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Studies on the ultrastructure of desmids and its relation to their taxonomy Gerrath, Joseph Fredrick 1968

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STUDIES ON THE ULTRASTRUCTURE OF DESMIDS AND ITS RELATION TO THEIR TAXONOMY by JOSEPH FREDRICK GERRATH B.A., University of B r i t i s h Columbia, 1959 B.Sc» (Hon.), University of B r i t i s h Columbia, I963 M.Scr, University of B r i t i s h Columbia, I 9 6 5 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department or by h ils r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . J . F. Gerrath Department o f Botany  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date June 27, 1968 i Abstract. The c e l l wall was studied i n 13 species i n 10 genera of the Desmidiaceae i n order to determine whether u l t r a s t r u c t u r a l differences ex i s t among the three subfamilies of the Desmidiaceae: Penieae. Closterieae, and Cosmarieae. The c e l l wall of the Penieae comprises two d i s t i n c t layers, an inner layer of c e l l u l o s e m i c r o f i b r i l s and an outer electron-dense layer. The semicell walls do not overlap at the isthmus region and the lack of pores i n the wall i s confirmed. The c e l l wall of the Closterieae i s one-layered without pores and the semicell walls overlap where they j o i n . The c e l l wall of the Cosmarieae i s one-layered with pores through which a pectic f i b r i l l a r sheath i s secreted. The semicell walls overlap at the isthmus. In the u n i c e l l u l a r Cosmarieae an i n i t i a l wall lacking pores i s present during elongation of the new semicell following c e l l d i v i s i o n . This i n i t i a l wall i s discarded a f t e r formation of the mature c e l l wall containing pores. In the filamentous Cosmarieae both i n i t i a l and mature c e l l walls are formed before semicell elongation occurs. In the c o l o n i a l desmid, Cosmocladium saxonicum, the connecting strands between c e l l s comprise several f i b r i l l a r subunit strands each associated with one of a group of pores near the isthmus of the c e l l . Cytoplasmic microtubules are present immediately below the plasma membrane i n the isthmus region of c e l l s i i fixed with glutaraldehyde. These microtubules are oriented perpendicular to the c e l l a x i s . A single c e l l at metaphase of Spondylosium nulchrum was examined. Evidence for the existence of l o c a l i z e d centromeres i n the chromosomes of th i s species i s presented. Bundles of spindle microtubules attach to the metaphase chromosomes at l o c a l i z e d regions and not at any other part of the chromosome. The r a r e l y c o l l e c t e d species Pleurotaenium spinulosum (Wolle) Brunei (Cosmarieae) has a c e l l wall with the same fine structure as i n the genus Penium (Penieae). The transfer of t h i s species to the genus Penium as Penium  spinulosum (Wolle) comb. nov. i s therefore proposed. Table of Contents i i i . Abstract i L i s t of Tables i v L i s t of Figures v Acknowledgment x i v Introduction 1 Terminology 3 Material and Methods .4 Results Penieae Penium margarltaceum 9 Penium splrostrlolatum 22 Penium spinulosum 30 Closterleae Closterlum llneatum 41 Cosmarieae Pleurotaenlum nodosum 47 Trlploceras v e r t l c l l l a t u m ... 6 l Bambuslna bre b i s s o n l l 76 Desmldlum s w a r t z l l 88 Desmldlum cyllndrlcum 90 Hyalotheca d l s s l l l e n s 101 Sphaerozosma laeve I l l Spondyloslum pulchrum 117 Cosmocladium saxonlcum 135 Discussion 147 Bibliography 160 L i s t o f T a b l e s T a b l e I . S o u r c e s o f C u l t u r e s o f D e s m i d i a c e a e S t u d i e d . Page• 6 V L i s t of Figures F i g . 1 Diagram o f c e l l of Cosmarlum (Cosmarieae) showing overlap of semicell walls, p. 8 F i g . 2 Diagram o f c e l l o f Closterlum (Closterieae). p. 8 F i g . 3 Diagram of c e l l o f Penium (Penieae). p; 8 F i g . 4 Diagram o f pore apparatus o f the Cosmarieae. p. 8 F i g . 5 L i v i n g c e l l o f Penium margar1taceum. p; 13 F i g . 6 Penium margarltaceum. Apex o f c e l l . p. 13 F i g . 7 Penium margarltaceum. Isthmus region o f c e l l . p. 13 F i g . 8 Penium margarltaceum. Longitudinal section o f c e l l w a l l . P? 15 F i g . 9 Penium margar1taceum. Oblique tangential section through c e l l w a l l , p i 15 F i g . 10 Penium margar1taceum. Longitudinal section through isthmus region, p. 1? F i g . 11 Penium margar1tac eum. Oblique section through c e l l wall and cytoplasm at isthmus region, p* 17 F i g . 12 Penium margarltaceum. Transverse section o f c e l l through a pyrenoid. p; 17 F i g . 13 Penium margarltaceum. Transverse section o f c e l l showing a x i a l chloroplast. p. 19 F i g . 14 Penium margar1taceum. Longitudinal section through part of an elongated mitochondrion, p. 19 F i g . 15 Penium margarltaceum. Transverse section of c e l l showing dictyosomes and mitochondrion, p. 21 v i Pig. 16 L i v i n g c e l l of Penium spirostriolatum. p. 2 5 F i g . 1 7 Penium spirostriolatum. Longitudinal section of c e l l w all, p i 2 5 F i g . 18 Penium spirostriolatum. Longitudinal section through c e l l wall at isthmus. P> 2 5 F i g . 1 9 Penium spirostriolatum. Oblique tangential section through c e l l w a l l , p i 2 ? F i g . 2 0 Penium spirostriolatum. Tangential section through outer c e l l wall layer, p. 2 7 F i g . 2 1 Penium spirostriolatum. Tangential section through inner c e l l wall layer, p i 2 9 F i g . 2 2 Penium spirostriolatum. Tangential section through outer c e l l w all layer a t isthmus, p i 2 9 F i g . 2 3 Penium splnulosum. L i v i n g c e l l with straight l a t e r a l margins, pt-F i g . 2k Penium splnulosum. L i v i n g c e l l with undulating l a t e r a l margins. p*«T 3 ^ F i g . 2 5 Penium splnulosum. Longitudinal section of c e l l wall through one of the spines, p. 3 ^ F i g . 2 6 Penium splnulosum. Tangential section through inner c e l l wall l a y e r . P i 3 ° F i g . 2 7 Penium splnulosum. Longitudinal section through c e l l apex, pi' 36 F i g . 28 Penium splnulosum. Tangential section through c e l l w a l l , p i 3 8 v i i P i g . 2 9 Penium splnulosum. Longitudinal section of c e l l w a l l at isthmus region, p." 4-0 F i g . 3 0 Penium splnulosum. Transverse section of c e l l showing a x i a l chloroplast. p. 4-0 F i g . 31 L i v i n g c e l l of Closterlum llneatum. p^ ' 4-4-F i g . 3 2 Closterlum llneatum. Longitudinal section through c e l l w a l l . pY 4 4 F i g . 3 3 Closterlum llneatum. Longitudinal section through apex of c e l l . PT 4 4 F i g . 34- Closterlum llneatum. Longitudinal section through isthmus region. P» 4-6 F i g . 3 5 Closterlum llneatum. Tangential section through c e l l w a l l ridges. P i 4-6 F i g . 3 6 L i v i n g c e l l s In l i g h t microscope of Pleurotaenlum nodosum, p*? 5 2 F i g . 3 7 Methyl v i o l e t stained c e l l of P. nodosum, p. 5 2 F i g . 3 8 Pleurotaenlum /nodosum. Longitudinal section of c e l l w a l l through a pore, p? 5 2 Pig* 3 9 Pleurotaenlum nodosum. Oblique section through c e l l w a l l , p i 5k F i g . 4-0 Pleurotaenlum nodosum. Longitudinal section through c e l l w a l l and pore apparatus, p i 54-F i g . 4-1 Pleurotaenlum nodosum. Tangential section of c e l l w a l l showing layers of m i c r o f i b r i l s , p. 5 6 F i g . 4-2 Pleurotaenlum nodosum. Longitudinal section through isthmus region of c e l l , p i 5 6 v i i i F i g . 43 Pleurotaenlum nodosum. Section of pyrenoid. p i 58 F i g . 44 Pleurotaenium nodosum. Transverse section of isthmus microtubules, pv 58 F i g . 45 Pleurotaenlum nodosum. Transversa section of c e l l through chloroplasts. pv 60 F i g . 46 Pleurotaenlum nodosum. Longitudinal section of isthmus microtubules, p. 60 F i g . 47 L i v i n g c e l l of Trlploceras v e r t l c i l i a t u r n , p. 65 F i g . 48 Trlploceras v e r t l c i l l a t u m . C e l l stained with methyl v i o l e t , p. 65 F i g . 49 Trlploceras v e r t l c i l l a t u m . Longitudinal section through protuberance. p» 65 F i g . 50 Trlploceras v e r t l c i l l a t u m . Cross section of pore. p.67 F i g . 51 Trlploceras v e r t l c i l l a t u m . Cross section of s o l i d cap i n the pore bulb. p« 67 F i g . 52 Trlploceras v e r t i c i l i a t u r n . Longitudinal section of c e l l w a l l through a pore. p. 69 F i g . 53 Trlploceras v e r t l c i l l a t u m . Longitudinal section through Isthmus region of d i v i d i n g c e l l , p; 69 F i g . 54 Trlploceras v e r t l c i l l a t u m . Section showing mitochondria and vacuoles, p. 71 F i g . 55 Trlploceras v e r t l c i l l a t u m . Section showing a dictyosome. p i 71 F i g . 56 Trlploceras v e r t l c i l l a t u m . Transverse section of c e l l through a pyrenoid. p. 73 i x P i g . 57 Trlploceras v e r t I c l l l a t u r n . Transverse section of c e l l through protuberances, p. 73 F i g . 58 Trlploceras v e r t I c l l l a t u r n . Section showing the isthmus microtubules. j>i 75 F i g . 59 Trlploceras vertic111aturn. I n i t i a l w a l l of new semicell i n divided c e l l . p. 75 P i g . 60 Trlploceras vertic111aturn. Section of new semicell w a l l i n c e l l with half-elongated new semicell. pv 75 F i g . 61 Bambuslna b r e b l s s o n i l . L i v i n g c e l l s . P*. 79 F i g . 62 Bambuslna b r e b l s s o n i l . Filament stained with methyl v i o l e t to show narrow sheath, p. 79 F i g . 63 Bambuslna b r e b l s s o n i l . Longitudinal section through l a t e r a l margin of c e l l apex. p. 79 F i g . 64 Bambuslna b r e b l s s o n i l . Longitudinal section of c e l l . P? 81 Fi g . 65 Bambuslna b r e b l s s o n i l . Lobe of chloroplast. P'» 83 F i g . 66 Bambuslna b r e b l s s o n i l . Pyrenoid. p. 83 F i g . 67 Bambuslna b r e b l s s o n i l . Longitudinal section of d i v i d i n g c e l l , p?° 85 F i g . 68 Bambuslna b r e b l s s o n i l . Longitudinal section through the f o l d region of the d i v i s i o n septum, p. 87 F i g . 69 Bambuslna b r e b l s s o n i l . Longitudinal section through unfolding walls of elongating semicells. p.87 F i g . 70 L i v i n g c e l l s of Desmldlum s w a r t z i l . p. 92 F i g . 71 Desmldlum s w a r t z i l . C e l l s stained with methyl v i o l e t . P'S4 92 X F i g , 72 Desmldlum s w a r t z l l . Apical view of c e l l stained with methyl v i o l e t . P. 92 F i g . 73 Desmldlum s w a r t z l l . Longitudinal section through c e l l at isthmus. p. 92 F i g . ?4 Desmldlum s w a r t z l l . Longitudinal section of two c e l l s joined at a p i c a l pads. P* 94 F i g . 75 Desmldlum s w a r t z l l . Longitudinal section of a p i c a l pad. p'.; 94 F i g . 76: T;desmldlum;swartzll. Transverse section of c e l l . p.96 F i g . 77 Desmldlum s w a r t z l l . Section through pores and sheath, p? 96 F i g . 78 Desmldlum cyllndrlcum. L i v i n g c e i l l s . P. 98 Fig# 79 Desmldlum cyllndrlcum. Sheath stained with methyl v i o l e t , p." 98 F i g . 80 Desmldlum cyllndrlcum. Surface view of c e l l s stained with methyl v i o l e t showing pores, p i 98 F i g . 81 Desmldlum cyllndrlcum. Section of c e l l wall.p. 98 F i g . 82 Desmldlum cyllndrlcum. Longitudinal section through c e l l apex, 100 F i g . 83 Desmldlum cyllndrlcum. Section through isthmus, pi100 F i g . 84 Desmldlum cyllndrlcum. Longitudinal section through d i v i s i o n septum, pi? 100 F i g . 85 Hyalotheca d i s s l l l e n s . L i v i n g c e l l s , p. 104 F i g . 86 Hyalotheca d i s s l l l e n s . Sheath stained with methyl v i o l e t , p. 104 x i F i g . 87 Hyalotheca d l s s l l l e n s . Longitudinal section through l a t e r a l margin of c e l l apex. .«„ 1 0 l | f F i g . 88 Hyalotheca d l s s l l l e n s . Enlarged a p i c a l r i n g separating two c e l l s . p. 106 F i g . 89 Hyalotheca d l s s l l l e n s . Narrow connection between adjacent a p i c a l walls. P« 106 F i g . 90 Hyalotheca d l s s l l l e n s . Overlapping semicell walls at isthmus. Pv 106 F i g . 91 Hyalotheca d l s s l l l e n s . Section through two pores and adjacent sheath, pv 108 F i g . 92 Hyalotheca d l s s l l l e n s . Tangential section through c e l l wall and adjacent sheath, p: 108 F i g . 93 Hyalotheca d l s s l l l e n s . Longitudinal section of sheath prisms. P*. 110 F i g . 94 Hyalotheca d l s s l l l e n s . Tangential section through sheath, p. 110 F i g . 95 Sphaerozosma laeve. Li v i n g c e l l s , p, 114 F i g . 96 Sphaerozosma laeve. Two l i v i n g c e l l s . P» 114 Fig* 97 Sphaerozosma laeve. Sheath stained with methyl v i o l e t . Pv 114 F i g . 98 Sphaerozosma laeve. Longitudinal section of c e l l through nucleus and protuberance. p> 114 F i g . 99 Sphaerozosma laeve. Longitudinal section through a p i c a l protuberance, p v 116 F i g . 100 Sphaerozosma laeve. Microtubules, p. 116 x i i P i g . 101 Sphaerozosma laeve. Section of c e l l w a l l . p.ll6 F i g . 102 L i v i n g c e l l s of Spongyloslum pulchrum. pv 122 F i g . 103 Spondyloslum pulchrum. Sheath stained with methyl v i o l e t . pf 122 F i g . 104 Spondyloslum pulchrum. C e l l s photographed i n polarized l i g h t . p? 122 F i g . 105 Spondyloslum pulchrum. Apical view of c e l l . p. 122 F i g . 106 Spondyloslum pulchrum. Section of c e l l w a l l through two pores. p. 124 F i g . 107 Spondyloslum pulchrum. Longitudinal section through a p i c a l pad. p.* 126 F i g . 108 Spondyloslum pulchrum. Tangential section through sheath. p.' 126 F i g . 109 Spondyloslum pulchrum. Tangential section through several pore bulbs. p';. 128 Fi g . 110 Spondyloslum pulchrum. Tangential section through c e l l w a l l and sheath c o l l a r s . p. 128 F i g . I l l Spondyloslum pulchrum. Section of metaphase spindle showing chromosomes. p. 130 F i g . 112 Spondyloslum pulchrum. Section of same metaphase spindle as F i g . 111. p. 130 F i g . 113 Spondyloslum pulchrum. Transverse section through centromere of metaphase chromosome, p. 132 F i g . 114 Spondyloslum pulchrum. Enlarged part of F i g . 111. j Spindle microtubules near mitochondria, p. 134 x i i i F i g . 115 Spondyloslum pulchrum. Nucleolar material near spindle pole, pv 134 F i g . 116 L i v i n g c e l l s of Cosmocladlum saxonlcum. P«' 138 F i g . 117 Cosmocladlum saxonlcum. Feathery structures i n methyl v i o l e t stained sheath. P« 138 F i g . 118 Cosmocladlum saxonlcum. Section of c e l l w a l l . p?l40 F i g . 119 Cosmocladlum saxonlcum. Apical view of c e l l . p. 140 F i g . 120 Cosmocladlum saxonlcum. Oblique section through connecting strand. 140 F i g . 121 Cosmocladlum saxonlcum. Section through Isthmus pore group, p.*5 140 F i g . 122 Cosmocladlum saxonlcum. Low magnification micrograph of c e l l s , pv* 142 F i g . 123 Cosmocladlum saxonlcum. Enlarged view of a strand subunit. vV 144 F i g . 124 Cosmocladlum saxonlcum. Longitudinal section through isthmus region showing microtubules. PT144 F i g . 125 Cosmocladlum saxonlcum. Enlarged view of nucleus and pyrenoid i n F i g . 122. pv 146 F i g . 126 Cosmocladlum saxonlcum. Longitudinal section of isthmus microtubules. p.' 146 x l v Acknowledgment This investigation was begun at Queen Mary College, University of London, and supported by a Commonwealth Scholarship provided by the B r i t i s h Council. The author wishes to express his gratitude to Dr. M. B. E. Godward of Queen Mary College for providing laboratory f a c i l i t i e s , to Dr. E. G. Jordan for suggestions concerning electron microscope techniques and to Mr. L. Shells of Carl Zeiss Ltd. for i n s t r u c t i o n i n the operation of the Zeiss electron microscope. This study was completed at the University of B r i t i s h Columbia under the d i r e c t i o n of Dr. J. R. Stein. The work was supported by two National Research Council Scholarships to the author and by National Research Council Grant No. A1035 to Dr. Stein. Dr. T. Bisalputra provided laboratory f a c i l i t i e s f or specimen preparation and permitted the use of h i s Zeiss electron microscope. The author expresses thanks to Dr. Stein, Dr. Bisalputra, Dr. R. F. Scagel and Dr. K. Cole for c r i t i c a l reading of th i s manuscript. 1 Introduction. Desmids are members of the green a l g a l order Zygnematales i n which sexual reproduction occurs by conjugation of non-flagellated gametes. The Zygnematales comprise at l e a s t three well-defined f a m i l i e s : the Zygnemataceae, or Sp irogyra-Zygnema group of filamentous algae; the Mesotaeniaceae, or saccoderm desmids; and the Desmidiaceae, or placoderm desmids. A fourth family, the Gonatozygaceae, i s sometimes segregated from the Mesotaeniaceae. The families Desmidiaceae and Mesotaeniaceae are distinguished on the basis of wall structure. In the Desmidiaceae the c e l l wall i s composed of two symmetrical halves which generally overlap at a constricted region. The wall i s usually ornamented with knobs, spines or s t r i a t i o n s , and may have pores through which a sheath of pectic material i s secreted. The Mesotaeniaceae have unconstricted c e l l s with the wall not composed of two halves, usually unornamented, and lacking pores. On the basis of l i g h t microscope investigations of the c e l l wall three subfamilies of the Desmidiaceae have been recognized (Krieger 1937)• The Penieae, with one genus, Penium, have elongated, straight c y l i n d r i c a l c e l l s . There may be a s l i g h t c o n s t r i c t i o n at the central region but not as pronounced as i n c e r t a i n other Desmidiaceae. The c e l l wall i s sometimes composed of several sections and the p o s i t i o n where c e l l d i v i s i o n w i l l occur cannot be 2 predicted (Lutkemuller 1 9 0 2 ) . The Closterieae, with one genus, Closterium, have elongated c e l l s narrowed at both ends and usually lunate or curved. There i s no central c o n s t r i c t i o n and the wall i s also often composed of several sections. The p o s i t i o n where c e l l d i v i s i o n w i l l occur i s variable but i s predictable since i t i s indicated by a wall thickening developing before c e l l d i v i s i o n . The t h i r d group, the Cosmarieae, or Cosmarium-type desmids, includes the majority of desmids. Bourrelly (1966) l i s t s 30 genera i n t h i s group. The c e l l of the Cosmarieae i s usually constricted at a central region where c e l l d i v i s i o n occurs. The taxonomy of the Desmidiaceae i s based on studies made with the l i g h t microscope (West and West 1904, 1905 1908, 1912; West, West and Carter 1923; Krieger 1937; Kossinskaya i 9 6 0 ) . The s i z e , shape and ornamentation of the c e l l wall are important c r i t e r i a f o r generic and s p e c i f i c separation but other characters such as chloroplast and zygote morphology are also employed. The introduction of the electron microscope to b i o l o g i c a l investigations has presented a method f o r obtaining new information on the structure of the desmid c e l l . Several electron microscope studies on desmids have^been published but most deal with the genera Micrasterias. Cosmarium. and Closterium (Brandham and Godward 1965; Chardard 1964, 1965; Chardard and R o u i l l e r 1957; Drawert and Metzner-Kiister I 9 6 I ; Drawert 3 and Mix I 9 6 l a-h, 1962a-c, 1 9 6 3 ; Kamiya 1 9 6 1 , 1 9 6 2 ; Leyon 1 9 5 4 ; Mix 1 9 6 6 , 1 9 6 8 ; Waddington 1 9 6 2 ) . The objectives of the present investigation were to survey the ultrastructure of several genera of the Desmidiaceae and to determine whether u l t r a s t r u c t u r a l differences e x i s t among the three subfamilies. Special emphasis i s given to the f i n e structure of the c e l l wall but observations are also presented on the cytoplasm of the c e l l s of several species. Terminology.' A number of specialized terms are employed i n the study of desmids. Some of the frequently used terms are defined here and i l l u s t r a t e d i n F i g . 1-4. The c e l l of a Cos mar i um-1ype desmid comprises two halves, or semicells, which are i d e n t i c a l or mirror images. The semicell walls overlap at a constricted region c a l l e d the isthmus (Fig. 1 ) . The two ends of the c e l l are the apices. The Penieae and Closterieae are s i m i l a r i n structure but some species have central sections c a l l e d g i r d l e bands inserted between the semicells (Fig. 2 , 3 ) . Pores are present i n the c e l l walls of most of the Desmidiaceae. The terms describing pore structure are derived from early work with the l i g h t microscope (Lutkemuller 1 9 0 2 ) and are retained by electron microscopists (Chardard and R o u i l l e r 1 9 5 7 ; Drawert and Metzner-Kiister I96I) . The term pore apparatus i s used to describe the whole pore complex which comprises three d i s t i n c t parts ( Fig. 4 ) . 4 The hole i n the c e l l wall i s the pore or pore filament (German: Porenfaden). On the inside of the c e l l wall the pore bulb (German: Porenzwiebel) i s situated under the pore; On the outside of the wall there i s a structure which i s c a l l e d the sheath c o l l a r i n the present investigation (German: Porenknopfchen)• The sheath i s composed of i n d i v i d u a l parts, sheath prisms (German; Gallertprismen), each associated with one pore apparatus. Material and Methods; Clonal u n i a l g a l cultures of the desmids shown i n Table I were established from c o l l e c t i o n s made by the author. The c o l l e c t i o n s i t e and i s o l a t i o n date are also indicated i n Table I.' A culture of Penium margarltaceum (LB 6 0 0 ) was obtained from the Indiana University culture c o l l e c t i o n (Starr 1 9 6 4 ) . A modified soil-water culture medium was used. Approximately 0 ; 5 cc each of s o i l and h o r t i c u l t u r a l peat were placed i n a test tube with 20 ml d i s t i l l e d water and the tube was autoclaved f o r 4 5 - 6 0 minutes. Cultures were grown at 20°C under a 16 hr / 8 hr l i g h t dark cycle with illumination at 2 0 0 - 2 5 0 f t . - c . ( 2 1 0 0 - 2 6 0 0 l u x ) ; Sheath and pore structure were studied with the l i g h t microscope by staining l i v i n g c e l l s with 1% methyl v i o l e t , and staining with 0 . 1 $ ruthenium red indicated the presence of pectic substances. Chlor-zinc-iodine and IKI-H^SOjj, were used to test f o r the presence of c e l l u l o s e i n 5 the c e l l w a l l . Methylene blue and Congo red were also used to s t a i n the sheath. For study with the electron microscope c e l l s were fixed at 20°C f o r one hour with 1% osmic acid or with 2 - 5 $ glutaraldehyde followed by p o s t f i x a t i o n f o r one hour with 1% osmic acid. 1 Phosphate buffer ( 0 . 1 5 M, pH 7 . 2 ) or cacodylate buffer ( 0 . 1 M, pH 7»0 and 5 v 8 ) were used. The f i x a t i o n s used are noted with -each micrograph. Dehydration i n an ethanol-propylene oxide series was followed by embedding i n Maraglas. C e l l s to be sectioned were selected by examining the block with low power of a compound microscope. Small pieces of the block were cut out with a coping saw and mounted for sectioning on a Porter Blum MT-1 microtome. Sections were cut with a glass or diamond knife and picked up on formvar-coated g r i d s . The sections were stained 30-60 minutes with uranyl acetate (saturated solution i n 70$ methanol) and 5-7 minutes with lead c i t r a t e (Reynolds I 9 6 3 ) i Micrographs were taken with Hitachi HU-11 and Zeiss EM-9A electron microscopes.' 6 TABLE I SOURCES OF CULTURES OF DESMIDIACEAE STUDIED Species Penieae Penium spirostriolatum Penium spinulosum Closterieae Closterium llneatum Cosmarieae ( u n i c e l l u l a r ) Pleurotaenium nodosum Isolation  Date 22/6/64 9/7/64 Trlploceras v e r t l c i l l a t u m Cosmarieae ( c o l o n i a l and filamentous) Bambusina br e b i s s o n i i Cosmocladium saxonicum Desmidium swartzll Desmldlum cyllndricum Hyalotheca d l s s i l i e n s Sphaerozosma laeve Spondylosium pulchrum Source* Mike L. (Haney) Placid L. (Haney) 26/8/65 Loon L. (Haney) 11/8/64 Placi d L. (Haney) 22/7/63 Mike L. (Haney) 3/7/67 Lost L. (Coquitlam) 26/8/65 Gwendoline L. (Haney) 26/8/65 Placid L. (Haney) I6/6/67 Pond 30 mi. south Kenora, Ontario 12/6/67 Pond 30 mi. south Kenora, Ontario 7/8/63 Dead Dog Cr. (north of F t . St. James) 26/8/65 Placid L. (Haney) * Name i n parentheses indicates nearest town i n B r i t i s h Columbia. 7 F i g . 1 Diagram of c e l l of Cosmarium (Cosmarieae) showing overlap of semicell walls (arrow), a = c e l l apex, i = isthmus. F i g . 2 Diagram of c e l l of Closterium (Closterieae). g ss g i r d l e band. F i g . 3 Diagram of c e l l of Penium (Penieae). g = g i r d l e band. F i g . 4 Diagram of pore apparatus of the Cosmarieae. w = c e l l wall, pm = plasma membrane, pb = pore bulb, sc = sheath c o l l a r , s = sheath. 9 Results. Subfamily Penieae The Penieae are distinguished by straight c y l i n d r i c a l c e l l s with l i t t l e or no isthmus c o n s t r i c t i o n and no pores i n the c e l l wall (Fig. 3 K In some species elongation by i n s e r t i o n of c y l i n d r i c a l c e l l wall pieces occurs. The s i t e of cytokinesis i s v a r i a b l e . Two or four a x i a l chloroplasts and a p i c a l vacuoles containing several granules are present. The nucleus i s situated at the middle of the c e l l between the chloroplasts. The subfamily contains only one genus Penium de Brebisson. Penium margaritaceum (Ehrenberg) de Brebisson Penium margaritaceum has elongate c y l i n d r i c a l c e l l s measuring 130-215 u long by 16-20 jm wide. The c e l l wall comprises several sections marked off by transverse l i n e s (Fig. 5)« The outer surface of the wall appears rough and covered with small granules. The iodine-H^SOij, c e l l u l o s e test gives a p o s i t i v e reaction for the inner part of the c e l l w a l l . Electron micrographs of the c e l l wall show that i t comprises two d i s t i n c t layers (Fig. 6-8). The inner layer i s ca. 300 mu thick and consists of a loose network of randomly oriented c e l l u l o s e m i c r o f i b r i l s 4 - 5 mu thick (Fig. 9 , 11).. The outer wall layer i s more electron-dense, extending out from the inner wall layer ca. 600 mu (Fig. 8) 10 and also extending down into the inner wall layer (Fig. 8 , 9)» The m i c r o f i b r i l s of the outer layer are also 4 - 5 mp. thick. Tangential sections through the outer wall layer show a perforated structure with holes 100-150 mu i n diameter (Fig. 9 ) . Each hole i s surrounded by a region of lower electron density than the remainder of the outer wall layer. The larger electron-dense areas seen in tangential sections correspond to thicker areas seen i n longitudinal sections and to the granules seen on the c e l l wall with the l i g h t microscope (Fig. 5» 6> 9 ) . The perforations i n the outer wall do not extend through the inner wall layer. The c e l l walls do not overlap at the isthmus as i n the Cosmarieae (Fig, 7 . 1 0 ) . The inner wall layer i s continuous across the isthmus and the outer wall layer has a furrovr at the isthmus region (Fig. 1 0 ) . Immediately below the plasma membrane i n the isthmus region there are microtubules approximately 20 mu i n diameter oriented perpendicular to the longitudinal axis of the c e l l (Fig. 1 1 ) . The microtubules and the m i c r o f i b r i l s of the inner wall layer do not have the same orientation. The a x i a l chloroplasts have several narrow lobes r a d i a t i n g from a ce n t r a l row of pyrenoids (Fig. 1 2 . 1 3 ) . The thylakoids are arranged i n stacks of variable thickness and of i n d e f i n i t e length (Fig. 1 2 ) . Many spherical electron-dense globules caf' 150 mu i n diameter are present among the bands of thylakoids, usually i n uniseriate rows (Fig. 1 2 ) . 11 Some of the bands of thylakoids traverse the chloroplast and others terminate near the middle of the chloroplast (Fig. 13). The large, elongate pyrenoids have an electron-dense granular matrix surrounded by a sheath of starch grains (Fig. 12). Chloroplast thylakoids pass through gaps between the starch grains. They either traverse the pyrenoid or end b l i n d l y within the matrix. The separation of the thylakoid membranes i n the pyrenoid i s ca. 13 mp, about the same as the separation i n the r e s t of the chloroplast. Mitochondria are generally very elongated with length up to 15 V- and width ca. 0.6 u (Fug. 14). They are found close to the chloroplast, usually occupying a pocket between chloroplast lobes (Fig. 15)• Dictyosomes are also situated i n the v i c i n i t y of the chloroplast lobes (Fig. 15). Dictyosomes comprise 7-8 f l a t cisternae about 30 mu thick by 2 u long and the dictyosome v e s i c l e s are ca. 100 mu i n diameter. Mnay ribosomes ca. 15 mu i n size are scattered i n the cytoplasm (Fig. 15). The large nucleus (12 u x 7 u) i s at the isthmus region and contains a central nucleolus with several less electron-dense channels i n i t (Fig. 7 ) . Penium margaritaceum F i g . 5 L i v i n g c e l l of P. margar1taceum. x930» Arrows mark boundaries of g i r d l e band. F i g . 6 Micrograph 4124. Apex of c e l l . Arrows indicate thicker parts of outer wall that appear as granules i n the l i g h t microscope. x5400. F i x a t i o n : k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . F i g . 7 Micrograph 4108. Isthmus region of c e l l . The nucleus (n) i s at the isthmus region. Arrow indicates isthmus furrow i n the outer c e l l wall layer. x5100. Fixation as i n F i g . 6 . 14 Penium margarltaceum F i g . 8 Micrograph 4503. Longitudinal section of c e l l w a l l . The outer wall layer (o) i s embedded i n the inner wall layer (1). x48000. e = embedded part of outer wall layer. F i x a t i o n : 4$ glutaraldehyde. PostfIxation: 1% osmic a c i d . Buffers cacodylate - pH 7.0. F i g . 9 Micrograph 4125* Oblique tangential section through c e l l wall showing inner wall layer (1), outer wall layer (o) and region of overlap (e). Arrows indicate thickened areas corresponding to granules v i s i b l e with the l i g h t microscope. xl6500. Fixa t i o n as i n F i g . 8. 16 Penium margar i tac eum F i g . 10 Micrograph 4102. Longitudinal section through isthmus region showing furrow (arrow) i n the outer wall layer. x20000. Fixat i o n : k% glutaraldehyde. P o s t f i x a t l o n : 1% osmic a c i d . Buffer: cacodylate - pH 7»0. F i g . 11 Micrograph 4092. Oblique section through c e l l wall and cytoplasm at isthmus region showing cytoplasmic microtubules (arrows). x42000. Fixa t i o n as i n F i g . 10. F i g . 12 Micrograph 4 5 0 7 . Transverse section of c e l l through a pyrenoid showing starch grains (s) around pyrenoid, grana i n chloroplast (g), mitochondrion (m), dictyosome (d) and osmiophilic globules i n chloroplast (arrow). x l 8 3 0 0 . Fixation as i n F i g . 1 0 . 17 18 Penium margaritaceum F i g . 13 Micrograph 4-4-95. Transverse section of c e l l showing a x i a l chloroplast, mitochondria (m) and dictyosome (d). x 6 3 0 0 . F i x a t i o n : k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . F i g . 14- Micrograph 4-104-. Longitudinal section through part of an elongated mitochondrion. x l 5 5 0 0 . F i x a t i o n as i n F i g . 1 3 . 19 20 Penium margarltaceum F i g . 15 Micrograph 4 5 2 0 . Transverse section of c e l l showing dictyosomes (d) and mitochondrion (m) situated between lobes of the chloroplast (ch). X 2 6 5 0 0 . F i x a t i o n : 4$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7.0. 21 Penium spirostriolatum Barker 22 C e l l s of Penium spirostriolatum measure 14-5-250 u long by 30-35 u wide. Longitudinal s t r i a t i o n s on the c e l l wall and the thicker c e l l s d i f f e r e n t i a t e t h i s species from Penium margaritaceum (Fig. 16). The elongate chloroplasts are a x i a l with a central row of pyrenoids. Electron micrographs of the c e l l wall of Penium  spirostriolatum are s i m i l a r to those of P. margaritaceum. Two d e f i n i t e wall layers are present, an inner f i b r i l l a r layer 4-00-600 mu thick and an outer one about 1000 mp. thick (Fig. 17 , 18). The outer layer i s denser than the corresponding layer i n P. margaritaceum and appears more granular than f i b r i l l a r , with the outer margin sharply defined. The granules and f i b r i l s are approximately 10 mu thick. In tangential section (Fig. 1 9 , 20) the outer layer i s perforated by holes 100-180 mu i n diameter with a space of about 300 mu between centres of the holes. The holes are surrounded by a region of lower electron density ca. 100 mu wide (Fig. 20)v The perforations i n the outer layer do not extend through the inner wall layer (Fig. 17 , 1 9 ) . The inner wall layer i s composed of a network of m i c r o f i b r i l s 6-8 mu wide (Fig; 21). Bands of 4--10 p a r a l l e l m i c r o f i b r i l s are present but the orientation of adjacent bands i s random. The outer c e l l wall layer at the isthmus region has a d e f i n i t e furrow (Fig. 16, 18) which i s e s p e c i a l l y noticeable i n tangential section (Fig. 22). The inner wall layer i s continuous across the isthmus, often with a difference i n thickness between the inner wall layer of the two semicells (Fig. 18). The part of the outer wall layer that i s overlapping the inner wall layer i s also continuous across the isthmus. 24 Penium spirostriolatum F i g . 16 L i v i n g c e l l showing isthmus furrow (f) and boundaries of g i r d l e bands (arrows), x 5 0 0 . F i g . 17 Micrograph 2 0 5 4 . Longitudinal section of c e l l w a ll showing inner layer ( i ) , outer layer (o) and region of overlap (e). x l 2 6 0 0 . F i x a t i o n : 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 » 2 . F i g . 18 Micrograph 2 0 5 8 . Longitudinal section through c e l l wall at isthmus, indicated by the furrow (f) i n the outer wall (o). ( i = inner wall) x 4 0 5 0 0 . Fixation as i n F i g . 1 7 . Penium spirostriolatum Pig. 19 Micrograph 2110. Oblique tangential section through c e l l wall showing inner wall ( i ) and areas where the outer wall layer i s embedded i n the inner layer (e). x l 6 5 0 0 . F i x a t i o n : 2.5$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 20 Micrograph 204-9. Tangential section through outer c e l l wall layer showing perforations surrounded by narrow areas of lower electron density than the remainder of the w a l l . X51500. Fixation as i n F i g . 19. Penium spirostriolatum F i g ; 21 Micrograph 2 0 5 2 . Tangential section through inner c e l l wall l a y e r . Arrows indicate bands of p a r a l l e l m i c r o f i b r i l s . x 5 2 5 0 0 . Fixations 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 22 Micrograph 2 3 0 7 . Tangential section through outer c e l l wall layer at isthmus region. The furrow (f) i n the outer wall layer at the isthmus i s shown. X I 3 5 0 0 . Fixation as i n F i g . 2 1 . 2 9 30 Penium spinulosum (Wolle) comb. nov. (=Pleurotaenium spinulosum (Wolle) Brunei) Although Brunei (1949) placed t h i s species i n Pleurotaenium (Cosmarieae) the present study indicates that i t belongs i n the genus Penium (Penieae). C e l l s of Penium spinulosum measure approximately 400 u long, 5 ° u wide at the semicell base and 42 u wide at the apex. The outline of the l a t e r a l semicell wall may be straight (Fig. 23) or undulating (Fig. 24) i n c e l l s of the same clone with the l a t t e r more common. There i s a d e f i n i t e c o n s t r i c t i o n at the isthmus. P. spinulosum d i f f e r s from a l l other Pleurotaenium and Penium species by having spines 4 - 8 u long covering the entire c e l l wall except for the apex (Fig. 2 3 , 24). The spines near the apex are usually s l i g h t l y longer and stouter. Methyl v i o l e t stains the spines but ruthenium red does not, although a wall layer below the spines stains with ruthenium red ind i c a t i n g the presence of pectic material. Some c e l l s from both cultures and f i e l d c o l l e c t i o n s have a c y l i n d r i c a l g i r d l e section interposed between the two semicells, a c h a r a c t e r i s t i c of the Penieae. In electron micrographs the c e l l wall comprises two d e f i n i t e layers s i m i l a r to those of the two Penium species already described (Fig. 2 5 , 2 7 ) . A coarse f i b r i l l a r network makes up the inner layer which i s 7 5 0 - 9 0 0 mu thick. 31 In oblique sections t h i s f i b r i l l a r network i s especially-clear (Fig. 26).' The m i c r o f i b r i l s are approximately 10 mu thick. There i s no d e f i n i t e pattern i n the orientation of the m i c r o f i b r i l s but bands of 4-8 p a r a l l e l m i c r o f i b r i l s are seen i n some areas (Fig. 26). The outer wall layer i s very dense and appears granular i n sections. It i s pa r t l y embedded i n the inner layer and appears less dense i n the overlapping area (Fig. 25» 28). The thickness of the outer wall layer i s 5 0 0 - 1 0 0 0 mu on the l a t e r a l c e l l wall and s l i g h t l y more at the apices (Fig. 2 7 ) . The spines on the c e l l are projections of the outer wall 8-9 ;u long (Fig; 25 )•'' They are i n c l i n e d toward the apex. In tangential sections the spine bases are dense areas about 1 u i n diameter (Fig; 28). Thick r i b s radiate from each spine base to j o i n adjacent spine bases. The intervening spaces are criss-crossed with smaller p a r t i t i o n s producing an ir r e g u l a r reticulum (Fig. 28). No pores are present i n the c e l l w a l l . Fine needle-like projections about 500 mu long extend outward from the smaller p a r t i t i o n s of the outer wall layer (Fig. 27) and i n cross sections appear as s o l i d dots (Fig. 28). The semicell walls do not overlap at the isthmus region as they do i n Pleurotaenium nodosum (Fig. 42). There i s a furrow i n the outer wall layer at the isthmus but the inner wall layer i s continuous across the isthmus (Fig. 2 9 ) . In some instances there i s a conspicuous 32 difference i n thickness between the inner wall layers of the two semicells. F i g . 29 shows an example where one inner wall layer i s ca.'' 660 mu thick and the other i s ca. 920 mu thick, r e f l e c t i n g a difference i n age between the two semicells. Cross sections of the c e l l reveal that the chloroplast i s a x i a l (Fig. 3 0 ) . However, the pyrenoids are not i n an a x i a l row but scattered randomly (Fig. 2 4 ) . The thylakoids are arranged into d e f i n i t e grana (Fig. 3 0 ) . The remainder of the cytoplasm i s f i l l e d with many small vacuoles (Fig. 2 7 , 3 0 ) . 33 Penium spinulosum Pig. 23 L i v i n g c e l l with almost straight l a t e r a l margins. Arrow indicates p o s i t i o n of isthmus. x440. F i g . 2k L i v i n g c e l l with undulating l a t e r a l margins from same clone as c e l l i n F i g . 2 3 . Arrow indicates p o s i t i o n of isthmus. Note scattered pyrenoids (py). xkkO. F i g . 25 Micrograph 1 113 . Longitudinal section of c e l l wall through one of the spines. (iw = inner wall layer, ow = outer wall l a y e r ) . x 2 1 5 0 0 . F i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . 34 35 Penium splnulosum Pig. 26 Micrograph 0 8 6 0 . Tangential section through inner c e l l wall layer. Arrows indicate a band of s i x p a r a l l e l m i c r o f i b r i l s . x 4 7 5 0 0 . F i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 » 2 . F i g . 27 Micrograph 1 2 0 0 . Longitudinal section through c e l l apex. Needle-like projections (arrow) are present on the outer wall (o). x 3 8 5 0 . F i x a t i o n as i n F i g . 2 6 . 36 37 Penium spinulosum F i g . 28 Micrograph 1222. Tangential section through c e l l w a l l . Large p a r t i t i o n s connect the spine bases (arrows) and smaller p a r t i t i o n s form a reticulum i n the outer layer. The inner wall layer (iw) i s f i b r i l l a r . x5300. Fix a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7.2. 38 39 Penium spinulosum I j r i g . 29 Micrograph 0 8 6 8 . Longitudinal section through c e l l wall at isthmus region. A furrow (f) i s present i n the outer w a l l . The inner wall of the semicell on the r i g h t i s thicker than that on the l e f t . X 1 5 0 0 0 . F i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 30 Micrograph 1 0 6 0 . Transverse section of c e l l showing a x i a l chloroplast containing grana (g). x 4 2 5 0 . Fixation as i n F i g . 2 9 . 4 0 41 Subfamily Closterieae This subfamily eontains one genus, Closterium Nltzsch, characterized by lunate c e l l s with no isthmus c o n s t r i c t i o n (Figv 2 , )v Pores are reported i n some species1.1' Intercalated g i r d l e sections e x i s t i n some species and the d i v i s i o n s i t e i s more regular than i n the Penieae, occurring at a speci a l i z e d l i n e on the c e l l wall (German: Ringfurche, Krieger 1937) which develops before each d i v i s i o n . The nucleus i s at the isthmus region and there are usually two chloroplasts. Apical vacuoles reported to contain gypsum c r y s t a l s are present. Closterium lineatum Ehrenberg Closterium lineatum has narrow, elongate curved c e l l s measuring 4 5 0 - 5 0 0 u long by 28-30 u wide at the isthmus and 10-12 u wide at the apex (Fig. 3 1 ) . Several f a i n t l o n g i t u d i n a l l i n e s are generally v i s i b l e on the c e l l wall i n the l i g h t microscope. The chloroplasts are a x i a l with a c e n t r a l row of pyrenoids. Only the c e l l wall was studied with the electron microscope. The c e l l wall appears granular i n electron micrographs with l i t t l e i n d i c a t i o n of f i b r i l l a r nature (Fig. 3 2 ) . At the isthmus region the wall i s 600-800 mu thick increasing to 1 2 0 0 - 2 0 0 0 mu thick at the apexv A diagonal suture l i n e 42 marks the overlap of the semicell walls at the isthmus (Fig. 34). The outer boundary of the wall i s limited by two dense l i n e s , the inner one approximately 10 mu thick and the outer one s l i g h t l y thinner (Fig. 32, 33 )• Low ridges extending out ca. 300 mu correspond to the l i n e s v i s i b l e on the c e l l wall i n the l i g h t microscope. These ridges are most conspicuous i n tangential section (Fig. 35) but are also seen i n longitudinal sections (Fig. 34). Although pores are reported to be present i n t h i s species (Krieger 1937) no structures corresponding to the pore apparatus of the Cosmarieae (Fig. 4) could be found i n electron micrographs. At the c e l l apex there are less electon-dense channels i n the wall which could serve as areas through which material i s secreted (Fig. 33)• These channels are about 100 mu wide and pass outward through the wall from an inner layer (200 mu thick) of low electron density. No connection with the outside of the c e l l wall was found but s e r i a l sections of the area were not obtained. Further work i s necessary to v e r i f y the presence or absence of a connection. 43 Closterlum lineatum F i g ; 31 L i v i n g c e l l of Closterium lineatum. The p o s i t i o n of the nucleus (n) and the a p i c a l vacuole (v) i s indicated. x 2 0 0 . F i g . 32 Micrograph 1247. Longitudinal section through c e l l w a l l . Arrow indicates two electron-dense l i n e s at outer boundary of the w a l l . x l 2 5 0 0 . F i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7«2. F i g . 33 Micrograph 276O. Longitudinal section through apex of c e l l showing possible channels f o r the passage of material through the c e l l : w a l l (arrows). X14000. Fixation as i n F i g . 3 2 . 45 Closterlum lineatum F i g . 34 Micrograph 6 9 - 9 . Longitudinal section through isthmus region showing overlap of semicell walls (arrows) and a ridge (r) on the c e l l w a l l . X 1 2 5 0 0 . F i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 35 Micrograph 2 7 4 4 . Tangential section through c e l l wall ridges ( r ) . x l 9 2 0 0 . F i x a t i o n as i n F i g . 3 4 . 46 47 Subfamily Cosmarieae This large subfamily contains genera that have a conspicuous constricted isthmus region connecting the semicells (Fig. 1). The c e l l wall contains pores and a pectic sheath i s present in most species. The d i v i s i o n s i t e i s always at the isthmus. Species may be u n i c e l l u l a r , filamentous or united i n globose colonies. Genus Pleurotaenium,Nageli This u n i c e l l u l a r genus has straight, elongate c y l i n d r i c a l c e l l s . The truncate apices may be naked or may have a r i n g of small knobs. A large vacuole containing several gypsum c r y s t a l s i s present at each apex. The chloroplasts may be p a r i e t a l bands or a x i a l bands. Pleurotaenium nodosum (Bailey) Lundell C e l l s of Pleurotaenium nodosum measure 2 5 0 - 3 5 0 u long by 45-48 u wide at the semicell base and 2 0 - 2 3 u wide at the apex (Fig. 3 6 ) . ' Four rings of i n f l a t i o n s are present on each semicell and a r i n g of short conical knobs i s present at the apex (Fig. 3 6)v Methyl v i o l e t staining reveals pores over the whole c e l l wall which are not arranged i n a regular pattern (Fig. 3 7 ) . Knobs of sheath 48 material are stained at the outside of each pore. In c e l l s which have divided and completely d i f f e r e n t i a t e d an i n i t i a l wall layer i s discarded from the outside of the c e l l ( Fig. 3 6 ) . This layer i s thought to be a primary wall that i s present only during the elongation of the new semicell following cytokinesisV The chloroplasts are p a r i e t a l bands and a spherical vacuole i s present at each apex. The nucleus i s i n the isthmus region between the chloroplasts of the two semicells. In electron micrographs the c e l l w all measures 1 -1 .5 u thick and comprises many thi n layers of c e l l u l o s e m i c r o f i b r i l s approximately 10 mu wide (Fig. 3 8 , 4 0 ) . The f i b r i l l a r nature of the wall i s c l e a r l y seen i n oblique sections (Fig. 3 9 , 41); M i c r o f i b r i l s of successive layers are oriented at an angle of 3 0 - 7 0 ° to each other but there i s no consistent angle difference throughout the wall. The c e l l wall does not comprise two d i s t i n c t layers as i n the Penieae; ; A narrow electron-dense layer marks the outer l i m i t of the wall (Fig. 3 8 . ' 40)'. At the isthmus region both semicell walls gradually become thinner and overlap (Fig. 42)tf The pores are c y l i n d r i c a l holes tin the c e l l wall with a lumen ca". 100 mu i n diameter (Fig. 3 8 , 3 9 , 40). A t h i n dense layer marks the margin of the pore and the m i c r o f i b r i l s of the wall surround th i s layer (Fig. 4 0 , 41). 49 Within the pore lumen there i s a thin peripheral layer of material continuous with the external sheath c o l l a r and the i n t e r n a l pore bulb (Fig. 38, 4 0 ) . The cen t r a l area of the pore lumen appears empty. The pore bulb has a cap-like structure under the pore surrounded by a network of f i b r i l l a r material (Fig. 39, 4 0 ) . The plasma membrane i s withdrawn i n the region of the pore bulb to form a hemispherical pocket. The sheath c o l l a r i s generally a funnel-shaped structure extending out ca. 300 mu from the c e l l wall and measuring about 700 mu at i t s widest point (Fig. 3 8 ) . A loose mass of f i b r i l s extends from the sheath c o l l a r a further 1000 mu. A thin sheath layer i s present close to the c e l l wall between the pores (Fig. 4 0 ) . As many as 12 chloroplast bands are present i n transverse sections of the c e l l (Fig. 4 5 ) . The thylakoids are arranged i n conspicuous grana and small spherical electron-dense bodies are present i n rows in the stroma. There are numerous small pyrenoids surrounded by starch sheath (Fig. 4 3 ) . Several thylakoids enter the dense pyrenoid matrix through gaps between the starch grains and many completely traverse the pyrenoid matrix.' Much of the cytoplasm i s occupied by small vacuoles, some appearing empty and others f i l l e d with f i b r i l l a r material ( F i g . 4 4 ) . Mitochondria are numerous i n the cen t r a l part of each semicell and close to the chloroplasts ( F i g . 4-5). L a r g e d i c t y o s o m e s 2-3 u l o n g a l s o o c c u r n e a r the c h l o r o p l a s t s ( F i g . 1 4-5). The n u c l e u s i s s i t u a t e d a t the i s t h m u s and c o n t a i n s a c e n t r a l n u c l e o l u s ( F i g . 4-2). I m m e d i a t e l y below the p l a s m a membrane i n the isthmus r e g i o n t h e r e a r e m i c r o t u b u l e s 1 5 - 2 0 mu i n d i a m e t e r g o i n g a r o u n d the i s t h m u s p e r p e n d i c u l a r t o the l o n g i t u d i n a l a x i s o f the c e l l ( F i g . 4-4-, 4-6). These m i c r o t u b u l e s a r e r e s t r i c t e d t o the i s t h m u s r e g i o n and do n o t o c c u r e l s e w h e r e i n the c e l l . 51 Pleurotaenium nodosum F i g . 36 L i v i n g c e l l s i n l i g h t microscope showing the isthmus c o n s t r i c t i o n (c) and a p i c a l knob (k). Lower c e l l i s discarding an i n i t i a l wall (arrow) from a recently formed semicell. xi | 4 5 . F i g . 37 Methyl v i o l e t stained c e l l i n surface view. The pores and sheath c o l l a r s have stained (arrow). x620. F i g . 38 Micrograph 1900. Longitudinal section of c e l l wall through a pore showing pore bulb (pb), sheath c o l l a r (sc), plasma membrane (pm), outer margin of c e l l wall (o) and t h i n layer of sheath above the c e l l wall ( s ) . X 2 6 5 0 0 . F i x a t i o n : 5$ glutaraldehyde; Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 ; 2 . 53 Pleurotaenium nodosum Fig* 39 Micrograph 1 8 3 8 . Oblique section through c e l l w a l l ; Arrow indicates dense margin of a pore; The pore bulb (pb) contains a network of m i c r o f i b r i l s . x 2 6 5 0 0 . F i x a t i o n : 5% glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 40 Micrograph 1 9 0 6 . Longitudinal section through c e l l wall and inner part of pore apparatus. The dense boundary of the pore i s indicated by an arrow. A cap-like structure i s situated under the pore (c) and the sheath (s) i s present above the w a l l . X 2 6 5 0 0 . Fixation as i n F i g . 3 9 . 55 Pleurotaenium nodosum Fi g , 41 Micrograph 4666. Tangential section through c e l l wall showing layers of p a r a l l e l m i c r o f i b r i l s . Arrow indicates dense margin of pore. x 5 7 0 0 0 . Fixations 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g ; 42 Micrograph 1 8 3 0 . Longitudinal section through isthmus region of cellsshowing overlap of semicells (arrow), large nucleus (n) and nucleolus (nu). X 3 5 0 0 . F i x a t i o n : 5$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 « 2 . 57 Pleurotaenium nodosum F i g . 43 Micrograph 1821. Section of pyrenoid. The pyrenoid matrix i s traversed by several thylakoids (t) and surrounded by starch grains ( s ) . x l 6 5 0 0 . F i x a t i o n : 5% glutaraldehyde. P o s t f i x a t i o n ; 1% osmic a c i d . Buffer: phosphate - pH 7*2. F i g . 44 Micrograph 1862. Transverse section of isthmus microtubules (arrow) below the plasma membrane (pm). Vacuole (v) contains f i b r i l l a r material. x 4 7 5 0 0 . Fixation as i n F i g . 4 3 . ' -58 59 Pleurotaenlum nodosum F i g . 45 Micrograph 1828. Transverse section of c e l l through several chloroplasts containing grana (g) and osmiophilic globules (arrow). Mitochondria (m) and a dictyosome (d) are also present close to the chloroplasts. x l 5 5 0 0 . F i x a t i o n : 5% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7.2. F i g . 46 Micrograph 1 8 1 6 . Longitudinal section of isthmus microtubules ( t ) . x 7 1 0 0 0 . Fixation as i n F i g . 4 5 . 61 Genus Triploceras Bailey This genus of u n i c e l l u l a r desmids has elongate c e l l s with several rings of protuberances and 2 - 4 -furcate apices (Fig. 4-7). The nucleus i s at the isthmus and the two chloroplasts are a x i a l with a central row of pyrenoids. Triploceras v e r t i c l l l a t u r n Bailey C e l l s of Trlploceras v e r t i c i l l a t u m measure 340 - 4 0 0 u long by 40 - 4 5 M wide. The protuberances are flat-topped near the base of the semicell but they become inclined and pointed near the apex (Fig. 4 7 ) . Methyl v i o l e t stains a small knob of sheath around each pore (Fig. 4 8 ) . The f i n e structure of the c e l l wall i s similar to that of Pleurotaenium nodosum. The wall i s 800-1300 mu thick and comprises many thi n layers of m i c r o f i b r i l s . The c e l l wall at a protuberance i s s l i g h t l y thicker (Fig. 4 9 ) . A thin electron-dense l i n e marks the outer boundary of the c e l l wall and-, there i s another narrow l i n e ca. 20 mu beyond the wall which i s continuous with the sheath material at the pores (Fig. 5 2 ) . The semicell walls overlap at the isthmus as in Pleurotaenlum nodosum. Pores i n the c e l l wall are about 200 mu i n diameter and are s i m i l a r to those of P. nodosum (Fig. 50, 5 2 ) . Below the pore a cap-like structure occupies part of the pore bulb (Fig. 51, 52) with a few m i c r o f i b r i l s occupying 62 the r e s t of the pore bulb (Fig. 51)• Within the pore lumen a layer of sheath material i s present around the edge of the pore (Fig. 5 2 ) . The sheath extends outward from the pore up to 1000 mu and consists of many fine m i c r o f i b r i l s . The chloroplast i s a x i a l with about seven lobes that radiate from a central area containing the pyrenoids (Fig. 5 6 , 5 7 ) . Each major lobe usually b i f u r c a t e s . The thylakoids are arranged into d e f i n i t e grana with a variable number of thylakoids i n each stack (Fig. 5 5)• Pyrenoid structure i s s i m i l a r to that of the pyrenoids of Pleurotaenium nodosum. Central less dense areas are present i n some pyrenoids (Fig. 5 6 ) . Electron-dense globules about 150 mu i n diameter are common in the chloroplast stroma and are often arranged i n groups (Fig. 5 7 ) . Mitochondria and dictyosomes are usually near the chloroplast lobes (Fig. 5 5)• Mitochondria are elongate and narrow, about 300 mu wide and up to 10 u long (Fig. 5 4 ) . Dictyosomes are 2-3 U long and have 6-12 cisternae (Fig. 5 5 ) • * » Vacuoles occupy a large part of the cytoplasm between the chloroplast lobes. Some appear empty and others are f i l l e d with a loose granular substance, scattered m i c r o f i b r i l s or a whorled arrangement of m i c r o f i b r i l s (Fig. 51» 5 5 ) . Large v e s i c l e s about 600 mu wide with an electron-dense central region surrounded by a less dense periphery are present near the chloroplast lobes (Fig. 5 4 ) . The endoplasmic reticulum i s represented by a few elongated v e s i c l e s (Fig. 5 5 ) . 63 A section of a c e l l i n which c e l l d i v i s i o n i s complete and where elongation of the young semicells has started i s shown i n F i g . 53* The parent semicell walls are separated at the isthmus and the new semicell walls are s t i l l t h i n . The d i v i s i o n septum i s ca. 500 mu thick and comprises two layers which can be distinguished near the outer margin (Fig. 5 3 , 5 9 ) . Between the newly developed c e l l wall and the plasma membrane there are ir r e g u l a r membranous and filamentous structures, some flattened against the inner surface of the c e l l wall (Fig. 5 9 )• In places the membranes are s t i l l connected with the plasma membrane (Fig. 6 0 ) . Large vacuoles i n the cytoplasm contain m i c r o f i b r i l s and membranes sim i l a r to those outside the cytoplasm (Fig. 5 9 ) . Small vacuoles s i m i l a r i n size to the dictyosome v e s i c l e s are numerous near the d i v i s i o n wall (Fig. 5 9 ) . The microtubules present at the isthmus i n interphase c e l l s are also present in divided c e l l s but only along the plasma membrane adjacent to the old semicell wall (Fig. 5 8 ) . F i g . 60 shows the c e l l wall of a young semicell that has elongated to approximately half of the mature s i z e . The wall i s ca. 200 mu thick and i s uniform and f i b r i l l a r with no evidence of pores. The outer surface of the young wall i s covered with a thin layer of electron-dense material (Fig. 60) and many vacuoles are seen coming out of the cytoplasm (Fig. 6 0 ) . 64 Trlploceras ver t i c 111a turn F i g . 4-7 Living c e l l of T. v e r t i c i l l a t u m . Arrow indicates isthmus c o n s t r i c t i o n . x280. F i g . 48 C e l l stained with methyl v i o l e t showing stained pores (arrow). x 5 4 0 . F i g . 49 Micrograph 2 9 7 5 . Longitudinal section through protuberance. Two pore bulbs (b) are present at the base of the protuberance. x l 7 0 0 0 . F i x a t i o n : k% glutaraldehyde. Po s t f i x a t l o n : 1% osmic aci d . Buffer: cacodylate - pH 7.0. ® 66 Trlploceras v e r t l c i l l a t u m F i g . 50 Micrograph 2 9 1 4 . Cross section of pore (p) near i n t e r n a l surface of c e l l wall (w). x 5 2 0 0 0 . Fixation: h% glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7»0» F i g . 51 Micrograph 2 9 1 5 . Cross section of s o l i d cap (arrow) i n the pore bulb. A network of m i c r o f i b r i l s (f) surrounds the cap. x550°0» A vacuole (v) near the pore bulb contains f i b r i l l a r material. Fixation as i n F i g . 5 0 . 68 Triploceras v e r t l c i l l a t u m F i g . 52 Micrograph 2 6 1 8 . Longitudinal section of c e l l wall (w) through a pore. A cap-like plug (arrow) i s situated under the pore and sheath material (s) i s coming out of the pore. x l 7 0 0 0 . Fixation: k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 » 0 . F i g . 53 Micrograph 2 9 7 4 . Longitudinal section through isthmus region of d i v i d i n g c e l l . The d i v i s i o n septum (s) i s s p l i t t i n g at the margin (arrow), n = nucleus. x 5 1 0 0 . Fixation as i n F i g . 5 2 . Trlploceras v e r t l c i l l a t u m F i g . 54 Micrograph 54-8. Section showing mitochondria (m), vacuoles ( v ) t electron-dense globules (gl) and vacuole with dense i n t e r i o r (arrow). x7800. Fixat i o n : 1% osmic a c i d . Buffers phosphate - pH 7 . 2 . F i g . 55 Micrograph 4 4 7 1 . Section showing dictyosome (d), mitochondrion (m)p vacuole (v), endoplasmic reticulum (er) and chloroplast granum (g). x40000. Fixat i o n : 4$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . 'Buffer: cacodylate - pH 7.0. 71 72 Trlploceras vertlc111aturn F i g . 56 Micrograph 4-515• Transverse section of c e l l through a pyrenoid. The pyrenoid has a central l e s s electron-dense region and i s surrounded by starch grains ( s ) . x 3 7 0 0 . F i x a t i o n : 4$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 « 0 . F i g . 57 Micrograph 4-518. Transverse section of c e l l passing through several protuberances of one r i n g ( r ) . Each protuberance contains one lobe of the chloroplast (ch). Electron-dense globules (g) are present i n the stroma. x 3 7 0 0 . Fixation as i n F i g . 5 6 . 74 Triploceras v e r t i c i l i a t u r n F i g . 58 Micrograph 2976. Section showing isthmus microtubules (arrow) below plasma membrane (pm). X95000. Fixation : 4 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . F i g . 59 Micrograph 2983. I n i t i a l wall of new semicell i n divided c e l l . The d i v i s i o n septum i s s p l i t t i n g at the margin (arrow). Vacuoles (v) containing f i b r i l l a r material and dictyosome v e s i c l e s (d) are present near the new wall. Membranous bodies (x) are present between wall and plasma membrane. x ! 3 5 0 0 . Fixation as i n F i g . 58 . F i g . 60 Micrograph 3425. Section of new semicell wall i n c e l l with half-elongated new semicell. The i n i t i a l w all ( i ) has a thin electron-dense layer (a) on i t s outer surface. Vacuoles (v) are coming out of the cytoplasm. x l 3 0 0 0 . Fixation as i n F i g . 5 8 . 75 76 Genus BambusIna Kutzing This filamentous desmid genus i s distinguished from a l l others except Desmidium by the formation of folds i n the d i v i s i o n wall (Fig. 6 1 ) . It d i f f e r s from Desmidium by having l o n g i t u d i n a l s t r i a t i o n s on the c e l l wall near the apex. The chloroplasts are s t e l l a t e i n a p i c a l view with one c e n t r a l pyrenoid. BambusIna b r e b i s s o n l l Kutzing C e l l s of Bambusina br e b i s s o n i i measure 25 u long, 22 u wide at the isthmus and 15 M wide at the apex (Fig. 61). Near the isthmus two i n f l a t i o n s are present on opposite side of the c e l l with a shallow isthmus groove between them (Fig. 61). A narrow sheath surrounds the filament (Fig. 6 2 ) . In electron micrographs the l a t r e r a l c e l l wall Is ca. 200 mu thick except for a thickened ridge (500 m)x) about 8 p. from the apex (Fig. 6 7 ) . The a p i c a l wall i s ca. 150 mn thick and adheres to the a p i c a l wall of the adjacent c e l l (Fig. 64). The l a t e r a l margin of the a p i c a l wall i s thickened and protrudes s l i g h t l y (Fig. 6 3 ) . Pores are present i n both a p i c a l and l a t e r a l c e l l walls (Fig. 6 7 ) . Pore diameter i s ca. 100 mu and the l a t e r a l pores near the isthmus t r a v e l obliquely through the c e l l wall (Fig. 6 7 ) . The pore bulb contains a network of 77 f i b r i l s and there i s a low sheath c o l l a r from which m i c r o f i b r i l s extend 1 . 5 - 2 . 0 u from the wall (Fig. 6 7 ) . Chloroplast structure i s sim i l a r to that of Triploceras with grana of variable size (Fig. 64, 6 6 ) . Osmiophilic granules are present and pyrenoid structure i s i d e n t i c a l with that of other desmids (Fig. 6 5)• Mitochondria about 1 u long and 0 . 5 P- wide and dictyosomes ca. 1 u long occur close to the chloroplast (Fig. 6 4 ) . Endoplasmic reticulum and many vacuoles are present i n the cytoplasm (Fig. 6 4 ) . The nucleus i s c e n t r a l l y located and has a large spherical nucleolus. Micrographs of c e l l s i n stages following cytokinesis were obtained. After cytokinesis a f o l d develops i n the new wall which gives the d i v i s i o n septum an H-shaped form (Fig 6 7)• A layer of less electron dense material occupying the center of the f o l d and extending out to the l a t e r a l margin of the c e l l i s interpreted as the i n i t i a l or primary wall (Fig. 6 8 ) . The secondary or mature wall i s l a i d down under t h i s i n i t i a l w a l l . In l a t e r stages the folded wall separates as the i n i t i a l wall i s torn apart by the elongation of the new semicell (Fig. 6 9 ) . The area between the folds remains i n contact as the a p i c a l walls of the mature semicells. In the mature wall the pos i t i o n of the f o l d remains as a s l i g h t depression (Fig. 64) and the outer corner of the d i v i s i o n septum i s i d e n t i f i e d as the thickened ridge on the wall mentioned e a r l i e r (Fig. 6 7 ) . 78 BambusIna br e b l s s o n i i F i g . 61 Living c e l l s , one with folded d i v i s i o n wall ( f ) . Isthmus (1) Is Indicated i n c e l l at l e f t . xl^OO. F i g . 62 Filament stained with methyl v i o l e t to show narrow sheath ( s ) . x 7 5 0 . F i g . 63 Micrograph 4619» Longitudinal section through l a t e r a l margin of c e l l apex showing thickened ridge i n wall at apex (ar). x l 6 0 0 0 . F i x a t i o n : k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 « 0 . 79 80 Bambusina b r e b i s s o n i l F i g . 64 Micrograph 4 6 0 2 . Longitudinal section of c e l l showing dictyosome (d), endoplasmic reticulum (er), chloroplast grana (g), nucleus (n), pyrenoid (p) and mitochondrion (m). X7100. F i x a t i o n : 4$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . 81 82 Bambusina br e b i s s o n i i Fig* 65 Micrograph 3896. Lobe of chloroplast showing grana (g) and dense globules (arrow). x51000. F i x a t i o n : k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . F i g . 66 Micrograph 3897 • Pyrenoid traversed by chloroplast thylakoids (t) and surrounded by starch grains ( s ) . X 5 1 0 0 0 . Fixation as i n F i g . 6 5 . 84 Bambusina b r e b l s s o n l l F i g . 67 Micrograph 3 9 0 0 . Longitudinal section of d i v i d i n g c e l l with H-shaped d i v i s i o n septum. Lateral pores (1) and a p i c a l pores (a) are present i n the c e l l w a l l . A,B,C,D indicate points on the wall that correspond to points on the d i v i s i o n septum i n F i g ; 6 8 ; x 6 0 0 0 . F i x a t i o n : k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . 85 86 Bambusina b r e b i s s o n i i F i g , 6 8 Micrograph 3 8 9 2 , Longitudinal section through the f o l d region of the d i v i s i o n septum. A,B,C,D indicate points on the septum corresponding to points on the mature c e l l wall indicated i n F i g . 6 7 . The i n i t i a l wall (iw) i s the le s s dense middle layer. A pore (p) i s developing i n the mature wall of the septum. xl8000. Fixa t i o n : Wfo glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7.0. Fi g . 6 9 Micrograph 4616. Longitudinal section through unfolding walls of elongating semicells (arrow). The i n i t i a l wall (iw) and mature wall (mw) d i f f e r i n density. x l 8 5 0 0 . Fixation as i n F i g . 68. 87 Genus Desmidium A. C. Agardh 88 This filamentous genus has c e l l s which are usually-broader than long (Pig. 70) and which are not ornamented with l o n g i t u d i n a l l i n e s at the apex as i n Bambusina. Replicate walls are formed a f t e r c e l l d i v i s i o n . Desmidium swartzii A.C. Agardh C e l l s of Desmidium swartzii measure 15 u long by 4-0 u wide and are triangular i n cross section (Fig. 7 2 ) . The c e l l s are joined together at three a p i c a l protuberances or pads and the c e l l s are arranged so that the filament appears twisted (Fig. 7 0 ) . A narrow sheath and several rows of pores are demonstrated by methyl v i o l e t staining (Fig. 71t 7 2 ) . The isthmus i s conspicuous only at the angles of the c e l l (Fig. 7 0 ) . A large, 5 - 6-lobed chloroplast i s present i n each semicell (Fig. 7 2 ) . The nucleus i s i n the middle of the c e l l between the chloroplasts. In electron micrographs the wall structure i s i d e n t i c a l with that described for Pleurotaenium nodosum and Triploceras  v e r t i c i l l a t u m . The wall i s 2 5 0 - 3 0 0 mu thick and composed of many layers of m i c r o f i b r i l s (Fig. 7 3 ) . At the isthmus the semicell walls overlap (Fig. 7 3 ) . The pores i n the l a t e r a l wall are ca. 100 mu i n diameter near the pore bulb and s l i g h t l y wider toward the outside of the wall (Pig. 7 7)• The pore bulb contains a network of m i c r o f i b r i l s and the sheath c o l l a r extends about 200 mu from the wall (Pig. 7 7 ) . The sheath i s composed of f i b r i l s extending out from the sheath c o l l a r up to 4 u from the c e l l wall (Fig. 7 6 ) . Pores are also present i n the a p i c a l wall and have the same structure as the l a t e r a l pores (Fig. 7 4 ) . The space between adjacent c e l l s i s f i l l e d with f i b r i l l a r material which appears i d e n t i c a l to the l a t e r a l sheath (Fig. 7 4 , 7 5 ) . The walls of adjacent c e l l s are c l o s e l y pressed together at the a p i c a l pads which are flattened protuberances of the a p i c a l wall (Fig. 7 4 , 7 5 ) . A thin layer of electron-dense material i s present between the pads and pores are present in the wall of the a p i c a l pad (Fig. 7 5 ) . One large, lobed, a x i a l chloroplast i s present i n each semicell (Fig. 7 6 ) . Usually two chloroplast lobes go into each angle of the c e l l . Several pyrenoids are present i n the chloroplast. Chloroplast and pyrenoid structure i s i d e n t i c a l with that of desmids already described (e.g. Bambusina). Mitochondria 1-4 u long and dictyosomes ca. 1 .5 u long occur close to the chloroplast lobes (Fig. 7 4 ) . 90 Desmidium cylindricum G r e v i l l e C e l l s of Desmidium cylindricum are approximately c y l i n d r i c a l but have l a t e r a l i n f l a t i o n s near the isthmus (Fig. 7 8 ) . C e l l s measure 22 u long by 4-0 u wide. Adjacent c e l l s are joined at the flattened apices. A broad sheath 10-15 u wide surrounds the filament and s t r i a t i o n s can be seen i n the sheath i n c e l l s stained with methyl v i o l e t (Fig. 7 9 ) . Several rows of pores on each semicell are revealed by staining (Fig. 8 0 ) . The chloroplast i s s t e l l a t e with four to s ix lobes. In electron micrographs the wall of D. cylindricum has e s s e n t i a l l y the same structure as that of D. s w a r t z i i . The l a t e r a l wall i s ca. 200 mu thick (Fig. 81) and the a p i c a l wall i s ca. 100 mu thick (Fig. 82). The overlapping of the semicell walls at the isthmus i s shown i n F i g . 8 3 . The l a t e r a l edge of the a p i c a l wall i s thickened and on the outside of the wall at the apex there i s a loose f i b r i l l a r layer which i s interpreted as the remnant of the i n i t i a l wall formed a f t e r c e l l d i v i s i o n (Fig. 82). Replicate or folded walls are formed following cytokinesis (Fig. 84-) as i n Bambusina with subsequent unfolding when the new semicell expands. Pores 1 5 0 - 2 0 0 mu wide are present and th e i r structure i s i d e n t i c a l to those of D." swartzii (Fig. 81). The sheath i s f i b r i l l a r with many m i c r o f i b r i l s coming from the sheath c o l l a r . 91 Desmidium swartzii F i g . 70 L i v i n g c e l l s i n l i g h t microscope showing the isthmus grooves ( i ) and a p i c a l pad (a). x 9 1 0 . F i g . 71 C e l l s stained with methyl v i o l e t to show pores (arrow). x l 2 5 0 . F i g . 72 Apical view of c e l l stained with methyl v i o l e t showing narrow sheath (s) and a x i a l chloroplast (ch). x 9 2 0 . F i g . 73 Micrograph 24-82. Longitudinal section through c e l l showing overlap of semicell walls at isthmus ( i ) , pore i n wall (p) and sheath ( s ) . Note thickened ridge on wall (arrow). x l 5 0 0 0 . F i x a t i o n : 2 . 5 $ glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . 92 93 Desmidium swartzii F i g . 74 Micrograph 2 3 2 3 . Longitudinal section through two c e l l s joined at a p i c a l pads (arrows). x 3 5 0 0 . a = pores i n a p i c a l w a l l . d = dictyosomes between chloroplast lobes. F i x a t i o n : 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 75 Micrograph 2481. Longitudinal section through a p i c a l pad. A pore (a) i s present in the wall of the pad and i s si m i l a r to the l a t e r a l pore (1). x l 9 0 0 0 . Fixation as i n F i g . 74. 94 95 Desmidium swartzii Pig. 76 Micrograph 4-348. Transverse section of c e l l showing a x i a l chloroplast with pyrenoids (arrow) i n the lobes. The c e l l i s surrounded by f i b r i l l a r sheath ( s ) . x 3 5 0 0 . F i x a t i o n : 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1$ osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 77 Micrograph 4-353. Section through pores and sheath (s), Pore bulb (b) and sheath c o l l a r (sc) are l a b e l l e d . x 2 9 0 0 0 . Fixation as i n F i g . 7 6 . 97 Desmidium cylindricum F i g . 78 L i v i n g c e l l s i n l i g h t microscope. Arrows indicate p o s i t i o n of isthmus. Note sheath boundaries v i s i b l e i n unstained c e l l s . x 4 5 0 . F i g ; 79 Sheath stained with methyl v i o l e t . Sheath s t r i a t i o n s are v i s i b l e close to the c e l l s (arrow). x 7 6 0 . F i g . 80 Surface view of c e l l s stained with methyl v i o l e t showing rows of pores (arrow) in c e l l w a l l . x ! 2 5 0 . F i g . 81 Micrograph 2 3 0 3 . Section of c e l l wall (w) and pore apparatus. x 2 0 0 0 0 . b = pore bulb, sc = sheath c o l l a r ; s = sheath. Fi x a t i o n : 2 . 5 $ glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . s 99 Desmidium cylindricum F i g . 82 Micrograph 2 3 0 4 . Longitudinal section through c e l l apex showing pore i n a p i c a l wall (a) and remnant of i n i t i a l wall (arrow). x 2 0 0 0 0 . Fixation: 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 83 Micrograph 2 2 8 7 . Section showing overlap of semicell walls at the isthmus (arrows). x l 6 5 0 0 . F i x a t i o n as i n F i g . 82. F i g . 84 Micrograph 4 6 5 7 . Longitudinal section through d i v i s i o n septum showing folded mature walls (mw) separated by i n i t i a l wall ( 1 ) . x l 6 5 0 0 . Fixation as i n F i g . 82. 100 Genus Hyalotheca Ehrenberg 101 This filamentous genus has c e l l s which are c y l i n d r i c a l with l i t t l e or no isthmus c o n s t r i c t i o n (Fig. 8 5 ) . Two a x i a l chloroplasts are present i n each c e l l around a c e n t r a l nucleus. Hyalotheca d i s s i l i e n s ( J . E. Smith) de Brebisson C e l l s of Hyalotheca d i s s i l i e n s measure 12-14 u long by 23-24 u wide and are surrounded by a broad sheath ca. 15 u wide (Fig. 85) . " Ruthenium red and methyl v i o l e t both s t a i n the sheath with sheath s t r i a t i o n s c l e a r l y v i s i b l e using the l a t t e r s t a i n (Fig. 8 6 ) . In electron micrographs the l a t e r a l c e l l wall i s ca. 250 mu thick and the a p i c a l wall i s ca. 200 nru thick (Fig. 8 7 ) . At the margin of the apex a r i n g of dense f i b r i l l a r material extends out to meet a s i m i l a r r i n g from the adjacent c e l l ( F ig. 8 7)• This a p i c a l r i n g measures about 800 mu wide and i s somewhat triangular i n section (Fig. 8 8 ) . A dense layer ca.' 50 mu thick i s situated between the a p i c a l rings of adjacent c e l l s . The a p i c a l walls of adjacent c e l l s are separated by a space 1 0 0 - 2 0 0 mu wide with narrow cross connections v i s i b l e i n some sections (Fig-; 8 9 ) . The semicell walls overlap at the isthmus (Fig. 9 0 ) . 102 Pores 100-150 mu i n diameter are present i n both a p i c a l and l a t e r a l walls (Fig; 1 8 7 ) . A network of m i c r o f i b r i l s i s situated under the pore and the sheath c o l l a r extends ca; 200 mu from the c e l l wall (Fig. 91).' In cross section the sheath c o l l a r i s about 600 mu i n diameter (Fig; 9 2 ) . Pores are present very close to the isthmus overlap (Fig;' 9 0 ) . The sheath i s composed of long m i c r o f i b r i l s ca. 10 mu wide extending from the sheath c o l l a r ( Fig. 9 3 ) . In the outer part of the sheath the m i c r o f i b r i l s become bead-like and the boundary f i b r i l s of each sheath prism also have t h i s appearance (Fig. 9 3 ) . Tangential sections through the sheath show the conspicuous i n d i v i d u a l sheath prisms (Fig. 9^)-. 103 Hyalotheca d i s s i l i e n s F i g . 85 L i v i n g c e l l s i n l i g h t microscope. The po s i t i o n of the isthmus ( i ) i s not conspicuous. x 8 5 0 . F i g . 86 Sheath (s) stained with methyl v i o l e t to show sheath s t r l a t i o n s . x 8 5 0 . F i g . 87 Micrograph 2280. Longitudinal section through l a t e r a l margin of c e l l apex showing a p i c a l r i n g (r) joining the c e l l s . Lateral (1) and a p i c a l (a) pores are present. x l 6 0 0 0 . F i x a t i o n : 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7.2. 104 105 Hyalotheca d l s s l l l e n s F i g . 88 Micrograph 2 2 7 7 . Enlarged a p i c a l r i n g (ar) separating two c e l l s . A dense connecting layer (c) i s present between the two parts of the r i n g . x 5 1 0 0 0 . F i x a t i o n : 2 . 5 $ glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 * 2 . F i g . 89 Micrograph 2 2 6 8 . Narrow connection (arrow) between adjacent a p i c a l walls. X54-500. Fixation as i n F i g . 8 8 . F i g . 90 Micrograph 2135• Overlapping semicell walls at isthmus (arrows) with two adjacent pores. x l 6 5 0 0 . Fixation as i n F i g . 8 8 . 1 0 6 Hyalotheca d i s s i l i e n s F i g . 91 Micrograph 2259*- Section through two pores and adjacent sheath ( s ) . x 5 0 5 0 0 . b = pore bulb, sc = sheath c o l l a r . F i x a t i o n : 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 92 Micrograph 3 9 1 9 . Tangential section through c e l l wall and adjacent sheath. Pores (p) and sheath c o l l a r (sc) are cut i n cross section. X4-2700. F i x a t i o n : k% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 5»8. 108 S Hyalotheca d i s s i l i e n s F i g . 93 Micrograph 2146. L o n g i t u d i n a l s e c t i o n of sheath prisms ( s p ) . F i b r i l s become b e a d - l i k e (arrow) i n outer p a r t of sheath. xl7000. F i x a t i o n : 2.5$ g l u t a r a l d e h y d e . P o s t f i x a t i o n : 1% osmic a c i d . B u f f e r : phosphate - pH 7.2. F i g . 94 Micrograph 3918. Tangential section through sheath showing i n d i v i d u a l sheath prisms (sp). xl4000. Fix a t i o n : 4$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 5»8. Genus Sphaerozosma Corda emend. Bourrelly 111 The di s t i n g u i s h i n g character of t h i s filamentous genus as defined by Bourrelly (1964-) i s the presence of asymmetrically arranged protuberances on the c e l l apices (Fig. 9 6 ) . There are two chloroplasts i n each c e l l each with one large pyrenoid. Sphaerozosma laeve (Nordstedt) Thomasson Ce l l s of Sphaerozosma laeve measure 14—16 u long by 20-25 p. wide with a broad sheath 12-15 u thick (Fig. 95» 97 )• Sheath s t r i a t i o n s are revealed by methyl v i o l e t staining (Fig. 97)• The a p i c a l protuberances are about 4- p. long and arranged so that they extend on opposite sides of the adjacent semicell (Fig. 9 6 ) . In electron micrographs the c e l l wall i s ca. 200 mp. thick (Fig. 101) . The a p i c a l protuberances are extensions of the c e l l wall 3-4- u long and 1 u wide (Fig. 9 8 ) . The center of the protuberance contains a narrow space (Fig. 9 9 ) . The overlap of semicell walls at the isthmus i s shown i n F i g . 98 . Microtubules are present under the plasma membrane at the isthmus (Fig. 100). ' The a p i c a l walls of adjacent c e l l s are not i n contact but are separated by ca. 0.5 u of sheath-like material (Fig;'1 98). Between the c e l l s i s a dense layer of material continuous with the sheath surrounding the a p i c a l protuberance (Fig. 98). This sheath material extends along the l a t e r a l c e l l wall, probably serving to keep the c e l l s of the filament together as the a p i c a l protuberances do not contact the adjacent c e l l (Fig. 99) Pores cav 150 m u i n diameter are present i n the c e l l wall (Fig. 101)• The pore bulb contains a cap-like structure s i m i l a r to that of Triploceras v e r t l c i l l a t u m . The sheath c o l l a r extends 200-250 mp. from the wall and sheath f i b r i l s attach to i t (Fig. 101). 113 Sphaerozosma laeve Fi g , 95 Liv i n g c e l l s with arrow ind i c a t i n g p o s i t i o n of deep isthmus c o n s t r i c t i o n . x800. Fi g , 96 Two l i v i n g c e l l s separated from filament to show a p i c a l protuberances (arrows). x800. F i g . 97 Sheath stained with methyl v i o l e t to show sheath s t r i a t i o n s ( s ) . xl600. F i g . 98 Micrograph I633. Longitudinal section of c e l l through nucleus (n) and a p i c a l protuberance (a). The c e l l contains many starch grains ( s ) . x5250. Fixat i o n : 1% osmic a c i d . Buffer: phosphate - pH 7«2. Sphaerozosma laeve F i g . 99 Micrograph 1 5 2 4 . Longitudinal section through a p i c a l protuberance. A central channel i s present (arrow) i n the protuberance. A sheath of dense material (s) surrounds the protuberance. xl8000. F i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g ; 100 Micrograph 1 6 3 7 . Microtubules (arrow) below plasma membrane (pm) at isthmus region. x 2 5 5 0 0 . F i x a t i o n as i n F i g . 9 9 . F i g . 101 Micrograph 1647. Section of c e l l wall (w) through a pore apparatus (arrow). xl8000. Fix a t i o n as i n F i g . 9 9 . 116 Genus Spondyloslum de Brebisson 11? C e l l s of Spondyloslum have a deep isthmus c o n s t r i c t i o n and are united at t h e i r apices into filaments (Fig. 102)., This genus i s distinguished from Sphaerozosma by the lack of a p i c a l protuberances, from Bambusina and Desmidium by the lack of a folded d i v i s i o n septum, and from Hyalotheca by the presence of c e l l s with a deep isthmus c o n s t r i c t i o n . Spondyloslum pulchrum (Bailey) Archer C e l l s measuring 40 u long, 75-80 u wide, and 25 M thick with a narrow isthmus (27 u) are joined at s l i g h t a p i c a l projections into filaments (Fig. 102). A broad sheath extends from the l a t e r a l wall ca. 40 u and sheath s t r i a t i o n s are conspicuous with methyl v i o l e t staining (Fig. 103). The ruthenium red t e s t for pectic sheath material i s p o s i t i v e with the inner part of the sheath staining more deeply. The c e l l wall stains with iodine-^SO^ and shows a marked birefringence with polarized l i g h t i n d i c a t i n g i t s c e l l u l o s i c nature (Fig. 104). Two p a r i e t a l chloroplasts are present i n each semicell (Fig. 105). In electron micrographs the c e l l wall i s ca. 400 mu thick and comprises many thin layers of m i c r o f i b r i l s (Fig. 106).' The a p i c a l projections l i e close together with an electron-dense substance occupying the space 118 between the c e l l s (Fig. 107). As i n a l l Cosmarieae the semicell walls overlap at the isthmus. Pores are numerous i n the c e l l wall, having a diameter of ca; 100 mu i n the inner part and slightly-wider i n the outer part of the wall (Fig. 106). In tangential sections the c i r c u l a r pore i s surrounded by an electron-dense area ca. 25 mu thick ( Fig. 110). The pore bulb i s composed of a network of f i b r i l l a r material. In cross sections through the pore bulb the inner area of m i c r o f i b r i l s i s c i r c u l a r i n arrangement and the outer area i s r a d i a l (Fig." 109). As i n other Cosmarieae the pore bulb l i e s i n a pocket between the wall and the cytoplasmic membrane (Fig. 106). The sheath c o l l a r extends outward from the pore ca. 300 mu and i s ca. 500 mu i n diameter at i t s widest point (Fig. 106).- In sections cut through the pore apparatus just outside the c e l l wall the sheath c o l l a r appears as an annular r i n g ca. 180 mu thick ( Fig. 110). In the central lumen of the pore c o l l a r there i s a mass of f i b r i l l a r material that can be followed into the pore lumen i n other sections (Fig. HO). 1 In sections through the sheath c o l l a r further from the c e l l wall the central lumen of the c o l l a r disappears (Fig; 1 110). The c o l l a r then appears as a uniformly granular c i r c u l a r area with the m i c r o f i b r i l s of the sheath ra d i a t i n g from the margin. Sections passing 119 above the sheath c o l l a r cut through the m i c r o f i b r i l s of the sheath which are shown i n longitudinal section i n Fig.' 106i' These m i c r o f i b r i l s form a compact bundle i n cross sections near the sheath c o l l a r but gradually diverge as they go out into the sheath (Fig. 108). The boundaries of adjacent sheath prisms are not v i s i b l e close to the wall but can be distinguished i n the outer part of the sheath (Fig. 108). Pores are also present i n the a p i c a l wall region where the c e l l s j o i n (Fig.* 1 0 7 ) . These pores are of the same diameter (ca.5 100 mp) as the l a t e r a l pores. However since the space between the c e l l s i s f i l l e d with an electron-dense substance, there i s no sheath c o l l a r and the pore ends b l i n d l y at the outer edge of the c e l l wall (Fig. 107) . The chloroplasts have conspicuous grana and many thylakoids traverse the pyrenoid ground substance as i n other Cosmarieaev A starch sheath surrounds the pyrenoid and several osmiophilic globules are present among the stroma lamellae.' The structure of mitochondria and dictyosomes corresponds to that of other desmids and both organelles are close to the chloroplasts. Vacuoles occupy a large part of the c e l l . A metaphase c e l l of Spondylosium pulchrum was sectioned. The chromosome number of t h i s c e l l was approximately 11; The chromosomes shown i n F i g . I l l , 112 120 are transversely sectioned and the microtubules of the spindle generally l i e i n the plane of the sections. The spindle microtubules do not converge at the poles but pass between many mitochondria that occupy the polar region (Fig. I l l , 114). In some sections mitochondria occur among the spindle microtubules close to the metaphase plate (Fig. 112)v Many small vacuoles are present i n the spindle (Fig; 111 , 1 1 2 ) . Chromosomal microtubules attach to the chromosomes at l o c a l i z e d centromeres (Fig. 113 )• The microtubules attach to electron-dense plates on each chromatid (Fig. 113)• Although a continuous series of sections could not be obtained only one region of microtubule attachment i s seen on each chromosome. Non-chromosomal microtubules pass between the chromosomes (Fig . 113 )• No nucleolar material i s present around the chromosomes but there i s a small amount near one pole of the spindle (Fig. 1 1 5 ) . Spondylosium pulchrum F i g . 102 L i v i n g c e l l s joined at a p i c a l protuberances (a). The deeply constricted isthmus region ( contains a conspicuous nucleus (n). x 5 6 0 . F i g . 103 Sheath stained with methyl v i o l e t to show sheath s t r i a t i o n s ( s ) . x64-0. F i g . 104- C e l l s photographed i n polarized l i g h t . C e l l walls b i r e f r i n g e n t . x 5 6 0 . F i g . 105 Apical view of c e l l . Two chloroplasts (ch) are present. x 6 l 0 . 123 Spondylosium pulchrum Pig. 106 Micrograph 5 2 - 1 7 . Section of c e l l wall (w) through two pores and adjacent sheath m i c r o f i b r i l s ( f ) . x 4 3 6 0 0 . b = pore bulb; sc = sheath c o l l a r . Fixations 2 . 5 $ glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . 1 124 125 Spondyloslum pulchrum F i g . 107 Micrograph 5 3 - 2 2 . Longitudinal section through a p i c a l pad showing dense material between c e l l s (arrow). A pore i s present i n the wall of the a p i c a l protuberance (a). x9750» F i x a t i o n : 2.$% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 108 Micrograph 53-24. Tangential section through sheath showing i n d i v i d u a l sheath prisms ( s ) . X 1 7 0 0 0 . Fixation as i n F i g . 1 0 7 . 12? Spondylosium pulchrum F i g . 109 Micrograph 52-14. Tangential section through several pore bulbs (b). C i r c u l a r l y arranged m i c r o f i b r i l s are surrounded by r a d i a l l y arranged m i c r o f i b r i l s . x 3 4 0 0 0 . F i x a t i o n : 2 . 5 $ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 110 Micrograph 5 2 - 1 1 . Tangential section through c e l l wall (w) and adjacent sheath c o l l a r s (sc). Pores are surrounded by an electron-dense area (arrow). x l 5 0 0 0 . Fixation as i n F i g . 1 0 9 . 128 129 Spondylosium pulchrum Pig. I l l Micrograph 3 2 6 ? . Section of metaphase spindle showing chromosomes (ch) and spindle microtubules ( t ) . Mitochondria (m) are concentrated at the spindle pole. x l 6 3 0 0 . F i x a t i o n : k% glutaraldehyde 1. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 « 0 . F i g . 112 Micrograph 3 0 1 8 . Section of same metaphase spindle as F i g . 111. Note mitochondria (m) situated close to the chromosomes (ch). X16500. Fixation as i n F i g . 111. 130 131 Spondyloslum pulchrum F i g . 113 Micrograph 3 3 9 2 . Transverse section through centromere region of metaphase chromosome. Chromosomal microtubules (arrows, top and bottom) attach to chromosome. Non-chromosomal microtubules (net) pass by chromosome without attaching. x 8 3 0 0 0 . F i x a t i o n : h% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH ? . 0 . 132 133 Spondylosium pulchrum Pig. 114 Micrograph 3 2 6 8 . Enlarged part of Pig. 111. Spindle microtubules (arrow) enter the region at the spindle pole containing mitochondria (m). X51000. F i x a t i o n : h% glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: cacodylate - pH 7 . 0 . F i g . 115 Micrograph 3313* Nucleolar material (arrow) near spindle pole. x 6 0 0 0 0 . Fixation as i n F i g . 114. Genus Cosmocladlum de Brebisson 135 The distinguishing features of t h i s c o l o n i a l genus are the connectingstrands which j o i n the c e l l s (Fig. 116). Globose colonies of two to many Cosmarium-like c e l l s are formed• Cosmocladlum saxonlcum de Bary Ce l l s of Cosmocladlum^saxonlcum are 28-32 u long by 22-24 ;u wide (Fig. 116). The strands uniting the c e l l s a r i s e from clusters of pores near the isthmus (Fig. 119)• These strands do not s t a i n with ruthenium red, methyl v i o l e t , congo red or methylene blue. There are 12-15 pores i n each isthmus cluster and two pore clusters per semicell ( F i g . 119)• The sheath surrounds the colony and stains with ruthenium red and methyl v i o l e t . Feathery structures are v i s i b l e using the l a t t e r s t a i n (Fig. 117). There i s one chloroplast with one pyrenoid i n each semicell. The c e l l w a l l i n electron micrographs Is 100-150 mu thick (Fig. 118). I t does not r e a d i l y s t a i n with uranyl acetate and lead c i t r a t e , often with only the outer part being stained. The overlap of semicell walls at the isthmus i s shown i n F i g . 124. Pores ca. 200 mu wide are present and have a structure s i m i l a r to those of other members of the subfamily. There i s no well-developed sheath c o l l a r 136 and, although f a n - l i k e structures are v i s i b l e i n stained colonies i n the l i g h t microscope, only scattered m i c r o f i b r i l s are v i s i b l e i n the sheath i n electron micrographs (Fig. 122). Strands connecting the c e l l s of a colony consist of several subunits (Fig. 120) each of which i s associated with one of the pores of the isthmus cluster (Fig. 122). The base of each strand subunit, about 180 mu i n diameter, i s inserted into a pore of the isthmus region (Fig. 123). The strand subunits expand, becoming d e f i n i t e l y f i b r i l l a r toward the middle (Fig. 123)t and overlapping the subunits which a r i s e from the other c e l l of the p a i r . The subunits d i v i d e as they leave the pore with the two parts going i n d i f f e r e n t d i r e c t i o n s (Fig. 121). M i c r o f i b r i l s attached to the plasma membrane come out into the pore bulb (Fig. 121). The chloroplast has several lobes extending from a large central pyrenoid ( F i g . 122). The pyrenoid matrix i s traversed by many thylakoids and the chloroplast contains obvious grana (Fig. 125). Large vacuoles occupy much of the c e l l (Fig. 122). Mitochondria and dictyosomes 1-3 u long are present close to the chloroplast (Fig. 122). The nucleus l i e s i n the isthmus region and has a central spherical nucleolus (Fig. 125). Microtubules below the plasma membrane perpendicular to the c e l l axis are present at the isthmus (Fig. 124, 126). 137 Cosmocladium saxonioum F i g . 116 L i v i n g c e l l s showing c o n n e c t i n g s t r a n d s ( a r r o w s ) j o i n i n g the c e l l s o f the c o l o n y . x 4 l 0 . F i g . 117 S t a i n i n g w i t h m e t h y l v i o l e t r e v e a l s f e a t h e r y s t r u c t u r e s (arrow) i n the s h e a t h t h a t r a d i a t e f r o m the c e l l ( c ) . x l 5 0 0 . 139 Cosmocladium saxonicum F i g . 118 Micrograph 2468. Section of c e l l wall (w). X 2 0 0 0 0 . F i x a t i o n : 2 .5$ glutaraldehyde. P o s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7»2. F i g , 119 Apical view of c e l l stained with methyl v i o l e t to show isthmus pore group (arrow), x2200. Fi g , 120 Micrograph 2382. Oblique section through connecting strand showing several strand subunits (su). x23000. Fixation as i n F i g . 118. F i g . 121 Micrograph 2467. Section through isthmus pore group. Note b i f u r c a t i o n of strand unit as i t leaves pore (arrow). x20000. Fixation as i n F i g . 118. 1 4 0 141 Cosmocladlum saxonicum F i g . 1 2 2 Micrograph 2 3 9 9 . Low magnification micrograph of c e l l s showing strand subunit (su) i n lon g i t u d i n a l section. x6400. d = dictyosome. m = mitochondrion, n s= nucleus, v = vacuole. s = sheath with scattered m i c r o f i b r i l s . Fixations 2 . 5 $ glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffers phosphate - pH 7 . 2 . 143 Cosmocladium saxonicum F i g . 123 Micrograph 2396. Enlarged view of strand subunit i n F i g . 122. The subunit i s dense and s o l i d as i t passes through the pore in the wall (w) but becomes f i b r i l l a r (arrow) near the middle of the strand. Note fi n e m i c r o f i b r i l s near the plasma membrane i n the pore bulb (b). x24000. Fix a t i o n : 2 .5$ glutaraldehyde. P o s t f i x a t i o n : 1$ osmic a c i d . Buffer: phosphate - pH 7.2. F i g . 124 Micrograph 2438. Longitudinal section through isthmus region showing the overlap of the semicell walls (large arrows) and the isthmus microtubules (small arrows) under the plasma membrane (pm). x72000. Fixation as i n F i g . 123. 144 Cosmocladlum saxonlcum F i g . 125 Micrograph 24-03. Enlarged view of part of F i g . 122. The nucleus (n) has a central nucleolus (nu) and the adjacent pyrenoid has a conspicuous sheath of starch ( s ) . X 1 3 0 0 0 . d = dictyosome. g = grana. m = mitochondrion. Fixation: 2 .5$ glutaraldehyde. Po s t f i x a t i o n : 1% osmic a c i d . Buffer: phosphate - pH 7 . 2 . F i g . 126 Micrograph 24-75• Longitudinal section of isthmus microtubules. X72000. Fixation as i n F i g . 125. 146 147 Discussion. The c e l l w a l l of the Desmidiaceae was considered to be two-layered on the basis of i t s staining reactions using l i g h t microscopy (Lutkemiiller 1 9 0 2 ) . The Desmidiaceae were subdivided into three subfamilies: the Penieae, with unconstricted c e l l s lacking pores and a regular s i t e of cytokinesis; the Closterieae, with unconstricted c e l l s lacking pores and with a regular s i t e of cytokinesis; and the Cosmarieae, with constricted c e l l s with pores present and a constant s i t e of cytokinesis. By means of electron microscopy Chardard and R o u i l l e r ( 1 9 5 7 ) and Drawert and Metzner-Kuster ( I 9 6 I ) showed only one layer i n the w a l l of the Cosmarieae. The present study confirms t h e i r observations. With the exception of Penium (Pleurotaenlum) spinulosum. which w i l l be discussed l a t e r , a l l Cosmarieae investigated have the c e l l w a l l composed of several t h i n layers of oriented m i c r o f i b r i l s . The difference i n the angle of .orientation of the m i c r o f i b r i l s i s r a r e l y 9 0 ° , but more often 6 0 - 7 0 ° . The staining of the w a l l with ruthenium red supports the view that pectic substances are present among the c e l l u l o s e m i c r o f i b r i l s of the w a l l (Drawert and Metzner-Kuster I 9 6 I ) . The c e l l u l o s i c nature of the w a l l i s shown by staining with chlor-zinc-iodine and iodine-HgSO^ reagents (Lutkemuller 1 9 0 2 ) . The c e l l w a l l of Closterlum lineatum (subfamily Closterieae) i s s i m i l a r to that of the Cosmarieae. The 148 w a l l i s n o t t w o - l a y e r e d as r e p o r t e d by l i g h t m i c r o s c o p i s t s . L u t k e m u l l e r (1902) d e s c r i b e d two w a l l l a y e r s i n s e v e r a l C l o s t e r l u m s p e c i e s , i n c l u d i n g C l . l i n e a t u m . based! on s t a i n i n g r e a c t i o n s u s i n g c h l o r - z i n c - i o d i n e and i o d i n e -HgSOj^. C h a r d a r d ( 1965) and C h a r d a r d and R o u i l l e r (1957) r e p o r t o n l y one l a y e r i n C l . a c e r o s u m . A narrow e l e c t r o n -dense l a y e r on t h e o u t s i d e o f t h e c e l l w a l l was i n t e r p r e t e d a s " a s u p e r f i c i a l d i f f e r e n t i a t i o n " ( C h a r d a r d I 9 6 5 ) . D r a w e r t and M e t z n e r - K i i s t e r ( I 9 6 I ) , u s i n g c h r o m i c a c i d t r e a t e d w a l l p r e p a r a t i o n s o f C l . m o n l l l f e r u m . o b s e r v e d two d i s t i n c t l a y e r s o f m i c r o f i b r i l s i n e l e c t r o n m i c r o g r a p h s . In t h e i n n e r l a y e r the m i c r o f i b r i l s a r e o r i e n t e d p a r a l l e l t o the c e l l a x i s and i n t h e o u t e r l a y e r the m i c r o f i b r i l s a r e t r a n s v e r s e l y o r i e n t e d . I t i s d i f f i c u l t t o c h a r a c t e r i z e t h e s u b f a m i l y C l o s t e r i e a e on t h e b a s i s o f e l e c t r o n m i c r o s c o p e s t u d y o f J u s t t h r e e s p e c i e s . However, t h e f o l l o w i n g t e n t a t i v e s t a t e m e n t s c a n be made: a ) The C l o s t e r i e a e r e s e m b l e the C o s m a r i e a e i n l a c k i n g a two l a y e r e d c e l l w a l l and i n h a v i n g a d e f i n i t e l i n e o f o v e r l a p between the two s e m i c e l l w a l l s . b ) The C l o s t e r i e a e d i f f e r f r o m the Cosmarieae l n the l a c k o f p o r e s : i n t h e c e l l w a l l and the f o r m a t i o n o f g i r d l e bands ( s e c t i o n s o f w a l l t h a t a r e formed by e l o n g a t i o n o f t h e c e n t r a l r e g i o n o f t h e c e l l ) i n s e v e r a l s p e c i e s . 149 The f i n e structure of the c e l l w a l l of the subfamily Penieae d i f f e r s sharply from that of the Closterieae and Cosmarieae. The presence of two d i s t i n c t layers i n the c e l l w a l l of Penium contrasts with the uniform w a l l i n the other two subfamilies. The lack of structures corresponding to the pore apparatus of the Cosmarieae as w e l l as the lack of a sheath are also distinguishing features of the Penium c e l l w a l l . Chardard (1964) reports the presence of a sheath i n P. margar1taceum. He used ruthenium red as an electron s t a i n a f t e r osmic acid f i x a t i o n and considered the stained outer w a l l layer to be the sheath. He also states that t h i s outer layer may be lacking and that i t s appearance depends on the physiological state of the c e l l . The outer w a l l layer was found i n a l l c e l l s sectioned In t h i s study and could be distinguished In the l i g h t microscope i n c e l l s from either young or old cultures. Mix (I968) studied w a l l structure i n P. margaritaceum. P. spirostriolatum and P. cyllndrus and observed two c e l l w a l l layers i n each. The outer layer i s interpreted as part of the c e l l w a l l i n the present study because of i t s greater density compared with the sheath material of the Cosmarieae and because i t s f i b r i l l a r structure reaches down into the inner w a l l layer. The chemical nature of the two w a l l layers was not investigated. The r e s u l t s of Chardard (1964) indicate the presence of pectic substances i n the outer layer based on i t s staining with ruthenium red. The inner layer stains 150 with the standard c e l l u l o s e reagents, chlor-zinc-iodine and iodine-HgSOjj, (Liitkemuller 1902). Pleurotaenlum splnulosum was c l e a r l y not c l a s s i f i e d correctly.' This rare species was o r i g i n a l l y placed In Docldlum (Wolle 1881) but was transferred to the genus Pleurotaenlum by Brunei (194-9) because i t resembled other members of the genus and because pores were reported to be present. Several points of evidence e x i s t f o r removing t h i s species from Pleurotaenlum and placing i t i n the genus Penium. The difference between the f i n e structure of the wall of PI. splnulosum and that of other Cosmarieae has already been Indicated. The c e l l wall of PI. splnulosum i s i d e n t i c a l i n structure with those of Penium spirostriolatum and P. margaritaceum. The lack of overlapping walls at the isthmus and the absence of pores i n the c e l l wall further support the view that PI. splnulosum should be transferred to the genus Penium. The chloroplasts of PI. splnulosum are s t e l l a t e and a x i a l while those of other Pleurotaenlum species are almost always p a r i e t a l bands. A new de s c r i p t i o n of the species Pleurotaenlum splnulosum (Wolle) Brunei i s necessary and t h i s species should now be c a l l e d Penium splnulosum (Wolle) comb. nov. The ultr a s t r u c t u r e of the pore apparatus of the Cosmarieae has been determined by Drawert and Mix (1961a) and Chardard and R o u l l l e r (1957)• The pore diameter i s 130-180 mu f o r Mlcrasterlas rotata (Drawert and Mix 1961a) 151 and approximately 140 mu for M. p a p l l l l f e r a (Chardard and R o u l l l e r 1957)• The present study confirms the general observations and measurements of these authors but there i s some v a r i a b i l i t y i n structure among the genera. The pore bulb i s described by Drawert and Mix (1961a) as resembling the plug of a wash basin f i t t i n g into the ; pore opening, A s i m i l a r structure i s observed i n Pleurotaenlum nodosum and Trlploceras v e r t l c i l l a t u m where a cap-shaped structure covers the pore opening. In other species studied such a s o l i d structure i s not seen, A loose f i b r i l l a r mass with a zonatlon into c i r c u l a r l y arranged and r a d i a l l y arranged.microfibrils i s present i n these species (e.g. Hyalotheca d i s s l l l e n s , Spondyloslum  pulchrum). Sheath structure i n the Cosmarieae i s variable with some species having l i t t l e sheath (e.g. Trlploceras v e r t l c i l - latum. Pleurotaenlum nodosum) and others having a broad sheath (e.g. Spondyloslum pulchrum). The sheath i s always composed of Individual parts, each situated over one of the pores. Drawert and Metzner-Kuster (1961) demonstrated the i n d i v i d u a l sheath prisms of Hyalotheca d i s s l l l e n s i n oblique sections through the sheath. The boundaries of the i n d i v i d u a l sheath prisms are seen i n some of the desmids i n the present study (e.g. Hyalotheca d i s s l l l e n s . Spondyloslum pulchrum)• The nature of the connecting strands of Cosmocladium Is n o t c e r t a i n . " The e a r l i e r a u t h o r s ( S c h r & d e r 1900, 1902, L u t k e m u l l e r 1902) c o n c l u d e d t h a t t h e s t r a n d s a r o s e f r o m a s p e c i a l g r o u p o f p o r e s on e i t h e r s i d e o f t h e i s t h m u s . Heimans (1935)» s t u d y i n g C . subramosum. s t a t e d t h a t t h e c o n n e c t i n g s t r a n d s were formed f r o m the i n i t i a l w a l l s d i s c a r d e d by young s e m i c e l l s f o l l o w i n g c e l l d i v i s i o n . The p r e s e n t s t u d y u s e d the same s p e c i e s as L u t k e m u l l e r and S c h r o d e r and c o n f i r m s t h e i r c o n c l u s i o n t h a t i n C . s a x o n l c u m s c o n n e c t l n g s t r a n d s J o i n w i t h the Isthmus g r o u p o f p o r e s . E a c h p o r e c o n t r i b u t e s one s t r a n d s u b u n i t w i t h t h e e n t i r e s t r a n d c o m p r i s i n g 12-15 s u c h s t r a n d s u b u n i t s . A r e s t u d y o f the s p e c i e s u s e d by Heimans i s recommended I n o r d e r t o d e t e r m i n e whether a d i f f e r e n t method o f s t r a n d f o r m a t i o n o c c u r s i n C . subramosum. The p r o b l e m o f how t h e c o n n e c t i n g s t r a n d s o f Cosmocladium a r e formed r e m a i n s t o be i n v e s t i g a t e d . T h r e e o r f o u r p a i r s o f c o n n e c t i n g s t r a n d s may a r i s e f r o m one c e l l b u t w i t h o n l y two p a i r s o f p o r e g r o u p s i t i s d i f f i c u l t t o imagine how the a d d i t i o n a l c o n n e c t i n g s t r a n d s a r i s e . The s t r a n d u n i t s a p p e a r t o s p l i t o u t s i d e t h e p o r e w i t h t h e two p a r t s g o i n g i n d i f f e r e n t d i r e c t i o n . The same p o r e group may be a b l e t o p r o d u c e more t h a n one s t r a n d . A d e f i n i t e c o n c l u s i o n c a n n o t be made f r o m the r e s u l t s o f t h e p r e s e n t s t u d y . C e l l d i v i s i o n i n t h e C o s m a r i e a e i n v o l v e s t h e inward g r o w t h o f a n a n n u l a r septum ( K r i e g e r 1937). The septum 153 i s a t t a c h e d t o a c y l i n d r i c a l w a l l s e c t i o n t h a t s e p a r a t e s t h e two o l d s e m i c e l l s . In t h e u n i c e l l u l a r Cosmarieae t h e young s e m i c e l l s expand u n t i l t h e mature s e m i c e l l morphology i s r e a c h e d . In t h e h a l f e l o n g a t e d s e m i c e l l o f T r l p l o c e r a s  v e r t l c i l l a t u m t h e w a l l i s t h i n and l a c k s p o r e s and must r e p r e s e n t t h e i n i t i a l w a l l t h a t i s l a t e r d i s c a r d e d a f t e r t h e mature c e l l w a l l i s f o r m e d . A s i m i l a r t h i n i n i t i a l w a l l i s f o u n d i n M l e r a s t e r l a s s p ; (Waddingtdn 1 9 6 2 ) . Mix ( I 9 6 6 ) showed t h a t t h e i n i t i a l w a l l s o f Tetmemorus and P l e u r o t a e n l u m a r e composed o f m i c r o f i b r i l s o r i e n t e d a t r a n d o m . M i c r o f i b r i l s a r e a l s o v i s i b l e i n s e c t i o n s o f t h e i n i t i a l w a l l o f T r l p l o c e r a s v e r t 1 c i l i a t u r n . The f o r m a t i o n o f the mature c e l l w a l l i n u n i c e l l u l a r Cosmarieae has n o t b e e n s t u d i e d and no m i c r o g r a p h s o f t h i s p r o c e s s were o b t a i n e d i n t h e p r e s e n t s t u d y . In t h e f i l a m e n t o u s Cosmarieae t h e d i s c a r d i n g o f a n i n i t i a l w a l l c a n n o t o c c u r o r the f i l a m e n t would d i s i n t e g r a t e i n t o s i n g l e c e l l s a f t e r c e l l d i v i s i o n . The f o r m a t i o n o f new s e m i c e l l w a l l s i s d i f f e r e n t i n t h i s g r o u p . In Desmldlum and Bambusina t h e c e l l w a l l Is l a i d down b e f o r e e l o n g a t i o n o f t h e new s e m i c e l l b e g i n s . Although e l e c t r o n m i c r o g r a p h s o f e a r l y s t a g e s i n c e l l d i v i s i o n were n o t o b t a i n e d t h e p r o c e s s may o c c u r i n the f o l l o w i n g way. An i n i t i a l d i v i s i o n septum i s formed a f t e r m i t o s i s and t h e c y l i n d r i c a l H - p i e c e i s f o r m e d . T h i s i n i t i a l w a l l p e r s i s t s 1 5 4 as the less electron-dense layer between the mature c e l l walls which are l a i d down on both sides. The position of pores i n the mature w a l l i s indicated by less dense areas but i t i s not clear how the location i s determined. When the new semicells elongate the walls separate at the outer edge of the d i v i s i o n septum and gradually the wa l l unfolds to form the mature semicell shape. Pieces of the I n i t i a l w a l l remain attached to the outside of the mature c e l l w a l l . The two new semicells remain i n contact i n the area i n t e r n a l to the c e l l w a l l f o l d . This description of c e l l elongation i n BambusIna bre b l s s o n l l d i f f e r s s l i g h t l y from that given by Hauptfleisch (1888) from l i g h t microscope observations and repeated without a l t e r a t i o n i n Krieger ( 1 9 3 7 ) . Hauptfleisch shows the separation of the folded parts of the wa l l before the l a t e r a l edge separates whereas i n the present study the reverse was found to occur. The structure of the mature semicell i n Bambusina can be related to the structure of the unexpanded semicell. The two l a t e r a l c e l l w a l l sections near the apex i n mature semicells correspond to the folded part of the d i v i s i o n w a l l with the edge of the f o l d marked by a s l i g h t depression i n the mature w a l l . The two narrower sections near the isthmus represent the d i v i s i o n w a l l outside the f o l d and the l a t e r a l part of the new semicell w a l l i n Pi g . 6 7 with the thickening i n the mature w a l l marking the 9 0 ° angle between the two sections. 155 C h l o r o p l a s t u l t r a s t r u c t u r e i n the D e s m i d i a c e a e i s u n i f o r m . The p r e s e n c e o f g r a n a i n desmid c h l o r o p l a s t s was r e p o r t e d by D r a w e r t and Mix (196ld, f ) f o r M i c r a s t e r l a s  r o t a t a and by C h a r d a r d (1964, 1965) f o r Cosmarium l u n d e l l l l . C l o s t e r l u m a c e r o s u m and Penium m a r g a r i t a c e u m . G r a n a have been found i n a l l s p e c i e s s t u d i e d i n the p r e s e n t i n v e s t i g a t i o n . The p r e s e n c e o f o s m i o p h l l l c g l o b u l e s s i n g l y o r i n c l o s e l y packed g r o u p s between the t h y l a k o i d s i s a c o n s t a n t f e a t u r e o f the d e s m i d s s t u d i e d . No c o r r e l a t i o n o f c h l o r o p l a s t u l t r a s t r u c t u r e t o s u b f a m i l i e s o f the D e s m i d i a c e a e can be made. P y r e n o i d s t r u c t u r e i s t h e same i n a l l desmids s t u d i e d up t o the p r e s e n t t ime (Drawert and Mix 1962a, C h a r d a r d 1964, I965t Kamiya 1961, 1962, L e y o n 1954). The m a t r i x a p p e a r s g r a n u l a r and e l e c t r o n - d e n s e w i t h g l u t a r a l d e h y d e -osmium f i x a t i o n and s e v e r a l t h y l a k o i d s p a s s t h r o u g h t h e m a t r i x . In some s p e c i e s ( e . g . T r l p l o c e r a s F i g . 56) t h e r e i s a c e n t r a l l e s s dense a r e a i n the p y r e n o i d t h r o u g h w h i c h many d f t h e t h y l a k o i d s p a s s . T h i s a r e a i s n o t an i n v a g i n a t i o n o f the c h l o r o p l a s t s t r o m a i n t o the p y r e n o i d m a t r i x b u t i s s u r r o u n d e d by t h e m a t r i x . I t i s n o t found i n a l l p y r e n o i d s o f a s i n g l e c e l l n o r i n a l l c e l l s o f one s p e c i e s . M i t o c h o n d r i a i n the desmids s t u d i e d may be s h o r t , e l o n g a t e d o r Y - s h a p e d (Drawert and Mix l96lh) b u t the i n t e r n a l s t r u c t u r e i s u n i f o r m . The m i t o c h o n d r i a o f 156 M l e r a s t e r l a s r o t a t a measure 5-35 P by 0.4 u (Drawert and Mix 1961h) and t h e e l o n g a t e d desmids l n the p r e s e n t s t u d y have m i t o c h o n d r i a up t o 15 u l o n g . D r a w e r t and Mix o b s e r v e d t h a t the o u t e r membrane o f t h e m i t o c h o n d r i o n i s n o t p r e s e r v e d w e l l (osmic a c i d f i x a t i o n ) and t h e same s t r u c t u r e i s o b s e r v e d i n the m i t o c h o n d r i a o f most s p e c i e s i n the p r e s e n t s t u d y . T h i s i s p r o b a b l y the r e s u l t o f p o o r f i x a t i o n s i n c e a w e l l d e f i n e d o u t e r m i t o c h o n d r i a l membrane i s p r e s e n t i n some c e l l s o f T r l p l o c e r a s v e r t l c i l l a t u m f i x e d w i t h g l u t a r a l d e h y d e - o s m i c a c i d . M i t o c h o n d r i a a r e l o c a t e d c l o s e t o t h e c h l o r o p l a s t l o b e s . In c r o s s s e c t i o n s o f d e s m i d s w i t h a x i a l c h l o r o p l a s t s ( e . g . P e n i u m . T r l p l o c e r a s ) t h e m i t o c h o n d r i a l i e i n the g r o o v e s between the c h l o r o p l a s t l o b e s • D i c t y o s o m e s a l s o a r e f o u n d c l o s e t o t h e l o b e s o f the c h l o r o p l a s t . Desmid d i c t y o s o m e s a r e among the l a r g e s t f o u n d i n p l a n t s , m e a s u r i n g up t o 5*5 P i n M l e r a s t e r l a s  r o t a t a (Drawert and Mix 196lg), 1.6-3.6 u i n M. d e n t l c u l a t a (Drawert and Mix 1963). 1-3 p i n C l o s t e r l u m acerosum and c a . 2 u i n Cosmarlum l u n d e l l i i ( C h a r d a r d 1965). In the p r e s e n t s t u d y d i c t y o s o m e s a r e u s u a l l y 1-3 u l o n g . A v a r i a b l e • number o f c i s t e r n a e make up the d i c t y o s o m e and s m a l l d i c t y o s o m e v e s i c l e s a r e p r e s e n t a t b o t h ends o f t h e s t a c k . C y t o p l a s m i c m i c r o t u b u l e s were f i r s t s e e n i n v a s c u l a r p l a n t s by L e d b e t t e r and P o r t e r (I96I) and have s u b s e q u e n t l y b e e n o b s e r v e d i n many o t h e r s ( M i i h l e t h a l e r 1967). The Isthmus m i c r o t u b u l e s o f t h e D e s m i d i a c e a e c o r r e s p o n d i n s i z e (23-2? mu) and f i x a t i o n p r o p e r t i e s t o t h o s e o b s e r v e d n e a r t h e p l a s m a membrane i n v a s c u l a r p l a n t s . These m i c r o t u b u l e s a r e s e e n i n g l u t a r a l d e h y d e - f i x e d c e l l s b u t n o t i n o s m i u m - f i x e d c e l l s . The m i c r o t u b u l e s o f the m i t o t i c a p i n d l e o f S p o n d y l o s i u m p u l c h r u m a r e s i m i l a r i n s i z e t o t h e i s t h m u s m i c r o t u b u l e s . M i c r o t u b u l e s n e a r t h e p l a s m a membrane a r e t h o u g h t t o f u n c t i o n i n t h e f o r m a t i o n and o r i e n t a t i o n o f t h e c e l l u l o s e m i c r o f i b r i l s o f the c e l l w a l l ( M u h l e t h a l e r 1 9 6 7 ) . T h i s c o n c l u s i o n i s b a s e d on the o b s e r v a t i o n t h a t the m i c r o t u b u l e s and m i c r o f i b r i l s o f t e n a r e o r i e n t e d i n t h e same d i r e c t i o n . In the c e l l w a l l o f t h e D e s m i d i a c e a e t h e m i c r o f i b r i l s a r e n o t o r i e n t e d i n t h e same d i r e c t i o n as t h e m i c r o t u b u l e s . The m i c r o t u b u l e s a r e o n l y p r e s e n t a t t h e i s t h m u s r e g i o n i n e l o n g a t i n g s e m i c e l l s . In s u c h c e l l s one would e x p e c t t h a t c e l l w a l l f o r m a t i o n i s o c c u r r i n g a t a r e a s away f r o m t h e i s t h m u s . However, no m i c r o t u b u l e s o t h e r t h a n t h o s e a t t h e i s t h m u s r e g i o n a r e p r e s e n t . Penium m a r g a r i t a c e u m , w h i c h does n o t have a c o n s t r i c t e d i s t h m u s , a l s o has m i c r o t u b u l e s whose o r i e n t a t i o n i s n o t the same as t h a t o f t h e c e l l w a l l m i c r o f i b r i l s . T h e r e f o r e , i s t h m u s m i c r o t u b u l e s a r e p r o b a b l y n o t i n v o l v e d i n c e l l w a l l d e p o s i t i o n i n t h e D e s m i d i a c e a e and a r e n o t o n l y a s s o c i a t e d w i t h c e l l s c o n t a i n i n g t h e i s t h m u s c o n s t r i c t i o n . 158 The chromosomes o f the desmids a r e r e p o r t e d as h a v i n g d i f f u s e o r n o n - l o c a l i z e d c e n t r o m e r e s ( K i n g i 9 6 0 ) b a s e d on t h e p a r a l l e l s e p a r a t i o n o f c h r o m a t i d s a t a n a p h a s e . In t h e r e l a t e d f a m i l y Zygnemataceae Godward (195*0 r e p o r t s t h a t some S p l r o g y r a s p e c i e s have chromosomes w i t h l o c a l i z e d c e n t r o m e r e s and o t h e r s have p o l y c e n t i c chromosomes o r chromosomes w i t h d i f f u s e c e n t r o m e r e s . S i r o g o n l u m , w h i c h i s s i m i l a r t o S p i r o g y r a , i s a l s o r e p o r t e d t o have p o l y c e n t r i c chromosomes (Hoshaw and Waer 1 9 6 7 ) . The c e n t r o m e r e i n e l e c t r o n m i c r o g r a p h s c o m p r i s e s two dense bands o r p l a t e s on e a c h s i d e o f t h e chromosome and b u n d l e s o f s p i n d l e m i c r o t u b u l e s a t t a c h t o t h e s e p l a t e s (Godward 1 9 6 5 ) . In chromosomes w i t h d i f f u s e c e n t r o m e r e t h e s p i n d l e m i c r o t u b u l e s a t t a c h s i n g l y a t many p o i n t s a l o n g t h e chromosome (Godward 1 9 6 5 ) . A t metaphase i n C l o s t e r l u m e h r e n b e r g l l the a b s e n c e o f b u n d l e s o f s p i n d l e m i c r o t u b u l e s a t t a c h i n g t o t h e chromosome was i n t e r p r e t e d a s e v i d e n c e f o r t h e e x i s t e n c e o f d i f f u s e c e n t r o m e r e s i n t h e chromosomes o f t h i s s p e c i e s (Brandham and Godward 1 9 6 5 . Godward I966). U n p u b l i s h e d m i c r o g r a p h s e x i s t o f metaphase i n M l e r a s t e r l a s showing s p i n d l e m i c r o t u b u l e s a t t a c h i n g s i n g l y a t s e v e r a l p o i n t s on t h e chromosome (Godward I 9 6 5 ) . In t h e metaphase chromosomes o f S p o n d y l o s l u m p u l c h r u m b u n d l e s o f s p i n d l e m i c r o t u b u l e s a t t a c h a t one r e g i o n on 159 t h e chromosome ( F i g . 113)• The s t r u c t u r e o f t h i s a t t a c h m e n t r e g i o n i s t h e same as t h a t o f t h e c e n t r o m e r e o f h i g h e r p l a n t and a n i m a l chromosomes. S i n c e no s p i n d l e m i c r o t u b u l e s a t t a c h t o o t h e r p o i n t s on the chromosome t h e chromosomes o f S . p u l c h r u m must have a l o c a l i z e d c e n t r o m e r e . In t h e D e s m i d i a c e a e and Zygnemataceae t h e n u c l e o l a r m a t e r i a l i s r e p o r t e d t o p e r s i s t d u r i n g m i t o s i s a s a d a r k s t a i n i n g s u b s t a n c e a r o u n d t h e chromosomes ( K i n g I960, Godward and J o r d a n 1965)* In e l e c t r o n m i c r o g r a p h s o f metaphase i n C l o s t e r i u m e h r e n b e r g i l (Brandham and Godward 1965) and i n S p o n d y l o s i u m p u l c h r u m o f t h i s s t u d y no n u c l e o l a r s u b s t a n c e i s p r e s e n t a r o u n d the chromosomes. Thus the p e r s i s t e n c e o f t h e n u c l e o l a r m a t e r i a l a r o u n d t h e chromosomes d u r i n g m i t o s i s i n d e s m i d s does n o t a p p e a r t o be a c o n s t a n t phenomenon. Bibliography. Bourrelly, P. 1964 Une nouvelle coupure generique dans l a f a m i l l e des Desmldlees: l e genre T e l l l n g l a . Rev. A l g o l . , n.s., 2 J 187-191• Bourrelly, P. 1966 Les Algues d»Eau Douce. I. Les Algues Vertes. P a r i s , N. Boubee & Gie. Brandham, P. and Godward, M.B.E. 1965 Ultrastructure of the nucleus of the green alga Closterlum ehrenbergll. J. Roy. Micr. Soc. 84: 499-507. Brunei, J. 1949 The rediscovery of the desmid Pleurotaenlum  splnulosum. with description of a new var i e t y from Madagascar. Contrib. Inst. Bot. Montreal 64: 3-19. Chardard, R. 1964 Etude de 1 'ultrastructure de deux algues Zygophycees: Mesotaenlum oaldarlorum et Penium  margaritaceum. Rev. Cytol. B i o l . Veget. 2£: 77-93. Chardard, R. I 9 6 5 Nouvelles observations sur 1 *infrastructure de deux Algues Desmldlees: Cosmarlum l u n d e l l l l et Closterlum acerosum. Rev. Cytol. B i o l . Vege't. 28: 15-30. Chardard, R and R o u i l l e r , C. 1957 L f u l t r a s t r u c t u r e de t r o l s Algues Desmldlees. Etude au microscope ellectronlque. Rev. Cytol. B i o l . Veget. 18: I 5 3 - I 7 8 . Drawert, H. and Metzner-Kuster, I. I96I L i c h t - und elektronenmikroskopische Untersuchungen an Desmidiaceen. I. Zellwand- und Gallertstrukturen bel elnigen Arten. Planta $6: 213-228. 161 Drawert, H. and Mix, M. 1961a L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. I I . H u l l g a l l e r t e und Schleimbildung bei Mlcroasterlas. Pleurotaenlum und Hyalotheca. Planta ^ 6 : 237-261. Drawert, H. and Mix, M. 1961b L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. I I I . Der Nucleolus im Interphasenkern von Mlerasterlas r o t a t a. Flora 150: 185-190. Drawert, H. and Mix, M. 1961c L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. IV. Beitrage zur elektronenmikroskopischen Struktur des Interphasenkernes von Mlerasterlas r o t a t a. Z. Naturforsch. 16b: 546-551. Drawert, H. and Mix, M. I 96 ld L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. V. Uber die Va r i a b i l i t ' a t der Chloroplastenstruktur bei Mlerasterlas r o t a t a . Planta jj>6: 648-665. Drawert, H.' and Mix, M. I96le Der Einfluss von An t i b i o t i k a auf die lichtmikroskoplsche und elektronenmikroskopische Struktur von Mlerasterlas. Ber. deutsch. bot. Ges. 2*ts ( ^ 6 ) - ( 4 7 ) . Drawert, H. and Mix, M. I 9 6 l f L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. VI. Der Einfluss von A n t i b i o t i k a auf die Chloroplastenstruktur bei Mlerasterlas r o t a t a . Planta 51-70. Drawert, Hv and Mix, M. I96lg L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. VII. Der Golgi-Apparat von Mlcrasterias rotata nach Pixierung mit Kaliumpermanganat und Osmiumtetroxyd. Mikroskopie 16: 207-212. Drawert, H. and Mix, M. 196lh L i c h t - und elektronen-mikroskopische Untersuchungen an 5-Desmidiaceen. V I I I . Die Chondriosomen von Mlcrasterias r o t a t a . Flora l g l : 4-87-508. Drawert, H. and Mix, M. 1962a L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. IX. Die Struktur der Pyrenoide von Mlcrasterias r o t a t a . Planta $8: 50-74-. Drawert, H. and Mix, Mi 1962b Zur Frage der Identitat von Karyoiden und Golgi-Apparat bei den Conjugaten. Naturwissenschaften 4j£: 353-354-. Drawert, H. and Mix, M. 1962c L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. X. Beitrage zur Kenntnls der "Hautung" von Desmidiaceen. Arch. Mikroblol. 4£: 96-109. Drawert, Hi and Mix, M. I 9 6 3 L i c h t - und elektronen-mikroskopische Untersuchungen an Desmidiaceen. XI. Die Struktur von Nucleolus und Golgi-Apparat bel Mlcrasterias dentlculata Breb. Portugaliae Acta B i o l i , Ser. A, 2 s 17-28. Godward, M . B . E . 1954 The d i f f u s e c e n t r o m e r e o r p o l y c e n t r i c chromosomes i n S p l r o g y r a . A n n . B o t . , n . s . , 18: 143-156. Godward, M . B . E . I 9 6 5 P r o b l e m s o f n u c l e a r d i v i s i o n w i t h s p e c i a l r e f e r e n c e t o t h e a l g a e . In J . D . C a r t h y and C L . D u d d i n g t o n , e d . ! , V i e w p o i n t s i n B i o l o g y 4: 29-53. Godward, M . B . E . I966 C h l o r o p h y c e a e . In M . B . E . Godward, e d . , The Chromosomes o f the A l g a e . L o n d o n , Edward A r n o l d L t d . Godward, M . B . E . and J o r d a n , E . G . I 9 6 5 E l e c t r o n m i c r o s c o p y o f the n u c l e u s o f S p l r o g y r a b r l t a n n l c a and S p l r o g y r a e l l i p s o s p o r a . J . R o y . M i c r . S o c . 84: 347-360. H a u p t f l e i s c h , P . 1888 Z e l l m e m b r a n und H u l l g a l l e r t e d e r D e s m i d i a c e e n . ' M i t t , n a t u r w i s s . V e r . Neuvorpommern Riigen 20: 59-136. Heimans, J . 1935 Das Genus C o s m o c l a d l u m . P f l a n z e n -f o r s c h u n g 181 1-132. Hoshaw, R.W. and Waer, R . D . I967 P o l y c e n t r i c chromosomes i n S l r o g o n l u m melanosporum. C a n . J . B o t . 45: 1169-1171. K a m i y a , T . I 9 6 I E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s on t h e c e l l s o f C o n j u g a t a e . B u l l . A i c h i G a k u g e i U n i v . , n a t . s c i . , 1(): 39-54. K a m i y a , T . I962 E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s on t h e c e l l s o f Conjugatae ( s u p p l e m e n t a r y r e p o r t ) . B u l l . A l c h i G a k u g e i U n i v . , n a t . s c i . , 11: 43-59. King, G.C. i960 The cytology of desmids: the chromosomes. New Phytol. 65-72. Kossinskaya, E.K. i960 Flora Plantarum Cryptogamarum UHSS. Vol. V. Conjugatae ( I I ) . Moscow, Acad. S c i . Krieger, W. 1937 Die Desmidiaceen Europas mit Beriick-slchtigung der aussereuropaisehen Arten. In L. Eabenhorst's Kryptogamenflora 1^  (!)• Leipzig, Akad. Verlag M.B.H. Ledbetter, M.C. and Porter, K.H. I 9 6 3 A "microtubule? i n plant c e l l f i n e structure. J . C e l l B i o l . 12: 239-250. Leyon, H. 1954 The structure of chloroplasts. I H f A study of pyrenoids. Exp. C e l l Res. 6: 497-505. Lutkemuller, J . 1902 Die Zellmembran der Desmidiaceen. B e i t r . B i o l . Pflanzen 8 : 347-414. Mix, M. 1966 L i c h t - und elektronenmikroskopische Untersuchungen an Demidiaceen. X I I . Zur Feinstruktur der Zellwande und M i k r o f i b r i l l e n einiger Desmidiaceen von Cosmarlum-Typ. Arch. Mikrobiol. ^5_: 116-133. Mix, M. I 9 6 8 Zur Feinstruktur der Zellwande i n der Gattung Penium (Desmidiaceae). Ber. deutsch. bot. Ges. 80: 715-721. Muhlethaler, K. I967 Ultrastructure and formation of plant c e l l w a l l s . Ann. Rev. Plant P h y s i o l . Ij8: 1-24. Reynolds, E.S. I 9 6 3 The use of lead c i t r a t e at high pH as an electron-opaque s t a i n i n electron microscopy. J . C e l l B i o l . 12: 208-212. Schroder, B. 1900 Cosmocladlum saxonlcum de Bary. Ber. deutsch. bot. Ges. 18: 15-23. Schroder, B. 1902 Untersuchungen uber Gallertbildungen der Algen. Verh. naturhlst.-med. Ver. Heidelberg 1'. 139 Starr, H.C. 1964 The culture c o l l e c t i o n of algae at Indiana University. Am. J . Bot. $ U 1013-1044. Waddington, CH. 1962 New Patterns i n Genetics and Development. New York, Columbia Univ. Press. West, W. and West, G.S. 1904 A Monograph of the B r i t i s h Desmidiaceae. V o l . I. London, Ray Society. West, W. and West, G.S. I 9 0 5 A Monograph of the B r i t i s h Desmidiaceae. Vol. 2. London, Ray Society. West, W. and West, G.S. 1908 A Monograph of the B r i t i s h Desmidiaceae. V o l . 3» London, Ray Society. West, W. and Wes;t, G.S. 1912 A Monograph of the B r i t i s h Desmidiaceae. Vol. 4. London, Ray Society. West, W;, West, G.S., and Carter, N. 1923 A Monograph of the B r i t i s h Desmidiaceae. Vol. 5* London, Ray Society. Wolle, F. 1881 American freshwater algae. B u l l . Torrey Bot. Club 8: 1-4. 

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