i v i s i o n process, although there i s no decreased s t a i n a b i l i t y . The diplotene stage i s perhaps the most d i f f i c u l t to d i s t i n g u i s h due to the exceedingly small s i z e of the chromosomes. Typical diakinesis configurations are also rarely distinguishable. -34-Consequently, diplotene and diakinesis have been regarded simply as prometaphase (Figs. 26, 27, 28 & 29). X-, V- and Y-shaped configurations have been recorded i n a few preparations and have been used to delimit diakinesis more accurately (Figs. 139 8s 140). During diplotene and diakinesis centromeres are distinguishable for the f i r s t time, but only i n the larger chromosomes. The nu c l e o l i and nuclear membranes have completely disappeared by di a k i n e s i s . Following the prometaphase stages the bivalents a l i g n themselves i n an equatorial plate (Figs. 30 8s 31). At metaphase I spindle apparati have been distinguished i n a number of preparations, but centrioles have never been seen. The homologues then move to opposite poles i n anaphase I. During telophase I the daughter nuclei revert to an interphase condition. Due to the s c a r c i t y of binucleate sporangia! c e l l s i n A.marginata, A.nana and A . t e n u i f o l i a i t appears that this interphase condition l a s t s for only a short time before the equational d i v i s i o n of meiosis occurs. Prophase II commences with the condensation of d i s t i n c t chromosomes (Fig. 32). These chromosomes are then aligned i n plates at metaphase II (Fig. 33). During anaphase II the daughter chromosomes separate i n t i g h t l y associated masses to t h e i r respective poles (Fig. 34). Following telophase II a four-nucleate sporangia! c e l l i s produced, each nucleus of which -35-i s presumed to be haploid. Three further mitotic d i v i s i o n s occur producing 32 nuclei i n the sporangium (Figs. 35, 36, 37, 40 & 41). After these 32 nuclei have passed into an interphase condition the cytoplasm of the sporangial c e l l i s cleaved i n such a way that 32 rounded spores are produced, each with a single nucleus and chloroplast. Although 32 spores per unilocular sporangium i s undoubtedly the most frequently occurring number, occasionally sporangia with 16 and 64 spores have been observed i n a l l the A l a r i a species examined. The dimensions of the mature, sporangium are approximately 10 x 45 microns. It has been possible to obtain chromosome counts at late prophase stages during the mitotic d i v i s i o n s i n the sporangium (Figs. 38 & 39). These counts have, as a rule, matched the bivalent counts made at prometaphase I and metaphse I of meiosis and the chromosome counts made during mitotic d i v i s i o n s i n the gametophytic generations. However, for the actual confirmation of a chromosomal alternation of generations i n t h i s genus, chromosome counts.had to be obtained from vegetative sporophytic c e l l s . The actual counts are presented i n section " f " of the r e s u l t s . In a single sorus i t i s possible to di s t i n g u i s h a l l stages of the development of sporangia. Sporangia do not mature i n a synchronised manner. Further, i t appears that there i s no synchrony of nuclear d i v i s i o n s between sporangia. Sporangial material fixed and examined at set int e r v a l s over 24 hour periods -36-shows a random range of d i v i s i o n stages. Such observations were, however, only made on A.marginata, A.nana and A . t e n u i f o l i a , and f i x a t i o n was always carried out on material l i v i n g under a r t i f i c i a l culture conditions. Within a single sporangium there i s synchronised d i v i s i o n of the n u c l e i . b. Development of gametophytes and the formation of gametangia; Gametophytie development and the formation of antheridia and oogonia were followed i n A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a , A.taeniata and A.grandifolia, and were found to be s i m i l a r . At maturity the zoospores are f o r c e f u l l y liberated from the unilocular sporangia. The empty sporangia then either break away or degenerate extremely quickly. The zoospores (Fig. 42) are elongate (approximately 8 x 4 microns), tapering a n t e r i o r l y . Depending on the orientation of these c e l l s under the microscope, some appear totbe somewhat pyriform i n shape. They are l a t e r a l l y b i f l a g e l l a t e , the anterior flagellum being the longer of the two. The zoospores remain a c t i v e l y motile for a variable lenth of time before losing t h e i r f l a g e l l a and rounding up (Fig. 43). Rounded-up spores of A.marginata have been observed within minutes of l i b e r a t i o n i n the same suspension i n which motile zoospores have been noted after 26 hours. The diameters of rounded-up spores have been measured for a l l the species examined and approximate 5 microns. No si z e difference i s apparent, either amongst the spores of one species or the spores -37-of a l l the species. The non-motile spores germinate by producing a small protuberance (Fig. 44) which subsequently elongates to become a d i s t i n c t germination tube. The spore nucleus divides m i t o t i c a l l y and one of the daughter nuclei migrates into the germination tube (Fig. 45) where i t i s cut off by a c e l l wall, forming the i n i t i a l c e l l of the gametophyte (Fig. 46). The remaining nucleus degenerates i n A.nana, A . t e n u i f o l i a , A . f i s t u l o s a and A.taeniata. In A.marginata, however, i t divides again (Fig.47) and one of the daughter nuclei of this d i v i s i o n i s cut off i n the formation of- the second c e l l of the gametophyte (Fig. 48). The remaining nucleus then degenerates within the empty spore case. Progressive mitotic d i v i s i o n s subsequently give r i s e to uniseriate, branched, filamentous gametophytes, the c e l l s of which remain uninucleate. A l l the species of A l a r i a examined are dioecious, forming morphologically d i s t i n c t male and female gametophytes (Figs. 49 & 50). The female gametophyte consistently has fewer and larger c e l l s than the male. The frequency of male and female gametophytes has been recorded i n cultures established from uniformsspore suspensions, and i t appears that the frequency of each i s approximately 50% (Table I I I ) . Since 7,500 gametophytes were counted, t h i s could indicate that the sex of the gametophytes i s genotypically determined. -38-The gametophytes mature afte r a variable length of time. Gametangia may be formed either when the gametophyte i s only one or two c e l l s long or aft e r i t has produced a considerable number of c e l l s . The factors responsible for the i n i t i a t i o n of antheridium and oogonium production are not c l e a r l y understood. During the course of th i s project i t has been noted that gametangia are readily produced i n cultures i n which the medium has remained unchanged for a considerable time, and that cultures established i n May and June produce gametangia without the prior establishment of extensive gametophytie systems. The vegetative growth of gametophytes on which gametangia are produced i s apparently c u r t a i l e d . Any c e l l of the female gametophyte i s a pote n t i a l oogonium. The f i r s t indications of oogonium production are an increased cytoplasmic density within the potential gametangium, the production of i r r e g u l a r protuberances from.-the c e l l , and an increase i n c e l l volume (Fig. 49). A single mitotic d i v i s i o n occurs within the oogonium (Figs. 54 & 55). One of the daughter nuclei degenerates and the other becomes the nucleus of the egg. Each oogonium produces a single egg c e l l (Fig. 56). The egg c e l l i s extruded through a rupture i n one of the i r r e g u l a r protuberances of the oogonium. Once extruded, the egg remains attached to the"neck" of the oogonium (Fig. 57), and i t i s i n thi s position that f e r t i l i s a t i o n takes place. Antheridium production involves numerous d i v i s i o n s of a single c e l l of the male gametophyte. The r e s u l t of these d i v i s i o n s i s a cl u s t e r of c e l l s rather than a uniseriate filament. Each c e l l of these clusters i s an antheridium. The c e l l wall of each antheridium becomes thickened proximally, leaving d i s t a l t h i n areas, which eventually rapture with the release of the antherozooids. The a n t h e r i d i a l clusters may be intercalary (Fig. 51), or terminal, or, as i n A.taeniata, terminal on short* l a t e r a l stalk filaments (Fig. 52). The antherozooids are released singly from the antheridia. Although the shape of the antherozooids i s seemingly quite variable, and ranges from an elongate form to a spherical form, the most common i s undoubtedly elongate (Fig. 53), with two unequal f l a g e l l a inserted i n a l a t e r i a l p o s i t ion. The anterior flagellum i s consistently! longer than the posterior. c. F e r t i l i s a t i o n : F e r t i l i s a t i o n i s heterogamous, involving the syngamy of the large (15-20 microns i n diameter), l a b i l e , spherical eggs and small ( 5 x 3 microns) b i f l a g e l l a t e antherozooids. Although several antherozooids have been seen swimmiaig i n the v i c i n i t y of extruded egg c e l l s , only one actually fuses with each egg. Once an antherozooid has become associated with an egg c e l l i t s f l a g e l l a are apparently shed and plasmogamy occurs. A - 4 U -f e r t i l i s a t i o n p a p i l l a remains on the surface of the egg, marking the point at which fusion took place (Figs. 58 & 59). In A.taeniata a f e r t i l i s a t i o n pore and tube appear to be produced within the cytoplasm of the egg just prior to karyogamy (Fig.57). This phenomenon has not been observed i n any other species examined. During plasmogamy the nuclei of both sex c e l l s remain i n an interphase condition, so that the r e s u l t of plasmogamy i s a binucleate c e l l i n which both nuclei are i n a re s t i n g stage (Fig. 60). Karyogamy then occurs with the fusion of the two resting nuclei and the zygote i s established. d. Development of the young sporophyte: The elongation of the zygote (Figs. 62, 63 8c 64), which i s s t i l l attached to the"neck" of the oogonium at t h i s stage, marks the i n i t i a t i o n of i t s germination. The elongation i s terminated by a mitotic d i v i s i o n giving r i s e to a two-celled sporophyte, (Fig. 65). The ontogeny of the young sporophyte most commonly involves the production of a uniseriate filament (Figs. 66 & 70) and the subsequent d i v i s i o n i n a second plane to produce a b i s e r i a t e filament. This secondary d i v i s i o n may occur when the filament i s only three or four c e l l s long (Figs. 67 8t 68), or i t may be delayed u n t i l the uniseriate filament i s ten or more c e l l s long (Fig. 71). Invariably the basal c e l l of the filament assumes the role of a r h i z o i d a l c e l l , i n i t i a l l y -41-acting as a means of anchoring the young sporophyte to the empty oogonium. Before d i v i d i n g t h i s r h i z o i d a l c e l l may become quite branched (Fig. 74). The b i s e r i a t e filament then divides i n a t h i r d plane, assuming the parenchymatous habit of a distromatic blade (Figs. 72 8s 73). The development of this distromatic blade has not been followed beyond a length of 4 - 5 m.m. There i s one major abnormality i n the process of sporophytic development. This i s the occurrence of parthenogenesis, and the production of embryonic stages of sporophytes without f e r t i l i s a t i o n taking place. There are two ways i n which "parthenosporophytes" are formed. Some are formed within the oogonium (Fig. 68), i n which case they can be readily detected, while others are produced subsequent to the extrusion of the egg, and cannot be readily distinguished from normal sporophytes. The only method of accurately determining whether the l a t t e r type i s parthenogenetic i n o r i g i n i s by examination of i t s chromosome complement. This has been done i n cultured material of A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a and A.taeniata (Figs. 247, 248, 249, 250, 251 8s 252), and i n fact i t appears that the chromosome complement of suspected "parthenosporophytes" i s haploid. e. Mitosis i n Gametophytes and young Sporophytes: -42-The mitotic process has been studied i n male and female gametophytes and extremely young sporophytes of A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a and A.taeniata and i s quite uniform for a l l species. The cultured plants of A.grandifolia did not respond to the staining techniques, making i t impossible to investigate them c y t o l o g i c a l l y . Interphase nuclei have a granular appearance (Fig. 75). The diameter of the male interphase nucleus varies from 2.5 to 4.0 microns; the female from 5.0 to 6.0 microns, and the sporophyte nucleus from 6.0 to 8.0 microns. A single nucleolus and nuclear membrane are v i s i b l e at t h i s stage. Several larger heterochromatic bodies are often discernable amongst the granules of the interphase nucleus. As the interphase nucleus moves into mitotic prophase i t becomes r e t i c u l a r . The granules appear to be linked together by fine chromatic threads (Fig. 76). At t h i s i n i t i a l stage of prophase, n u c l e o l i are seldom observed. The contraction and condensation of the chromatic threads give r i s e to larger granules and the f i r s t indications of d i s t i n c t chromosomes (Fig. 77). In a few preparations of A l a r i a  marginata, A.nana and A . t e n u i f o l i a d i s t i n c t chromatids have been observed, although i n most cases these are not distinguishable, presumably on account of t h e i r exceedingly small si z e (Figs. 78 & 79). . Prophase continues with the progressive contraction of the chromosomes (Fig. 80), u n t i l they appear as - 4 3 -d i s t i n c t separate bodies (Figs. 8 1 , 82, 83 8s 84) i n a prometaphase stage. At t h i s stage i t i s possible to d i s t i n g u i s h the centromeres of the larger chromosomes, and chromosome counts can be made. The chromosomes then a l i g n themselves i n an extremely t i g h t l y associated metaphase plate (Fig. 8 4 ) . The chromosomes are so t i g h t l y compacted together at t h i s stage that i t has proved a very unsatisfactory source of chromosome counts. The separation of crescent-shaped chromatid masses i n anaphase i s also a very t i g h t l y compacted stage of mitosis. In anaphase configurations (Fig. 86) separate daughter chromosomes cannot be distinguished at a l l . Neither spindles nor centrioles have been observed during mitosis. In telophase a new c e l l wall i s l a i d down and the resultant nuclei revert to an interphase condition. f. Chromosome numbers,and s i z e : The haploid chromosome numbers for A.marginata, A.nana, A. t e n u i f o l i a , A . f i s t u l o s a , A.taeniata and A.grandifolia have been obtained from diplotene, diakinesis and metaphase I configurations of meiosis (Table IV). In A.marginata bivalent counts range from 13 to 16 (Figs. 8 7 , 8 8 , 8 9 , 9 0 , 9 1 , 9 2 , 9 3 , 9 4 , 9 5 , 9 6 , 97 8s 9 8 ) ; i n A.nana from 12 to 16 (Figs. 9 9 , 1 0 0 , 1 0 1 , 1 0 2 , 1 0 3 , 1 0 4 , 1 0 5 , 1 0 6 , 1 0 7 , 1 0 8 , 1 0 9 8s 1 1 0 ) ; i n A. tenuif o l i a from 12 to 18 (Figs. 1 1 1 , 1 1 2 , 1 1 3 , 1 1 4 , 1 1 5 , 1 1 6 , 1 1 7 , 1 1 8 , 1 1 9 , 1 2 0 , 1 2 1 8s 1 2 2 ) ; -44-i n A . f i s t u l o s a from 12 to 16 (Figs. 123,124,125,126,127,128,129, 130,131,132,133 & 134); i n A.grandifolia from 17 to 26 (Figs. 13 9,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154, 155,156,157 8s 158) and i n A.taeniata, where only two counts have been made due to a lack of suitable material, the haploid number i s 14 (Figs. 135, 136, 137 & 138). In A.marginata, A.nana A . t e n u i f o l i a and A . f i s t u l o s a the modal number i s 14, whereas i n A.grandifolia i t i s 24 ( f i g . 253).- In A . t e n u i f o l i a two counts of 21 and 26 were omitted from the computations on the basis;: that they probably represent asynaptic or p a r t i a l l y asynaptic configurations. Chromosome cosnts have also been made from prometaphase stages of mitosis i n the gametophytes of a l l species but A. . gr a n d i f o l i a (Table V). In A.marginata these counts ranged from 13 to 16 (Figs. 159,160,161,162,163,164,165,166,167 & 168); i n A.nana from 12 to 17 (Figs. 169,170,171,172,173,174,175 & 176); i n A . t e n u i f o l i a from 12 to 17 (Figs. 177,178,179,180,181 & 182); i n A.taeniata from 12 to 18 (Figs. 183,184,185,186,187,188,189, 190 8s 191) and i n A. f i s t u l o s a from 12 to 17 (Figs. 192,193,194, 195,196,197 8s 198). The modal number for a l l of these species i s 14 (Fig. 254). The d i p l o i d chromosome complements of young cultured sporophytes have been determined from prometaphase stages of mitosis for a l l species except A.grandifolia (Table VI). In A.marginata the numbers have ranged from 26 to 29 (Figs. 204,205, 206,207,208,209,210,211,212 8s 213); i n A.nana from 26 to 30 (Figs. 214,215,216,217,218,219,220,221,222 & 223); i n A. t e n u i f o l i a from 24 to 31 (Figs. 224,225,226,227,228,229,230 & 231); i n A.taeniata from 24 to 31 (Figs. 232,233,234,235,236,237,238,239, 240 & 241) and i n A . f i s t u l o s a from 23 to 31 (Figs. 242,243,244, 245 8s 246). In a l l cases the modal number i s 28. (Fig. 255). From these chromosome counts i t appears that the haploid number i s approximately 14 and the d i p l o i d number approximately 28 for A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a and A. taeniata. In A.grandifolia the haploid number i s approximately 24, although t h i s number could only be confirmed from meiotic material (Table VII). The chromosomes representing the haploid complement of a l l six species can be grouped approximately into three s i z e groups, when measured at prometaphase I. In A.marginata, A.nana, A. t e n u i f o l i a , A . f i s t u l o s a and A.taeniata s i x chromosomes are 0.5 to 1.0 microns, four, 1.25 to 1.6 microns and four, 1.8 to 2.0 microns. In A.grandifolia eleven chromosomes are'0.5 to 1.0 microns, nine, 1.2-1.6 microns and four, 1.8 to 2.0 microns. -46-DISCUSSION In A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a , A.taeniata and A.grandifolia the immature zoosporangia are the s i t e of meiosis. This corresponds with every other investigation on members of the Laminariales since the time of Kylin*s i n i t i a l description of meiosis i n Chorda filum i n 1918. However, descriptions of the meiotic process d i f f e r widely. Some of the differences may r e f l e c t d i s s i m i l a r i t i e s between species; others, the d i f f e r e n t techniques and interpretations used by various authors. For example K y l i n (1918), Myers (1928), McKay (1933), Hollenberg (1939) and a l l the Japanese workers i n this f i e l d have used a haemotoxylin staining reaction for the examination of chromosomes and d i v i s i o n stages. During t h i s current project i t was found that haemotoxylin i s not a s p e c i f i c nuclear s t a i n , since i t not only stains chromosomes and nuclei but also chloroplasts and other miscellaneous cytoplasmic int r u s i o n s . A Feulgen technique was f i r s t introduced to the c y t o l o g i c a l studies of the Laminariales by Walker (1952) and Magne (1953). Although t h i s procedure"undoubtedly results i n a DNA s p e c i f i c s t a ining reaction, i t i s extremely d i f f i c u l t to use on the Laminariales and has been superceded by the application of Belling*s (1926) aceto-carmine technique by Naylor (1956), Kemp & Cole (1961), Cole (1962) and Evans (1963a, 1965). In addition, a l l authors i n this f i e l d , with the exception of Evans -47-(1965), have r e l i e d upon sectioned material for the examination of meiotic stages. This method has obvious li m i t a t i o n s and has been superceded by a squash technique used i n th i s current project i n conjunction with a softening procedure. The interphase nuclei of the sporangial mother-cells of A l a r i a spp. appear granular, as do those of Nereocystis  luetkeana (Kemp 8c Cole 1961), Laminaria d i g i t a t a , Laminaria  saccharina, Laminaria hyperborea, Laminaria ochroleuca, A l a r i a  esculenta, Chorda filum and Sacchoriza polyschides (Evans, 1965). Other authors have reported a r e t i c u l a r interphase nucleus. The fact that nuclear membranes and nucle o l i are seldom v i s i b l e at t h i s stage i n A l a r i a may be explained by the rigorous squashing technique used. In the majority of cases i n which nuclear membranes and n u c l e o l i have been reported as e a s i l y v i s i b l e at interphase, a sectioning technique has been used. In most species studied up u n t i l this present time the synaptic zygotene stage has been considered synonomous with the congregation of chromonemata to one side of the nucleus, and although chromatin "knots" have been noted by Evans (1965), the actual process of pairing has never been described. In A l a r i a spp. the presence of this u n i l a t e r a l congregation of chromatin" loops has not been observed, nor have chromatin "knots". These differences between zygotene i n A l a r i a and zygotene i n other members of the Laminariales might possibly indicate that either -48-the arduous squash technique used on A l a r i a has obliterated the u n i l a t e r a l congregation of loops or that t h e i r absence indicates a genetic difference between A l a r i a spp. and other Laminarian species. In A l a r i a , however, there i s convincing evidence of the pairing of chromomeres, presumed to be homologous chromosomes, even although i t cannot be said at which region of the chromosomes pairing commences or at what rate it? occurs. Today the concept that chromosomes are joined end-to-end i n pachytene and diplotene i n a "spireme" i s neither c y t o l o g i c a l l y nor genetically conceivable (Darlington, 1965). This i s es p e c i a l l y so i n Laminaria angustata (Nishibayashi & Inoh, 1956; Ohmori, 1967), Costaria costata (Nishibayashi & Inoh, 1957; Ohmori, 1967), Undaria undarioides (Nishibayashi 8s Inoh, 1960b, 1960c; Ohmori, 1967), Chorda filum (Evans, 1965; Nishibayashi 8s Inoh, 1961a, 1961b; Ohmori, 1967), Ecklonia s t o l o n i f e r a and Ecklonia cava (Ohmori, 1965, 1967), Undaria p i n n a t i f i d a (Inoh 8s Nishibayashi, 1954, 1955, 1960; Ohmori, 1967), Pterygophora  c a l i f o r n i c a (McKay, 1933), Ecklonia radicosa, Laminaria  longipedalis, Laminaria yendoana, Eisenia b i c y c l i s , Ecklonia  kurome (Ohmori, 1967), where "0-"shaped bivalents have been described i n d i a k i n e s i s . Although the concept of a "spireme" i n pachytene and diplotene has been seriously questioned i n Nereocystis luetkeana by Kemp 8s Cole (1961) on the basis of the -49-i m p r a c t i c a b i l i t y of observing the continuous nature of the chromatin, every other author from the time of Kylin's i n i t i a l d e s cription of meiosis i n Chorda filum (1918) has either maintained that a spireme exists (Abe, 1939; Inoh & Nishibayashi, 1954, 1955, 1960; McKay, 1933; Myers, 1928; Nishibayashi & Inoh, 1956, 1957, 1960b, 1960c, 1961a, 1961b; Ohmori, 1965, 1967; Yabu, 1957, 1958, 1964b, 1965; Yabu & Tokida, 1963), or has f a i l e d to describe these phases of meiptic prophase completely (Evans, 1965). In the species of A l a r i a studied i n t h i s project there i s no reason at a l l to suspect that a spireme exists i n pachytene and diplotene, since there i s a condensation of d i s t i n c t chromosomes during these stages. Although i t i s impossible to:, say exactly how much contraction of the chromosomes occurs during prophase I, i t i s considerable. The p a r t i a l separation of the homologues, described i n diplotene bivalents of higher organisms, has not been seen i n A l a r i a , due presumably to the small s i z e of the chromosomes. Although X-, Y- and V-shaped bivalent configurations, s i m i l a r to those reported by every other author i n th i s f i e l d , have occasionally been observed i n A l a r i a spp., diplotene and diakinesis are most often indistinguishable and have been regarded simply as prometaphase I. Centromeres can be distinguished i n the larger chromosomes from constrictions between chromomeres at t h i s stage. Metaphase I has been reported as a t i g h t l y compacted stage of meiosis by Nishibayashi & Inoh (1956, 1960a, 1961a), Inoh & -50-Nishibayashi (1954, 1955, 1960), Ohmori (1965, 1967), Yabu (1957, 1958, 1964b, 1965), Yabu & Tokida (1963), Magne (1953) and Kemp & Cole (1961). Chromosomes i n the species studied by these authors have been so t i g h t l y associated at metaphase I that chromosome counts could not be made. The squash technique used by Evans (1965) on Laminaria d i g i t a t a , Laminaria saccharina, Laminaria hyperborea, Laminaria ochroleuca, A l a r i a esculenta, Chorda filumt and Sacchoriza polyschides and by the author i n thi s current project on A.marginata, A.nana, A . t e n u i f o l i a , A. f i s t u l o s a , A.taeniata and A.grandifolia has, however, successfully flattened and separated metaphase I bivalents, so that chromosome counts could be made at this stage. A number of the so-called metaphase I configurations described by Evans (1965) are extremely s i m i l a r to prometaphase I and even e a r l i e r prophase I squashes of A l a r i a spp.. Metaphase I bivalents i n the A l a r i a species c h a r a c t e r i s t i c a l l y have the "dumbell" shape previously reported at metaphase I i n the species studied by Nishibayashi & Inoh (1956, 1957, 1960a, 1960b, 1960c, 1961a, 1961b), Inoh & Nishibayashi (1954, 1955, 1960), Ohmori (1965, 1967), Yabu (1957, 1958, 1964b, 1965), Yabu & Tokida (1963), Magne (1953) and Kemp SE Cole (1961) . This shape i s not reported by Evans (1965), further substantiating the p o s s i b i l i t y that he has been describing prophase I chromosomes as metaphase bivalents. -51-Although the orientation of the spindle apparatus can be inferre d from the position of the metaphase plate i n A l a r i a spp., a spindle has very seldom been seen. The appearance of spindles may simply be a function of the staining reaction used, since a l l workers using haemotoxylin report t h e i r presence. They have also reported either one or two "centrosomes':' at the spindle poles. These structures were not observed at a l l by Evans (1965), and were observed very occasionally by Kemp & Cole (.1961) but were interpreted as d i v i s i o n abnormalities. They have not been observed at a l l i n the A l a r i a species studied i n this project. It should be stated that before the presence or absence of spindles and centrosomes can be confirmed, u l t r a -structure studies of the nucleus of the Laminariales are required. Anaphase I and telophase I apparently follow the same pattern i n A l a r i a spp. as i n other members of the Laminariales, with the exception that indications of non-disjunction have not been observed i n these species as they have i n Nereocystis  luetkeana (Kemp & Cole, 1961). Prophase II follows telophase I very quickly aft e r a very b r i e f interphase stage i n A.marginata, A.nana and A . t e n u i f o l i a . This i s i n agreement with the observation of Evans (1965) i n A l a r i a esculenta, Chorda filum, Sacchoriza polyschides and 4 spp. of Laminaria and of Kemp 8c Cole (1961) on Nereocystis luetkeana. Whether or not this i s true for other members of the order i s not known, since no other author has commented on the speed of d i v i s i o n at a l l . The equational meiotic d i v i s i o n and subsequent mitotic d i v i s i o n s i n ' the aporangia of the A l a r i a spp. appear to be no d i f f e r e n t from the equivalent d i v i s i o n s i n other members of the Laminariales that have been studied c y t o l o g i c a l l y . In the A l a r i a spp. di v i s i o n s i n a single sporangium are synchronous while i n Nereocystis luetkeana t h i s i s not necessarily so (Kemp & Cole, 1961). With the exception of Chorda filum, which has 16, (Nishibayashi & Inoh, 1961a, 1961b; Ohmori, 1967) and Sacchoriza bulbosa, which has 128, (Sauvageau, 1915a) the f i n a l number of nuclei (and spores) i n each sporangium i s 32 i n the Laminariales. The occurrence of rounded-up spores i n suspensions of freshly liberated zoospores of A l a r i a spp. has not been previously reported for any member of the Laminariales. A possible i n t e r p r e t a t i o n of thi s phenomenon i s that some zoospores are liberated p r i o r to the formation of f l a g e l l a . This may either be an inherent c h a r a c t e r i s t i c of some zoospores or i t may simply occur due to the rupturing of immature sporangia. The l a t t e r may be caused by the bursting of neighbouring mature c e l l s or the turgor changes induced a r t i f i c i a l l y to promote spore release i n the laboratory. The germination of zoospores of A.marginata, A.nana, A. t e n u i f o l i a , A . f i s t u l o s a , A.taeniata and A.grandifolia by way oi a germ tube i s quite consistent with other members of the Laminariales as i s the single mitotic d i v i s i o n i n the spore case of a l l the A l a r i a spp., except A.marginata. In A.marginata two d i v i s i o n s occur i n the spore case, a single daughter nucleus of each d i v i s i o n being cut off i n the i n i t i a l two c e l l s of the gametophyte. The production of morphologically d i s t i n c t male and female gametophytes i n A l a r i a spp. also occurs i n a l l other members of the order except Chorda tomentosa, which has a monoecious gametophytic generation (Sundene, 1963). The proportions of male and female gametophytes formed from uniform spore suspensions of a l l the A l a r i a spp. would seem to indicate that t h e i r sex i s genotypically determined. However, such a statement can only be made with serious reservations. Before d e f i n i t e genotypic determination of sex can ben established i t must be demonstrated that the products of single sporangium produce 50% male and female gametophytes. o In A l a r i a species i t has been determined that a 5 C. the development of the gametophytes and the formation of gametangia are i n h i b i t e d . It has also been demonstrated that there may be a seasonal e f f e c t on the production of gametangia, s i m i l a r to that reported by McKay (1933) for Pterygophora c a l i f o r n i c a , and that nutrient depletion may e f f e c t the formation of gametangia. In addition i t has been found that the vegetative growth of A l a r i a gametophytes i s c u r t a i l e d by the production of gametangia. -53- b Although the l i t e r a t u r e concerning the environmental regimes that e f f e c t gametophyte growth and the production of gametangia i s often c o n f l i c t i n g , the observations on A l a r i a are i n agreement with Kain*s (1964) general observation that the ranges of each environmental factor are greater for the support of vegetative growth than for the production of gametangia. Since gametophytic growth and the production of gametangia are obviously cl o s e l y associated i n A l a r i a spp., i t might be construed that the early production of gametangia and the consequential termination of gametophytic growth r e f l e c t s a tendency towards the loss of the gametophyte generation altogether. Svedelius (1927) maintained that the l i f e cycles of the Phaeophyta represent a phylogenetic series from an alternation of isomophic generations (Ectocarpales) through an alternation of heteromorphic generations XLaminariales) to a l i f e - c y c l e i n which there was no gametophytic generation at a l l (Fucales). Therefore, based upon the assumption that such a phenetic series actually r e f l e c t s phylogeny, i t might be argued that t h i s reduction of the gametophyte generation r e f l e c t s the t r a n s i t i o n from a Laminarian-type l i f e - c y c l e to a Fucales-type l i f e - c y c l e . However, before such a hypothesis can be substantiated i n the absence of a good f o s s i l record, i t i s necessary to know the exact physiological e f f e c t of environmental variables on growth of the gametophyte and the production of gametangia. Further, i t would ' be desirable to know the history or these environmental factors -54-i n the oceans. In the Phaeophyta ontogeny may well recapitulate phylogeny, but not necessarily so. The formation of oogonia and antheridia i n A.marginata, A. nana, A . t e n u i f o l i a , A . f i s t u l o s a , A.taeniata and A.grandifolia i s indistinguishable from these processes i n other members of the Laminariales. However, the actual process of egg formation i s quite d i f f e r e n t , since a mitotic d i v i s i o n i n the oogonium precedes egg formation, one of the resultant nuclei being incorporated into the egg c e l l . In Pterygophora c a l i f o r n i c a (McKay, 1933), Egregia menziesii (Myers, 1928) and Nereocystis luetkeana (Kemp & Cole, 1961), for example, the entire contents of the oogonium are rounded up as the egg c e l l without a nuclear d i v i s i o n . F e r t i l i s a t i o n i n A.marginata, A. nana, A . t e n u i f o l i a , A . f i s t u l o s a and A.taeniata occurs while both male and female nuclei are i n interphase. This i s consistent with the observations of Kemp & Cole (1961) on Nereocystis luetkeana. In Pterygophora c a l i f o r n i c a (McKay, 1933) and Eisenia arborea (Hollenberg, 1939), however, th i s process occurs while the nuclei are i n prophase. The occurrence of a " f e r t i l i s a t i o n pore" and "tube" i n the egg c e l l s of A l a r i a  taeniata i s unique. Neither have been reported i n any other member of the Laminariales. It i s possible that the " f e r t i l i s a t i o n pore" i s i n fact a "micropyle" and the "tube" represents the pathway followed by the male nucleus preceding -55-karyogamy. On the other hand both may simply be remnants of incomplete cytoplasmic cleavage during the d i v i s i o n preceding 1 egg formation. The early developmental stages of the young sporophytes of the A l a r i a spp. studied i n this project are very s i m i l a r to those described for other species of the Laminariales. Si m i l a r l y the mitotic process as i t occurs i n the gametophytes and young sporophytes of these species corresponds with the descriptions of mitosis made by Naylor (1956), Kemp & Cole (1961) and Evans (1965). Previously the occurrence of parthenogenesis has been described i n Laminaria d i g i t a t a and Laminaria hyperborea by Schreiber (1930), i n Laminaria r e l i g i o s a , Laminaria japonica, Laminaria angustata, Laminaria angustata var. longissima, Laminaria ochotensis, Laminaria d i a b o l i c a , A l a r i a c r a s s i f o l i a , Undaria p i n n a t i f i d a and Arthrothamnus bif i d u s by Yabu (1964a) and i n Nereocystis luetkeana by Kemp & Cole (1961) . In the current project parthenosporophytes have been observed i n a l l A l a r i a species and, as i n Nereocystis luetkeana by Kemp & Cole (1961), these abnormal sporophytes are haploid. It i s not known whether the occurrence of parthenosporophytes i s a naturally occurring phenomenon or whether i t i s r e s t r i c t e d to cultured plants. If they occurred under normal f i e l d conditions, acheived maturity and were able to sporulate then t h e i r presence might assume substanial s i g n i f i c a n c e . However, -56-since s o l e l y mitotic sporangia have never been reported i n mature plants, and since the proportions of male and female gametophytes i n cultures established from one sporophyte are i n d i c a t i v e of a normal meiotic segregation, i t appears most l i k e l y that these young parthenosporophytes do not mature at a l l . Nevertheless since no author has attempted to culture these abnormalities to maturity i t i s obvious that further investigations are required to determine t h e i r f a t e . Ghromosome numbers throughout the Laminariales show a wide range which may r e f l e c t genetic differences between species. On the other hand, since widely c o n f l i c t i n g counts for the same species do occur i n the l i t e r a t u r e , they may r e f l e c t the possible discrepancies detailed below, and a c e r t a i n amount of s u b j e c t i v i t y that must necessarily be applied to the counting of extremely small chromosomes. From Table II i t can be seen that the haploid number of c. 14 i n A.marginata, A.nana, A . r e n u i f o l i a , A . f i s t u l o s a and A.taeniata i s considerably lower than those of most other members of the Laminariales, whereas the haploid number of c.24 for A.grandifolia more closely approximates the haploid numbers of other species of the order. The numbers that are reported here for A l a r i a spp. do not conform with numbers of other members of the genus. However, assuming that the d i f f e r e n t chromosome numbers do r e f l e c t genetic differences between species and not discrepancies i n technique, i t i s conceivable that a polyploid series around the numbers 13 or 14 -57-exists i n the genus A l a r i a . Such a polyploid series may even apply to the family Alariaceae, or to the entire order. Kemp & Cole (1961) have tenta t i v e l y suggested that a polyploid series around the number 10 might .exist i n the Laminariales. These authors emphasised, and i t should be mentioned again, that before such a system can be f u l l y accepted i t i s necessary that most numbers be confirmed and many more species examined. It should o{ cLrror also be recognised that most of the possible s o u r c e s A i n counting chromosomes would tend to produce a f a l s e l y high number. Perhaps, as has been suggested by Cole (1967), the actual chromosome numbers of the Laminariales are lower than those reported. The small size of the chromosomes of the Laminariales, and the extreme d i f f i c u l t y involved i n separating them succ e s s f u l l y ^ makes i t very d i f f i c u l t to d i s t i n g u i s h each chromosome separately. In addition to this are the problems involved i n distinguishing chromatids from chromosomes, when both simply appear as tiny chromatic masses almost at the ultimate resolving power of the l i g h t microscope. S i m i l a r l y there i s d i f f i c u l t y i n distinguishing the arms of a chromosome on either side of the centromere from two entire chromosomes l y i n g close together, and the homologues of a bivalent at meiosis, from two bivalents l y i n g together. In obtaining chromosome numbers many authors have not defined -58-c l e a r l y what they are counting as chromosomes i n terms of the above al t e r n a t i v e s . There has been a tendency for authors to simply count each chromatin mass as a separate chromosome. What i s more confusing i s the fact that often the stage of d i v i s i o n , at which counts are made, i s not c l e a r l y defined, or i s even indefinable because of the small size of the chromosomes. This obviously leads to discrepency - for example i f a count were made at early prophase i t would probably be of chromomeres whereas at count made at very late prophase would be of entire chromosomes or bivalents. In t h i s current study of A l a r i a , chromosomes have been counted at either metaphase or very d e f i n i t e late prophase stages to avoid these possible discrepancies. In mitotic counts two chromatin bodies l y i n g i n juxtaposition have been regarded as parts of the one chromosome -either arms of the one chromosome or chromatids. In meiosis, also, chromatin bodies i n close association with one another have been regarded as parts of a single bivalent. In A l a r i a , as i n other members of the Laminariales that have been examined c y t o l o g i c a l l y , most of the chromosomes f a l l within the si z e range 0.5 to 2.0 microns at late prophase stages of meiosis and mitosis (Evans, 1965; McKay, 1933). An exception to t h i s has been demonstrated i n Sacchoriza polyschides (Evans, 1965) with the discovery of heteromorphic sex chromosomes i n which the X chromosome i s 4-6 microns long and i s much longer than the Y. Although Evans (1965) suspects a s i m i l a r mechanism i n -59-Laminaria d i g i t a t a , Laminaria saccharina, Laminaria hyperborea, Laminaria ochroleuca, Chorda filum and A l a r i a esculenta, i t i s not at a l l obvious from his measurements and micrographs. No apparent X/Y sex chromosomes have been detected at a l l i n any of the A l a r i a spp. studied i n th i s project. Although A.marginata. A.nana, A . t e n u i f o l i a , A . f i s t u l o s a , A.taeniata, and A.grandifolia are morphologically distinguishable (Widdowson, 1964), the only d i s t i n c t i o n between species that can be made on the basis of chromosome numbers i s that A.grandifolia has a d i f f e r e n t haploid chromosome number from A.marginata, A.nana, A . t e n u i f o l i a , A . f i s t u l o s a and A.taeniata. Not only are the chromosome numbers of these f i v e species the same, but the chromosome s i z e and the actual d i v i s i o n processes are indistinguishable. Although there are some small developmental differences between these species, such as the germination of zoospores i n A.marginata and the presence of a possible " f e r t i l i s a t i o n pore" and "tube" i n the egg c e l l s of A.taeniata, i t i s apparent that e c o l o g i c a l investigations are urgently required i f a complete understanding of the genus i s to be obtained. -60-SUMMARY The morphological and cytologi'cal phases of the l i f e - c y c l e s of A l a r i a marginata, A l a r i a nana, A l a r i a t e n u i f o l i a , A l a r i a  f i s t u l o s a , A l a r i a taeniata and A l a r i a g r a n d i f o l i a have been investigated. The morphological l i f e - c y c l e s of a l l s i x species are represented by an alternation of macroscopic sporophytic generations with microscopic dioecious gametophytic generations. Chromosomal alternations correspond with the morphological i n that the sporophyte i s d i p l o i d and the gametophyte, haploid. The f i r s t and second d i v i s i o n s i n the immature zoosporangia of mature sporophytes are meiotic. Meiosis has been c r i t i c a l l y investigated i n a l l s i x species and compared with t h i s process i n other members of the Laminariales. In a number of cases i t was found to be s i m i l a r , although the concepts of the d i v i s i o n processes presented by a number of authors were found to be questionable. In addition to th i s the majority of authors have completely ommitted comprehensive descriptions of meiosis and mitosis. The development of gametophytes and gametangia, the process of f e r t i l i s a t i o n and the development of the young sporophytes have been followed. Gametophytic development i s s i m i l a r to that i n other members of the Laminariales, although the germination of zoospores of A.marginata i s apparently unique i n that i t involves two mitotic d i v i s i o n s instead of the normal one d i v i s i o n i n the spore case. The male and female -61-gametophytes are morphologically d i s t i n c t and genotypic sex determination i s suspected. F e r t i l i s a t i o n i s oogamous as i n other members of the order, involving the fusion of b i f l a g e l l a t e antherozooids with extruded non-motile egg c e l l s . Egg formation i s somewhat d i f f e r e n t , however, since i t involves a mitotic d i v i s i o n within the oogonium prior to the d i f f e r e n t i a t i o n of the egg. In addition the presence of a possible " f e r t i l i s a t i o n pore" and "tube" i n the eggs of A.taeniata i s apparently unique. The development of young sporophytes from the zygotes of A l a r i a spp. i s indistinguishable from t h i s process i n other members of the Laminariales. The occurrence of parthenogenesis and the formation of haploid "parthenosporophytes" have been demonstrated. The haploid numbers of the six species of A l a r i a have been obtained from diplotene, diakinesis and metaphase I stages of meiosis. The haploid number of A.marginata, A.nana, A. t e n u i f o l i a , A . f i s t u l o s a and A.taeniata i s approximately 14, whereas i n A.grandifolia i t i s approximately 24. In a l l species, except A.grandifolia the haploid number has also been confirmed from prometaphase stages of mitosis i n the gametophytes. A d i p l o i d number of approximately 28 has been obtained from mitotic d i v i s i o n s i n the young sporophytes of A.marginata, A.nana, A. t e n u i f o l i a , A . f i s t u l o s a and A.raeniata. The use of nuclear cytology i n the taxonomy of these members of the genus has obvious - 6 2 -l i m i t a t i o n s . Only A.grandifolia could be distinguished on the basis of chromosome number. The chromosome numbers and sizes of the A l a r i a species have been compared and contrasted with those of other members of the Laminariales. 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The development of Costaria, Undaria and Laminaria. Ann. Bot. Lond. 25 : 691-715. t -74-Appendix I Key to the species of the genus A l a r i a (Widdowson, 1964) Dimensions are taken from wet f e r t i l e plants of the species, and treated s t a t i s t i c a l l y . 1. Midrib f i s t u l o s e (Furnished with a i r chambers) at int e r v a l s A. f i s t u l o s a 1. Midrib s o l i d throughout 2 2. At least some l a t e r a l branching ( f e r t i l e or s t e r i l e ) with midrib A. paradisea 2. Lat e r a l branches completely devoid of midribs, usually f e r t i l e 3 3. Stipe v i r t u a l l y non-existant; lower trunk bearing two ranks of persistent sporophyll petioles or adapted hapters A. ochotensis 3. Stipe present 4. 4. Stipe more than 15.4 cm long 5. 4. Stipe less than 15.4 cm long 6. 5. Sporophylls less than 2.8 cm wide A. g r a n d i f o l i a 5. Sporophylls more than 2.8 cm wide A. t e n u i f o l i a 6. Stipe less than 3.0 cm long A. taeniata 6. Stipe more than 3.0 cm long 7. 7. Sporophylls more than 2.8 cm wide 8. 7. Sporophylls less than 2.8 cm wide 10. 8. Stipe c y l i n d r i c a l , 0.3 cm or less wide A. marginata 8. Stipe sometimes flattened, 0.3 cm or more wide. 9. -75-9. Sporophylls less than 4.5 cm wide A. praelonga 9. Sporophylls more than 4.5 cm wide.... A. p y l a i i 10. Sporophylls tending to be fa s c i c u l a t e with more than one rank on each side 11. 10. Sporophylls one-ranked at point of attachment 12. 11. Sporophylls c y l i n d r i c a l i n cross section close to the petiole very thick and narrow A. cris p a 11. Sporophlls flattened i n cross-section A. angusta 12. Rachis evenly tapering d i s t a l l y A. nana 12. Rachis not tapering although sometimes constricted at t r a n s i t i o n zone 1$. 13. Two kinds of sporophylls; one, most common i n summer, dark, jjhick, s t e r i l e ; the other, more common i n winter, l i g h t e r i n colour, thin and f e r t i l e A. c r a s s i f o l i a 13. Sporophylls usually a l l one kind; i f some are thickened, of unequal thickness i n d i f f e r e n t parts A. esculenfa. -76-Appendix I I . S e m i - a r t i f i c i a l and a r t i f i c i a l culture media (Starr', 1956) 1. "Erdschreiber" Medium To prepare 2 l i t r e of th i s medium, "X ; l i t r e s of sea water were f i l t e r e d three times through a 12" glass column, packed with o glass wool. This f i l t e r e d sea water was then heated to 70 C. and allowed to cool. To prepare a s o i l extract, 500 gms of garden s o i l were brought to the b o i l i n one l i t r e of tap water and simmered for 40 minutes. After allowing the s o i l to s e t t l e i t was f i l t e r e d through two layers of number 2 Whatman F i l t e r Paper. lOOcc of thi s extract was then brought to the b o i l and when just o f f the b o i l 0.020 gms. NaNO and 0.040 gms of Na 3 2 HPO^ were added. F i n a l l y the s o i l extract, with added s a l t s , was added to the cold f i l t e r e d sea water and allowed to cool again, a f t e r which the medium was ready for use. A l l glassware was heat s t e r i l i s e d and the prepared medium o was stored i n dark glass bottles at 10 C. 2. Controlled enrichment medium. For the preparation of thi s medium, vitamins, n i t r a t e and organic phosphate were added to s t e r i l i s e d sea water i n the following concentrations: NaN03 0.3 gm/1 Na Glycerophosphate 0.1 gm/1 Vitamin B 1 9 0.00001 mg/1 -77-B i o t i n 0.00001 mg/1 Thiamine 1.0 mg/1 3. Modified ASP medium (West, personal communication). Stock solutions: No. 1. NaCl Weigh out 240 gms. and dissolve i n 1 l i t r e of double d i s t i l l e d (DD) water. Add 100 ml/1 medium. No. 2. MgS04.7H20 DisseiLie 40 gms in, 200 ml DD water. Add 20 ml/l medium. No. 3. Basal s a l t s Dissolve 7 gms KC1, 2 gms CaCl and 3 gms NaNOg separately i n DD water and make up to 100 ml. Add 10 ml/1 medium. No. 4. Vitamin B]^ > Dissolve 0.1 mg, i n 100 ml DD water. Add 1 ml of t h i s solution to 99 ml DD water. Add 1 ml of t h i s f i n a l d i l u t i o n to each l i t r e of medium. No. 5. B i o t i n Same as for vitamin Bj2 No. 6. Thiamine Dissolve 100 mg of thiamine i n 100 ml DD water, and add 1 ml/l medium. No. 7. Trace metals Weigh out 5 gms NTA ( n i t r i l o - t r i a c e t i c acid) i n 50 ml DD water -78-(Dilute NaOH). A. Dissolve 10 gms FeClg i n 100 ml DD water. B. Dissolve 2.5 gms ZnClg i n 100 ml DD water. C. Dissolve 10 gms MnClg i n 100 ml DD water. D. Dissolve 100 mg CuCl i n 100 ml DD water. E. Dissolve 1 gm H B0. i n 100 ml water. 3 4 F. Dissolve 2.5 gms Na2Mo04.H20 i n 100 ml DD water. G. Dissolve 100 mg CoCl 2 i n 100 ml DD water. Add 1 ml of each to the NTA, except the B0g - add 10 ml of thi s solution, and bring to 100 ml with DD water. Add 1-3 ml of this s o l u t i o n to each l i t r e of medium. Next adjust thepH of the medium to 7.5 - 7.8 and add stock solutions 8 and 9. No. 8. Hydroxy-methylamino-methane (Tris.) Dissolve 10 gms of T r i s . buffer to 100 ml DD water and add 10 ml/ 1 of medium. No. 9. Sodium Glycerophosphate Dissolve 1 gm i n 100 ml DD water and add 10 ml/1 of medium. -79-Appendix III Fixing, mordanting, softening and staining procedures. 1. Aceto-carmine squash technique for sorus material. A. Section sorus into approximately 1 mm x 5 mm pieces. B. Fix i n 3:1 absolute ethanol:glacial acetic acid for 12 - 24 hours u n t i l completely discoloured. C. Washthoroughly i n d i s t i l l e d water. (Material can be stored for a short period at t h i s stage i n a re f r i g e r a t o r . ) D. Mordant i n FeCl^/EDTA (ethylenediaminetetraacetic acid) solu t i o n for 24 hours. E. Wash thoroughly i n d i s t i l l e d water. F. Place i n 1 M Lithium Chloride for 15 minutes-2 hours. G. Place i n d i s t i l l e d water for 6 hours. H. Squash i n a drop of aceto-carmine. I. Heat gently and continue to i r r i g a t e s l i d e with aceto-carmine. (Darlington & LaCour, 1962). J . Float o f f cover-glass i n 95%yiethanol. K. Pass cover-glass with attached material into 100% ethanol for 15 min.. L. Mount cover-glass on a s l i d e with Euparal. 2. Aceto-carmine staining technique for gametophytes and microscopic sporophytes. A. Place s l i d e s or cover-glasses with attached material i n - » u -3:1 asbolute ethano l : g l a c i a l acetic acid f i x a t i v e f or 24 hours. B. Wash well i n d i s t i l l e d water. C. Mordant i n FeCl /EDTA soluti o n for 24 hours. D. Wash thoroughly i n d i s t i l l e d water. E. Stain i n warm aceto-carmine. , F. D i f f e r e n t i a t e s t a i n b r i e f l y i n 45%„acetic acid. G. Dehydrate completely i n 100% ethanol. H. Mount with Euparal. Acetic-iron-haemotoxylin technique for sorus material, (after Wittmann, 1962) A. Fix i n 3:1 f i x a t i v e for 12 - 24 hours, u n t i l completely discoloured. B. Mordant and macerate i n the following solution for 10 minutes: Iodic acid 3 gms. Aluminium alum 3 gms. Chrome alum 3 gms. 95% ethanol 90 c.c. Concentrated HCl 90 c.c. C. Post f i x i n 3:1 f i x a t i v e for 10 minutes. D. Squash i n acetic-iron-haemotoxylin Haemotoxylin 2 gms. Iron alum 0.5 gms. 45% acetic acid 50 c.c. -81-E. Heat gently during squashing and continue to i r r i g a t e s l i d e with s t a i n . F. D i f f e r e n t i a t e s t a i n b r i e f l y i n a 2% solu t i o n of iron alum i n 45% acetic acid. G. Float o f f cover-glass i n 95% ethanol. H. Pass cover-glass with attached material into 100% ethanol for 15 minutes. I. Mount on a s l i d e with Euparal. -82-Appendix IV Pretreatment procedures. 1. Colchicine i n modified ASP culture medium. Cultures were established i n the following concentrations of colchicine i n culture medium: 2%, .1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% and 0.01%. Material was fixed from a l l concentrat-ions af t e r 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 48 hours. 2. Paradichlorobenzene i n modified ASP medium. Two cultures were established i n modified ASP culture medium. In one culture the medium was 100% saturated with paradichloro-benzene, and i n the other i t was 50% saturated. Material was fixed from both cultures a f t e r 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 48 hours. 3. Bacto-phytohemagglutinin M i n modified ASP medium. Cultures were established i n 1%, 0.5%, 0.25%, 0.1%, 0.05%, and 0.01% Bacto-phytohemagglutinin M, and material was fixed from each culture a f t e r 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 48 hours. 4. Bacto-phytohemagglutinin M i n a 0.1% solut i o n of colchicine i n modified ASP culture medium. The concentrations of Bacto-phytohemagglutinin M were 1.0%, 0.5%, 0.25%, 0.1%, 0.05% and 0.01%. Fixations were again made aft e r 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, -83-8 hours, 12 hours, 24 hours and 48 hours. A l l four pretreatments were applied to established gameto-phyte cultures of A l a r i a marginata (10 days old) and a c t i v e l y germinating zoospores of A l a r i a nana (48 hours o l d ) . -84-Appendix V Tables I - VI -85-Table I MEMBERS OF THE LAMINARIALES IN WHICH THE LIFE CYCLE HAS BEEN INVESTIGATED SPECIES CHORDACEAE Chorda filum Chorda tomentosa  Chorda sp. LAMINARIACEAE Laminaria angustata Laminaria angustata var, longissima  Laminaria yendoana  Laminaria cichorioides  Laminaria d i g i t a t a AUTHOR Kylin, 1918 Kanda, 1938 Evans, 1965 Sundene, 1963 Williams, 1921 Kanda, 1941a Yabu, 1964a Yabu, 1964^ Kanda, 1938 Kanda, 1938 Drew, 1910 Kyl i n , 1916 Schreiber, 1930 Harries, ,1932 Walker, 1954 Naylor, 1956 Sundene, 1958, 1962 Evans, 1965 -86-Laminaria f l e x i c a u l i s Laminaria ochroleuca Laminaria saccharina Laminaria hyperborea Laminaria cloustoni Laminaria japonica Laminaria s i n c l a i r i i Laminaria d i a b o l i c a Laminaria r e l i g i o s Laminaria ochotensis Laminaria yezoensis Laminaria p a l l i d a Laminaria sp, Sauvageau, 1916a,1916b Naylor, 1956 Evans, 1965 Drew, 1910 Sauvageau, 1916a,1916b Kuckuck, 1917 Pascher, 1918 Schreiber, 1930 Harries, 1932 Naylor, 1956 Evans, 1965 Schreiber, 1930 Evans, 1965 Harries, 1932 Kanda, 1936 Yabu, 1964a Myers, 1925 Yabu, 1964a Yabu, 1964a Yabu, 1964a Kanda, 1938 Papenfuss, 1942 Yendo, 1911 Williams, 1921 -87 Sacchoriza polyschides  Sacchoriza bulbosa Pleurophycus gardneri  Agarum 8ribros.um  Costaria costata Costaria turneri Kjelimanie1la c r a s s i f o l i a Arthrothamnus b i f i d u s LESSONIACEAE Nereocystis luetkeana Poste l s i a palmaeformis  Macrocystis i n t e g r i f o l i a Macrocystis p y r i f e r a Pelagophycus porra Evans, 1965 Thuret, 1850 Sauvageau, 1915a, 1915b Angst, 1929 Kanda, 1941a Angst, 1927 Kanda, 1936 Yendo, 1911 Kanda, 1938 Kanda, 1936 Yabu, 1964a Hartge, 1928 Kemp & Cole, 1961 Myers, 1925 Walker, 1952 Cole, 1959, 1967 Brandt, 1923 Delf & Levyn, 1926 Levyns, 1933 Papenfuss, 1942 Neushul, 1963 Herbst & Johnstone, 1937 -88 ALARIACEAE A l a r i a c r a s s i f o l i a A l a r i a esculenta Pterygophora c a l i f o r n i c a  Undaria undarioides  Undaria p i n n a t i f i d a Undaria p i n n a t i f i d a f . distans  Undaria peterseniana  Ecklonia cava  Ecklonia s t o l p n i f e r a  Ecklonia maxima  Eckloniopsis radicosa  Eisenia b i c y c l i s  Eisenia arborea Egregia menziesii Kanda, 1936 Yabu, 1964 Sauvageau, 1916b,1916c Evans, 1965 McKay, 1933 Segi & Kida, 1957, 1958 Yendo, 1911 Kanda, 1936 Yabu, 1964 Kanda, 1941a Kanda, 1941b Kanda, 1941b Papenfuss, 1942 Kanda, 1941b Kanda, 1941b Clare & Herbst, 1938 Hollenberg, 1939 Myers, 1928 Table II Chromosome numbers i n the Laminariales SPECIES DIVISION M M E I I T 0 O S s 1 I s s Chorda filum x x x x x x x Laminaria angustata x x Laminaria angustata var. longissima x Laminaria d i g i t a t a x x x x x Laminaria f l e x i c a u l i s x x Laminaria ochroleuca x STAIN F A H E C A U E E L T M G 0 0 E c T N A 0 R X M Y I L N I E N X X X X X X X X X X X X X X X X X X METHOD N 2N AUTHOR S S Q E U C A T S I H 0 N x 20 40 Kylin, 1918 x 30 Nishibayashi & Inoh 1961a, 1961b. x c.30 Ohmori, 1967 x c.30 Evans, 1963a x c.28 c.56 Evans, 1965 x 22 Nishibayashi & Inoh 1956 x c.22 Ohmori, 1967 x 30 Yabu, 1965 x 16 Walker, 1954 x 27-31 Naylor, 1956 x 31 Evans, 1963a x 31 62 Evans, 1965 x 13 Magne, 1953 x 27-31 Naylor, 1956 x 31 Evans, 1963a Table Il/Continued SPECIES DIVISION STAIN Laminaria ochroleuca x x x Laminaria saccharina x x x X X X X X X X X Laminaria hyperborea x x x x x Laminaria cloustoni x x Laminaria japonica x x Laminaria d i a b o l i c a x x Laminaria longipedalis x x Laminaria yendoana x x Sacchoriza polyschides x x x x x Agarum cribrosum x x Costaria costata x x x x Kje1lmanie11a gyrata x x Arthrothamnus bif i d u s x x Nereocystis luetkeana x x x Macrocystis i n t e g r i f o l i a x x x x Al a r i a c r a s s i f o l i a x x Al a r i a praelonga x x Al a r i a esculenta x x X X X Pterygophora c a l i f o r n i c a x x x Undaria undarioides x x x x N 2N AUTHOR 31 62 Evans, 1965 13 Magne, 1953 27-31 Naylor, 1956 31 Evans, 1963a 31 62 Evans, 1965 31 Evans, 1963a 31 62 Evans, 1965 11 22 Walker (unpublished) c.22 Abe, 1939 22 Yabu, 1958 28-32 Ohmori, 1967 27-31 Ohmori, 1967 c.30 Evans, 1963a 31 62 Evans, 1965 22 Yabu, 1964b c.8=0 Nishibayashi 8s Inoh, 1957 27-31 Ohmori, 1967 22 Yabu, 1965 22 Yabu & Tokida, 1963 31 58-62 Kemp 8s Cole, 1961 16 32 Walker, 1952 14-16 28-32 Cole, 1967 22 Yabu, 1957 22 Yabu, 1964b c.30 Evans, 1963a 28 56 Evans, 1965 13 26 McKay, 1933 29 Nishibayashi 8s Inoh, 1960a c.30 Ohmori, 1967 Table I I / continued SPECIES Undaria p i n n a t i f i d a Ecklonia cava Ecklonia s t o l o n i f e r a Eisenia arborea  Eisenia b i c y c l i s  Egregia menziesii DIVISION STAIN x x x x x x x x X X X X X X X X X X X X X X * in Vtal lcer , Wga METHOD N 2N AUTHOR x 22 Inoh 8s Nishibayashi 1954 x c.30 Inoh 8s Nishibayashi 1955, 1960a x c.30 Ohmori, 1967 x c.30 Ohmori, 1965 x 29-32 Ohmori, 1967 x c.3'3; Ohmori, 1965 x 30-32 Ohmori, 1967 x 15 30 Hollenberg, 1939 x 27+ Ohmori, 1967 x 8 16 Myers, 1928 -92-Table III Frequency of occurrence of Male and Female gametophytes i n cultures established from uniform spore suspensions. ( A l l cultures approximately one month old at time of counting.) Number of Male Number of Female Gametophytes Gametophytes A. marginata Culture 1 262 238 Culture 2 257 243 Culture 3 281 219 Total 800 700 PERCENTAGE 53.3% 46.7% A. nana ~ Culture 1 241 259 Culture 2 268 232 Culture 3 251 249 Total 760 740 PERCENTAGE 50.7% 49.3% A. t e n u i f o l i a Culture 1 270 230 Culture 2 244 256 Culture 3 262 238 Total 776 724 PERCENTAGE 51.7% 48.3% A. f i s t u l o s a Culture 1 251 249 Culture 2 263 237 Culture 3 254 246 1 Total 768 732 PERCENTAGE 51.2% 48.8% A. taeniata Culture 1 228 272 Culture 2 248 252 Culture 3 260 240 Total 736 764 PERCENTAGE 49,1% 50.9% -93-Table Ill/Continued Number of Male Number of Female Gametophytes Gametophytes A. g r a n d i f o l i a Culture 1 288 212 Culture 2 246 254 Culture 3 267 233 Total 801 699 PERCENTAGE 53.4% 46.1 - 9 4 -Table IV Bivalent counts made during meiosis at diplotene, diakinesis or metaphase I. SPECIES COUNTS A l a r i a marginata 13, 13, 14(Figs. 87 & 8 8 ) , 14 (Figs 89 & 9 0 ) , 14(Figs. 9 1 , 92 8s 9 3 ) , 3*4 (Figs.97 & 9 8 ) , 14, 1 4 , 14, 1 4 , 14, 14, 1 4 , 1 5 , 1 5 , 1 6 ( F i g s . 9 4 , 95 & 96) , 1 6 . A l a r i a nana 1 2 ( F i g s . 1 0 2 , 103 & 1 0 4 ) , 12 ( F i g s . 108, 109 & 110) 1 2 , 13, 14 ( F i g s . 105 106 & 1 0 7 ) , 14, 14, 14, 1 5 , 1 6 . A l a r i a t e n u i f o l i a 1 2 ( F i g s . 1 1 8 , 119 8s 1 2 0 ) , 1 2 , 13 (Figs. I l l & 1 1 2 ) , 1 3 ( F i g s . 121 8s 1 2 2 ) , 1 3 , 1 3 , 1 3 , 14 ( F i g s . 113 8s 1 1 4 ) , 14, 1 4 , 14, 14, 14, 14, 14, 14, 1 5 , 15, 16, 16, 18, 2 0 , 2 1 (Figs. 1 1 5 , 116 & 1 1 7 ) . A l a r i a f i s t u l o s a 1 2 ( F i g s . 1 2 3 , 124 8s 1 2 5 ) , 1 2 ( F i g s . 126 8s 1 2 7 ) , 13 (Figs. 128 8s 1 2 9 ) , 13 (Figs. 130 & 1 3 1 ) , 13 (Figs. 1 3 2 , 133 8s 1 3 4 ) , 1 3 , 14, 14, 14, 14, 14, 14, 1 5 , 16. A l a r i a taeniata 14 (Figs. 135 & 1 3 6 ) , 14 (Figs. 137 8s 1 3 8 ) . A l a r i a grandi f o l i a 17 (Figs. 1 5 5 , 156, 157 8s 1 5 8 ) , 2 0, 24 (Figs. 144, 1 4 5 , 8s 1 4 6 ) , 24 (Figs. 147, 148, 149, 150, 151 8s 1 5 2 ) , 24, 2 4 , 2 4 , 25 (Figs. 139 8s 1 4 0 ) , 25 (Figs. 153 8s 1 5 4 ) , 2 5 , 26 (Figs. 141, 142 8s 1 4 3 ) , 2 6 . -95-Table V Haploid chromosome counts made at prometaphase of mitosis i n male and female gametophytes. SPECIES COUNTS A l a r i a marginata 13(Figs. 165 & 166), 13, 13, 14 (Figs. 159, 160 & 161), 14(Figs. 167 & 168), 14, 14, 14, 14, 15, 15, 15, 15, 16(Figs. 162, 163 & 164), 16. A l a r i a nana 12, 14(Figs. 174, 175 & 176), 14, 14, 14, 14, 14, 15(Figs. 169 & 170), 15 (Figs. 171, 172 & 173), 17. A l a r i a t e n u i f o l i a 12, 13(Figs. 179 & 180), 13(Figs. 181 & 182), 13, 13, 14, 14, 14, 14, 14, 14, 15(Figs. 177 & 178), 15, 16, 17. A l a r i a taeniata 12(Figs. 187, 188, 189, 190 & 191) 13, 14, 14, 14, 15(Figs. 183, 184, 185 & 186), 18. A l a r i a f i s t u l o s a 12, 12, 13, 13, 14(Figs. 192, 193, 194 & 195), 14(Figs. 196, 197 & 198), 14, 14, 14, 14, 14,15, 16, 17. -96-Table VI Dip l o i d chromosome counts made at prometaphase of mitosis i n very young sporophytes. SPECIES COUNTS A l a r i a marginata 26(Figs. 210, 211, 212 8s 213), 26, 26, 28(Figs. 204, 205, 206, 207, 208 8s 209), 28, 28, 28, 28, 28, 29. Al a r i a nana 23(Figs. 220, 221, 222 & 223), 26, 26, 28(Figs. 214, 215, 216, 217, 218 8s 219), 28, 28, 28, 28, 28, 30. A l a r i a t e n u i f o l i a 24 (Figs. 224, 225, 226 8s 227), 24 (Figs. 228, 229, 230 8s 231), 26, 28, 28, 28, 28, 28, 30, 31. Al a r i a taeniata 24(Figs. 238, 239, 240 8s 241), 24 (Figs. 238, 239, 240 8s 241), 24, 26 (Figs. 232, 233, 234, 235, 236 8s 237), 26, 26, 28, 28, 28, 28, 28, 28, 28, 29, 30, 31. A l a r i a f i s t u l o s a 23 (Figs. 242, 243, 244, 245, 8s 246), 25, 28, 28, 28, 30, 31. -97-Table VII Chromosome numbers of six species of the genus, Alaria , Species M G M S M e a i p i i m t o t 0 e o r o S t S O S 1 o i p i s p s h s h y y t t e e A. marginata c.14 c. 14 c. 28 A. nana c.14 c. 14 c. 28 A. t e n u i f o l i a c.14 c. 14 c. 28 A. f i s t u l o s a c.14 c. 14 c. 28 A. taeniata C.14* c. 14 c. 28 A. g r a n d i f o l i a c.24 * - Based on two observations only. -98-Table VIII Voucher specimens of A l a r i a used i n investigation, Species A l a r i a marginata Location W i f f i n Spit, Sooke, Vancouver Island Glacier Point Vancouver Island Jordan River Vancouver Island Herbarium and No, UBC 29468 UBC 29469 UBC 12534 UBC 29831 Beaver Point Saltspring Island Indian Beach, Ecola State Park, Oregon Short Sands Beach, Oregon. Volga Island Alaska. UBC 29832 UBC 21619 UBC 24912 UBC 24656 UBC 24911 UBC 25151 UBC 27946 UBC 25139 UBC 12165 A l a r i a nana Glacier Point, Vancouver Island UBC 13060 UBC 12370 UBC 12633 Pescadero Point, San Mateo Co., C a l i f o r n i a Wigham Island, Alaska. UBC 24568 UBC 20952 A l a r i a t e n u i f o l i a Goose Island, UBC 20788 UBC 20785 Brockton Point, Vancouver. UBC 11513 -99-Species Location A l a r i a f i s t u l o s a Cape Muzon, Alaska Port Conclusion. Alaska A l a r i a taeniata Wigham Island, Volga Island, Cape Spencer, Cross Road, Alaska. A l a r i a g r a n d i f o l i a Aats Bay, Coronation Island, Alaska. Herbarium and No. UBC 20799 UBC 20709 UBC 20588 UBC 21007 UBC 23076 UBC 23078 UBC 20783 UBC 27948 UBC 25146 UBC 23677 UBC : Phycological Herbarium of the University of B r i t i s h Columbia. -100-Appendix Plates 1 - 3 6 -101-Plate 1 Figure 1: Habit drawing of A l a r i a marginata Postels et Ruprecht. x 0.5 -102-Plate 2 Figure 2: Habit drawing of A l a r i a nana Schrader. Actual s i z e . -103-Plate 3 Figure 3: Habit drawing of A l a r i a t e n u i f o l i a S e t c h e l l . x 0.5 -104-Plate 4 Figure 4: Habit drawing of A l a r i a f i s t u l o s a Postels Ruprecht. x 0.5 -105-Plate 5 Figure 5: Habit drawing of A l a r i a taeniata Kjellman. x 0.75 -106-Plate 6 Figure 6: Habit drawing of A l a r i a g r a n d i f o l i a J . Agardh. x 0.75 - 1 0 7 -Plate 7 Figure 7 : C o l l e c t i n g stations on the P a c i f i c coast of North America from which specimens of A l a r i a were obtained. Scale: 1 : 1 2 , 5 0 0 , 0 0 0 W i n g h a m I s l a n d C a p e Spencer -G o o s e Is Ian. V o l g a I s l a n d C o r o n a t i o n I s l a n d P o r t C o n c l u s i o n C a p e M u z o n B r o c k t o n P o i n t L o n g B e a c h B e a v e r P o i n t J o r d a n R i v e r G l a c i e r P o i n t W i f f i n S p i t I nd ian B e a c h S h o r t S a n d s B e a c h N A L A S K A 1 : 1 2 , 5 0 0 , 0 0 0 B R I T I S H C O L U M B I A W A S H I N G T O N O R E G O N C A L I F O R N I A P e s c a d e r o P o i n t -108-Plate 8 Figure 8: Interphase nucleus of an immature sporangium of A.marginata. x 4,000 Figure 9: Diagrammatic representation of F i g . 8. Figure 10: Leptotene of meiosis i n an immature zoosporangium of A. marginata. x 4,000 Figure 11: Diagrammatic representation of F i g . 10. Figure 12: Zygotene of meiosis i n an immature zoosporangium of A l a r i a marginata. x 4,000 Figure 13: Diagrammatic representation of Fig.12. Figure 14: Zygotene of meiosis i n an immature zoosporangium of A. marginata. x 4,000 Figure 15: Diagrammatic representation of F i g . 14. Figure 16: Pachytene chromosomes i n an immature zoosporangium of A. marginata. x 4,000 Figure 17: Diagrammatic representation of F i g . 16. Figure 18: Pachytene chromosomes i n an immature zoosporangium of A. nana, x 4,000 Figure 19: Diagrammatic representation of F i g . 18. Legend: Nc Pc R - Nucleolus - Pairing chromomeres - Beaded thread of reticulum. -109-Plate 9 Figure 20: Figure 21: Figure 22: Figure 23: Figure 24: Figure 25: Figure 26: Figure 27: Figure 28: Figure 29: Figure 30: Figure 31: Pachytene chromosomes i n an immature zoosporangium of A. t e n u i f o l i a . x 4,000 Diagrammatic representation of F i g . 20. Pachytene chromosomes i n an immature zoosporangium of A. f i s t u l o s a . x 4,000 Diagrammatic representation of F i g . 22. Pachytene chromosomes i n an immature zoosporangium of A. g r a n d i f o l i a . x 4,000 Diagrammatic representation of F i g . 24. Diplotene chromosomes i n an immature zoosporangium of A. g r a n d i f o l i a . x 4,000 Diagrammatic representation of F i g . 26. Diakinesis chromosomes i n an immature zoosporangium of A. nana. x 4,000 Diagrammatic representation of F i g . 28. Metaphase I equatorial plate showing 13 bivalents i n A. f i s t u l o s a . x 4,000 Diagrammatic representation of F i g . 30. z Legend: Nc - Nucleolus C - Centromere -110-Plate 10 Figure 32: Prophase II i n a young zoosporangium of A. nana, x 3,000 Figure 33: Metaphase II i n a young zoosporangium of A. t e n u i f o l i a . x 3,000 Figure 34: Anaphase II i n a young zoosporangium of A. nana, x 2,300 Figure 35: Prophase of four-nucleate sporangium of A. nana, x 2,500 Figure 36: Prophase i n eight-nucleate sporangium of A. nana, x 2,000 Figure 37: Mit o t i c metaphase plates i n eight-nucleate sporangium of A. nana (four metaphase plates shown), x 2,000 Figure 38: Late prophase configurations i n eight-nucleate sporangium, showing d i s t i n c t chromosomes. A. g r a n d i f o l i a . x 3,000 Figure 39: Late prophase configurations i n eight-nucleate sporangium of A. marginataj showing d i s t i n c t chromosomes, x 3,000 Figure 40: Sixteen-nucleate sporangium of A. marginata. x 2,500 Figure 41: Thirty-two-nucleate sporangium of A. nana, x 2,000 - I l l -Plate 11 Figure 42: Figure 43: Figure 44: Figure 45: Figure 46: Figure 47: Figure 48: Figure 49: B i f l a g e l l a t e motile zoospore of A . marginata. x 1,000 Rounded-up, non-motile zoospore of A. marginata. x 1,000 I n i t i a l stage of germination of zoospore of A . marginata. x 1,000 Production of germination tube, and migration of nucleus into the tube i n A . marginata. x 1,000 The formation of the i n i t i a l c e l l of the gametophyte of A . taeniata. x 1,000 Second d i v i s i o n of the spore nucleus i n A . marginata. x 1,000 Two-cell gametophyte of A . marginata. x 1,000 Female gametophytes of A . marginata. Some c e l l s have produced protuberances [typical to oogonia. x 1,000 s c - Spore case GT - Germination tube CY = cytoplasm V - Central vacuole N - Nucleus OP - Oogonial protuberance -112-Plate 12 Figure 50: Male gametophyte of A, marginata. x 1,000 Figure 51: Male gametophyte of A.marginata, bearing i n t e r c a l a r y antheridia. x 1,000 Figure 52: Male gametophyte of A. taeniata, bearing intercalary stalked clusters of antheridia, x 1,000 Figure 53: B i f l a g e l l a t e antherozooids of A. marginata, x 1,000 Legend: A - Antheridia -113-Plate 13 Figure 54: Oogonium of A. marginata. Cytoplasm has drawn away from the c e l l wall, x 3,000 Figure 55: Mit o t i c d i v i s i o n prior to the formation of the egg i n an oogonium of A. marginata. x 3,000 Figure 56: Extrusion of the egg from the oogonium i n A. marginata. x 3,000 Figure 57: Extruded egg c e l l of A. taeniata, showing " f e r t i l i s a t i o n pore" and " f e r t i l i s a t i o n tube", x 3,000 Figure 58: Extruded egg of A. marginata showing attachment of antherozooid. x 3,000 Figure 59: Extruded egg of A. taeniata showing the point of attachment of the antherozooid. x 3,000 Legend: FT - " F e r t i l i s a t i o n tube" FP - " F e r t i l i s a t i o n pore" ON - Oogonial "neck" AP - Antherozooid p a p i l l a -114-Plate 14 Figure 60: Figure 61: Figure 62: Figure 63: Figure 64: Figure 65: Binucleate egg c e l l a f t e r plasmogamy i n A. marginata. x 3,000 Karyogamy i n A. marginata. x 3,000 Elongation of the zygote of A. marginata pri o r to i t s germination, x 3,000 Elongation of the zygote of A. taeniata prior to i t s germination, x 3,000 Elongation of the zygote i n A. nana, x 3,000 Two-celled sporophyte of A. nana, x 3,000 Legend: N - Nucleus Nc - Nucleolus -115-Plate 15 Figure 66: Four-celled sporophyte of A. marginata. x 1,000 Figure 67: Formation of a b i s e r i a t e filament at the three-c e l l e d stage i n A., tenuif o l i a . x 1,000 Figure 68: Formation of a b i s e r i a t e sporophyte filament at the four-celled stage i n A. marginata. x 1,000 Figure 69: Formation of a parthenosporophyte within an oogonium of A. taeniata. x 1,000 Figure 70: Nine-celled uniseriate sporophyte of A. marginata showing the formation of a r h i z o i d a l basal c e l l , x 1,000 Figure 71: Formation of a b i s e r i a t e young sporophyte at ten c e l l stage i n A. nana, x 1,000 Legend: N - Nucleus -116-Plate 16 Figure 72: Young sporophyte of A. marginata s t i l l attached to empty oogonium, x 1,000 Figure 73: Young sporophyte of A. t e n u i f o l i a . x 1,000 Figure 74: Branched r h i z o i d a l basal c e l l of young sporo-phyte. x 1,000 -117-Plate 17 Figure 75: Figure 76: Figure 77: Figure 78: Figure 79: Figure 80: Figure 8.1: Figure 82: Figure 83: Figure 84: Figure 85: Figure 86: Granular interphase nucleus i n zygote of A . taeniata. x 3,000 Reticular early prophase stage of mitosis i n female gametophyte of A . marginata. x 3,000 Mid-prophase of mitosis i n female gametophyte of A . marginata showing the condensation of d i s t i n c t chromosomes, x 3,000 Mid-prophase of mitosis i n female gametophyte of A . t e n u i f o l i a showing pairs of chromatids, x 4,000 Diagrammatic representation of F i g . 78. Late prophase of mitosis i n male gametophyte of A . f i s t u l o s a . x 4,000 Late prophase of mitosis i n female gametophyte of A . f i s t u l o s a showing d i s t i n c t chromosomes, x 4,000 Three progressive f o c a l views of late prophase of mitosis i n female gametophyte of A . taeniata. Centromeres can be distinguished at t h i s stage, x 4,000 Metaphase plate of chromosomes i n mitosis of male gametophyte of A . marginata. x 3,000 Mit o t i c anaphase i n germination tube of A . nana, x 3,000 ~ Legend: N - Nucleus Nc - Nucleolus HG - Heterochromatic granules CD - Chromatids CM - Centromere - 1 1 8 -Piate 18 Figure 87: Figure 88: Figure 8 9 : Figure 90: Figure 9 1 : Figure 92: Figure 9 3 : Figure 94: Figure 9 5 : Figure 96: Figure 97: Figure 98: Diplotene configuration of bivalents i n an immature zoosporangium of A. marginata'. 14 bivalents can be counted, x 4,000 Diagrammatic representation of F i g . 8 7 . Prometaphase stage of meiosis i n A. marginata i n which 14 bivalents can be counted, x 4,000 Diagrammatic representation of F i g . 89. Progressive f o c a l views of polar aspect of metaphase I i n an immature zoosporangium of A. marginata. 14 bivalents. x 4,000 Diagrammatic representation of Figs. 91 8s 9 2 . Progressive f o c a l views of diplotene/diakinesis configuration i n an immature zoosporangium of A. marginata. 16 bivalents. x 4,000 Diagrammatic representation of Figs 94 8s 9 5 . Metaphase I plate i n an immature zoosporangium of A. marginata, showing 14 bivalents. x 4,000 Diagrammatic representation of F i g . 9 7 . -119-Plate 19 Figure 99: Figure 100: Figure 101: Figure 102: Figure 103: Figure 104: Figure 105: Figure 106: Figure 107: Figure 108: Figure 109: Figure 110: Progressive f o c a l viewsof a polar aspect of metaphase I i n an immature zoosporangium of A. nana. 15 bivalents. x 4,000 Diagrammaticrepresentation of Figs. 99 & 100. Diplotene configuration i n immature zoospor-angium of A. nana, showing 12 bivalents. x 4,000 Diagrammatic representation of Figs. 102 & 103, Progressive f o c a l views of prometaphase I i n an immature zoosporangium of A. nana. 12 bivalents. x 4,000 Diagrammatic representation of Figs. 105 & 106, Progressive f o c a l views of prometaphase I i n an immature zoosporangium of A. nana. 12 bivalents. x 4,000 Diagrammatic representation of Figs. 108 & 109, -120-Plate 20 Figure 111: Metaphase plate i n meiosis I i n an immature zoosporangium of A. t e n u i f o l i a . 13 bivalents, x 4,000 Figure 112: Diagrammatic representation of F i g . 111. Figure 113 Metaphase I configuration i n an immature zoosporangium of A. t e n u i f o l i a . 14 bivalents x 4,000 Figure 114: Diagrammatic representation of F i g . 113. Figure 115: Figure 116: Figure 117: Figure 118: Figure 119: Figure 120: Figure 121: Figure 122: Asynaptic stage of la t e prophase I i n an immature zoosporangium of A. t e n u i f o l i a , at which 21 chromosomes can be counted, x 4,000 Diagrammatic representation of Figs. 115 & 116, Progressive f o c a l views of metaphase I con-f i g u r a t i o n i n an immature zoosporangium of A. t e n u i f o l i a . 12 bivalents. x 4,000 Diagrammatic representation of Figs. 118 8& 119, Side view of metaphasel plate of bivalents i n an immature zoosporangium of A. t e n u i f o l i a . 13 bivalents. x 4,000 Diagrammatic representation of F i g . 121. -121-Plate 21 Figure 123: Figure 124: Figure 125: Figure 126: Figure 127: Figure 128: Figure 129: Figure 130: Progressive f o c a l views of metaphase I i n am immature zoosporangium of A. f i s t u l o s a . 12 bivalents. x 4,000 Diagrammatic representation of Figs. 123 & 124. Metaphase I plate of bivents i n an immature zoosporangium of A. f i s t u l o s a . 12 bivalents. x 4,000 Diagrammatic representation of F i g . 126. Metaphase I plate of bivalents i n an immature zoosporangium of A. f i s t u l o s a . 13 bivalents. x 4,000 Diagrammatic representation of F i g . 128. Metaphase I plate of bivalents i n an immature zoosporangium of A. f i s t u l o s a . 13 bivalents. x 4,000 Figure 131: Diagrammatic representation of F i g . 130. Figure 132: Figure 133: Figure 134: Progressive f o c a l views of a metaphase I plate of bivalents i n an immature zoosporangium of A. f i s t u l o s a . 13 bivalents. x 4,000 Diagrammatic representation of Figs. 132 & 133. -122-Plate 22 Figure 135: Diplotene configuration i n an immature zoo-sporangium of A. taeniata. 14 bivalents. x 4,000 Figure 136: Diagrammatic representation of F i g . 135. Figure 137: Diakinesis configuration i n an immature zoo-sporangium of A. taeniata. 14 bivalents. x 4,000 Figure 138: Diagrammatic representation of F i g . 137. Figure 139: Diakinesis configuration i n an immature zoo-sporangium of A. g r a n d i f o l i a . 25 bivalents. x 4,000 Figure 140: Diagrammatic representation of F i g . 139. Figure 141:Progressive f o c a l views of diplotene of meiosis Figure 142: i n an immature zoosporangium of A. g r a n d i f o l i a . 26 bivalents. x 4,000 Figure 143: Diagrammatic representation of Figs. 141 & 142. Figure 144: Progressive f o c a l views of a diplotene con-Figure 145: f i g u r a t i o n i n an immature zoosporangium of A. g r a n d i f o l i a . 24 bivalents. x 4,000 Figure 146: Diagrammatic representation of Figs. 144 & 145. -123-Plate 23 Figure 147: Figure 148: Progressive f o c a l views of a diakinesis con-Figure 149: f i g u r a t i o n i n an immature zoosporangium of Figure 150: A. g r a n d i f o l i a . 24 bivalents. x 4,000 Figure 151: Figure 152: Diagrammatic representation of Figs. 147, 148, 149, 150 & 151. Figure 153: Diplotene configuration i n an immature zoo-sporangium of A. g r a n d i f o l i a . 25 bivalents. x 4,000 Figure 154: Diagrammatic representation of F i g . 153. Figure 155: Progressive f o c a l views of a diplotene con-Figure 156: f i g u r a t i o n i n an immature zoosporangium of Figure 157: A. g r a n d i f o l i a . 17 bivalents. x 4,000 Figure 158: Diagrammatic representation of Figs. 155, 156 & 157. -124-Plate 24 Figure 159: Figure 160: Figure 161: Figure 162: Figure 163: Figure 164: Figure 165: Figure 166: Figure 167: Figure 168: Figure 169: Figure 170: Progressive f o c a l views of late prophase of mitosis i n a male gametophyte of A', marginata. 14 chromosomes, x 4,000 Diagrammatic representation of Figs. 159 & 160. Progressive f o c a l views of la t e prophase of mitosis i n a female gametophyte of A. marginata, 16 chromosomes, x 4,000 Diagrammatic representation of Fi g s . 162 & 163. Late prophase of mitosis i n a males gameto-phyte of A. marginata. 13 chromosomes1, x 4,000 Diagrammatic representation of F i g . 165. Late prophase of mitosis i n a female gameto-phyte of A. marginata. 14 chromosomes, x 4,000 Diagrammatic representation of F i g . 167. Late prophase of mitosis i n a female gameto-phyte of A., nana. 15 chromosomes, x 4,000 Diagrammatic representation of F i g . 169. -125-Plate 25 Figure 171: Figure 172: Figure 173: Figure 174: Figure 175: Figure 176: Figure 177: Figure 178: Figure 179: Figure 180: Figure 181: Figure 182: Progressive f o c a l views of late prophase of mitosis i n a female gametophyte of A. nana. 15 Chromosomes, x 4,000 Diagrammatic representation of Figs. 171 & 172. Progressive f o c a l views of late prophase of mitosis i n a male gametophyte of A. nana. 14 chromosomes, x 4,000 Diagrammatic representation of Figs. 174 & 175. Late prophase of mitosis i n a female gameto-phyte of A. t e n u i f o l i a . 15 chromosomes, x 4,000 Diagrammatic representation of F i g . 177. Late prophase of mitosis i n a male gameto-phyte of _A. t e n u i f o l i a . 13 chromosomes, x 4,000 Diagrammatic representation of F i g . 179. Late prophase of mitosis i n a male gameto-phyte of A. t e n u i f o l i a . 13 chromosomes. x 4,000 Diagrammatic representation of F i g . 181. -126-Plate 26 Figure 183: Progressive f o c a l views of la t e prophase of Figure 184: mitosis i n a female gametophyte of A . taeniata. Figure 185: 15 chromosomes, x 4,000 Figure 186: Diagrammatic representation of Figs. 183, 184 8s 185. Figure 187: Progressive f o c a l views of late prophase of Figure 188: mitosis i n a male gametophyte of A . taeniata. Figure 189: 12 chromosomes, x 4,000 Figure 190: Figure 191: Diagrammatic representation of Figs. 187, ,188, 189 & 190. Figure 192: Progressive f o c a l views of la t e prophase of Figure 193: mitosis i n a female gametophyte of A . f i s t u l o s a . Figure 194: 14 chromosomes, x 4,000 Figure 195: A diagrammatic representation of Figs. 192. 193 & 194. Figure 196: Progressive f o c a l views of late prophase of Figure 197: mitosis i n a female gametophyte of A . f i s t u l o s a . 14 chromosomes, x 4,000 "~" Figureel98: Diagrammatic representation of Figs. 196 & 197. -127-Plate 27 Figure 199: Figure 200: Figure 201: Figure 202: Figure 203: Figure 204: Figure 205: Figure 206: Figure 207: Figure 208: Progressive f o c a l views of late prophase of the d i v i s i o n of the female gametophyte of A. taeniata prior to egg formation. 19 chromosomes, x 4,000 Diagrammatic representation of Figs. 199, 200, 201, & 202. Progressive f o c a l views of late prophase of mitosis i n the f i r s t d i v i s i o n of the zygote of A. marginata. 28 chromosomes, x 4,000 Figure 209: Diagrammatic representation of Figs. 204, 205, 206, 207 & 208. -128-Plate 28 Figure 210: Progressive f o c a l views of late mitotic prophase Figure 211: i n the f i r s t d i v i s i o n of the zygote i n A. marginata, Figure 212: 26 chromosomes, x 4,000 Figure 213: Diagrammatic representation of Figs. 210, 211, & 212. Figure 214: Progressive f o c a l views of late mitotic prophase Figure 215: i n the f i r s t d i v i s i o n of the zygote of A. marginata, Figure 216: 28 chromosomes, x 4,000 fig u r e 217: Figure 218: Figure 219: Diagrammatic representation of Figs. 214, 215, 216, 217 & 218. -129-Plate 29 Figure: 220: Progressive f o c a l views of late mitotic prophase Figure 221: i n the f i r s t d i v i s i o n of the zygote i n A. nana. Figure 222: 23 chromosomes, x 4,000 Figure 223 : Diagrammatic representation of Figs. 220, 221 & 222. Figure 224: Progressive f o c a l views of la t e mitotic Figure 225: prophase i n the f i r s t d i v i s i o n of the zygote Figure 226 : i n A. t e n u i f o l i a . 24 chromosomes, x 4,000 Figure 227: Diagrammatic representation of Figs. 224, 225 & 226. Figure 228: Progressive f o c a l views of late mitotic Figure 229: prophase i n the f i r s t d i v i s i o n of the zygote i n Figure 230: A. t e n u i f o l i a . 24 chromosomes, x 4,000 Figure 231: Diagrammatic representation of Figs. 228, 229 8s 230. -130-Plate 30 Figure 232: Late mitotic prophase i n a three-celled sporo-Figure 233: phyte of A. taeniata (progressive f o c a l views) Figure 234: 26 chromosomes, x 4,000 Figure 235: Figure 236: Figure 237: Diagrammatic representation of Figs. 232, 233, 234, 235 & 236. Figure 238: Late mitotic prophase i n a binucleate sporo-Figure 239: phytic c e l l of A. taeniata (progressive f o c a l Figure 240: views). 24 chromosomes v i s i b l e i n both complements, x 4,000 Figure 241: Diagrammatic representation of Figs. 238, 239 & 240. -131-Plate 31 Figure 242: Figure 243: Figure 244: Figure 245: Late mitotic prophase i n the i n i t i a l d i v i s i o n of the zygote of A. f i s t u l o s a (progressive f o c a l views). 23 chromosomes, x 4,000 Figure 246: Diagrammatic representation of Figs. 242, 243, 244 8s 245. Figure 247: Figure 248: Figure 249: Figure 250: Figure 251: Late mitotic prophase i n a two-celled partheno-sporophyte of A. taeniata. 12 chromosomes, x 4,000 Figure 252: Diagrammatic representation of Figs. 247, 248, 249, 250 8s 251. Plate 32 Figure 253 FIGURE 253 Graphic representation of chromosome counts made during meiosis in Alar i a spp. CHROMOSOME NUMBERS OBTAINFP rt rt •H faJD rt e <l <l o •H 3 C CU <l rt o rH 3 (fi •H < o •r-l TJ C rt u SPECIES Plate 33 Figure 254: FIGURE 254 Graphic representation of chromosome counts made during mitosis in male and female gametophytes of A l a r i a spp. oi w ca s Z w s o w o s o 0£ JE U o u z w S3 w OS rH 12 11 10 9 . 6 . 4 -2 . 16 12 17 12 CHROMOSOME NUMBERS OBTAINED a +-> a c • i H rt O •r-l 3 C SPECIES a +-> •H c cu rt rt cn O r—I 3 +-> (fl •H < Plate 34 F i g u r e 255 FIGURE 255 G r a p h i c r e p r e s e n t a t i o n of chromosome counts made d u r i n g m i t o s i s i n young c u l t u r e d s p o r o p h y t e s of A l a r i a s p p . co OS w CQ S W s o w o s o ca 55 o fa o >" u 55 w w ca fa 12. 11. 10. 9 8-7" 6 5 4-3 2 1 29 23 30 24 31 24 CHROMOSOME NUMBERS OBTAINED 31 23 31 +-> S3 C •H hD rH S cS o «H •i-l 3 C OJ <l SPECIES ui o r-l 3 cn •H <H <l -135-Plate 35 Figure 256: The aff e c t of temperature on gametophytic growth i n A. g r a n d i f o l i a . Graphic representation of range of gametophyte size and mean s i z e . o A - Male gametophytes at 10 6. B - Female gametophytes at 10 °C. C - Male gametophytes at 5°C. D - Female gametophytes at 5°C. F i g u r e 2 56 0 B C C U L T U R E S D -136-Plate 36 Figure 257: The aff e c t of temperature on gametophytie growth i n A. f i s t u l o s a . Graphic representation of range of gametophyte si z e and mean s i z e . A - Male gametophytes at 10°C. B - Female gametophytes at 10°C. C - Male gametophytes at 5 ° C D - Female gametophytes at 5°C. Figure 257 w H X a o a ^ 200 -w o A B C D cultures 

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