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A cytomorphological study of Pelvetiopsis limitata (Setchell) Gardner Zanon , Carol Aileen Rosemary (Hennigan), 1961

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A CYTOMORPHOLOGICAL STUDY OF PELVETIOPSIS LIMITATA (SETCHELL) GARDNER by CAROL AILEEN HENNIGAN B. S c , Mount Saint Vincent College, 1958 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of BIOLOGY AND BOTANY We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1961 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed-without my w r i t t e n permission. Department of Biology and Botany The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. D a t e March 21, 1961. i i ABSTRACT A cytomorphological study of P e l v e t i o p s i s l i m i t a t a ( S e t c h e l l ) Gardner confirmed a c y t o l o g i c a l a l t e r n a t i o n of generations i n the plant. Approximately thirty-two chromosomes i n the haploid phase alternate with si x t y - f o u r chromosomes i n the d i p l o i d phase. P e l v e t i o p s i s i s oogamous and monoecious. Antheridia and oogonia are produced i n flask-shaped conceptacles. Nuclear d i v i s i o n i s regular i n developing gametangia. Reduction of chromosome number appears to take place during the f i r s t nuclear d i v i s i o n i n the antheridium and oogonium. No centrosomes or chromophilous spherules are d i s c e r n i b l e during nuclear d i v i s i o n . In the oogonium the two d i v i s i o n s of the meiotic sequence are followed by a m i t o t i c d i v i s i o n producing eight n u c l e i . Two eggs of unequal siz e are formed i n the oogonium. The larger egg i s uninucleate and f u n c t i o n a l , while the smaller i s seven-nucleate. At maturity k to 16 chromocenters may be observed i n the nucleus of the large egg p r i o r to i t s l i b e r a t i o n . Before the release of the large egg from the oogonium approximately 32 chromosomes d i f f e r e n t i a t e as the nucleus enters m i t o t i c prophase. The smaller egg formed i n the oogonium u s u a l l y d i s i n t e g r a t e s a f t e r extrusion from the conceptacle. The inner layers of the wall of the oogonium are soon d i s -carded a f t e r l i b e r a t i o n . Aberrant eggs occur. Two eggs of equal s i z e may form or there may be as many as 5 eggs of various s i z e s . Binucleate eggs are sometimes present. In the antheridium h m i t o t i c d i v i s i o n s follow meiosis producing 6k n u c l e i . The cytoplasm d i f f e r e n t i a t e s around each nucleus and Gk spermatozoids are formed. These are released by the rupture of the outer lay e r , and by g e l a t i n i z a t i o n of the inner layer of the w a l l of the antheridium. Approximately 30 - 32 chromosomes were observed at the second nuclear d i v i s i o n i n the a n t h e r i d i a l i n i t i a l . i i i Oogamy i n P e l v e t i o p s i s restores the d i p l o i d complement of approximately 6k chromosomes. F e r t i l i z a t i o n occurs a f t e r the gametes have been l i b e r a t e d from the conceptacle. The egg i s naked at the time of f e r t i l i z a t i o n . The f i r s t d i v i s i o n of the zygote i s transverse to the plane i n which the r h i z o i d develops. One, sometimes two, primary r h i z o i d s form. Several other septate r h i z o i d s develop l a t e r . The anatomy of the t h a l l u s of P e l v e t i o p s i s i s s i m i l a r to other Fucaceae. Cryptostomata are rare and caecostomata are absent. S t e r i l i z e d f i l t e r e d seawater cooled to 6° to 9°C provides a sui t a b l e medium i n which P e l v e t i o p s i s l i m i t a t a may be grown i n cul t u r e . A minimum l i g h t i n t e n s i t y of 250 foot-candles promotes s a t i s f a c t o r y development. i v TABLE OF CONTENTS Topic Page Acknowledgements v A. Introduction 1 1. Objectives of the i n v e s t i g a t i o n 1 2. P e l v e t i o p s i s and r e l a t e d genera 1 B. L i t e r a t u r e Survey h 1. General k 2. Conceptacle formation k 3. Cryptostomata and caecostomata 5 k. Reproductive structures 6 a. Production of eggs 7 b. Production of spermatozoids 10 5. L i b e r a t i o n of eggs and spermatozoids 12 6. F e r t i l i z a t i o n 13 7. H y b r i d i z a t i o n ik 8. Embryo development 15 C. Materials and Methods 17 D. Observations 20 1. Cultures 20 2. Anatomy of the t h a l l u s 20 3. Conceptacle formation 21 h. Oogonia and antheridia 22 a. Oogonia 22 b. Antheridia 26 5. L i b e r a t i o n of gametes 29 6. F e r t i l i z a t i o n 29 7. Embryo development 30 E. Discussion 32 1. Culture conditions and natural environment 32 2. E a r l y development of the conceptacle i n i t i a l 32 3. M i t o s i s and meiosis 33 k. Chromocenters i n the mature egg 3^ -5. Variations i n the production of eggs 35 6. Comparison of gametangia and gametes of Pe l v e t i o p s i s l i m i t a t a with other species of the Fucales 36 7. Development of the l i b e r a t e d eggs and spermatozoids 39 8. The systematic p o s i t i o n of P e l v e t i o p s i s kl F. Summary kk G. L i t e r a t u r e c i t e d h6 H. Tables 53 I. Figures 54 V ACKNOWLEDGEMENTS The author wishes to express her gratitude to the following organizations and i n d i v i d u a l s for assistance i n making possible the research reported i n t h i s t h e s i s : to the Archbishop McCarthy Assembly, Fourth Degree Knights of Columbus, Ha l i f a x , Nova Scotia for award of a post graduate scholarship (1958 - 1959); to the National Research Council for award of a bursary (1959 - i960) and summer supplement (i960); to Dr. K. M. Cole for her invaluable c r i t i c i s m ; to Dr. R.F. Scagel fo r h i s h e l p f u l suggestions; to Mr. T. B. Widdowson for h i s assistance i n c o l l e c t i n g experimental material; and to the Department of Biology and Botany of the Un i v e r s i t y of B r i t i s h Columbia f o r use of f a c i l i t i e s . 1 A Cytomorphological Study of P e l v e t i o p s i s  l i m i t a t a ( S e t c h e l l ) Gardner. A. Introduction 1. Objectives of the Investigation. Although many members of the Fucales have been investigated thoroughly, there i s very l i t t l e l i t e r a t u r e concerning P e l v e t i o p s i s  l i m i t a t a ( S e t c h e l l ) Gardner. Since the development of antheridia and oogonia of P e l v e t i o p s i s has not been thoroughly discussed, a c y t o l o g i c a l study of the l i f e cycle of one form, P e l v e t i o p s i s l i m i t a t a f. l i m i t a t a , has been undertaken. The data c o l l e c t e d are compared to those presented by other investigators f o r various other members of the family Fucaceae and the order Fucales. An attempt i s made to e s t a b l i s h the p o s i t i o n of Pe l v e t i o p s i s l i m i t a t a amongst r e l a t e d genera with regard to i t s c y t o l o g i c a l and morphological c h a r a c t e r i s t i c s i n order that the l i f e cycle may be better understood. 2. P e l v e t i o p s i s and r e l a t e d genera. The genus P e l v e t i o p s i s was established by Gardner i n 1910. P e l v e t i o p s i s was merely a new combination for the plant S e t c h e l l had f i r s t described i n 1905 as Pe l v e t i a f a s t i g i a t a f. l i m i t a t a ( C o l l i n s , Holden and S e t c h e l l , 1905). In t h i s e a r l y paper, Gardner described b r i e f l y the mor-phology of the plant. Further morphological d e t a i l s were l a t e r presented (Gardner, 1913; S e t c h e l l and Gardner, 1925)- A somewhat s u p e r f i c i a l d e s c r i p t i o n of the vegetative tissues and reproductive structures had been made e a r l i e r by Holtz (1903) who i d e n t i f i e d the plant as Pe l v e t i a f a s t i g i a t a (J. Ag.) DeToni. 2 Although P e l v e t i o p s i s and P e l v e t i a are s i m i l a r i n external vegeta-t i v e c h a r a c t e r i s t i c s , cytoplasmic d i v i s i o n s within the oogonium occur i n a d i f f e r e n t manner i n the two genera, (Gardner, 1910, 1913)- P e l v e t i o p s i s resembles Hesperophycus harveyanus i n i t s sexual development, but the two plants are quite d i f f e r e n t i n gross morphology (Gardner, 1913)- Thus i t can be seen that the establishment of the genus P e l v e t i o p s i s as d i s t i n c t from other genera was necessary. The genus P e l v e t i o p s i s includes two species, P. l i m i t a t a ( S e t c h e l l ) Gardner, and P. arborescens Gardner (Gardner, 1910, 19^0). P. l i m i t a t a has two recognized forms, P. l i m i t a t a f. l a t a (Gardner, 1913) and P. l i m i t a t a f. l i m i t a t a (Gardner, 1913; Scagel, 1957)' P e l v e t i o p s i s l i m i t a t a f. l i m i t a t a occurs from Hope Island, B.C. to Carmel, C a l i f o r n i a (Scagel, 1957)« P. l i m i t a t a f. l a t a grows i n the area from Tomales Point to Monterey, C a l i f o r n i a (Smith, 19^)• P- arborescens i s known only from Carmel, C a l i f o r n i a (Gardner, 19^0). P e l v e t i o p s i s l i m i t a t a f . l i m i t a t a i s a small plant, 1-12 cm. high when mature. I t i s o l i v e to brownish green i n c o l o r , and bushy i n habit (Fig. 1 and 2). The plant i s d i f f e r e n t i a t e d into three areas, the holdfast, the s t i p i t a t e region and the branches. The holdfast i s i r r e g u l a r l y c o n i c a l i n shape and about 6 - 9 Him. i n diameter. One or more stipes may a r i s e from the holdfast. There i s no midrib. Each branch consists of a lower f l a t t e n e d portion a r i s i n g from the repeatedly dichotomous s t i p i t a t e region and a d i s t a l l y extended portion containing mucilage. The receptacles are usually bilobed. The receptacles become much enlarged during reproductive periods. The gametangia are borne i n conceptacles located within the receptacles. Related genera of the Fucaceae mentioned i n the following discussion include Ascophyllum, Fucus, Hesperophycus, P e l v e t i a , Phyllospora and Xiphophora. 3 Included also are a few representative genera from other f a m i l i e s : Hormosira and Notheia from the Notheiaceae; B i f u r c a r i a , Cystophyllum, Cystoseira and Halidrys from the Cystoseiraceae; Carpophyllum, Coccophora, Sargassum and Turbinaria from the Sargassaceae; and Himanthalia from the monotypic Himanthaliaceae. B. L i t e r a t u r e Survey 1. General Members of the Fucales do not produce spores, and have only a c y t o l o g i c a l a l t e r n a t i o n of generations; i n these respects the Fucales are comparable to the Metazoa (Strasburger, 1897; Farmer and Williams, I898; Yamanouchi, 1909; Svedelius, 1929). In the Fucales the extremely reduced haploid generation develops -within the conceptacles of the d i p l o i d generation. The d i p l o i d chromosome number i s restored at f e r t i l i z a t i o n and maintained i n the macroscopic generation. The haploid generation i s represented by only the few n u c l e i i n the oogonium and antheridium produced by meiosis and the m i t o t i c d i v i s i o n succeeding i t (Fig. 3)' Such haploid n u c l e i occur i n the antheridium from the two- to s i x t y - f o u r -nucleate stage, i n the oogonium from the two- to eight-nucleate stage, and i n the mature spermatozoids and eggs (Yamanouchi, 1909). Smith (1956), however,maintains that the t h a l l i of the Fucales are sporophytes. Accord-ing to h i s i n t e r p r e t a t i o n of the l i f e c ycle, spores which are produced i n u n i l o c u l a r sporangia function as gametes. 2. Conceptacle formation In the Fucaceae, conceptacles are i n i t i a t e d within receptacles located on the upper l i m i t s of the dichotomies. There are about 150 to 200 conceptacles i n each branch of the bilobed receptacles i n P e l v e t i o p s i s  l i m i t a t a (Holtz, 1903). Several workers have described the development of the conceptacle and i t s contents. Bower (1880) was the f i r s t to suggest that the conceptacle of Fucus has i t s o r i g i n from an epidermal c e l l of the receptacle; he wrongly concluded, however, that the i n i t i a l c e l l l a t e r d i s i n t e g r a t e s and c o r t i c a l c e l l s complete'the conceptacle. Holtz (1903) also concluded that c e l l s from the 5 cortex of P e l v e t i o p s i s l i m i t a t a p a r t i c i p a t e i n the development of the concep .-tacl-e.. I t was soon established (Nienburg, 1913) that the cortex i s not involved in^conceptacle formation of Fucales, but that the conceptacle i s i n i t i a t e d by a single epidermal c e l l which becomes sunken i n the t h a l l u s . Further study showed that the flask-shaped conceptacle i n i t i a l c e l l divides into two c e l l s by a transverse or curved w a l l . The outer c e l l so formed i s known as the tongue c e l l , the inner c e l l i s known as the basal c e l l . The f i r s t d i v i s i o n of the conceptacle i n i t i a l of P e l v e t i a c a n a l i c u l a t a , however, i s l o n g i t u d i n a l and i s followed by a transverse d i v i s i o n producing two basal c e l l s (Nienburg, 1913/ Moore, 1928). Nienburg (1913) considered the tongue c e l l filament, which occurs i n some of the Fucales, to be a manifestation of v e s t i g i a l t r i c h o t h a l l i c growth. In the Fucaceae, the tongue c e l l grows no further, and does not contribute to the formation of the conceptacle. 3. Cryptostomata and caecostomata. I t was noted by Simons (1906) that conceptacles and h a i r p i t s ( c a v i t i e s i n the receptacle containing h a i r - l i k e structures) of Sargassum  f i l i p e n d u l a are homologous structures. Both structures are derived from i n d i v i d u a l epidermal c e l l s . I t was noted i n Fucus that h a i r p i t s are capable of becoming sexual (Roe, I916). Cryptostomata ( s t e r i l e h a i r p i t s ) have been observed i n Fucus vesiculosus (Baker and Bohling, 1916), Sargassum f i l i p e n d u l a (Simons, 1906), S. tenerrimum (Rao, 1946), Turbinaria turbinata (Blomquist, 19^5), o c c a s i o n a l l y i n P e l v e t i a f a s t i g i a t a (Moore, 1928), and r a r e l y i n Fucus  p a r k s i i (Gardner, 19^0). In Notheia anomala, cryptostomata have been observed i n the young plant, but they l a t e r disappear as the plant matures (Williams, 1923)- No cryptostomata have been noted i n Ascophyllum nodosum ( F r i t s c h , 19^5'), 6 B i f u r c a r i a tuberculata (Rees, 1933), Halidrys spp. ( F r i t s c h , 19 -^5) or Pelve t i a c a n a l i c u l a t a (Gardner, 1910). Cryptostomata have been reported to occur i n young plants of P e l v e t i o p s i s l i m i t a t a (Gardner, 1913)- Rao (19^6) saw oogonial i n i t i a l c e l l s occurring i n what he considered to be cryptostomata i n Sargassum tenerrimum and concluded that these represent degenerate and non-functional conceptacles. Caecostomata (completely closed c a v i t i e s which lack h a i r s ) are cl o s e l y r e l a t e d to cryptostomata and conceptacles (Powell, 1957b). Gardner (1922) noted caecostomata i n Fucus furcatus (= F. d i s t i c h u s subsp. edentatus (De l a Pyl.) Powell). Since that time caecostomata have been found i n a l l North American sub-species of Fucus d i s t i c h u s , and are now considered to be more common i n the A t l a n t i c sub-species (Powell, 1957a, 1957b). There are no caecostomata i n the C a l i f o r n i a species Fucus p a r k s i i (Gardner, igkO). k. Reproductive structures. Bower (1880) noted that male and female reproductive structures are morphologically i d e n t i c a l i n t h e i r e a r l y development. In Pe l v e t i o p s i s  l i m i t a t a as i n other Fucaceae i t has been noted that the antheridia and oogonia develop from c e l l s which form the wall of the conceptacle (Holtz, 1903)- Williams (1923) suggested that the oogonium i s sporangial i n nature and that Notheia anomala i s always d i p l o i d . Neither she nor M i t c h e l l (1893) found any i n d i c a t i o n of antheridia or of reduction d i v i s i o n i n the oogonium of Notheia. Williams (1923) seems to have been unaware of the work of Barton (1899) i n which antheridia were i d e n t i f i e d . 7 4a. Production of eggs In the Fucales the oogonia are u s u a l l y stalked, and are seldom borne on paraphyses (Thuret, 1854). In some species, such as Sargassum  tenerrimum, there i s no sta l k c e l l (Rao, 19^6). Each oogonium of Fucales species has a w a l l composed of at l e a s t three layers before l i b e r a t i o n of eggs takes place (Thuret and Bornet, 1878; Farmer and Williams, 1898). The outer portion of the oogonial w a l l of P e l v e t i a f a s t i g i a t a i s p e c t i c , and the inner portion contains c e l l u l o s e (Moore, 1928). Holtz (1903) claimed that the oogonial w a l l of P e l v e t i o p s i s l i m i t a t a has only two la y e r s . I t has been reported that discharged Fucus oogonia have f i v e - l a y e r e d walls (Resuhr, 1935). Three of these layers are firm, and they alternate with two softer l a y e r s . In P e l v e t i a c a n a l i c u l a t a (Subrahmanyan, 1957b) an outer layer of the oogonial w a l l and four inner layers have been i d e n t i f i e d . The outer layer g e l a t i n i z e s a f t e r the oogonium i s released i n t o the sea. From her studies of stained material M i t c h e l l (1941) concluded that there are at l e a s t f i v e layers i n some regions of the w a l l of the oogonium of Xiphophora. Naylor (1954), however, studied l i v i n g specimens of Xiphophora and concluded that there are only three layers i n the oogonial w a l l . The most p r i m i t i v e form of egg development i s found i n the genus Fucus where eight eggs are produced (Thuret, 1854; Thuret and Bornet, 1878). In a l l members of the Fucales, the nucleus of the oogonial i n i t i a l divides three times producing eight p o t e n t i a l egg n u c l e i . The f i r s t two d i v i s i o n s are reduc t i o n a l (Strasburger , 1897J Farmer and Williams, 1898) and comprise the meiotic sequence (Yamanouchi, 1909). Following a short period of r e s t , the t h i r d nuclear d i v i s i o n ensues (Farmer and Williams, 1898). This l a s t d i v i s i o n i s m i t o t i c (Yamanouchi, 1909)• Not a l l of these r e s u l t i n g eight n u c l e i 8 d i f f e r e n t i a t e into eggs within the mature oogonium (Oltmanns, 1889b; Thuret and Bornet, 1878). In most genera fewer eggs are produced. In P e l v e t i a two eggs are produced. In P. c a n a l i c u l a t a the d i v i s i o n of the oogonium i n t o two eggs i s p a r a l l e l to the longer axis of the oogonium, and the s i x supernumerary n u c l e i are cast out on the surface (Oltmanns, 1889b). In P. f a s t i g i a t a and P. w r i g h t i i the d i v i s i o n of the oogonium into two eggs i s p a r a l l e l or oblique to the shorter axis of the oogonium and the extra n u c l e i are cast out between the eggs (Yendo, 1907; Gardner, 1910; Moore, 1928). D e t a i l s on the number of eggs produced i n c e r t a i n other Fucales are presented i n Table I. Three nuclear d i v i s i o n s were observed i n oogonia of Hesperophycus  harveyanus and P e l v e t i o p s i s l i m i t a t a (Gardner, 1910). Gardner claimed that the m i t o t i c d i v i s i o n i s not simultaneous i n a l l n u c l e i i n the oogonium. Of the eight r e s u l t i n g n u c l e i , seven migrate to that end of the egg which i s cl o s e s t to the conceptacle w a l l , and a septum forms, c u t t i n g them o f f from the l a r g e r , now uninucleate, portion. Thus, two egg c e l l s are formed, one large and uninucleate, the other smaller with seven n u c l e i . Holtz (1903) was able to f o l l o w only the d i v i s i o n of the oogonial mother c e l l nucleus to the four-nucleate stage i n P e l v e t i o p s i s l i m i t a t a . He thought that the two eggs which form i n the oogonium are equal i n s i z e , and are separated by a transverse w a l l . Both eggs escape from the oogonium. The two eggs of Pel v e t i a spp. are retained within a thick-walled mucilaginous oogonium which protects them from dry p h y s i c a l conditions enabling the plant to s u c c e s s f u l l y reproduce i n the upper i n t e r t i d a l zone (Oltmanns, 1889a; Isaac, 1933). 9 Some of the e a r l i e s t chromosomal studies of Fucus and r e l a t e d genera were made by Farmer and Williams i n 1896 and 1898. In t h e i r c l a s s i c work they reported that the haploid number of chromosomes was approximately 10 - 12 i n the oogonia of Fucus serratus and F. vesiculosus . They considered the number to be between 26 - 30 i n the oogonium of Ascophyllum nodosum and Ik - 15 i n the egg i t s e l f . At l a t e prophase of f i r s t d i v i s i o n meiosis, Yamanouchi (1909) counted 6k chromosomes i n oogonia of Fucus vesiculosus. At metaphase of the t h i r d d i v i s i o n 32 chromosomes were observed, and also i n the four n u c l e i at the end of meiosis I I (Yamanouchi, 1909). Kunieda (1926, 1928) claimed that 16 was the haploid complement of chromosomes i n Sargassum horneri oogonia, but i t was l a t e r shown by Okabe (1929a) that there are 32 chromosomes. Nuclear d i v i s i o n i n the somatic c e l l s was described i n a l a t e r paper, and the d i p l o i d number was found to be 6k (okabe, 1930). The r e s u l t s of other investigations of the chromosome numbers of various Fucales are presented i n Table II. The cytology of the Fucales, p a r t i c u l a r l y that of the Sargassaceae, has been investigated by several workers. The f i r s t nuclear d i v i s i o n i n the oogonium of Sargassum i s re d u c t i o n a l (Tahara and Shimotomai, 1926). Synapsis and di a k i n e s i s are t y p i c a l i n Sargassum horneri (okabe, 1929a). In meiotic d i v i s i o n s the nuclear membrane u s u a l l y disappears when the metaphase p l a t e forms. This i s d i s t i n c t from the m i t o t i c d i v i s i o n s i n the young embryo where the nuclear membrane does not disappear u n t i l anaphase (Okabe, 1930). There i s no septum d i s c e r n i b l e at the r a r e l y observed two-nucleate stage; the spindle apparatus i n intranuclear (Shimotomai, 1928). The second d i v i s i o n of the meiotic sequence follows soon a f t e r the f i r s t d i v i s i o n i s completed. The two daughter n u c l e i divide simultaneously i n Sargassum (Tahara and Shimotomai, 1926). 10 kb. Production of Spermatozoids. The antheridium, l i k e the oogonium, a r i s e s as a p r o j e c t i o n from one of the c e l l s which form the w a l l of the conceptacle. The antheridium i s sometimes stalked as i n Sargassum horneri (Kunieda, 1928), Fucus spp., Ascophyllum nodosum, P e l v e t i a spp., and Himanthalia sp. (Thuret and Bornet, 1878). In Pe l v e t i o p s i s the a n t h e r i d i a may he borne s i n g l y on a ba s a l h a i r , or located on a branching h a i r . One to s i x a n t h e r i d i a may be found on each branching h a i r (Holtz, 1903). Antheridia of Ascophyllum, Fucus, Himanthalia (Thuret and Bornet, 1878), Hormosira (osborn, 19^9); and Pe l v e t i a (Isaac, 1933) have two-layered w a l l s , but those of B i f u r c a r i a , Cystoseira and Halidrys have only one layer (Thuret and Bornet, 1878). Meiotic and mi t o t i c d i v i s i o n s w i t h i n the a n t h e r i d i a of Fucales have been c l e a r l y outlined by several i n v e s t i g a t o r s . The f i r s t two d i v i s i o n s of the a n t h e r i d i a l i n i t i a l nucleus comprise the meiotic sequence. In Sargassum  p i l u l i f e r u m a s y n i z e s i s - l i k e f i g u r e appears i n lat e prophase of the f i r s t nuclear d i v i s i o n (inoh and Hiroe, 195^-a). T y p i c a l l y , four m i t o t i c d i v i s i o n s follow meiosis i n the a n t h e r i d i a of Fucales. This has been demonstrated i n Cystophyllum spp. (Shimotomai, 1928), Fucus vesiculosus (Yamanouchi, 1909), P e l v e t i a c a n a l i c u l a t a (Subrahmanyan, 1957a), P. w r i g h t i i (Yabu and Imai, 1957), Phyllospora comosa (Williams, 1923), Sargassum horneri (Kunieda, 1926, 1928), S. p i l u l i f e r u m (inoh and Hiroe, 195^ +c) and S. tenerrimum (Rao, 19^6). Kunieda (1926) reported that these d i v i s i o n s are simultaneous i n S. ho r n e r i . At the thirty-two to sixty-four-nucleate stages i n P e l v e t i a c a n a l i c u l a t a no cross-walls are formed i n the cytoplasm (Subrahmanyan, 1957a)• The formation of cross-walls i n a n t h e r i d i a l cytoplasm has been reported i n Fucus (Yamanouchi, 1909; Kylin , 1 9 l 6 ; Richard, 1932), and Pe l v e t i a f a s t i g i a t a (Moore, 1928). 11 Smith (igkk) appears to be the only investigator who claims that there are one-hundred twenty-eight, rather than sixty-four "microspores" (spermatozoids) formed i n the "microsporangium" (antheridium) of P e l v e t i o p s i s  l i m i t a t a and P e l v e t i a "fastigiata. Holtz (1903) presumed that there were sixt y - f o u r n u c l e i produced i n the.antheridium of P e l v e t i o p s i s although he counted approximately only f o r t y himself. The spermatozoid of Fucus i s uninucleate. Guignard (1889) was among the f i r s t to see the spermatozoid of Fucus, which he described as a pear-shaped c e l l . I t was f i r s t thought that a t h i n cytoplasmic envelope surrounded the nucleus ( K y l i n , 19l6, 1920; Richard, 1932). K y l i n reported that the main body of the spermatozoid has a blepharoplast, plastomere and chromatophore. I t has been demonstrated recently that contrary to previous thought, the nucleus does not occupy most of the body of the spermatozoid (Manton and Clarke, 1951). The mature spermatozoid i s k - 5 microns long and 2.3 - 2.5 microns wide ( K y l i n , 1916). Two f l a g e l l a of unequal length are l a t e r a l l y inserted i n the cytoplasm. The shorter flagellum i s d i r e c t e d forward (Richard, 1932). The longer of the two f l a g e l l a i s d i r e c t e d p o s t e r i o r l y . In Hormosira banksii the anterior flagellum i s 10 microns long, and the other, 13-5 microns (Osborn, 19^9)- Manton and Clarke (1951) i n t h e i r remarkable electron micro-scope study demonstrated the t i n s e l l a t i o n of the anterior flagellum of Fucus  serratus. The f i r s t count of the number of chromosomes i n the spermatozoid nucleus of Fucus was made by Strasburger (l897)' He estimated that there were 30 chromosomes i n the spermatozoid nucleus of Fucus platycarpus (F. s p i r a l i s L. var. platycarpus). Yamanouchi (1909) counted 32 chromosomes 12 i n the spermatozoid nucleus of Fucus vesiculosus, as also d i d Yabu and Imai(l957) i n the m i t o t i c d i v i s i o n s following meiosis i n the a n t h e r i d i a l n u c l e i of Pe l v e t i a w r i g h t i i . Subrahmanyan (1957a), however, reported 22 chromosomes as the haploid number i n the a n t h e r i d i a l nucleus of P. c a n a l i c u l a t a . 5. L i b e r a t i o n of eggs and spermatozoids. The mechanism responsible f o r l i b e r a t i o n of sexual products i s not c l e a r . I f receptacles of P e l v e t i o p s i s are d r i e d somewhat and then placed i n sea water, l i b e r a t i o n of eggs and spermatozoids w i l l ensue (Gardner, 1910). Turgor pressure and the pressure of the swelling mucilage of the inner layers of the oogonial w a l l may be the v i t a l f a c t o r s (Oltmanns, 1889a; Resuhr, 1935)' Pierce (1902) stated that l i b e r a t i o n i n Fucus was induced by mechanical pressure which developed within the plant i t s e l f , and was not dependent on drying followed by immersion. In P e l v e t i a f a s t i g i a t a gametes are shed i f plants are brought into the l i g h t a f t e r a period of darkness. As many as 10,000 eggs per receptacle have been released i n a half-hour period ( j a f f e , 195k). L i b e r a t i o n of the egg fascinated many of the e a r l y workers such as Thuret (185^) and Farmer and Williams (1898). Release of the eggs of Fucus begins with the rupture of the exochiton, the outermost layer of the oogonial w a l l (Thuret, 185^). The mesochiton (medial layer) then bursts and s l i d e s back over the eggs, and the endochiton (the innermost layer surrounding the egg) dis s o l v e s . According to Subrahmanyan (1957b), the ent i r e oogonium of Pe l v e t i a c a n a l i c u l a t a i s released from the conceptacle, although Delf (1935) reported that i n other Fucales the exochiton may rupture while the oogonium i s s t i l l attached. 13 The spermatozoids are released by the rupture of the exochiton while the antheridium i s s t i l l attached to the sta l k c e l l . The endochiton dissolves a f t e r discharge from the conceptacle i n t o the sea. In Hesperophycus harveyanus, however, the e n t i r e antheridium i s released into the surrounding medium when the conceptacles are induced to l i b e r a t e . Immature as w e l l as mature antheridia are released (Walker, 1931)• 6. F e r t i l i z a t i o n F e r t i l i z a t i o n u s u a l l y occurs a f t e r the eggs are released from the oogonium. In P e l v e t i a c a n a l i c u l a t a , however, f e r t i l i z a t i o n takes place within the oogonium (Subrahmanyan, 1957b). The eggs of several species increase i n size a f t e r t h e i r r e l e a s e . In Fucus spp. spermatozoids often surround the egg i n numbers large enough to cause i t to rotate at h-0 - 50 gyrations per minute (Thuret, 195^; Farmer and Williams, 1898). One spermatozoid penetrates the egg c e l l membrane and the male nucleus s l i p s through the membrane of the egg nucleus (Yamanouchi, 1909). Soon a f t e r t h i s the f e r t i l i z a t i o n membrane forms. The f e r t i l i z a t i o n membrane of eggs of Fucus s p i r a l i s and F. vesiculosus i s known from the studies of Levring (19V7). The spermatozoid which f e r t i l i z e s the egg causes a contraction of the surface of the egg. He postulated that the membrane of the u n f e r t i l i z e d egg acts as a base upon which the f e r t i l i z a t i o n membrane forms, and i s a part of i t . F e r t i l i z a t i o n i n Fucus may occur up to one hour a f t e r the sperma-tozoid has penetrated the egg (Yamanouchi, 1909). I t has been reported that a second centrosome i s associated with the entrance of the spermatozoid f o r i t appears at the spot where the spermatozoid entered the egg (Strasburger, 1897; Yamanouchi, 1909). Farmer and Williams (1898) claimed that the centrosomes Ik which occur i n the f e r t i l i z e d oogonia of Fucus serratus, F. vesiculosus, and Ascophyllum nodosum are not associated with the spermatozoid. Polyspermy has been observed i n Ascophyllum, Fucus and Hespero-phycus (Farmer and Williams, 1898; Yamanouchi, 1909; Walker, 1931)• Damman ( l 9 3 l ) observed spindles with three and four poles i n Fucus and suggested that these are evidence that polyspermy has taken place. 7. Hybridization The p o s s i b i l i t y that h y b r i d i z a t i o n occurs between c e r t a i n algae has been investigated. As ea r l y as 185^ Thuret introduced hybrid formation by f e r t i l i z i n g eggs of Fucus vesiculosus with Fucus serratus spermatozoids. In 1908, Sauvageau reported the occurrence of the hybrid i n nature. This cross and i t s r e c i p r o c a l have since been made with over 90$ successful germinations i n both cases (Burrows and Lodge, 1951)• In l899> Williams reported that a hybrid had been induced i n the laboratory between Fucus  serratus and Ascophyllum nodosum. Whether or not t h i s hybrid occurs i n nature i s not known as there are no further d e t a i l s on the appearance, structure, or, behavior of the intergeneric hybrid. Whitaker (1931) established that there i s no c r o s s - f e r t i l i z a t i o n between F. vesiculosus and A. nodosum, nor between F. evanescens and F. vesiculosus. A cross between F. platycarpus (= F. s p i r a l i s L.) and F. ceranoides was found i n nature (Gard, 1910), and i t was noted that the female organs i n the hybrid were abnormal. Hybridization has also been reported as occurring between F. platycarpus (= F. s p i r a l i s ) and F. l u t a r i u s (Sauvageau, 1909), and between F. s p i r a l i s and F. vesiculosus (Burrows and Lodge, 1951)• 15 8. Embryo development Farmer and Williams (1898) claimed that d i v i s i o n of the Fucus zygote occurs twenty to twenty-four hours a f t e r the spermatozoid has penetrated the egg, although i n some Instances only t h i r t e e n hours elapse i n F. serratus. The newly f e r t i l i z e d egg elongates and divides transversely to the plane of the developing r h i z o i d (Decaisne and Thuret, 18^5; Oltmanns, 1889a). Three transverse walls are formed i n the Fucus zygote d i v i d i n g i t into four c e l l s . The c e l l d i s t a l to the d i f f e r e n t i a t i n g r h i z o i d then divides into four c e l l s by two l o n g i t u d i n a l d i v i s i o n s (oltmanns, 1889a; Nieriburg, 193l)« Segmentation of the embryo i s f a i r l y uniform i n Fucus spp., and d i f f e r s from other genera (Thuret, l851+; Oltmanns, 1889b; Wienburg, 1910). P o l a r i t y of the egg i s determined at f i r s t d i v i s i o n (Whitaker, 1931). In most cases the nucleus of the f e r t i l i z e d egg divides f i r s t and the r h i z o i d then appears. However, sometimes the r h i z o i d appears f i r s t and the zygote assumes a pear-shape before karyokinesis occurs (Farmer and Williams, 1898). Abe (1938) studied the d i v i s i o n of the f e r t i l i z e d egg of Sargassum. He discovered that further d i v i s i o n of the zygote nucleus takes place at the periphery of the c e l l rather than at the center. The segmentation d i v i s i o n s are not a l l simultaneous i n the embryo of Sargassum p i l u l i f e r u m (inoh and Hiroe, 195^a). The number of primary r h i z o i d s v a r i e s i n d i f f e r e n t species. The number of c e l l s p a r t i c i p a t i n g i n r h i z o i d formation i s r e l a t e d to the siz e of the egg (inoh, 1932). The upper c e l l s of the embryos of Fucales undergo further a n t i c l i n a l and p e r i c l i n a l d i v i s i o n s a f t e r the formation of a c e n t r a l core of c e l l s and a p e r i p h e r a l layer (oltmanns, 1889b; Nieriburg, 1931). Although LeTouze (1912) 16 claimed that there were only two tis s u e s i n the t h a l l u s , there are three d i s t i n c t regions i n the mature plant, the epidermis, the cortex and the medulla ( F r i t s c h , 19^5)• The a p i c a l meristem of members of the Fucales has i t s o r i g i n i n a sin g l e c e l l of the t h a l l u s (Woodworth, 1888). In Fucus, a c e l l at the apex of the young plant sinks i n t o the t h a l l u s as the c e l l s around i t continue to di v i d e , forming a groove (Oltmanns, 1889b). The c e l l becomes d i f f e r e n t i a t e d i n t o a h a i r with meristematic a c t i v i t y (Rienburg, 1931)' Other c e l l s i n the a p i c a l depression then produce h a i r s (Nienburg, 1931)> g i v i n g a t u f t e d appearance to the/young embryo. A l l of these h a i r s eventually d i s i n t e g r a t e with the exception of the basal c e l l of the f i r s t -formed h a i r which becomes the a p i c a l c e l l (Oltmanns, 1889b). In other f a m i l i e s of the Fucales, the a p i c a l . i n i t i a l c e l l remains three-sided. In the Fucaceae, however, i t becomes four-sided at an ea r l y stage (Woodworth, 1888; Oltmanns 1889b). In P e l v e t i a and Ascophyllum there are no hairs formed i n the a p i c a l groove (Oltmanns, 1889b). Increase i n width i s accomplished by d i v i s i o n s of the much l e s s -active c e l l s of the epidermis, or meristoderm, as i t i s sometimes known (Oltmanns, 1889b). The dichotomies of Fucus a r i s e as the r e s u l t of the d i v i s i o n of the a p i c a l i n i t i a l into two l a t e r a l i n i t i a l s , each of which then assumes the function of an a p i c a l c e l l . In Ascophyllum where the t h a l l u s i s much branched, several i n i t i a l s are cut o f f from d e r i v a t i v e s of the a p i c a l i n i t i a l . The growth and d i v i s i o n of the surrounding c e l l s cause them to become sunken i n the t h a l l u s . These l a t e r a l i n i t i a l c e l l s then grow and div i d e , thus g i v i n g r i s e to the branches of the t h a l l u s (Oltmanns, 1922). 17 C. Materials and Methods Specimens of Pe l v e t i o p s i s l i m i t a t a f . l i m i t a t a were c o l l e c t e d near Botany Beach, Port Renfrew, B r i t i s h Columbia i n June and November, 1959> a n-d i n May i960. M a t e r i a l was al s o obtained from Clayoquot, B.C. during the summer of 1959> a n& from Long Beach and Amphitrite Point, B.C. i n March, i960. Some plants from each c o l l e c t i o n were kept i n culture i n the cold chamber at the U n i v e r s i t y of B r i t i s h Columbia. Other plants were preserved i n one of the following s o l u t i o n s : - (a) a 10% s o l u t i o n of formalin i n sea water; (b) 3:1 f i x 3 parts 95% e t h y l a l c o h o l to one part g l a c i a l a c e t i c a c i d ; (c) a modified (Fensholt, 1955) Karpetchenko 1s s o l u t i o n of chrom-acetic formalin, c o n s i s t i n g of 2 solutions mixed i n equal proportions j u s t p r i o r to use. When material f i x e d f o r 8 to 2k hours i n 3:1 s o l u t i o n was not used immediately i t was stored i n 70% e t h y l a l c o h o l to which a small amount of f e r r i c acetate had been added. Mature and immature plants were cultured i n a cold chamber i n which the temperature was maintained at 6° - 9 ° C , with 12 hours of l i g h t and 12 hours of darkness per 2U-hour day. Each plant was located so that i t would be exposed to the same i n t e n s i t y of l i g h t each day. D i f f e r e n t sections of the culture area provided l i g h t i n t e n s i t i e s of 16, 50, 125, and 25O f o o t -candles. U n s t e r i l i z e d f i l t e r e d sea water was used i n i t i a l l y , but f i l t e r e d sea water, s t e r i l i z e d i n an autoclave at a presure of 15 pounds per square inch for 20 minutes, and cooled to 6° - 9 ° C , provided a more sui t a b l e medium. Sea water was changed i n the cultures every 5 - 7 days. Fungal contamination was c o n t r o l l e d by quic k l y dipping i n f e c t e d plants i n 95% e t h y l a l c o h o l , or i n a weak s o l u t i o n of calcium hypochlorite. 18 In order to determine i f material containing antheridia and oogonia was undergoing nuclear d i v i s i o n and was p r o f i t a b l e f o r future study, hand sections were made, both of l i v i n g and f i x e d material, according to the technique ou t l i n e d by Naylor (1957)• When i t was decided that the specimens were sui t a b l e f o r further examination, k i l l e d material was embedded i n Tissuemat with a melting point 56° - 58°C. Embedded material was sectioned with a rotary microtome. Sections varying i n thickness from k - 12 microns were mounted on glass s l i d e s . The mounted sections were dewaxed, hydrated i n a xylene, ethanol, water s e r i e s , then stained, dehydrated and mounted i n gum damar from xylene. Several stains were employed, among them, acetic-lacmoid, aceto-carmine, and Heidenhain's haematoxylin. A Feulgen technique as out l i n e d by Naylor (1958) was also attempted. A s a t i s f a c t o r y procedure for sectioned material was a modified aceto-carmine technique i n which both the mordant, 2fo f e r r i c alum ( f e r r i c ammonium s u l f a t e ) , and the aceto-carmine were heated to 60°C. The s l i d e s were mordanted 5 - 1 5 minutes, dipped i n d i s t i l l e d water to remove excess mordant, then stained i n the warm aceto-carmine f o r 10 -20 minutes. F e r t i l e conceptacles were encouraged to l i b e r a t e according to the method of Gardner (1910). The receptacles were permitted to dry f o r a few hours and were then immersed i n sea water. The l i b e r a t e d oogonia were c o l l e c t e d on s l i d e s . The mucilaginous nature of the conceptacle contents caused the eggs to adhere to the glass. These s l i d e s were then kept i n culture f o r further observation, or else f i x e d i n 3:1 a l c o h o l - a c e t i c - s o l u t i o n . 19 Photomicrographs were taken using Kodak High Contrast Copy 35 mm. f i l m , which was developed i n Kodak D - l l , and f i x e d i n Amfix. P r i n t s were made on Kodabromide F -3 single weight paper, developed i n Cobrol f o r 2 minutes and f i x e d i n Amfix. Photographs of camera l u c i d a drawings were made with Adox R lk 120 f i l m , and pr i n t e d on Kodak velox F-3 paper. 20 D. Observations 1. Cultures Specimens of P e l v e t i o p s i s l i m i t a t a f. l i m i t a t a grew favorably when cultured i n f i l t e r e d , s t e r i l i z e d sea water i n a col d chamber where the temperature was maintained at 6° - 9°C at the U n i v e r s i t y of B r i t i s h Columbia. Those plants exposed to the highest l i g h t i n t e n s i t y (250 foot-candles) were hea l t h i e r than plants grown i n the same chamber which received only 16, 50 or 125 foot-candles i l l u m i n a t i o n . Growth rates of specimens exposed to lower l i g h t i n t e n s i t i e s were not s a t i s f a c t o r y and the contamination by diatoms and fungi were encouraged. 2. Anatomy of the t h a l l u s In the mature receptacles of P e l v e t i o p s i s l i m i t a t a three d i s t i n c t t issues are present, the epidermis, cortex and medulla ( F i g . h and 5 ) ' The epidermis i s one layer thick, and i s protected by a mucilaginous layer, the " c u t i c l e " . The c e l l s of the epidermis appear rectangular i n cross-section ( F i g . 5)- They contain many ovoid chromatophores and fucosan v e s i c l e s . In a mature part of tire receptacle the epidermal c e l l averages 28 x 16 microns i n size and possesses a nucleus of about 5 microns i n diameter. The c e l l s of the cortex are c l o s e l y compressed and appear somewhat rounded i n cross-section, with f i v e to s i x sides ( F i g . 5 and 6 ) . The cortex i s four to f i v e c e l l s t h ick and ovoid chromatophores are p a r t i c u l a r l y abundant i n the outer two layers of the cortex. These c e l l s vary i n siz e from 16 x 12 microns to 28 x 22 microns. Most c o r t i c a l c e l l s are about 25 x 20 microns with n u c l e i from 5 to 6 microns i n diameter. Approximately 6^ chromosomes were seen i n one c e l l ( F i g. 7 a n d 8 ) . 21 There i s no cl e a r d i s t i n c t i o n between c o r t i c a l and medullary t i s s u e . The change i s gradual rather than abrupt. The medullary c e l l s form a network, although there are no hyphae i n the receptacle medulla. The c e l l s of the medulla are elongated v e r t i c a l l y ( F i g . 9 and 10) and have the appearance of sieve-tubes of higher plants. Simple p i t connections occur ( F i g . 9)- There i s abundant mucilage i n the i n t e r c e l l u l a r spaces. The n u c l e i of medullary c e l l s are about 6 microns i n diameter. 3» Conceptacle formation Many conceptacles are embedded within •- the receptacles of Pel v e t i o p s i s l i m i t a t a ( F ig. 11). During periods of f e r t i l i t y the receptacles become swollen and bloated, and sometimes are larger than the remainder of the plant. In a single cross-section of the receptacle of a f r u i t i n g specimen, as many as t h i r t e e n conceptacles may be observed; the average number per cross-section i s about s i x . The conceptacle i s i n i t i a t e d , as i n other Fucaceae, by a sing l e epidermal c e l l . The conceptacle i n i t i a l has a retarded rate of c e l l d i v i s i o n , and becomes embedded i n the t h a l l u s , remaining behind when the neighboring c e l l s adjoining i t grow and div i d e . As the surrounding c e l l s continue t h e i r growth, they separate from the conceptacle i n i t i a l . A groove i s thus formed with the i n i t i a l c e l l at the base (Fig. 12). The i n i t i a l c e l l divides transversely, forming an inner base c e l l and an outer tongue c e l l . The upper c e l l does not divide any further. I t may detach from the basal c e l l and d i s i n t e g r a t e l a t e r . The c e l l s l i n i n g the f l o o r of the conceptacle are derived from the i n i t i a l c e l l . The wall of the conceptacle may be three or four c e l l s t h ick as the r e s u l t of c e l l d i v i s i o n ( F ig. 11). The n u c l e i of the wall c e l l s are approximately 5 microns i n diameter. 22 The mature conceptacle i s a flask-shaped c a v i t y which contains oogonia, antheridia and paraphyses (hair filaments) ( F i g . 11). These paraphyses are p a r t i c u l a r l y abundant at the o s t i o l e , but do not project through i t . Cryptostomata were observed i n only a few instances, and i n very immature receptacles. A l l samples containing these cryptostomata also con-tained conceptacles with mature oogonia and a n t h e r i d i a . No caecostomata were observed. k. Oogonia and antheridia Oogonia and antheridia of P e l v e t i o p s i s l i m i t a t a were seen i n both l i v i n g and f i x e d material from a l l c o l l e c t i o n s . Oogonia and antheridia a r i s e as p a p i l l a e from c e l l s which form a part of the conceptacle w a l l . (Fig. 13 and ik). The c e l l nucleus divides m i t o t i c a l l y and one daughter nucleus remains i n the lower part of the p a p i l l a while the other assumes a p o s i t i o n near the d i s t a l end (Fig. 15 and 51.). A cross-wall then develops between them. The lower c e l l thus formed becomes the st a l k c e l l , and the upper c e l l becomes an oogonial or a n t h e r i d i a l mother c e l l . ha. Oogonia A study of f i x e d material c o l l e c t e d i n the summer of 1959 at Port Renfrew, B.C. y i e l d e d observations which were not p a r a l l e l e d i n any preceding or subsequent c o l l e c t i o n . The eggs of t h i s material were at a much more mature stage than those of any other material examined, and d e t a i l s of nuclear a c t i v i t y p r i o r to f e r t i l i z a t i o n were noted. D i f f e r e n t i a t i o n of the oogonium of P e l v e t i o p s i s l i m i t a t a begins as the oogonial i n i t i a l c e l l which i s attached to the w a l l of the conceptacle, enlarges. The nucleus may increase from 6.4 up to 15•5 microns i n diameter. 23 Two d i v i s i o n s of the nucleus ensue, presumably comprising the meiotic sequence. This cannot be stated d e f i n i t e l y as no chromosome count could be made during the f i r s t d i v i s i o n . As few oogonia were seen i n the two-nucleate stage i t was concluded that these nuclear d i v i s i o n s take place i n rapid sequence. (Fig. 16 - 20). There i s a b r i e f r e s t i n g stage between the f i r s t and second nuclear d i v i s i o n s when the nuclear membrane reforms (Fig. 17). At t h i s r e s t i n g stage the cytoplasm i s very dense, and the chromatin material i n the interphase nucleus i s very weakly stained. The chromatin i s d i f f u s e at t h i s stage, and i n d i v i d u a l chromosomes can no longer be distinguished. In some cases a single nucleolus i s the only deeply s t a i n i n g body observable i n the nucleus. The second nuclear d i v i s i o n of meiosis occurs simultaneously i n both of the n u c l e i i n the oogonium ( F i g . 18). The nuclear membrane d i s -appears e a r l y i n prophase I I , and the chromosomes pass r a p i d l y from t h i s stage into metaphase. No chromophilous spherule was observed. The chromosomes are very condensed at t h i s stage, and form the plate i n the equatorial region i n a regular manner. The metaphase I I spindle structure i s about 10 microns from pole to pole of the two simultaneously d i v i d i n g n u c l e i ( Fig. 19 and 20). The e q u a t o r i a l plate i s 7«2 microns at i t s widest point. In metaphase II the two n u c l e i are not orientated with regard to one another i n the oogonium. The spindle f i b e r s are very d i s t i n c t . No centrosomes occur, and there i s no i n d i c a t i o n of a s t r a l ray formation. There i s no precocious movement of chromosomes towards the polar regions. At the metaphase II stage of d i v i s i o n the oogonium measures 35'8 x 23-6 microns. 2k No septa are formed at either the two-nucleate or four-nucleate stage i n the developing oogonium. There i s a b r i e f r e s t i n g period at the four-nucleate stage p r i o r to the f i n a l d i v i s i o n into eight n u c l e i ( F i g . 21). At t h i s time the n u c l e i are i n close a s s o c i a t i o n . They s t a i n very l i g h t l y with aceto-carmine and become very translucent. The nucleolus i s the most d i s t i n c t body i n the nucleus, at t h i s stage. The t h i r d d i v i s i o n within the oogonium i s m i t o t i c and r e s u l t s i n the formation of eight n u c l e i ( F i g . 22). At t h i s time once again the n u c l e i are c l o s e l y associated. At f i r s t they s t a i n darkly with aceto-carmine, then the chromatin tends to become almost i n v i s i b l e as seven of the n u c l e i migrate to the end of the oogonium c l o s e s t to the st a l k c e l l ( F i g . 23). During d i v i s i o n stages of the n u c l e i , the cytoplasm of the oogonium occas i o n a l l y absorbs some of the s t a i n . A cross-wall forms i n the basal part of the oogonium ( F i g . 2k and 25). The plane of cleavage i s u s u a l l y perpendicular to the longer axis of the oogonium, but i t i s sometimes oblique ( F i g . kk). I t f i r s t appears as a t h i n l i n e i n the c e n t r a l region of the lower quarter of the oogonium. This cross-wall gradually thickens and eventually extends across the lower part of the oogonium. As the oogonium matures the two eggs produced separate from one another where the cross-wall formed ( F i g . 27 - 29). The space between the two eggs may be 3«1 microns wide. The larger egg formed by the cleavage of the cytoplasm has an e c c e n t r i c a l l y placed nucleus 10 microns i n diameter. The smaller egg contains seven n u c l e i of equal s i z e , ranging from 5*7 10 microns i n diameter, and averaging 6.8 microns. 25 The small egg i s l i m i t e d i n volume and the seven n u c l e i are n e c e s s a r i l y c l o s e l y situated to one another within the c e l l ( F i g. 30)- Often a single nucleolus i s d i s c e r n i b l e i n each of the n u c l e i . The nucleolus may be 1.9 - 2.1 microns i n diameter. Sometimes the sta l k c e l l of the mature oogonium i s p a r t i a l l y embedded i n the wall of the conceptacle. The sta l k c e l l averages 31 x 16 microns (Fig. 25). The wall of the oogonium has at l e a s t four and perhaps f i v e layers ( F i g . 26). The outer w a l l , or exochiton, i s firm, and the other layers are thinner. The size of a mature oogonium varies from 102.3 x 55-8 microns to 111.6 x 69-8 microns. There may be as few as one, or as many as thirty-two oogonia per conceptacle. The average number per conceptacle i s s i x oogonia. The eggs of P e l v e t i o p s i s l i m i t a t a vary i n size before and af t e r l i b e r a t i o n from the conceptacle. Before i t s release, the large mature egg i s 71-3 x ^9.6 microns to 86.8 x 6k microns, while the smaller egg i s from 15-5 x 26.k microns to 21.7 x 37-2 microns. While i t i s s t i l l contained and compressed within the oogonium i n the conceptacle, the smaller seven-nucleate egg often appears wedge-shaped i n l o n g i t u d i n a l section ( F ig. 25). A f t e r i t s release from the oogonium the large uni-nucleate egg may increase i n size from 90 to an average diameter of 108 microns (Fig. 75)- The large egg may be as much as 155 microns i n diameter a f t e r i t s l i b e r a t i o n . The nucleus may increase from 9 - l 6 microns i n diameter. A f t e r l i b e r a t i o n the smaller egg i s less compressed, becomes rounded (Fig. 28 and 29), and increases from 33 - 50 microns' i n diameter, averaging about 43 microns. The n u c l e i of the seven-nucleate eggs are 5-6 - 7-8 microns i n diameter both before and a f t e r l i b e r a t i o n ( F ig. 30). 26 The nucleus of the mature egg migrates from the center of the oogonium while i t i s i n the r e s t i n g condition. ( F i g . 31)> and i s s t i l l attached to the s t a l k c e l l . When i t reaches the periphery of the egg , several granular, densely s t a i n i n g bodies may be observed ( F i g . 32 - 37)- The number of these chromocenters varies from 4 - l 6 . The s i z e of these p a r t i c l e s varies; there may be discerned several smaller, d o t - l i k e granules as w e l l as one or two larger bodies ( F i g . 33)' There i s a tendency of these clumps of chromatin to e x h i b i t p o l a r i z a t i o n s i m i l a r to s y n i z e s i s ( Fig. 33 and 3*0' A number of d i s t i n c t granules was also seen (Fig. 3^ - 37)- At a l a t e r stage a number of chromosomes appear as the egg enters prophase i n preparation for d i v i s i o n even before f e r t i l i z a t i o n occurs. Approximately 32 chromosomes were counted i n the n u c l e i of mature eggs, by c a r e f u l examination of the material, and from photomicrographs which were taken at several l e v e l s i n the plane of v i s i o n (Fig. 38 and 39). While cleavage of the cytoplasm within the oogonium us u a l l y r e s u l t s i n the formation of a large uninucleate egg and a smaller egg with seven n u c l e i , aberrant forms have been noted. In some oogonia two equally-sized eggs were formed (Fig. 40 - 42). Three, four and p o s s i b l y f i v e eggs of various sizes have also been observed i n one oogonium ( F i g . 45 - 50). In addition, binucleate eggs occur (Fig. 43 and 44). 4b. Antheridia The antheridium of P e l v e t i o p s i s l i m i t a t a i s i n i t i a t e d i n the same way as the oogonium. It may be recognized at an e a r l y stage i n i t s development by i t s a f f i n i t y f o r aceto-carmine s t a i n . The antheridium i s stalked, a r i s i n g s i n g l y from the w a l l of the conceptacle, or on branched paraphyses (Fig. 51 - 53). 27 The w a l l of the antheridium i s composed of two l a y e r s which are united at the base of the antheridium. The mature antheridium i s from 3U.1 x 17.0 microns up to 57.2 x 23.0 microns i n s i z e . At the d i s t a l end of the antheridium there i s a space of h - 7 microns between the w a l l and the cytoplasm i n which the n u c l e i are embedded. In the maturing antheridium t h i s space has the appearance of an a p i c a l cap. Antheridia are more p l e n t i f u l than oogonia i n the conceptacle. Sometimes the an t h e r i d i a are small and immature while the eggs appear to be ready f o r l i b e r a t i o n . As many as t h i r t e e n a n t h e r i d i a may be observed on the same branch system, but f i v e per h a i r i s the average number (Fig . 7U). The f i r s t three d i v i s i o n s i n the antheridium are very s i m i l a r to those i n the oogonium. The f i r s t and second d i v i s i o n s follow r a p i d l y on one another and are probably meiosis I and I I ( F i g . 5U - 59)• Before d i v i s i o n the a n t h e r i d i a l i n i t i a l nucleus measures 5.0 - 6.2 microns i n diameter. The metaphase I spindle apparatus i s about 10 microns long. The chromosomes are very small and are compactly arranged. At prophase II the n u c l e i are I 4 .2 — 5«U microns i n diameter. At metaphase of second d i v i s i o n approximately 30 chromosomes may be counted i n polar view (Fig„ 58 and 59). No chromophilous spherules or chromocenters were observed i n connection with d i v i s i o n s i n the antheridium. The chromosomes of the n u c l e i of the oogonium and antheridium can be counted with accuracy only at metaphase II i n polar view, and i n the preparatory prophase of the mature egg. At metaphase the chromosomes form a s o l i d p late which i s small i n area. 28 A f t e r the formation of four n u c l e i i n the antheridium, no r e s t i n g stage occurs. Each of the n u c l e i measures approximately h.6 microns i n diameter. The four n u c l e i divide simultaneously and equationally i n t o eight n u c l e i about k microns i n diameter ( F i g . 60 and 6 l ) . Further d i v i s i o n s follow from eight to sixteen, sixteen to thirty-two, thirty-two to s i x t y - f o u r n u c l e i ( F i g . 62 - 72). The s i z e of the n u c l e i i n the antheridium varies at d i f f e r e n t stages of development. They are l a r g e s t at the one-nucleate stage, and decrease with each successive d i v i s i o n , becoming smallest at the sixty-four-nucleate stage, when they are about 2 microns i n diameter. D i v i s i o n of the n u c l e i i s usually, but not always, simultaneous (F i g . 56). In anaphase of mit o s i s , the chromosomes separate i n t o two groups (Fig. 65). These groups of chromosomes are often curved at the outer edge of the spindle apparatus, g i v i n g a crescent-shaped appearance as the chromosomes are p u l l e d to the poles. No i n d i v i d u a l chromosome was observed p u l l i n g away from the main group. In anaphase each of these clumps has the appearance of a' f l a t p l ate with curved edges. Cross-walls do not form i n the cytoplasm a f t e r the nuclear d i v i s i o n s have taken place. Cross-walls are not distinguishable at the thirty-two and sixty-four-nucleate stages. The sixty-four n u c l e i and surrounding cytoplasm d i f f e r e n t i a t e into spermatozoids within the antheridium before release of gametes takes place. The spermatozoid of P e l v e t i o p s i s l i m i t a t a i s about 2.0 -" 2.2 microns long. I t has a pear-shaped body composed l a r g e l y of nucleus. A chromatophore can be distinguished. No eyespot was seen. There are two f l a g e l l a of unequal s i z e which are l a t e r a l l y i n s e r t e d ( F i g . 73). The shorter flagellum i s d i r e c t e d a n t e r i o r l y . I t could not be discerned i f the f l a g e l l a are t i n s e l l a t e d . 29 5. L i b e r a t i o n of gametes Gametes of P e l v e t i o p s i s l i m i t a t a are l i b e r a t e d i n groups at d i f f e r e n t times. When receptacles, which have been kept i n the dark and dr i e d f o r four to twelve hours, are immersed i n sea water, l i b e r a t i o n of eggs and spermatozoids takes place. Some immature as w e l l as mature gametes are released. The exochiton of the oogonium bursts and the two unequal eggs are released from the conceptacle with the inner layers of the oogonial w a l l p e r s i s t i n g . They pass through the o s t i o l e one at a time. A second layer p u l l s back over the eggs and i s discarded. The t h i r d layer of the wa l l ruptures. The fourth membrane diss o l v e s and the two eggs are released ( F i g . 29, 30 and 75 )• The two eggs then separate from one another. The freed eggs i n some cases attach to the outer wall of the receptacle. The spermatozoids are released from the antheridium by the rupture of the exochiton and are expelled from the conceptacle i n a j e l l y - l i k e mass. The endochiton dissolves and the spermatozoids swim away. 6. F e r t i l i z a t i o n L i v i n g material of P e l v e t i o p s i s l i m i t a t a was induced to l i b e r a t e gametes and examined i n order to study f e r t i l i z a t i o n . In some cases gyration of the egg i s d i s c e r n i b l e i n the process. One spermatozoid may enter the egg, and within h a l f an hour a f e r t i l i z a t i o n membrane forms. There are no centrosomes formed i n the egg which could be c o r r e l a t e d with the entrance of the sperm. 30 7. Embryo development The f i r s t d i v i s i o n of the f e r t i l i z e d egg of P e l v e t i o p s i s  l i m i t a t a occurs within twenty-four hours a f t e r the release of gametes. The zygotes sink to the bottom of the culture medium and attach to s l i d e s previously placed there. There i s a tendency f o r embryos to associate i n groups. In a few cases embryos were observed growing on the creceptacle surface. P o l a r i t y of the zygote i s determined at the f i r s t nuclear d i v i s i o n . The zygote becomes pyriform before d i v i s i o n of the cytoplasm takes place ( F i g . 76). The f i r s t two cytoplasmic d i v i s i o n s f o llowing karyokinesis are perpendicular to the plane of the developing r h i z o i d (Fig. 77)« Another d i v i s i o n i n the same plane follows. Then the upper c e l l of the a p i c a l part of the embryo divides a n t i c l i n a l l y by two d i v i s i o n s i n t o four c e l l s ( F ig. 78) forming an e i g h t - c e l l e d embryo. V e r t i c a l d i v i s i o n of the uppermost c e l l may occur at the two-, three-, or f i v e - c e l l stage. In some instances the f i r s t d i v i s i o n of the zygote occurs before the primary r h i z o i d appears. A primary r h i z o i d i n i t i a l i s u s u a l l y d i f f e r e n t i a t e d before the zygote undergoes cytokinesis ( F i g . 76). The r h i z o i d divides a few times before more r h i z o i d s appear ( F i g . 80). Within the f i r s t four days follow-ing f e r t i l i z a t i o n the embryo undergoes p e r i c l i n a l and a n t i c l i n a l d i v i s i o n s which cause i t to elongate ( F i g . 79 and 80). Although at f i r s t there i s only one r h i z o i d i n P e l v e t i o p s i s  l i m i t a t a u s u a l l y seven more may be found at a l a t e r stage ( F i g . 8l). 31 In older embryos there i s sometimes a b i f u r c a t i o n or t r i f u r c a t i o n of one or more of these r h i z o i d s (Fig. 80 and 8 l ) . In some cases two primary r h i z o i d s appear ( F i g . 82). In normal embryos, r h i z o i d s grow on the side of the embryo which i s f a r t h e s t removed from the source of i l l u m i n a t i o n . When several embryos are grouped together t h i s i s not observed. As c e l l d i v i s i o n occurs, and the embryo grows, i t loses i t s s p h e r i c a l shape and becomes elongated. The upper end of the embryo always remains larger ( F i g . 79 and 80). Embryos r a i s e d to the age of four months i n the cold chamber under previously mentioned culture conditions do not show any hairs i n the a p i c a l d i s t a l region, although an a p i c a l groove i s d i s t i n g u i s h a b l e . At t h i s stage i t i s impossible to determine the number of r h i z o i d s as they are a l l branched and much intermeshed. The upper c e l l s of the r h i z o i d c l o s e s t to the main body of the maturing embryo divide p e r i c l i n a l l y . The c e l l s of the embryo are much elongated. The nucleus i s about 6 microns i n diameter and the c e l l contents are l e s s concentrated than those i n the c e l l s of the main body of the embryo. Often the walls separating the c e l l s of the r h i z o i d s are oblique rather than at r i g h t angles to the l o n g i t u d i n a l axis of the embryo (Fig. 79)« Each c e l l of the upper surface of the embryo has a c e n t r a l l y -located nucleus and dense cytoplasm containing chromatophores and fucosan v e s i c l e s . Unusual growth of the seven-nucleate egg of P e l v e t i o p s i s l i m i t a t a occurs. This s o - c a l l e d "non-functional" egg u s u a l l y d i s i n t e g r a t e s a f t e r extrusion from the oogonium. In some cases, however, i t appeared to have divided to produce an abnormal embryo (F i g . 83), which d i d not p e r s i s t past the f i v e - c e l l stage. 32 E. Discussion 1. Culture conditions and natural environment The slow growth of P e l v e t i o p s i s kept i n culture i n the cold chamber with a constant temperature of 6° - 9°C- i s believed to be a t y p i c a l -I t i s reasonable to expect that the organism would not t h r i v e under culture conditions as i t would have done under natural conditions. In i t s natural environment P e l v e t i o p s i s i s exposed to a maximum l i g h t i n t e n s i t y of 7500 foot-candles, whereas the highest l i g h t i n t e n s i t y i n culture was 250- foot-candles. Factors such as the r i s e and f a l l of the t i d e s , ocean spray and seasonal v a r i a t i o n could not be imitated i n the cold chamber. In i t s h a b i t a t , P e l v e t i o p s i s i s submerged only 1% - 5% of the time (Widdowson, personal communication, 1959); however, i n culture i t was submerged for the major part of i t s existence. Since P e l v e t i o p s i s grows i n the higher i n t e r t i d a l zone, maximum emergence i s favored to the extent that the organism may almost be considered to be more t e r r e s t r i a l than marine. 2. E a r l y development of the conceptacle i n i t i a l In P e l v e t i o p s i s , as i n other Fucaceae, the conceptacle i n i t i a l i s a flask-shaped c e l l . In most. Fucales the conceptacle i n i t i a l u s u a l l y divides by a transverse d i v i s i o n i n t o an outer tongue c e l l and an inner base c e l l . P e l v e t i o p s i s l i m i t a t a i s very s i m i l a r to Fucus i n that the tongue c e l l does not elongate or divide to form a filament (Wienburg, 1913)' Pelvetiopsis' d i f f e r s from P e l v e t i a f a s t i g i a t a i n that the f i r s t d i v i s i o n .of the conceptacle i n i t i a l i nto two c e l l s i s transverse rather than v e r t i c a l and no tongue c e l l i s formed (Moore, 1928). 33 3. Mitosis and meiosis M i t o s i s and meiosis i n somatic and reproductive c e l l s appear to be quite regular i n P e l v e t i o p s i s as in other Fucales. The metaphase plate i s very small and compact. The d i v i s i o n of the chromosomes takes place without any apparent abnormalities. No laggards or precocious chromosomes were observed. A s t r a l rays and centrosomes, t y p i c a l of a more p r i m i t i v e state, are l a c k i n g i n the nuclear d i v i s i o n s of the oogonium and antheridium, i n d i c a t i n g an advancement on the evolutionary scale. Centrosomes have been observed i n the n u c l e i of developing oogonia of Ascophyllum nodosum (Farmer and Williams, 1898), Cystophyllum sisymb rib ides (Shimotomai, 1928), Fucus evanescens (inoh, 1935), F. serratus, F. vesiculosus (Strasburger, 1897), P e l v e t i a w r i g h t i i (inoh, 1935), Sargassum f i l i p e n d u l a (Simons, 1906), S. horneri (Okabe, 1929a), and enerve (Tahara and Shimotomai, 1926). They have also been observed i n antheridia of H i z i k i a  fusiformis (inoh and Hiroe, 195^ -b) and Sargassum horneri (Hiroe and Inoh, 195^a) and embryos of S. horneri (Okabe, 1930) and P e l v e t i a c a n a l i c u l a t a (Subrahmanyan, 1957b). No centrosomes, however, have been seen i n the antheridia of Sargassum confusum (Abe, 1933), p i l u l i f e r u m (inoh and Hiroe, 195^c), or S. t o r t i l e (Hiroe and Inoh, 1956), i n the oogonia of Coccophora  l a n g s d o r f i i (Tahara, 1929) and Hesperophycus harveyanus (Walker, 193i) or i n the embryo of Sargassum p i l u l i f e r u m (inoh and Hiroe, 195^ +a)- Walker ( l93l) reported observation of a s t r a l rays i n Hesperophycus. The s o - c a l l e d "chromophilous spherule" was not seen i n the n u c l e i of antheridia or oogonia of P e l v e t i o p s i s . Although chromophilous spherules have not been seen i n oogonia of Fucus evanescens (inoh, 1935), or i n antheridia of Halidrys s i l i q u o s a (Naylor, 1958), they have been observed i n contact with the nucleolus and aggregating chromosomes i n some other species. Chromophilous spherules have been seen i n d i v i d i n g oogonia of Coccophora l a n g s d o r f i i (Tahara, 1929), Carpophyllum flexuosum (Dawson, 19^+0), Halidrys s i l i q u o s a (Naylor, 1958) a n d Sargassum horneri (Okabe, 1929 a ) ' These structures have also been reported i n antheridia of Sargassum p i l u l i f e r u m (inoh and Hiroe, 1954c), S. horneri (Hiroe and Inoh, 1954a), and H i z i k i a fusiformis (inoh and Hiroe, 1954b). 4. Chromocenters i n the mature egg The many dark, densely-staining bodies within the membrane .of r e s t i n g n u c l e i of P e l v e t i o p s i s are probably chromocenters. Chromocenters are portions of the chromatin network which s t a i n during interphase. Darlington and LaCour ( l 9 4 l ) observed very s i m i l a r bodies i n the l i l i a c e o u s species F r i t i l l a r i a pudica. These granules e x h i b i t i n g heteropyenosis are composed of heterochromatin, and are believed to represent i n e r t parts of chromosomes. Naylor (1958) noted the s i m i l a r i t y of her observations on Halidrys s i l i q u o s a to those of Darlington and LaCour. Photomicrographs of P e l v e t i o p s i s showing chromocenters are very s i m i l a r to Naylor's photo-micrographs of Halidrys. Chromocenters were observed only i n oogonia of Pe l v e t i o p s i s c o l l e c t e d i n November, 1959; at Port Renfrew. I t i s possible, therefore, that the egg nucleus remains i n the r e s t i n g stage i n the l a t e f a l l months. The nucleus of the mature f u n c t i o n a l egg then enters prophase preparing for the f i r s t d i v i s i o n of the zygote before the spermatozoid enters. This i s i n agreement with the observations made on Halidrys (Naylor, 1958). A condition s i m i l a r to t h i s e x i s t s i n Pinus spp. where the egg nucleus enters the pre-liminary stages of mitosis before the po l l e n tube reaches the egg (Ferguson, 1901). In both P e l v e t i o p s i s and Halidrys the number of chromocenters 35 varies and i s not r e l a t e d to the number of chromosomes. The author's i n v e s t i g a t i o n s , however, show several differences between P e l v e t i o p s i s and Halidrys. In P e l v e t i o p s i s , unlike Halidrys, there are no chromocenters i n the a n t h e r i d i a l n u c l e i . The chromocenters p e r s i s t i n the oogonium of P e l v e t i o p s i s u n t i l prophase, whereas i n the oogonium of Halidrys they disappear before prophase begins. In Halidrys 2 - 9 chromocenters were dist i n g u i s h a b l e , while h- 16 were seen i n P e l v e t i o p s i s . 5- Variations i n the production of eggs The number of eggs produced i n P e l v e t i o p s i s may vary from two to f i v e , and the eggs may also d i f f e r i n s i z e . These observations have been p a r a l l e l e d i n P e l v e t i a (Gardner, 1910), and i n Ascophyllum (Farmer and Williams, 1896). In P e l v e t i a spp., two eggs of equal si z e are u s u a l l y formed, but three, four and f i v e eggs are known i n P e l v e t i a f a s t i g i a t a (Gardner, 1910; Moore, 1928). Four eggs have also been seen i n a t e t r a d i n P. c a n a l i c u l a t a and P. w r i g h t i i (Gardner, 1910; Yabu and Imai, 1957)' Binucleate eggs occur i n P e l v e t i a as well as i n P e l v e t i o p s i s (Gardner, 1910). In Ascophyllum four eggs are usually produced, but a f i f t h was seen by Farmer and Williams (1896). There are two possible explanations which may account for the observation of two eggs of equal s i z e i n the oogonium. Sometimes the c l e f t between the two eggs of P e l v e t i o p s i s i s oblique rather than h o r i z o n t a l . In l o n g i t u d i n a l section, only a part of the oogonium i s observable, and there i s only one view of the oblique c l e f t between the eggs. In t h i s s i t u a t i o n the c l e f t might appear to divide the oogonial contents into two equal eggs. In r e a l i t y , one may be examining a section which has passed through the upper end of the oblique d i v i s i o n between the two eggs. I f s e r i a l sections have been 36 made, t h i s s i t u a t i o n can be corrected by observing preceeding and subsequent sections. As t h i s , however, does not account for the appearance of three and four eggs i n the oogonium of P e l v e t i o p s i s , i t i s more probable that these are aberrant forms of eggs. The v a r i a t i o n s from the accepted normal condition of two unequally-sized eggs i n P e l v e t i o p s i s might, on f i r s t glance, be considered as an error i n d i v i s i o n of the cytoplasm caused by some external f a c t o r . Every one of these "abnormalities" occurred i n a l l experimental material which was examined, but most frequently i n cultured material. I t i s suggested, there-fore, that these unusual oogonial d i v i s i o n s are phenotypic differences only r e s u l t i n g from various responses of genotypes to environmental conditions. Environmental agents such as l i g h t , heat, nourishment, and substrate, may contribute to the establishment of threshold conditions by which a character, such as the tendency to form extra cross-walls i n the oogonium, may be expressed. Not enough data are known to substantiate these ideas, and they are beyond the scope of t h i s i n v e s t i g a t i o n . I t i s suggested that c o n t r o l l e d experiments and s t a t i s t i c a l analysis of samples of P e l v e t i o p s i s would be of value i n e l u c i d a t i n g the cause or causes of t h i s phenomenon. I t should not be assumed that a l l v a r i a t i o n s i n cytokinesis are the r e s u l t s of external modifying agents, however, cytokinesis i s more susceptible to environmental changes than karyokinesis. As f a r as the author has been able to d i s c e r n , l i t t l e i s known of the c r i t e r i a . of analysing genetic v a r i a t i o n s among the marine algae. 6. Comparison of gametangia and gametes of P e l v e t i o p s i s l i m i t a t a with other species of Fucales. The development of oogonia i n the Fucales i s s i m i l a r u n t i l the eight-nucleate stage. The f i r s t two nuclear d i v i s i o n s i n an oogonial i n i t i a l 37 c e l l of a member of the Fucales comprise the meiotic sequence and are followed by a m i t o t i c d i v i s i o n to produce eight n u c l e i . The oogonial w a l l i s composed of at l e a s t three layers and four were seen i n P e l v e t i o p s i s . P e l v e t i o p s i s ( F i g . Qk) and Hesperophycus are the only genera i n which two eggs of unequal siz e are produced (Gardner, 1910). In other genera as few as one and as many as eight eggs are produced (see Table l ) . The occurrence of a small "non-functional" egg with seven n u c l e i i n P e l v e t i o p s i s can be compared to the extrusion of supernumerary n u c l e i i n Pe l v e t i a and Ascophyllum (Oltmanns, 1889a). The oogonia and eggs of P e l v e t i o p s i s are of medium size compared to those of r e l a t e d species of the Fucales (Tables IV and V). The oogonium i s smaller than that occurring i n Sargassum (Fensholt, 1955), Turbinaria (Blomquist, 19^5), Hormosira (Osborn, 19^+9), and Carpophyllum•(Dawson, 19^0) spp., but larger than Cystoseira (Fensholt, 1955) and Notheia spp. (Barton, 1899)' The large egg of P e l v e t i o p s i s before l i b e r a t i o n i s about the size of a Fucus egg, being 87 x 6k microns. The large egg increases to as much as 155 microns, and the smaller up to 50 microns i n diameter a f t e r t h e i r release. Both large and small eggs are greater i n size and volume than the r e l a t e d Hesperophycus harveyanus i n which the large egg measures 11 microns i n diameter, and the smaller, 2.6 microns (Walker, 1931)' The eggs are l i b e r a t e d i n much the same fashion i n a l l genera. The release of the large egg of P e l v e t i o p s i s from the inner membranes of the oogonium wall i s very s i m i l a r to Himanthalia (Gibb, 1937)- In both genera a f t e r the two outer layers of the wall of the oogonium have disappeared and only the inner layer p e r s i s t s , a protuberance develops on one side of the periphery of the remaining oogonial membrane. A small rupture occurs and the c e l l contents pour through the opening leaving the membrane behind (Gibb, 1937). 38 The development of antheridia i n P e l v e t i o p s i s ( F i g . 84) as i n other Fucales follows the same sequence i n a l l genera. The si z e of antheridia seems very s i m i l a r i n many genera. Antheridia of P e l v e t i o p s i s , B i f u r c a r i a , and Hormosira are between 30 - 57 microns long x 17 - 23 microns wide, while those of Himanthalia are longer and narrower (Table VI). In the Fucales (Yamanouchi, 1909) as i n P e l v e t i o p s i s the f i r s t two nuclear d i v i s i o n s of the a n t h e r i d i a l i n i t i a l nucleus are meiotic, followed by four m i t o t i c d i v i s i o n s producing s i x t y - f o u r n u c l e i . Each spermatozoid i s formed by the mod i f i c a t i o n of one of these s i x t y - f o u r n u c l e i and the cytoplasm surrounding i t . The spermatozoids show consider-able v a r i a t i o n i n s i z e amongst the various genera (Table VI). The length of the spermatozoid of P e l v e t i o p s i s (2 - 2.2 microns), i s amongst the smallest recorded i n the Fucaceae, and i s more s i m i l a r to the spermatozoid of the d i s t a n t l y - r e l a t e d Halidrys i n s i z e than to the c l o s e l y - r e l a t e d Fucus. Spermatozoids of P e l v e t i o p s i s do not have eyespots. Eyespots have been observed i n spermatozoids of Fucus spp. ( K y l i n , 1916, 1920), Coccophora l a n g s d o r f i i (Shimotomai, 1928) and P e l v e t i a c a n a l i c u l a t a (Subrahmanyan, 1957°) but not i n Cystoseira spp. (Sauvageau, 1911). In most genera of the Fucaceae, i n c l u d i n g P e l v e t i o p s i s , the release of spermatozoids from the two-layered a n t h e r i d i a l w a l l begins with the rupture of the exochiton while the antheridium i s s t i l l i n the concept-a c l e . In Hesperophycus, however, i t occurs a f t e r the antheridium has escaped from the conceptacle (Walker, 1931). In most Fucaceae (Thuret & Bornet, 1878), as i n Pe l v e t i o p s i s , the inner w a l l of the antheridium then g e l a t i n i z e s and the spermatozoids are freed. 39 7« Development of the l i b e r a t e d eggs and spermatozoids The l i b e r a t i o n of gametes from the conceptacles of P e l v e t i o p s i s seems to be a reaction to 2 s t i m u l i , l i g h t and water (Oltmanns, 1889a; Pierce, 1902; Resuhr, 1935)• As soon as the eggs are l i b e r a t e d they begin to swell. In remarking that the eggs of some species are larger than others, Inoh (1932) suggested that those species with larger eggs are higher i n the evolutionary scale. The swelling of the l i b e r a t e d egg i s probably of advantage i n at l e a s t two ways. I t exposes a larger surface to which the spermatozoid may be attracted, thus increasing the p r o b a b i l i t y that f e r t i l i z a t i o n w i l l take place. Also as the f e r t i l i z e d eggs contain chromatophores there w i l l be an increase i n photosynthesis and a subsequent increased food supply. As has been observed i n Fucus serratus (Manton and Clarke, 1951), the spermatozoids of P e l v e t i o p s i s l i m i t a t a are not motile when they are f i r s t released from the antheridium. Within a very b r i e f period the f l a g e l l a u n c o i l and the spermatozoid i s able to move about f r e e l y . In most Fucales f e r t i l i z a t i o n occurs soon a f t e r sexual products have been released. In P e l v e t i a c a n a l i c u l a t a , Subrahmanyan (1957b) has observed f e r t i l i z a t i o n taking place within the oogonium. Spermatozoids of P e l v e t i o p s i s c l u s t e r about an egg, one penetrates the c e l l membrane, and i t s nucleus fuses with the egg nucleus as i n Fucus (Yamanouchi, 1909). The f e r t i l i z e d eggs of P e l v e t i o p s i s develop i n the same manner as other Fucaceae. I t seems that more than one c e l l i s capable of responding to conditions which favor the growth of r h i z o i d s . Usually one primary r h i z o i d occurs i n P e l v e t i o p s i s but sometimes two form. In t h i s l a t t e r case, i t i s suggested that some factor or factors was a l t e r e d i n the e a r l y growth of the f i r s t r h i z o i d . I t has been found that several agents may influence the ko developmental axis of embryos of the Fucaceae. Farmer and Williams (1898) made the basic observation that r h i z o i d s of Fucus were formed on the side of the embryo which was f a r t h e s t away from the source of i l l u m i n a t i o n . Proximity to nearby eggs causes the embryo r h i z o i d s to grow towards these neighbouring c e l l s (Whitaker, 1931). I t has also been found that temperature gradients can a f f e c t the p o l a r i t y of eggs which have been grown i n the dark; the r h i z o i d s appear on the warm side of the zygotes (Lowrance, 1937). Centrifugation of f e r t i l i z e d eggs of Pe l v e t i a f a s t i g i a t a and Fucus serratus grown i n the dark causes some to form r h i z o i d s near the c e n t r i f u g a l poles (Lowrance and Whitaker, 19^0). J a f f e (1958) found that zygotes of P e l v e t i a and Fucus have a "polarotropic response". Rhizoids w i l l grow 90° - 135° away from a source of p o l a r i z e d l i g h t . As many as 50fo of the embryos r a i s e d under these conditions have r h i z o i d s growing at both poles. Other f a c t o r s a f f e c t i n g p o l a r i t y include u n i l a t e r a l l i g h t , pH gradient, and e l e c t r i c a l current (duBuy and Olson, 1937). I t has been established that p o l a r i t y of the egg i s not af f e c t e d by the spermatozoid i n Sargassum  p i l u l i f e r u m , as eggs can develop parthenogenetically a f t e r treatment with b u t y l i c a c i d and calcium carbonate i n sea water (Hiroe and Inoh, 195^)-In Table I I I the number of primary r h i z o i d s formed i n various genera i s recorded. I f larger than one or two, the number appears to be a multiple of four. The number of primary r h i z o i d s i s d i r e c t l y c o r r e l a t e d with the si z e of the egg and complexity of t h a l l u s structure (inoh, 1932). In t h i s respect P e l v e t i o p s i s has a more p r i m i t i v e type of development as only one primary r h i z o i d , sometimes two, occurs'. Other Fucaceae may have as many as four primary r h i z o i d s . In the Cystoseiraceae, the number may be as high as thirty-two. kl In some unusual cases, the small egg of P e l v e t i o p s i s developed a r h i z o i d . In those embryos examined, u s u a l l y only two or three n u c l e i could be discerned i n the main body c e l l of the embryo. Perhaps t h i s state follows f u s i o n of the spermatozoid nucleus with one of the seven n u c l e i of the small egg before the d i s i n t e g r a t i o n of a l l of the s i x remaining n u c l e i . This condition would be somewhat analogous to that i n Sargassum spp. where the spermatozoid nucleus fuses with one of the eight n u c l e i of the large egg and the other seven d i s i n t e g r a t e (Kunieda, 1926; Tahara and Shimotomai, 1926). Two other p o s s i b i l i t i e s e x i s t . The small egg might undergo parthenogenetic development, and most of the n u c l e i d i s i n t e g r a t e with the exception of the one or ones functioning. The other p o s s i b i l i t y i s that spermatozoid nucleus may not yet have fused with the egg nucleus. • Unfortunately no chromosome counts are a v a i l a b l e to confirm or deny these postulations. As t h i s abnormal embryo soon aborts and d i e s , i t may be stated that the small eggs cannot grow under the standard culture conditions employed i n t h i s i n v e s t i g a t i o n . 8. The Systematic P o s i t i o n of P e l v e t i o p s i s P e l v e t i o p s i s i s c l o s e l y r e l a t e d to three genera i n the Fucaceae, P e l v e t i a , Hesperophycus and Fucus (Table V T l ) . In gross morphological structure, P e l v e t i o p s i s i s s i m i l a r to P e l v e t i a i n the shape of the t h a l l u s and lack of a midrib. Cryptostomata found occ a s i o n a l l y on immature P e l v e t i o p s i s sometimes occur i n P e l v e t i a f a s t i g i a t a , but not at a l l i n P. c a n a l i c u l a t a . In P e l v e t i a as w e l l as i n P e l v e t i o p s i s there may be one or two primary r h i z o i d s , except i n P e l v e t i a w r i g h t i i when there are commonly four (inoh, 1935). C y t o l o g i c a l l y P e l v e t i o p s i s and P e l v e t i a have s i m i l a r features i n oogonial development. The septa form transversely or obliquely except i n the oogonium 42 of P. w r i g h t i i where they form v e r t i c a l l y (Yendo, 1907). Differences i n the two genera occur. The t h a l l u s of P e l v e t i o p s i s i s much smaller than P e l v e t i a and i s l i g h t e r i n c o l o r . There are two eggs of unequal s i z e , one i s u n i -nucleate while the other i s seven-nucleate (Gardner, 1913), while i n P e l v e t i a (Oltmanns, 1889a), both eggs are equal i n s i z e and uninucleate. In P e l v e t i a the s i x supernumerary n u c l e i are extruded from the cytoplasm (Oltmanns 1889a; Gardner, 1910). Centrosomes occur i n P e l v e t i a (Subrahmanyan, 1957b), but i n P e l v e t i o p s i s no centrosomes were observed. Although chromosome counts of 2n = 64 have been recorded i n P e l v e t i a w r i g h t i i and P e l v e t i o p s i s l i m i t a t a , a count of 2n = 44 has been reported for P. c a n a l i c u l a t a (Tables I I , VII). The cytomorphological aspects of the l i f e cycle of P e l v e t i o p s i s are s i m i l a r to Hesperophycus i n several ways (Table VII). There i s one primary r h i z o i d formed i n both genera. The cross-walls which divide the contents of the oogonium into 2 unequally-sized eggs are transverse and sometimes oblique. In both genera the large egg has one nucleus and the smaller egg has seven n u c l e i . Wo centrosomes have been observed i n d i v i d i n g n u c l e i of either genus. Several important differences e x i s t between these two genera. The t h a l l u s of P e l v e t i o p s i s i s shorter and l i g h t e r i n colour than Hesperophycus. Also, Hesperophycus has a midrib and p l e n t i f u l cryptostomata (Walker, 1931). Both of these structures are l a c k i n g i n P e l v e t i o p s i s . The two eggs of P e l v e t i o p s i s are much larger than those of Hesperophycus, f i n a l l y the chromosome numbers of these two species d i f f e r , as 2n = approxi-mately 64 i n P e l v e t i o p s i s ( F i g . 7 and 8) while 2n = l4 - 18 i n Hesperophycus (Tables I I , VII). 3^ P e l v e t i o p s i s i s much l i k e Fucus with regard to the l i g h t tan color and small s i z e of the t h a l l u s , the occurrence of one primary r h i z o i d (sometimes two) i n the embryo, and the d i p l o i d chromosome complement of approximately 6k. However, the d i s s i m i l a r i t i e s are much more s t r i k i n g . In P e l v e t i o p s i s there are no midribs or caecostomata, and a few cryptostomata, a l l of which are found i n Fucus. Eight eggs of equal s i z e occur i n oogonia of Fucus (Thuret and Bornet, 1878; Oltmanns 1889a,b), whereas only two unequally-sized eggs form i n P e l v e t i o p s i s . There are no centrosomes i n d i v i d i n g n u c l e i of P e l v e t i o p s i s . In Fucus the spermatozoids are larger than those found i n P e l v e t i o p s i s (Table VII). From these p o i t s i t can be seen that P e l v e t i o p s i s i s most l i k e P e l v e t i a from which Gardner (1910) separated i t with regard to the gross morphology of the plant. The c y t o l o g i c a l structure of the t h a l l u s i s s i m i l a r to that of Hesperophycus. The r e l a t i o n s h i p to Fucus i s more remote. Therefore, i t can be seen that P e l v e t i o p s i s i s c l e a r l y r e l a t e d to P e l v e t i a ,  Hesperophycus, and Fucus i n more than one respect, but i t i s d e f i n i t e l y a d i s t i n c t e n t i t y . kk F. Summary A cytomorphological study of P e l v e t i o p s i s l i m i t a t a was under-taken using both l i v i n g and f i x e d material. Mature plants and embryos were kept i n culture i n s t e r i l i z e d f i l t e r e d sea water i n a cold chamber where the temperature varied from 6° - 9°C Growth of plants was most s u i t a b l e with a l i g h t i n t e n s i t y of 250 foot-candles. Sectioned material stained with warm aceto-carmine proved most s a t i s f a c t o r y f o r c y t o l o g i c a l study. Conceptacle formation i s i n i t i a t e d by a sing l e epidermal c e l l , as i n other Fucaceae. There are no caecostomata or cryptostomata i n the mature plant. There are three t i s s u e s i n the t h a l l u s , the epidermis, cortex, and medulla. The l i f e cycle i s s i m i l a r to that of other Fucales. Oogonia and antheridia are formed i n the conceptacles and the gametes are formed within the oogonia and ant h e r i d i a . The oogonia grow attached to the wa l l of the conceptacle by a s t a l k c e l l . Three nuclear d i v i s i o n s , the f i r s t two of which are probably meiotic, occur i n the oogonium producing eight n u c l e i . There i s a b r i e f r e s t i n g stage between each d i v i s i o n . Two unequal eggs are formed i n the oogonium. The la r g e r egg formed i s uninucleate while the smaller egg has seven n u c l e i . Both eggs are l i b e r a t e d from the oogonium and increase i n s i z e . Approximately 32 chromosomes were counted i n the nucleus of the mature uninucleate egg. Aberrations, such as the occurrence of two equally-sized eggs, and of three and four eggs of unequal s i z e were found. The antheridia grow s i n g l y from a sta l k c e l l , or from a branching paraphysis. Meiosis probably takes place during the f i r s t two d i v i s i o n s of the a n t h e r i d i a l i n i t i a l c e l l . Approximately 30 - 32 chromosomes were counted h5 at metaphase of the second d i v i s i o n i n the antheridium. Each a n t h e r i d i a l i n i t i a l undergoes d i v i s i o n s i x times to produce si x t y - f o u r n u c l e i . The l a s t four d i v i s i o n s are m i t o t i c . The sixty-four n u c l e i and surrounding cytoplasm are modified into spermatozoids within the antheridium. Mature gametangia were induced to l i b e r a t e , and i t was noted that immature as well as mature eggs and spermatozoids were released. The mature egg i s naked at the time f e r t i l i z a t i o n takes place. F e r t i l i z a t i o n of the egg by one spermatozoid restores the 2n chromosome number. There i s , therefore, only a c y t o l o g i c a l a l t e r n a t i o n of generations i n P e l v e t i o p s i s  l i m i t a t a . The f i r s t cytoplasmic d i v i s i o n of the zygote occurs a f t e r p o l a r i z a t i o n has been established. The f i r s t three cross-walls formed i n the zygote are usually transverse to the developing r h i z o i d , and are soon followed by c e l l d i v i s i o n s i n the other two planes when the primary r h i z o i d has become two or three c e l l s long. The mature t h a l l u s i s d i p l o i d . Approximately 6k chromosomes were counted i n c e l l s of the cortex. 46 G. L i t e r a t u r e C i t e d Abe, K. 1933- Mitosen im Antheridium von Sargassum confusum Ag. S c i . Repts. Tohoku Imp. Univ. Ser. IV, 8 (3): 259 - 262. 1938. Uber die Befruchtung und die i h r folgende e r s t Kernteilung bei Sargassum. S c i . Repts. Tohoku Imp. Univ. Ser. IV, 13 (3)*-253 - 257-Baker, S.M. and Bohling, M.H. 1916. On the Brown Seaweeds of the S a l t Marsh. I I . Their Systematic Relationships, Morphology and Ecology. Journ. Linn. Soc. London, Bot. 4_3_: 325 - 380. Barton, E.S. 1899* On Notheia anomala Harv. et B a i l . Journ. Linn. Soc. 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I I I . Some inve s t i g a t i o n s on Xiphophora chondrophylla. (R. Br. ex. Turner) Harv. Journ. Bot. 79_: 49""- 56, 65 - 71. Moore, L.B. 1928. P e l v e t i a f a s t i g i a t a . Bot. Gaz. 86 (4): 4 l 9 - 434 Moss, B.L. and E l l i o t , E. 1957« Observations on the Cytology of Halidrys s i l i q u o s a . Ann. Bot. n.s. 21: l43 - 151-Naylor, M. 1954. -A note on Xiphophora chondr-aphylla var. maxima J . Ag. New Phytol. 53: 155 -159* 1957- An Acetocarmine Squash Technique f o r the Fucales. Nature 180: 46-1958. The cytology of Halidrys s i l i q u o s a . Ann. Bot. 22 (86): 205 - 217-Nienburg, W. 1910. Die Oogonentwicklung b e i Cystoseira und Sargassum. Fl o r a 101: 167 - l80« 1913. Die Konzeptakelentwicklung b e i den Fucaceen. Z e i t s . Bot. 5: 1 - 2 7 . 1929. Zur Entwicklungsgeschichte der Fucus -Keimlinge. Ber. deuts. bot. Ges. 47 (8): 527 - 529-1931. Die Entwicklung der Keimlinge von Fucus vesiculosus und ihre Bedeutung fii r die Phylogenie der Phaeophyceen. Wiss. 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Studies i n the Genus Fucus L. I. Fucus d i s t i c h u s L. emend.Powell. Journ. Mar. B i o l . Ass. U.K. 36 (2): kOl - 432. 1957b. Studies i n the Genus Fucus L. I I . D i s t r i b u t i o n and Ecology of Forms of Fucus d i s t i c h u s L. emend. Powell i n B r i t a i n and Ireland. Journ. Mar. B i o l . Ass. U.K. 36: 663 - 693« Rao, C.S.P. 1946. A Contribution to the Morphology and L i f e History of Sargassum tenerrimum J . Ag. Proc. Indian Acad. S c i . Section B 23 ( 1 ) : 39 - 51. Rees, E.M. 1933. Some Observations on B i f u r c a r i a tuberculata Stackh, Ann. Bot. 4j_: 101 - 115. Resuhr, B. 1935* Uber den Bau und den Offnungsmechanismus der Fucus-oogonien• F l o r a 122 (3): 336 - 3^6. Richard, J . 1932. Origine et Structure du Spermatozoide de Fucus. CR. Soc. B i o l . 110 (21): 436 - 438. Roe, M.L. 1916. The Development of the Conceptacle i n Fucus. Bot. Gaz. 6 l : 231 - 246. Sauvageau, C 1908. 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Befruchtung und Kernteilung b e i Coccophora l a n g s d o r f i i (Turn.) Grev. S c i . Repts. Tohoku Imp. Univ. IV, T: 43 - kl. Walker, R.I. 1931. F e r t i l i z a t i o n and Embryo Development i n Hesperophycus  Harveyanus. La C e l l u l e 40 (2): 175 - 188. Whitaker, D.M. 1931* Some Observations on the Eggs of Fucus and upon Their Mutual Influence i n the Determination of the Developmental Axis. B i o l . B u l l . Mar. B i o l . Lab. 6l (3): 294 - 308. Williams, J.L. 1899. New Fucus Hybrids. Ann. Bot. 13: I87 - 188. Williams, M.M. 1923« A Contribution to Our Knowledge of the Fucaceae. Proc. Linn. Soc. New South Wales 48: 634 - 646. Woodworth, W.M. 1888. The A p i c a l C e l l of Fucus. Ann. Bot. 1: 203 - 211. Yabu, H. and Imai, A. 1957* On Nuclear D i v i s i o n i n the Antheridium of Fucus evanescens and Pe l v e t i a w r i g h t i i , and on the Four-Egged Oogonium of P e l v e t i a W r i g h t i i . B u l l . Jap. Soc. Phycol. 5_: 44 - 49. Yamanouchi, S. 1909. M i t o s i s i n Fucus. Bot. Gaz. 47: 173 - 197. Yendo, K. 1907» The Fucaceae of Japan. Journ. C o l l . S c i . Imp. Univ., Tokyo, Japan. Vol. 21: 1 - 174. 53 H. Tables Table I Comparison of Oogonial C h a r a c t e r i s t i c s of Certain Fucales Species •H •H I—I 0) <L> <3 r-< CQ to C o flj O bO bo o o bO ti bD'H S -P w -P T3 U o o o ' o •• h • ci No & O ,£> s 5 o o Reference FUCACEAE: Pe l v e t i o p s i s l i m i t a t a 8 Ascophyllum nodosum 8 Fucus spp. 8 Hesperophycus harveyanus 8 P e l v e t i a c a n a l i c u l a t a 8 P e l v e t i a f a s t i g i a t a 8 P e l v e t i a w r i g h t i i 8 Phyllospora comosa 8 Xiphophora chondrophylla 8 0 k 6 6 6 7 h 2u 1 Gardner, 1910, 1913 he k Oltmanns, 1889b Farmer and Williams, 1898. Thuret and Bornet, 1878 8e 8 Farmer and Williams, 1898. Oltmanns, 1889a,b. Thuret and Bornet, 1878. 2u 7s 2e 2e 2e kr 1 he 1 Gardner, 1910. 2 Thuret and Bornet, 1878. Oltmanns, 1889a. 2 Gardner, 1910. Moore, 1928. 2 inoh, 1935-1 Williams, 1923. k Barton, 1899. M i t c h e l l , 19^1. 5 4 Table I cont. Species •H •H CD <U H •d H O (D O o -P M 0 O O O o M o ,£> S3 OH CQ bO bO tJ o o CO . to a bO o bO -H PM -P CJ E H O Reference CYSTOSEIRACEAE: B i f u r c a r i a tuberculata 8 7 l 1 Rees, 1933. Thuret and Bornet, 1878. B i f u r c a r i a laevigata 8 4 4e 4b Laing, 194l Cystoseira osmundacea 8 7 1 1 Gardner, 1910 Halidrys s i l i q u o s a 8 7 1 1 Naylor, 1958 NOTHEIACEAE: Hormosira banksii 8 4 4e 4 Gruber, 1896 Getman, 19l4. Notheia anomala 8 0 8e 8 Barton, 1899 Williams, 1923. SARGASSACEAE: Sargassum, many spp. 8 7 1 l Kunieda, 1926, 1928. Tahara and Shimotomai, 1926 Simons, 1906 Abe, 1933. Turbinaria turbinata 8 7 1 l Blomquist, 1945. Abbreviations; b .... binucleate eggs occur e .... equal d i v i s i o n r .... r a r e l y u .... unequal d i v i s i o n 7s ... smaller egg has 1 n u c l e i 55 Table I I Chromosome Counts of Fucales Species FUCACEAE: Ascophyllum nodosum Fucus evanescens Fucus platycarpus ( = F . ~ s p i r a l i s J ~ Fucus serratus Fucus vesiculosus Hesperophycus harveyanus 2n 26-30 no" 26-28 64 14-18 P e l v e t i a c a n a l i e u l a t a 44ap. P e l v e t i a w r i g h t i i HIMANTHALIACEAE: Himanthalia lorea NOTHEIACEAE: Hormosira b a n k s i i CYSTOSEIPACEAE: Cystophyllum sisymbrioides 28 2k 32 30 30 32 20 + 32 12 n9 14-15 32 30 30 14-15 32 22 32 Reference Farmer and Williams, 1898 Inoh, 1935. Yabu and Imai, 1957• Strasburger, 1897• Stra sburger, 1897• Farmer and Williams, 1898 Yamanouchi, 1909• Walker, 1931. Subrahmanyan, 1956, 1957a, 1957b. Inoh, 1935. Yabu and Imai, 1957-Farmer and Williams, 1898 0shorn, 19^9• 32 Shimotomai, 1928. 56 Table I I Cont. Species 2n Halidrys s i l i q u o s a 55 4-SARGASSACEAE: Coccophora l a n g s d o r f i i 64 H i z i k i a f u s i f o r m i s  Sargassum confusum  Sargassum enerve Sargassum horneri 32 64 Sargassum patens 64 Sargassum p i l u l i f e r u m 64 Sargassum t o r t i l e nd" n°-8 30ap. 28ap. 32 32 16 32 32 32 32 32ap. 16 32 Reference Moss and E l l i o t , 1957 Naylor, 1958 Tomita, 1932 Tahara, 1929 Inoh and Hiroe, 195^ -b Abe, 1933 Tahara and Shimotomai, 1926 Kunieda, I926, 1928 Okabe, 1929a Okabe, 1930 Hiroe and Inoh, 195^a Hiroe and Inoh, 195kb Inoh and Hiroe, 195^ +a Inoh and Hiroe, 195^ -c Hiroe and Inoh, 1956" ap. = approximately 57 Table I I I The Number of Primary Rhizoids Formed i n Embryos of Certain Fucales Species FUCACEAE: Ascophyllum nodosum  Fucus evanescens  Fucus serratus  Fucus vesiculosus P e l v e t i a c a n a l i c u l a t a • Pe l v e t i a w r i g h t i i CYSTOSE LRACEAE: Cystoseira barbata  Cystophyllum hakodatense  Cystophyllum sisymbrioides SARGASSACEAE: Sargassum confusum  Sargassum hemiphyllum  Sargassum horneri  Sargassum enerve  Sargassum patens  Sargassum p i l u l i f e r u m Sargassum t o r t i l e Number 2 1 1 1 (2)1, k 1 (2) 4b h 32 8 8 8 16 16 16 16 Reference Oltmanns, 1889b Oltmanns, 1889b Inoh, 1935 Thuret and Bornet, 1878 Oltmanns, 1889a,b Oltmanns, 1889b inoh, 1935 inoh, 1935 Inoh, 1935 Okabe, 1929b Inoh, 1932 Inoh, 1932 Inoh, 1932 Inoh, 1932 Inoh, 1932 Inoh., 1932 Inoh, 1932 58 Table I I I cont. The Number of Primary Rhizoids Formed i n Embryos of Various Fucales Species Number Reference SARGASSACEAE: Turbinaria turbinata 4 , 8 , 1 6 ,or Blomquist, 19^5-32 Numbers i n brackets () r e f e r to the number of primary r h i z o i d s which may occur, b ... may be bifurcated. 59 Table IV Oogonium Size i n Certain Fucales Species Sargassum muticum  Turbinaria turbinata  Carpophyllum flexuosum  I-Iormosira banks i i  C ystoseira geminata  Cystoseira osmundacea Notheia anomala Size ( i n microns) 176 x ikO Yjk x IkO 188 x 137 160 x 110 71 - 7 5 1 70 - 7 5 1 75 x 20 Reference Fensholt, 1955 Blomquist, 1945 Dawson, 1940 0shorn, 1949 Fensholt, 1955 Fensholt, 1955 Barton, 1899 . . . diameter 60 Table V Egg Size i n Certain Fucales Species Himanthalia lorea  Sargassum microantheum  Cystophyllum sisymbrioid.es  Sargassum horneri  Carpophyllum flexuosum Sargassum hemiphyllum  P e l v e t i a c a n a l i c u l a t a  P e l v e t i a w r i g h t i i  P e l v e t i a f a s t i g i a t a Hormosira b a n k s i i Fucus furcatus Fucus serratus  Fucus vesiculosus  Fucus evanescens  Hesperophycus harveyanus 1 . . . diameter (L) . . Large egg (S) . . Small egg Size ( i n microns) 300 - 500 384 x 275 321 x 229 264 x 198 150 x 110 to 178 x 137 125 x 105 111 - 137 1 84 1 82 - 113 1 64 - 7 1 1 to 126 x 112 65 - 9 0 1 60 - 90 ] 52 - 70 ] 6 0 1 111 ( L ) 2 . 6 1 (s) Reference Gibb, 1937 Inoh, - 1932 inoh, 1935 inoh, 1932 Dawson, 1940 inoh, 1932 Subrahmanyan, 1957b inoh, 1935 Lowrance and Whitaker, 1940 Osborn, 1949 Lowrance and Whitaker, 1940 Beams, 1937 Whitaker, 1931 inoh, 1935 Walker, 1931 61 Table VI Antheridium and Spermatozoid Size i n Certain Fucales Species Sargassum horneri Hormosira b a n k s i i  B i f u r c a r i a tuberculata  Fucus spp. Himanthalia lorea  Halidrys s i l i q u o s a Antheridium Size 42 x 17 (20) 30 (40) long • 0 • 42 (60) x 9 (12) Spermatozoid Size 6 (7) long 5 (6) x 2 a • e 4 (5) x 2.3 (2.5) 3 (4) Reference Kunieda and Suto, 1940 Osborn, 1949 Rees, 1933 K y l i n , 1916 Gibb, 1937 Naylor, 1958 . . . diameter Brackets ( ) indi c a t e maximum measurement Table VII Comparison of Data on Pelvetiopsis with Data Reported i n the L i t e r a t u r e on P e l v e t i a , Hesperophycus and Fucus. Character Gross siz e ( i n centimeters) Color Midrib Primary Rhizoids Cryptostomata Caecostomata Size of spermatozoids ( i n microns) Pe l v e t i o p s i s 7 - 12 P e l v e t i a Light tan None 1 (2 sometimes) In young parts None 2 - 2.2 Pf .Pw 15 70 Dark green None Pc - k Pw 1 some-times 2 Pf - present Pc - none Hesperophycus 20 - kO Dark green-brown Present 1 Present Fucus 7 - 1 2 some spp. Light Present 1 sometimes Present Present some spp. k ( 5 ) x 2-3 (2.5) Reference S e t c h e l l & Gardner, 1925 Smith, 19I+4 Inoh, 1935 Smith, 19M Gardner, 1910 Oltmanns, 1889b Walker, 1931 Inoh, 1935 Gardner, 1910 Baker 85 Bohling, 1916 Moore, 1928 Gardner, I9I+0 Powell, 1957b K y l i n , 1916 Table VII cont'd. Character P e l v e t i o p s i s P e l v e t i a Size of eggs 90 (155 ) X L 80 (120) x ( i n microns) 33 ( 5 0) 1 S 113 (l60) Cross walls Transverse, Pc, Pf Trans-i n oogonium sometimes verse oblique Pw v e r t i c a l Number of eggs 2 2 formed Fate of supermini- Expelled i n Expelled from erary n u c l e i small egg cytoplasm Centrosomes None Present Chromosome 2n = 64 Pc - 2n = 44 number Pw - 2n = 64 Pc P e l v e t i a canalieulata Pf P e l v e t i a f a s t i g i a t a Pw P e l v e t i a w r i g h t i i L Large egg S Small egg 1 Diameter Hesperophycus Fucus Reference l l h 2.6 1S Transverse, sometimes oblique Expelled i n small egg 52 - 90 Octagonal None present Walker, 1931 Inoh, 1935 Beams, 1937 Subrahmanyan, 1957b Oltmanns, 1889a Yendo, I9O7 Gardner, 1910 Moore, 1928 Oltmanns, 1889a Gardner, 1910 Oltmanns, 1889a Gardner, 19IO None, but a s t r a l rays present 2n = 14 - 18 Present Yamanouchi, I9O9 Walker, 1931 Subrahmanyan, 1957b 2n = 64 Yamanouchi, 1909 Walker, 1931 Subrahmanyan, 1957b 6k I„ Figures Abbreviations used i n Figures ApiCQl • o « » « » o * a e * « * o o o o a o e « o « « « a a « o o e « o • A C C03?teX • o e e « a * a o « « e o o e o e o a o « 9 * * o * o e 0 * « e « ( > e o e 0 C"Q"tiClG oo»«o«e«ooo«oeo«eo«o0eeBOO*»*o««e«tt0o CU Epid-SITrii S o * o o o o » » Q o e 9 0 0 o « e o * 0 6 » * a o o « o * o « o » o E Medulla e * o e s o o o « o o o e o f t O * e s » « a * « o e « o a e o * o e o » MD MSHltljrQnG o a e « o » a * » o o « 0 a o o B O O « « « « a * o « * e o » o o o « MM M C t S p t l S SG e o o o a » o o a « o o o » o a e « « * « » o » « B O e * « o f t e « M Metaphase Plate » < > « • « • o« MP N l l C l e O l l l S « o o « * « © « o o * o « o o o » » < o o a * » o 9 » 9 O « o o » 0 » ITU NtlCleUS o o e a o « « a e a o e o e » 0 » e * o « * o « 9 o o o o » * » o o o o N P i t e « * « o » e o » o « o o » O D o e * o o o o o » B « * o o o « o a o o o * o « P SeptUHl o o 0 e e o * » o « o 9 O * a a e o o o e o e e o « 0 O « o o o t t o » » o S« Stalk C e l l « » o « © » o o o o o o e c « » o o » o < > o o » o < » « o e « © < > * « SC 65 F i g . 1 - 2 . Habitat and Morphology of P e l v e t i o p s i s l i m i t a t a . F i g . 1 - P e l v e t i o p s i s l i m i t a t a f. l i m i t a t a at Port Renfrew, B.C., June, 1959- x 1/5. F i g . 2 - Mature P. l i m i t a t a . x 2/3-F i g . 1 - 2 . from l i v i n g m aterial. 66 67 F i g . 3' Generalized Fucales L i f e Cycle. A macroscopic d i p l o i d generation alternates with a microscopic haploid generation which produces gametes. There i s no f r e e -l i v i n g haploid generation. 68 FIG. 3 GENERALIZED FUCALES LIFE CYCLE MACROSCOPIC 2N GENERATION SYNGAMY MICROSCOPIC N GENERATION " 6 4 SPERMATOZOIDS PRODUCED 1-8 EGGS FORMED 69 F i g . 4 - 12. Anatomy of the Thallus of P e l v e t i o p s i s l i m i t a t a . F i g . 4 - Cross section of t h a l l u s showing epidermis, cortex and medulla, x 48. F i g . 5 - Surface layers of the t h a l l u s . x 430. F i g . 6 - C o r t i c a l c e l l with chomatin material v i s i b l e , x 2000. F i g . 7 - M i t o t i c prophase i n a c e l l of the cortex. Approximately 64 chromosomes v i s i b l e , x 2200. F i g . 8 - Camera l u c i d a drawing of F i g . 7 showing the p o s i t i o n s of the chromosomes, x 2200. F i g . 9 - Longitudinal section of medulla showing elongated c e l l s with p i t connections, x 1100. F i g . 10 - Camera luc i d a drawing of c e l l s of the medulla showing l o n g i t u d i n a l elongation, x 400. F i g . 11 - Conceptacle containing gametangia. x 100. F i g . 12 -Young conceptacle with remnant of i n i t i a l c e l l at base, indi c a t e d by arrow, x 500. F i g . 4 - 1 2 from f i x e d m aterial. TO 71 F i g . 13 - 21. Development of Eggs i n P e l v e t i o p s i s l i m i t a t a . F i g . 13 - P a p i l l a e of conceptacle wall c e l l s , x 800. F i g . 14 -P a p i l l a showing nucleus, x 1300. F i g . 15 - M i t o t i c d i v i s i o n of c e l l has produced two'nuclei. Top nucleus i s the oogonial i n i t i a l , x 1000. F i g . 16 - Metaphase of f i r s t nuclear d i v i s i o n i n the oogonial i n i t i a l (probably meiosis i ) . x 3150. F i g . 17 -Resting stage. The two n u c l e i are stained very l i g h t l y , x 670. F i g . 18 - Camera l u c i d a drawing of metaphase of second d i v i s i o n (probably meiosis II) i n the oogonium. D i v i s i o n i s simultaneous, x 1540. F i g . 19 - Metaphase I I nucleus as i n r i g h t of F i g . 18. x 56OO. F i g . 20 - Metaphase II nucleus as i n l e f t F i g . 18. x 56OO. F i g . 21 - Interphase between meiosis I I and m i t o s i s . Nuclei are grouped and s t a i n l i g h t l y . Nucleolus v i s i b l e , x 670. F i g . 13 - 21 from f i x e d m a t e r i a l . 72 73 F i g . 22 - 30. Development of Eggs i n P e l v e t i o p s i s l i m i t a t a . F i g . 22 - Eight n u c l e i i n close a s s o c i a t i o n i n the oogonium a f t e r m i t o s i s , x QkO. F i g . 23 - Nuclei migrating to end of oogonium cl o s e s t to stalk c e l l ( indicated by arrow), x 530. F i g . 2h -Seven n u c l e i at the lower end of the oogonium, and one nucleus i s c e n t r a l l y situated, x 590. F i g , 25 - Maturing eggs i n oogonium. Note septum between eggs, x 410. F i g . 26 - At l e a s t four layers are present i n the wall of the oogonium, x 400. F i g . 27 - Large and small eggs. Two n u c l e i are v i s i b l e i n the smaller egg. x 880. F i g . 28 - Two eggs shortly a f t e r t h e i r release from the conceptacle. Smaller egg i s f l a t t e n e d , x 370. F i g . 29 - Eggs surrounded by inner w a l l membranes. The smaller egg has become rounded, x 375• F i g . 30 - Small egg with seven n u c l e i v i s i b l e , x 1125. F i g . 22 - 27 and F i g . 30 from f i x e d material. F i g . 28, 29 from l i v i n g m a t e r i a l . 75 F i g . 31 - 39' Chromocenters i n the Egg of P e l v e t i o p s i s l i m i t a t a . F i g . 31 - Threads of chromatin network v i s i b l e while the nucleus i s i n the center of the egg. x 5160. F i g . 32 - Condensed chromatin threads, x 4850. F i g . 33 - Mass of chromatin material, and 3 - 4 chromocenters above i t . Nucleus i s now situated near the d i s t a l periphery of the egg. x 4750. F i g . 3 4 - 7 chromocenters v i s i b l e , x 4850. F i g . 3 5 - 8 chromocenters v i s i b l e , x 4850. F i g . 36 -Nucleus contains about 11 chromocenters. x 4850. F i g . 37 - About 16 chromocenters. x 4850. F i g . 38 - Egg nucleus with some of i t s 32 chromosomes v i s i b l e , x 5840. F i g . 39 - Composite drawing of F i g . 38, showing 32 chromosomes, x 5840. F i g . 31 - 39 from f i x e d m a t e r i a l . 76 FIG . 3 1 - 3 9 CHROMOCENTERS IN THE EGG OF PELVETIOPSIS LIMITATA H i 77 F i g . ho - 50. Aberrant Eggs Formed i n P e l v e t i o p s i s l i m i t a t a . F i g . hO - Two equal eggs, note v e r t i c a l septum, x 1100. F i g . hi - Two equal eggs. 3 n u c l e i may be observed i n lower egg. x 590. F i g . h2 - Two equal eggs. Lower egg i s elongated. Both appear to be uninucleate, x 58O. F i g . U3 - Binucleate egg. x 5^0. F i g . hh - Oblique septum forming. Large egg i s binucleate. x 610. F i g . 45 - Three eggs developing, lower two are smaller, x 58O. F i g . h6 - Four eggs, x 58O. F i g . 7^ - Four eggs, n u c l e i v i s i b l e i n three of them, x 565. F i g . hQ - D i f f e r e n t focus of F i g . hi, showing that one of the eggs i s probably binucleate (ind i c a t e d by arrow), x 565. F i g . h$ - Three eggs. Two l i n e s of cleavage cut across the egg s i m i l a r to F i g . hi and hQ, but there does not appear to be a v e r t i c a l septum, x 58O. F i g . 50 - Four, perhaps f i v e eggs. Numbers re f e r to septa, x 630. F i g . ho - 50 from f i x e d m a t e r i a l . 78 FIG. 4 0 - 5 0 ABERRANT EGGS FORMED IN PELVETIOPSIS LIMITATA 79 F i g . 51 - 62.. Development of Spermatozoids i n P e l v e t i o p s i s l i m i t a t a F i g . 51 - Wall c e l l of conceptacle has given r i s e to two n u c l e i . The upper nucleus i s the a n t h e r i d i a l i n i t i a l , x 169O. F i g . 52 - The nucleus indicated by the arrow i s undergoing d i v i s i o n to form a new antheridium, which w i l l branch to the r i g h t of the h a i r filament, x 178O. F i g . 53 - Uninucleate a n t h e r i d i a l i n i t i a l showing the s t a l k c e l l , x 1210. F i g . 5^ - Metaphase of the f i r s t nuclear d i v i s i o n i n the antheridium (probably meiosis I ) , x 3200. F i g . 55 - Two n u c l e i i n a n t h e r i d i a l i n i t i a l c e l l , x 1430. F i g . 56 - Second nuclear d i v i s i o n i n the antheridium (probably meiosis I I ) . One nucleus appears to be i n prophase while the other i s i n metaphase. x i860. F i g . 57 - Metaphase of second nuclear d i v i s i o n , probably meiosis I I . x 3200. F i g . 58 - Meiosis I I . Approximately 30 chromosomes v i s i b l e i n polar view of metaphase on r i g h t , x 2200. F i g . 59 - Camera luc i d a drawing of F i g . 58, with 30 chromosomes shown on r i g h t , x 2800. F i g . 60 - D i v i s i o n I I I . Four n u c l e i i n m i t o t i c prophase, x 3040. F i g . 6 l - Eight-nucleate stage i n the antheridium. x 1400. F i g . 62 - Metaphase of D i v i s i o n IV from eight to sixteen n u c l e i , x i860. F i g . 51 - 62 from f i x e d material. 80 F I G . 51 - 6 2 DEVELOPMENT OF SPERMATOZOIDS IN PELVETIOPSIS LIMITATA 81 F i g . 63 - 74. Development of Spermatozoids i n P e l v e t i o p s i s l i m i t a t a . F i g . 63 - Eight n u c l e i i n mi t o t i c metaphase i n antheridium, some i n polar view. Antheridium i s not so elongated as i n F i g . 60. x 2380. F i g . 6k - Eight n u c l e i i n metaphase, top four i n polar view, x 2380. F i g . 65 - Eight n u c l e i i n anaphase. Note crescent e f f e c t at edge of spindle, x 2300. F i g . 66 - Sixteen-nucleate stage, x 1825. F i g . 67 - Sixteen n u c l e i i n prophase, x 1665. F i g . 68 - Sixteen n u c l e i i n metaphase. x 2000. F i g . 69 - Sixteen n u c l e i d i v i d i n g , some of the lower n u c l e i appear to be i n anaphase. Note a p i c a l cap. x 2000. F i g . 70 - Thirty-two-nucleate stage, x 2^00. F i g . 71 - Camera luc i d a drawing of thirty-two n u c l e i d i v i d i n g , x 3200. F i g . 72 - 6k n u c l e i , x 1165. F i g . 73 - Drawing of l i b e r a t e d spermatozoid with shorter anterior flagellum. x 3180. F i g . 74 - Antheridia a r i s i n g on a branching h a i r system from the wal l of the conceptacle. x kOQ. F i g . 63 - 72 and F i g . 7^  from f i x e d material. F i g . 73 from l i v i n g m a terial. 82 F I G . 63 - 74 DEVELOPMENT OF SPERMATOZOIDS IN PELVETIOPSIS LIMITATA 83 F i g . 75 - 83. Embryology of P e l v e t i o p s i s l i m i t a t a . F i g . 75 - Liberated naked egg 10 minutes a f t e r l i b e r a t i o n from the conceptacle. x 390* F i g - 76 - Pear-shaped zygote, 2k hours a f t e r l i b e r a t i o n . No d i v i s i o n of the' cytoplasm has taken place, x 390. F i g . 77 - Line drawing of 3-celled zygote, 1 day o l d . Septa (SE 1, SE2) have formed. A primary r h i z o i d i n i t i a l has been established below SE^. x 297' F i g . 78 - Line drawing showing d i v i s i o n of young embryo by 3 transverse walls. Top c e l l has divided v e r t i c a l l y by 2 d i v i s i o n s into k c e l l s , 2 of which are shown here, 2 days old. x 290. F i g . 79 - Line drawing showing further growth i n the embryo, 3 days old. x 290. F i g . 80 - Line drawing showing a second r h i z o i d appearing,k days o l d . x 210. F i g . 8 l - Normal embryo with several r h i z o i d s , one month old. x 135• F i g . 82 - Embryo with r h i z o i d s growing from two areas, one month o l d . x 135- F i g . 83 - Line drawing of abnormal embryo with 3 n u c l e i i n upper c e l l , 5 days o l d . x 516. F i g . 75 - 76 from l i v i n g m a terial. F i g . 77 - 83 from both l i v i n g and f i x e d material. Oh FIG, 7 5 - 8 3 EMBRYOLOGY OF PELVETIOPSIS LIMITATA 85 F i g . 8k. L i f e History of P e l v e t i o p s i s l i m i t a t a ( S e t c h e l l ) Gardner. The mature plant with a d i p l o i d chromosome complement of 6k ( i ) has many receptacles ( i i ) producing gametangia i n the f l a s k -shaped conceptacles ( i i i ) . The f i r s t two nuclear d i v i s i o n s i n the oogonium comprise the meiotic sequence which reduces the chromosome number to approximately 32 ( i v , v, v i ) . These are followed by a m i t o t i c d i v i s i o n which produces 8 n u c l e i i n the oogonium ( v i i ) . Seven n u c l e i migrate to one end of the oogonium and are cut o f f from the l a r g e r , now uninucleate portion as a septum develops ( v i i i , i x , x). Two eggs are formed. The larger one i s f u n c t i o n a l ( x i ) , while the smaller egg ( x i i ) soon aborts. In the antheridium the f i r s t 2 d i v i s i o n s are probably meiotic ( x i i i , x i v ) reducing the chromosome number to approximately 30 - 32. Four m i t o t i c d i v i s i o n s follow (xv, x v i , x v i i , x v i i i , i x ) which produce 6k n u c l e i . Sixty-four spermatozoids are released into the surrounding medium by rupture of the antheridium w a l l . One sperm nucleus (xx) fuses with one egg nucleus r e s t o r i n g the the o r i g i n a l chromosome number of approximately 6k ( x x i ) . The embryo develops ( x x i i ) and grows in t o a new plant, thus completing the l i f e cycle ( i ) . Magnifications of drawings: i (x 7/10); i i ( x l ) ; i i i (x25); i v , v i (x250); v (xlko); v i i , v i i i , i x (x200); x, x i i (x280); x i , x x i (x l66); x i i i , x i v , xv, x v i (x600); x v i i , x v i i i , x i x (x700); xx (xl600); x x i i (x 70). 86 F I 6 . 8 4 LIFE HISTORY OF PELVETIOPSIS  LIMITATA (SETCHELL) GARDNER 

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