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University M icrofilms International
III! M /[(HMD A N N AHHUH MI-1H11H; 8207199
Hoops, Harold John, 111
ULTRASTRUCTURAL STUDIES OF SELECTED COLONIAL VOLVOCALEAN ALGAE
The Ohio State University Rh.D. 1981
University Microfilms International300 N. Zeeb Road. Ann Arbor. MI 48106 PLEASE NOTE:
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University Microfilms International ULTRASTRUCTURAL STUDIES OF
SELECTED COLONIAL VOLVOCALEAN ALGAE
DISSERTATION
Presented In Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy in the Graduate
School of The Ohio State University
by
Harold John Hoops I I I , B .S ., M.S.
* a * * *
The Ohio State University
1981
Reading Commitee: Approved By
Dr. Gary L. Floyd
Dr. Daniel J. Crawford
Dr. Michael L. Evans
Dr. Thomas N. Taylor f Advisor f Department of Botany To Debbie
ii ACKNOWLEDGMENTS
X would like to thank Gary L. Floyd, my major professor, for the support and encouragement he has given me during my tenure at
Ohio State, but even more for what he has taught me, both academically and otherw ise.
1 would also like to express my appreciation for the helpful
Input of Dr. Charles 0 "Kelly and the members of my committee,
Drs. Daniel J. Crawford, Michael L. Evans, and Thomas N. Taylor.
Thanks also to Carol Stuessy who gave of her time and talentB In preparing the drawings which are so valuable in communicating the findings of these studies to others.
I gratefully acknowledge my mother, who among other things taught me persistence, my father, who taught me the value of logic and the scientific method, Dr. Robert Mayer who gave me encouragement, and my wife Debbie, ;»ho has made my accomplishments p o ssib le . VITA
April 3, 1954 Born-New York, New York
1976 B.S., C arroll College, Wuakesha, Wisconsin
1976-1981 Teaching and Research Associate, The Ohio State U niversity, Columbus, Ohio
1978 M.S., The Ohio State University, Columbus, Ohio
1981 Fresdentlal Fellow, The Ohio State University
PUBLICATIONS
Hoops, H.J. and G.L. Floyd* 1979. U ltrastructure of the cen tric diatom, Cyclotella meneghinlana: Vegetative cell and auxospore development. Phycologia 18, 424-435.
Floyd, G.L., H.J. Hoops and J.A. Swanson. 1980. Fine structure of the zoospore of Ulothrlx belkae with emphasis on the flagellar apparatus. Protoplasma 104, 17-31•
Hoops, H .J., G.L. Floyd and J.A. Swanson. 1981. U ltrastructure of the biflagellate motile cells of Ulvaria oxysperma (Kutz)Blidlng and phylogenetic relationships among Ulvaphycean algae. Amer. J. Bot. (in press).
Hoops, H.J. and G.L. Floyd. Ultrastructure of the flagellar apparatus of Pyrobotrys (Chlorophyceae) . (submitted to J. Phycol.)
Hoops, H.J. and G.L. Floyd. U ltrastructure and the taxonomic position of the rare Volvocalean alga Chlorcorona bohemlca (Chlorophyceae). (submitted to J. Phycol.)
Hoops, H.J. and G.L.Floyd. Mitosis, cytokinesis, and colony formation in Astrephomene (Chlorophyceae, Volvocales). (submitted to Brit, phycol. J.)
iv ABSTRACTS
Hoops, H.J. and G.L. Floyd.1978. Preliminaryultra s tru c tu ra l observations on the development of the auxospore of the freshwater diatom Cyclotella meneghinlana. Ohio J. Sci. 78, 26.
Hoops, H.J. andG.L. Floyd.1978. Preliminaryultrastructural observations on the development of the auxospore of the freshwater centric diatom Cyclotella meneghinlana. J. Phycol 14(Suppl.), 41.
Floyd, G.L. and H.J. Hoops.1979. U ltrastructure of the zoospore and germling of Ulothrix belkae. J. Phycol. 15 (Suppl*), 23.
Hoops, H.J. andG.L. Floyd.1980. Motile cellultrastru c tu re of Ulvarla oxysperma (Chlorophy ta , Ulvales) and Its pylogenetlc sig n ifican ce. Ohio J. Sci. 80, 28.
Hoops, H .J., G.L. Floyd and D.F. Chappell. 1980. Tannic acid fixation reveals covering on the "wall less" green alga Asteromonas gracilis. J. Phycol. 16 (Suppl.), 20.
Floyd, G.L. and H.J. Hoops. 1980. Schizomerls lelbleinil revisited: Ultrastructure of the flagellar apparatus. J. Phycol. 16 (Suppl.), 20.
Hoops, H.J. and G.L. Floyd. 1981. The ultraB tructure of the fla g e lla r apparatus of two colonial Volvocalean algae, Uva and Chlorcorona. Ohio J. Sci. 81, 30.
Hoops, H.J. 1981. An u ltra s tru c tu ra l study of fla g e lla r apparatus development in Astrephomene gubernaculifera. J. Phycol. 17 (Suppl.), 3.
Hoops, H.J. and G.L. Floyd. 1981. UltraBtructure of the fla g e lla r apparatus of Pyrobotrys and Chlorcorona (Volvocales). J. Phycol 17 (Suppl.), 5.
V TABLE OF CONTENTS
DEDICATION...... ii
ACKNOWLEDGMENTS...... i l l
VITA...... iv
LIST OF FIGURES...... v ii
INTRODUCTION...... 1
CHAPTER 1. ULTRASTRUCTURE OF THE FLAGELLAR APPARTUS OF PYROBOTRYS...... 4
CHAPTER 2. ULTRASTRUCTURE AND TAXONOMIC POSITION OF THE RARE VOLVOCALEAN ALGA CHLORCORONA BOHEMICA...... 25
CHAPTER 3. ULTRASTRUCTURE OF THE FLAGELLAR APPARATUS AND FLAGELLAR MOTION IN ASTREPHOMENE GUBERNACULIFERA...... 35
CHAPTER 4. THE DEVELOPMENT OF THE FLAGELLAR APPARATUS IN ASTREPHOMENE...... 59
CHAPTER 5. MITOSIS, CYTOKINESIS AND COLONY FORMATION IN ASTREPHOMENE GUBERNACULIFERA...... 86
CHAPTER 6. OVERVIEW...... Ill
LIST OF REFERENCES...... 120 LIST OF FIGURES
Scanning electron micrograph (SEM) of Pyrobotrys caslnoensis ...... 17
SEM of stellata ...... 17
Papilla and flagellar insertion in P^. caslnoensls ...... 17
Suggested reconstruction of the Pyrobotrys flagellar apparatus ...... 16
Top view of Pyrobotrys flagellar apparatus, first section ...... 19
Top view of Pyrobotrys flagellar apparatus, second section ...... * ...... 19
Top view of Pyrobotrys flagellar apparatus, third section ...... 19
Top view of Pyrobotrys flagellar apparatus, fourth section...... 19
Top view of Pyrobotrys flagellar apparatus, fifth section ...... 19
Top view of Py robot rys flagellar apparatus, sixth section...... 19
Oblique top view of Pyrobotrys flagellar apparatus, first section...... * ...... 21
Oblique top view of Pyrobotrys flagellar apparatus, second section...... 21
Ventral rootlets of Pyrobotrys, first section. 21
Ventral rootlets of Pyrobotrys, second section 21
vii 15. Ventral rootlets of Pyrobotrys . third section ...... 21
16. Pyrobotrys flagellar apparatus sectioned at right angles to flagellum, first section .21
17. Pyrobotrys flagellar apparatus sectioned at right angles to flagellum, second section ...... 21
18. Side view of Pyrobotrys flagellar apparatus, first section ...... 23
19. Side view of Pyrobotrys flagellar apparatus, second section ...... 23
20. Pyrobotrys striated fiber cross section...... 23
21. Pyrobotrys striated fiber longitudinal section...... 23
22. Association of the smaller rootlet of Pyrobotrys with a SMAC, first section ...... 23
23. Association of the smaller rootlet of Pyrobotrys with a SMAC, second section ...... 23
24. Chlorcorona bohemlca colony ...... 32
25. Chlorcorona side view ...... 32
26. Squashed colony of Chlorcorona ...... * ...... 32
27. SEM of Chlorcorona ...... 32
28. SEM of individual cell of Chlorcorona.*...... 3 2
29. TEM of Chlorcorona c e ll ...... 33
30. Cell to cell connections in Pyrobotrys ...... 33
31. Cell to cell connections in Chlorcorona ...... 33
32. Flagellar apparatus of Chlorcorona ...... 33
33. Microtubular rootlets in Chlorcorona ...... 33
34. Diagramatic representation of the flagellar apparatus of the mature cell of Astrephomene ...... 4 9
35. Top view of Astrephomene flagellar apparatus, first section ...... 50
vi i i 36. Top view of Astrephoreene flagellar apparatus, second section ...... 30
37. Top view of Astrephomene flagellar apparatus, third section ...... 50
38. Lateral view of Astrephomene flagellar apparatus, fourth section ...... 50
39. Lateral view of Astrephomene flagellar apparatus, first section...... 52
40. Lateral view of Astrephomene flagellar apparatus, second section ...... 52
41. Lateral view of Astrephomene flagellar apparatus, third section ...... 52
42. Astrephomene; cross section of the accessory basal body associated with functional basal body 2...... 54
43. Striated fiber of Astrephomene...... 54
44. Astrephomene; strut near the vicinity of the basal body...... 54
45. Region proximal to the basal body in As t rephomene ...... 54
46. Basal body region of Astrephomene ...... 56
47. Astrephomene flagellar motion ...... 57
48. Single frame of Astrephomene flagellar motion from cine film ...... 58
49. Very early stage in flagellar apparatus development in Astrephomene , first section...... 72
50. Very early stage in flagellar apparatus development in Astrephomene, second section...... 72
51. Very early stage in flagellar apparatus development in Astrephomene , third section...... 72
52. Very early stage in flagellar apparatus development in Astrephomene , fourth section...... 72
1'X Very early stage in flagellar apparatus development in Astrephomene, fifth section..* 72
Very early stage in flagellar apparatus development in Astrephomene, sixth section... 72
Chlamydomonad-stage of flagellar apparatus development in Astrephomene. first section... 74
Chlamydomonad-stage of flagellar apparatus development in Astrephomene, second section.. 74
Chlamydomonad-stage of flagellar apparatus development in Astrephomene, third section... 74
Chlamydomonad-stage of flagellar apparatus development in Astrephomene, fourth section*. 74
Larger rootlet of the chlamydomonad-stage of Astrephomene ...... 74
Smaller rootlet of the chlamydomonad-stage of Astrephomene ...... 74
Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, first section...... 76
L ateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, second sectio n ...... 76
Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, third s e c tio n ...... 76
Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, fourth sectio n ...... 76
Reconstruction of the chlamydomonas—stage of fla g e lla r development in Astrephomene ...... 78
Developing flagellar apparatus of Astrephomene, first section ...... 79
Developing flagellar apparatus of Astrephomene, second section ...... 79
x 68* Developing flagellar apparatus of Astrephomene, third section ...... 79
69. Developing flagellar apparatus of Astrephomene, fourth section ...... 79
70. Developing flagellar apparaus of Astrephomene, fifth sectio n ...... 79
71. Developing flagellar apparatus of Astrephomene, sixth section ...... 79
72. Nearly mature flagellar apparatus of Astrephomene, first section ...... 81
73. Nearly mature flagellar apparatus of Astrephomene , second s e c t i o n ...... 81
74. Nearly mature flagellar apparatus of Astrephomene, third section ...... 81
73. Nearly mature flagellar apparatus of Astrephomene , fourth s e c t i o n ...... 81
76. Diagrammatic representation of flagellar apparatus development in Astrephomene ...... 83
77. Diagrammatic comparison of flagellarmotion in Chlamydomonas and Astrephom ene ...... 85
78. Vegetative u l t r a s t r u c t u r e of Astrephomene gubemacullfera...... 96
79. Section through the flagellar pit of Astrephomene ...... 96
80. Interphase nucleus of Astrephomene ...... 98
81. Centrioles of interphase cell in As t rephomene...... 98
82. Parental flagellum of Astrephomene ...... 98
83. Prophase nucleus of Astrephomene ...... 100
84. Intranuclear virus-like particles in Astrephomene...... * ...... 100
85. Late prophase nucleus in Astrephomene ...... 100
86. Metaphase spindle of Astrephomene ...... 102
xi 87. Spindle pole of Astrephomene ...... 102
88. Early anaphase In Astrephomene ...... 104
89. Kinetochore of anaphase chromosome of Astrephomene...... 104
90. First pair of polar centrloles of Astrephomene ...... 104
91. Second pair of polar centrloles of Astrephomene ...... 104
92. Late anaphase, early telophase In Astrephomene ...... 106
93. Telophase In Astrephomene...... 108
94. Phycoplast of Astrephomene ...... 108
95. Developing daughter colony of Astrephomene ...... 110
xi i INTRODUCTION
The order Volvocales includes members of the Chlorophyceae which remain motile for much of their life cycle. Unicellular members of the Volvocales are normally blflagellate, although quadriflagellate cells also occur (e.g.* Carterla, Polytomella) . These algae possess a wide v ariety of morphological forms. Many have been extensively studied ultrastructurally, and are often used as experimental organisms.
The colonial members of the order are normally blflagellate
( Spondylomorum, a rep o rted q u a d rif la g e lla te colonial form now appears to have been based on inaccurate observations of the blflagellate Pyrobotrys (Prlngshelm, 1969)). It is believed that members of this group are descended from unicellular members of the class* although some consider the colonial habit to have arisen more than once (Crow, 1918; F ritsch , 1935; Lang, 1963; Plckett-H eaps, 1975;
Ettl* 1976). The alga most often suggested as being like the ancestor of this group is Chlamydomonas (ibid), although Haematococcus is often mentioned as a possible ancestor to some forms as well (Crow,
1918; F ritsch , 1935; E ttl, 1976). In spite of the presumed common origin of the colonial forms, colony morphology is diverse in the different genera. It ranges from a flat plate with flagella on one
1 (Gonium) or both (Platydorlna) sides, to grapelike clusters of cells (Pyrobotrys, Pascherlella), to spherical colonies with the cells roughly parallel to one another (Stephanosphaera), or radially arranged (Pandor ina , Volvu1ina . Eudo r ina , Pleodorlna ,
Volvox, Astrephomene) • They may (Volvox , Pleodorlna,
Astrephomene), or may not (most other species) show marked cell differentiation• The development of many of these organisms, especially Volvox, (Ja n et, 1912; 1923; Pocock, 1933; Darden, 1966;
Kochert, 1968; S ta rr, 1968; 1969; 1970; McCracken and S tarr, 1970;
VandeBerg and S tarr, 1971; Karn, S tarr and Hudock, 1974; Kockert,
1981), but also Eudorlna (Goldstein, 1964), Volvullna (Pocock,
1953; Stein, 1958), and Astrephomene (Pocock, 1953; Stein, 1958) have been carefully followed by means of the light microscope* The features described above provide the opportunity to study the effects of colonial morphology on flagellar motion (determined by high speed flash clnephotomlcrography) and the structure of the flagellar apparatus (determined by careful analysis of serial micrographs)*
Such studies have the potential to determine aspects of certain structure-functlon relationships not easily documented by other means.
Although a complete comparison of all the genera in this group is not possible here, special emphasis is given to two genera of markedly different colonial types; Pyrobotrys, which has a colony reminiscent of a cluster of grapes, and Astrephomene, chosen to represent those
spherical colonies which have radially arranged cells* In each case
the flagellar apparatus is very different from that typical for the
Chlorophyceae, apparently in response to the constraints placed on 3 fla g e lla r and organlsmal motion by the morphology of the colony.
In addition, the Influence of the colonial development on the flagellar apparatus is examined in detail in Astrephomene. This type of analysis has the potential to correlate the morphogenetic events with evolutionary pathways (a subcellular ontogeny recapitulates phyiogeny sequence), as well as suggesting additional relationships between the structure of the flagellar apparatus and the way In which i t functions.
Thirdly, the structure of the flagellar apparatus of various genera In this group is used to suggest certain taxonomic and evolutionary relationships among these organisms. Specifically the flagellar apparatuses of Pyrobotrys, Chlorcorona and
Astrephomene are compared w ith each o ther, the p a rtia lly characterized flagellar apparatuses of Volvox (Olson and Kochert
1970; Pickett-Heaps, 1970; Deason and Darden, 1971; Birchem and
Kochert, 1979b) and Eudorlna (Hobbs, 1971), and especially the unicellular forms such as Chlamydomonas (Ringo, 1967; Moestrup,
1978; Katz and McLean, 1979; Goodenough and Weiss, 1979) Gloeomonas
(Schnepf, Delchgraber and E ttl, 1976), and Carterla (Lembl, 1975).
Lastly, mitosis, cytokinesis and colony formation are examined In
Astrephomene. M itosis and c y to k in e sis are compared with these processes In both unicellular and colonial green algae, and aspects of colony development are compared to those of other colonial Volvocales. CHAPTER 1
ULTRASTRUCTURE OF THE FLAGELLAR
APPARATUS OF PYROBOTRYS
Although the colonial members of the Volvocales are among the most extensively examined green algae, detailed ultrastructural studies, especially of the flagellar apparatus, are few In number.
This void Is critical In view of the phylogenetic importance placed on the detailed fine structure of the flagellar apparatus among many other green algae (Melkonlan, 1980a; Floyd, Hoops and Swanson, 1980;
Hoops, Floyd and Swanson, In press). U ltrastru ctu ral studies on a number of algae placed In the Volvocaceae and Astrephomenaceae (Lang,
1963; Deason, Darden and Ely, 1969; Pickett-Heaps, 1970; Olson and
Kochert, 1970; Deason and Darden, 1971; Hobbs, 1971; Blrchem and
Kochert, 1979b) have shown that there are some similarities in the
flagellar apparatus between some of these organisms and
Chlamydomonas, an organism widely perceived to be sim ilar to the probable ancestor of these groups.
The Spondylomoraceae, another family of colonial Volvocales has been used in nutrition and photosynthesis research (e.g., Wiesssner,
1965; Goulding and M errett, 1967; Thiede, 1976), but the flag ellar apparatus of no member of the family has been ultrastructurally
4 analyzed> Careful examination of a number of colonial Volvocales will lead to a better understanding of phylogenetic relationships betweeen the families, as well as increasing our knowledge of the relationship between form and mode of motion and the d e tails of the flagellar apparatus structure* With this in mind the study of the flagellar apparatus of members of the colonial Volvocales has been
Initiated with two species of Pyrobotrys.
MATERIALS & METHODS
Pyrobotrys castlnoensls (P layfair) Silva was found growing in a eutrophic pond on the campus of The Ohio State University during
August and September of 1980. It was fixed In IX glutaraldehyde in pond water for 7-10 min and transferred d irectly to IX glutaraldehyde
In 0.2M sodium cacodylate buffer at pH 7*2 for 1 hr at room temperature. After washing in buffer the sample was postflxed In 1% osmium tetroxide in buffer for 1 hr, washed in water, dehydrated in acetone and embedded In Spurrs resin between teflon coated glass slides. Colonies were selected, sectioned and picked up on formvar coated slot grids. After staining with uranyl acetate and lead citrate the serials were viewed with a Hitachi H300 transmission electron microscope (TEM). In addition, soil samples were collected near the pond and used to prepare soll-water cultures. Pyrobotrys
Btellata Korsikov, as well as _P. casinoensls, grew In some of the cultures that had been fortified with pea cotyledon. This alga was fixed in the same way as P. casinoensls except that supernatant from 6 soil-water medium was uBed In place of pond water.
Samples for the scanning electron microscope (SEM) were fixed and dehydrated in the same manner as the TEM samples, critical point dried and gold coated. They were viewed with a Hitachi S500 SEM.
RESULTS
Identity of the alga
The generic name of these algae has been questioned (Prlngsheim,
1960, 1969; Bourrelly, 1962; Fott, 1967; Dillard and DaPra, 1971).
Silva (1972) has shown, however, that the correct name is Pyrobotrys rather than Uva or ChlamydobotryB. Of the two species discussed in this paper, the first Is composed of 16 cells and resembles both
Pyrobotrys elongata Korsikov and JP. casinoensls. In light of the observation by Prlngsheim (1960) regarding the variability of the characteristics of this genus, placement in the latter taxon is appropriate. The Becond isolate, an eight celled specieB, was assigned to Pyrobotrys stellata (Korsikov)Korsikov.
Although both species of Pyrobotrys were present in the same general area, no colonies were found representing morphological lntergradation between the two. 7 Form of the colonies
Figures 1-3 show that the colonies of Pyrobotrys are composed of eight or sixteen cells arranged In alternating tiers of four cells each, with the long axis of the cells either parallel to one another
(the posterior three tiers of P. casinoensls) or with the flagellar ends of the cell convergent (the anterior tier of P. castlnoensis and both tiers of stellata). In the posterior tier of cells In
P. castlnoensis, connections are present between cells of the same tier (Fig. 1). Cell connections are also present between the cells of one tie r and cells of the other tie rs above or below (Figs. 1-3).
The flagella are inserted Into a small papilla at the cell apex, but they do not project forward, rather each pair extends laterally from the cell toward the outside of the colony (Fig. 3). This condition
Is unusual in the Chlorophyceae whose flagellar apparatus Is symmetrical (180 rotational symmetry as used by Floyd, et al. 1980) as well as being Inserted apically. To avoid confusion in the following discussion a terminology similar to that proposed for
Chlamydomonas (Ettl, 1976) is used to describe the position of structures within the cell. The long axis of the cell will have an anterior, flagellar and a posterior end. The dorsal side of the cell
Is designated as that side which is toward the inside of the colony, while the ventral side faces the outside. These terms describe the c e ll and not the fla g e lla r apparatus per se, which as discussed by
Ringo (1967), will have a distal direction (away from the base of the flagellum) and a proximal direction (towards the base of the 8 flagellum )■ As described below and In contrast to the well known case of Chlamydomonas, the d is ta l region of the flag ellar apparatus is not the most anterior In the cell.
Details of the flagellar apparatus
The flagellar apparatus of Pyrobotrys Is complex, and unique In those green algae studied to date. Figure A Is a diagrammatic drawing of the flagellar apparatus. Although It Is based on
P. casinoensls, there are no differences in the details of the flagellar apparatus of the two algae as judged from serial sectioning of both species. Therefore the figures shown In this paper are selected from either of the two species. As discussed in Floyd et al. (1980), the correct absolute orientation Is maintained in all figures and the diagram. The two basal bodies are arranged in a
V-shaped configuration (Figs. 5-11,17-19). Unlike the other
Chlorophyceae studied to date, the V is tilted so that the flagella exit the cell to the ventral side of the cell rather than directly forward from the apical papilla. In addition to the two basal bodies that extend into flagella, two accessory basal bodies are present
(Figs. 9,10). These accessory basal bodies are short (about 125 um) and tend to stain more lightly than the functional basal bodies.
They possess a basal cartwheel structure (not shown), but no tra n sitio n region. They possess the fu ll complement of microtubules.
The four rootlets that run toward the apical end of the cell alternate between four and two microtubules (Figs. 13-15). The four-membered rootlet is present In a three over one configuration at
the basal bodies (Fig. 13), but as the rootlet extends posteriorly
the mlcrotubule that was underneath the others moves to a position alongside the other three (Figs. 14,15). Each of the rootlets
alternate with the functional and accessory basal bodies
(Figs. 5-10). The two rootlets that are present on the ventral side
are relatively close to one another and to the ventral accessory
basal body. These rootlets pass to each side of the ventral
accessory body and attach to its base (Figs. 8-10). The rootlets are
straight until they Insert into the flagellar apparatus. Both
rootlets on the dorsal side are arched and further separated from
each other and from the accessory basal body (Figs. 6-10). Between
the two basal bodies that extend into flagella and the dorsal
accessory basal body is an amorphous, globular structure
(F igs. 7-9,18,19). This stru c tu re is composed of two regions on the
basis of differential stainability (Figs. 18,19). It appears
amorphous in every plane of section.
Also associated with the flagellar apparatus are various
striated and non-striated fibers. At the anterior-most region of
each cell, an apparently non-striated fiber crosses from one
functional basal body to the other (Fig. 5). This fiber terminates
at a dense bar that overlies each basal body (Fig. 5). In a
different plane this dense structure appears as a curved plate which
arches over part of the aforementioned fiber and covers the insertion
of the two microtubule rootlets from the dorsal side (Fig. 19).
Further ventrally, a striated fiber also connects the two functional 10 basal bodies with each other (this fiber is obliquely sectioned in
Fig. 5, but is more clearly seen in Figs. 11,16, and 18). This connecting fiber has numerous fine filaments running the length of the fiber and darker strlations running across the narrow dimension.
Although the curvature of this structure makes it difficult to get good banding appearance in a single section, it is apparent that three broader bands are present, equally spaced along the fiber* In addition there is a dark, narrow band on the basal body side of the broader bands (Fig. 18).
A shorter fiber also connects the two functional basal bodies toward the posterior side of the basal bodies. This fiber is located close to, but under the ventral mlcrotubule rootlets (Fig. 7).
In addition to the fibers associated only with the basal bodies, there are other fibers associated with the rootlets* One, a large, distinctly striated structure, is associated with the larger rootlet near its insertion into the basal body complex. This fiber is best seen near the rootlet which comes from the dorsal side of the cell
(Figs. 7,8,20,21). It attaches near one of the functional basal bodies, and abuts both the amorphous m aterial around the dorsal accessory basal body and the accessory basal body Itself
(Figs. 7,20,21). It is characterized by numerous fine filaments running in the direction of the fiber with four broad strlations perpendicular to its long axis. The fiber runs along the larger rootlet, extends posteriorly for a short distance on the underside of the rootlet and appears "flag-like" in cross section (Figs* 10,13).
Due to the asymmetry of the flag ellar apparatus, the fiber associated 11 with the opposite major rootlet is much more difficult to precisely delimit, but it is seen on the posterior side of the basal body in appropriate serial sections (Figs. 11,12). Posterior to the end of the flag-like portion of these fibers, there is electron dense material around the rootlet that is limited in size and does not appear striated in any plane of section (Fig* 14). In contrast to the large rootlets, the attachment of the two membered rootlets to the basal body complex is not striated (Fig. 7). However, there is a periodicity of about 30 nm associated with the material that surrounds these rootlets (Fig. 22). This material matches the description of a SMAC as defined in Floyd et al. (I960).
Descending from each of the functional basal bodies is a structure with the general appearance of a very small rhlzoplast
(system II fiber, Melkonian, 1980a) (Fig. 17). Although the rh iz o p last-lik e structure does not have well defined cross periodicities and thus does not completely fulfill the requirements of a rhlzoplast as suggested by Floyd et al. (1980), the position of the structure and its composition of fine longitudinal fibers make it lik ely that it is a homologous stru ctu re. Fixation has been shown to have a major effect on the appearance of undoubted rhizoplasts
(Salisbury and Floyd, 1978). It is not known whether the very fine appearance of the rhizoplast-like structure in Pyrobotrys is due to fixation artifact, but it is notable that this structure did not appear prominant in any of the different fixations attempted with this organism (not shown). 12 DISCUSSION
The ultrastructure of the flagellar apparatus of green algae has been shown to be of major phylogenetic interest (Moestrup, 1978;
Stewart and Mattox, 1978; Melkonian, 1980a; Hoops et a l. in press).
As emphasized by Moestrup (1978), the phylogenetic value of the variability in flagellar apparatuses is based on the presence and arrangement of various compoments such as rootlets and fibers, and not on the flagellar axoneme itself* All three classes of the green algae sensu Stewart and Mattox (1978) can be recognized on the basis of fine structural details of the flagellar apparatus (Melkonian,
1979,1980a; Slulman, Roberts, Stewart and Mattox, 1980; Roberts,
Slulman, Stewart and Mattox, 1980; Hoops et al* in press). For this reason an analysis of the flagellar apparatuses of several algae of various positions within the colonial Volvocales has been Initiated.
Both the Ulvaphyceae and the Chlorophyceae are characterized by the apical insertion of flagella* However, the motile cells of the
Charophyceae, like the flagellated cells of land plants, are characterized by subaplcal or lateral flagellar attachment. The majority of algae belonging to the Chlorophyceae and Ulvaphyceae are also characterized by having a flagellar apparatus that is
"symmetrical". The type of symmetry displayed in the Chlorophyceae and Ulvaphyceae is described by the concept of 180 rotational symmetry (and not by the term mirror image; Floyd et al 1980). In contrast, the flagellar apparatus of the Charophyceae 1 b asymmetrical. 13 At first glance the situation in Pyrobotrys appears ambiguous.
£j The flagellar apparatus does not show 180 rotational symmetry, and
the flagella project out of the cell in a manner that might be called lateral. Unlike the Charophyceae however, the flagellar apparatus of
Pyrobotrys is apical and does not contain the multilayered structure
(MLS) characteristic of that class. In features other than symmetry and direction of flagellar insertion, the flagellar apparatus of
Pyrobotrys shows an unmistakable affinity for the Chlorophyceae. The presence of four rootlets that alternate between two and four microtubules each, is a common situation in the Chlorophyceae, although it has been reported in the Ulvaphyceae as well (Moestrup,
1978; Melkonian, 1980a; Slulman et al. 1980; Hoops et al. in press).
In addition, the striated fiber connecting the two functional basal bodies is common in the Chlorophyceae, but absent in the
Charophyceae. Although the flagella project to the side of each cell, they are Inserted into an apical papilla rather than subapically as in the Charophycean motile cell. To describe the flagellar insertion as lateral would induce confusion with the situation as in the Charophyceae, therefore the term tangential might be appropiately used for this configuration.
It has been suggested that Pyrobotrys along with the other members of the Spondylomoraceae evolved from algae that resembled the present day Chlatnydomonas ( E ttl, 1976). Chlamydomonas was o rig in ally reported to have four rootlets all with four microtubules each
(Rlngo, 1967), but it has more recently been shown that Chamydomonas, like Pyrobotrys, has alternating two and four membered rootlets 14 (Moestrup, 1978; Goodenough and Weiss, 1978; Katz and McLean, 1979).
This by itself is not sufficient evidence for a close relationship, as members of both the Chlorophyceae and Ulvaphyceae have a similar arrangement of microtubule rootlets. However the large striated
fiber that connects the two functional basal bodies of Pyrobotrys is
similar to the distal fiber of Chlamydomona b , and to other algae of
the "chlamydomonad-type" (Melkonian, 1980a). The possibility of a relationship is further strengthened by the ultrastructure of the wall of Pyrobotrys, which like that of the closely related
Chlorcorona, is similar to the wall of certain species of
Chlamydomonas and related algae (Chapter 2)• The striated fibers associated with the larger rootlets have an appearance similar to certain sections of the proximal fibers of Chlamydomonas. However
the striated proximal fibers in Chlamydomonas do not appear to be
associated with the rootlet and travel from the proximal region of
one basal body to the proximal area of the second basal body (Rlngo,
1967; Goodenough and Weiss, 1978). Thus, the fibers in Pyrobotrys may be d ifferen t from the proximal fibers in Chlamydomonas. They
resemble striated fibers associated with the larger rootlets of
U lothrlx (Floyd et a l. 1980), Cylindrocaspa (Hoffman, 1976) and
Schlzomerls (Floyd and Hoops, in prep). Taken together these facts
suggest that although there is a relationship between Chlamydomonas and Pyrobotrys, there are also differences, and Pyrobotrys cannot be
thought of as simply a colonial Chlamydomonas.
The type of arrangement of flag ellar components in Pyrobotrys
is unusual for the Chlorophyceae. Unlike some of the praslnophytes 15 and the Charophyceae, the flagellar apparatus has the general appearance of being balanced, that la, although the flagellar apparatus does not show 180* rotational symmetry, many of the structures present on one side of the flagellar apparatus are present on the other side as well, even If their position and appearance are altered• Although the rootlets are not evenly arranged around the basal bodies, they do alternate between similar sized rootlets* In addition, the large striated fibers are associated with the two larger rootlets and with both functional basal bodies* Further, the large non-striated fiber on the anterior side of the basal bodies Is mirrored by a smaller connecting fiber further posteriorly. This o would suggest that the lack of 180 rotational symmetry in
Pyrobotrys is derived from a more symetrlcal ancestor. o If we assume that the ancestral cell type possesed 180 rotational symmetry and had flagella that extend forward from the apical papilla, then perhaps the following evolutionary mechanism might be used to explain the structure of the flagellar apparatus of
Pyrobotrys. The placement of the distal fiber indicates that the flagellar apparatus has been "tipped over" on its side so that it now faces towards the outside of the colony and is nearly at right angles to the cell axis. The placement of the four rootlets indicates that these structures are pulled in that direction also, although the alteration in position is not as pronounced. The non-striated fibers on each side of the distal fiber appear to be in the same relative position as the two proximal fibers of Chlamydomonas. If this interpretation is correct, then the extended length of the anterior 16 most fiber could indicate that, in addition to a change in the
orientation of the flagellar apparatus itself, each of the two basal
bodies has rotated in opposite directions. Since isolated axonemes
of Chlamydomonas can beat with a polarized beat (Bessen, Fay and
Wltman, 1980), the flagellar axoneme may have a structurally
determined plane of beat. Accordingly, the rotation of each of the
basal bodies would allow an effective stroke in a different
direction. Although cinephotomlcrographlc analysis of these cells is
difficult, there is some suggestion that the flagellar beat is down
and along the side of the cell (not shown), which is consistent with
the direction of presumed rotation. It is not difficult to imagine
the selection pressure that would result in this type of modification
of the flagellar apparatus. Since the cells of Pyrobotrys are
parallel or convergent at their anterior ends, a typical
Chlamydomonas-like breaststroke would not work. Either the flagella
of neighboring cells would become tangled or one of the flagella from
each of the cells would project to the inside of the colony. This
suggests that the unusual structure of the flagellar apparatus is
associated with the mode of colony formation In this organism.
Further it implies that the modification of the flagellar apparatus
and the development of the colonial habit are related events that may have evolved together. 17
Fig. 1. Scanning electron micrograph (SEM) of Pyrobotrys caslnoensis. The cells are either parallel to one another or , as in the top tier, the anterior ends of the cells are slightly convergent. Scale in microns.
Fig. 2. SEM of P. stellata. The cells are arranged with their flagellar ends convergent. Scale in microns.
Fig. 3. Papilla and flagellar insertion in j>. caslnoensis. The flagella are located at the apex of each cell and extend outward. Scale In microns. 18
AM
Fig. 4. Suggested reconstruction of the Pyrobotrys flagellar apparatus. The two basal bodies that extend into flagella, and the dorsal accessory basal body (DBB) Insert into amorphous material (AM). A s tr ia te d fib e r (DF) connects the two fu n c tio n a l b asal bodies distally. Two non-striated fibers (NF) also connect the functional basal bodies. The four membered rootlets (R4) are associated with a second striated fiber (SF). A striated microtubule-associated component (SMAC) is associated with each two membered rootlet (R2). A rhizoplast-like structure (RLS) descends from each functional basal body. Note the ventral accessory basal body (VBB) and the "flag-like" extensions from each R4. Fig. 5. Top view of Pyrobotrys flagellar apparatus, first section. The anterior-most non-striated fiber extends from over one basal body to over the second.
Fig. 6. Top view of Pyrobotrys flagellar apparatus, second section. A s t r i a t e d fib e r (DF) extends between the two b asal b o dies, and both the dorsal rootlets (R2, R4) insert at this level*
Fig. 7, Top view of Pyrobotrys flagellar apparatus, third section. The proximal ends of both functiona1 flagella insert into amorphous m a te ria l (AM). The p o s te rio r n o n -s tria te d fib e r (NF) is also v i s i b l e .
Fig. 8. Top view of Pyrobotrys flagellar apparatus, fourth section. All four rootlets (R2, R4) descend from the flagellar apparatus.
Fig. 9. Top view of Pyrobotrys flagellar apparatus, fifth section. Electron dense connections are present between the rootlets and the basal bodies* Also note the dorsal acessory basal body (DBB).
Fig. 10. Top view of Pyrobotrys flagellar apparatus, sixth section. Note the flag-like extension descending from the dorsal 4 membered rootlet (arrow), and both the ventral (VBB) and dorsal (ABB) accessory basal bodies. Scale “ 0.25pm for Figs. 5-10.
19 20 Fig. 11. Oblique top view of Pyrobotrys flagellar apparatus, first section. Note the striated fiber (OF).
Fig. 12. Oblique top view of Pyrobotrys flagellar apparatus, second section. Note the ventral striated fiber (SF) associated with the four membered rootlet (R4). Figs. 11 and 12 are from a serial so orientation can be obtained by comparison of the two. Scale “ 0.25 pm for Figs. 11,12.
Fig. 13. Ventral rootlets of Pyrobotrys, first section. This section Is taken from an area near the flagellar apparatus. Note the alternation between the four membered rootlet, here present In the 3 over 1 configuration and the 2 membered rootlet. Also note the electron dense material about both rootlets.
Fig. 14. Ventral rootlets of Pyrobotrys, second section. This section Is posterior to the one above. The bottom microtubule in the larger rootlet is now under the outermost microtubule in the 3 membered row.
Fig. 15. Ventral roolets of Pyrobotrys, third section. At this level, which is posterior to above Figs. 13 and 14, the four microtubules of the larger rootlet are arranged in a row. Also note that there is less electron dense material associated with this rootlet. Scale “ 0.25 pm for Figs. 13-15.
Fig. 16. Pyrobotrys flagellar apparatus sectioned at right angles to flagellum, first section. A rhizoplast-like structure (RLS) descends from one of the functional basal bodies.
Fig. 17. Pyrobotrys flagellar apparatus sectioned at right angles to flagellum, second section. The dorsal rootlets insert above the functional basal bodies and in association with the anterior non-striated fiber (arrow). Scale “ 0.25 um for Figs. 16,17.
21 22 Fig* 18. Side view of Pyrobotrys flagellar apparatus, first section. The functional basal bodies are connected by a striated fiber (DF).
Fig. 19. Side view of Pyrobotrys flagellar apparatus second section. This section, which is a serial to the previous figure, shows the anterior non-striated fiber CNF) covering the rootlets. Scale * 0.25 Um for Figs. 18,19.
Fig. 20. Pyrobotrys striated fiber, cross section. The striated fiber (SF) is associated with the 3 over 1 rootlet. Scale ■ 0.25 un-
Fig. 21. Pyrobotrys striated fiber, longitudinal section. This fiber (SF) descends from the rootlet and runs along the amorphous material that is in association with the functional basal bodies. Scale - 0.25 um.
Fig. 22. Association of the smaller rootlet with a SMAC, first section. The two membered rootlet (R2) Inserts over the basal body in association with electron dense material.
Fig.23. Association of the smaller rootlet with a SMAC, second section. The SMAC is associated with a mlcrotuble rootlet more clearly seen in the previous figure. Scale ~ 0.25 um for Figs. 22,23).
23
CHAPTER 2
ULTRASTRUCTURE AND TAXONOMIC POSITION OF THE RARE
VOLVOCALEAN ALGA, CHLORCORONA BOHEMICA
Chlorcorona Is an unusual green alga of uncertain taxonomic affinity. It was originally described from a pond In Czechoslovakia and named Corone bohemlca (Fott, 1950). He placed this organism in its own family, the Coronaceae. After discovering that the generic name was occupied by a f o s s i l diatom , the alga was renamed
Chlorcorona bohemlca (Fott) Fott (1967). The alga has apparently not been reported since. Taxonomically, this alga has also been placed in the family Spondylomoraceae (Bourrelly, 1972), and has alternately been thought to have some affinities with the Volvocaceae (Starr,
1980). The recent rediscovery of Chlorcorona gave an opportunity for an ultrastructural study to determine its structural features and taxonomic affinities.
MATERIALS AND METHODS
Chlorcorona bohemlca was collected from a small pond on the campuB of The Ohio State University in Columbus, Ohio. The pond was covered with a thick mat of Lemna (duckweed). Sparse populations of 26 the alga were located in shallow water near the shore and were found
only on sunny days during the fall of 1980. The colonies of
Chlorcorona were outnumbered by a large variety of euglenoids as well
as the more abundant Pyrobotrys■ The samples were fixed and embedded
as described in Chapter 1. Individual colonies were selected,
remounted and sectioned. In most cases the cells were stained with
uranyl acetate and lead citrate, however, some sections were stained
with potassium permanganate followed by lead citrate (Bray and
Wagenaar, 1978) to increase contrast.
RESULTS
The colonies of Chlorcorona invariably consist of eight cellB.
The cells are arranged in two parallel layers of four cells each
(Figs. 24,25). The four cells that comprise each of the parallel
layers are arrranged in a rhombodlal configuration (Fig. 24). In
side view the cells in one layer alternate with those in the second
layer (Figs. 25,27). No surrounding matrix is present (Figs* 24-28).
Each cell is oval with the protoplast pulled back from the cell wall,
especially at the posterior end. Each cell has a cup-shaped
chloroplast (Fig. 25) with an eyespot (not shown) and la biflagellate without a papilla (Fig. 28). On examination with the light
microscope there appears to be an ill-defined pyrenold in the
chloroplast, but analysis of electron micrographs did not reveal one.
The apparent pyrenold seen under the light microscope is probably an
accumulation of starch which does occur in the chloroplast (Fig. 29). 27 Cells of the colony are held together by elongated, clear connections between adjacent cells In a layer and between cells of each layer (Figs* 24-27). These connections are firm and do not bend as the alga swims* When the colony Is flattened under a coversllp the nature of the connections Is clearly seen (Fig. 26). Each connection has a lin e running across i t at a spot midway between the cells. When the connections break they do so at this line (Fig. 26).
Under the transmission electron microscope (TEM) these connections are outgrowths of the cell wall (Fig. 27). The ultrastructure of the connections is identical to that of the wall in other places. The wall In either area Is composed of two regions
(Figs. 29,31). The outermost region consists of a thin bounding layer on the outside and a fine periodic component abutting this layer on the Inside. This periodic component presumably represents the crystalline material that is present In a variety of Volvocalean algae (Roberts, 1974). The Inner region of the cell wall is a fine fibrillar matrix located in a layer appressed to the outer region. A variable amount of course granular to fibrillar material is located between the Inner wall region and the plasmalemma (not extensive in the pictures shown here, but some can be seen in Fig. 29). A similar type of wall construction is also present in Pyrobotrys casinoensis
(Playfair) Silva (Fig. 30), included here for comparison. In this case the protoplast is more closely appressed to the cell wall, but the same two wall regions are present, as is the material between the wall and the cell membrane. 28 In Chlorcorona. the lines present in the connections as observed by light microscopy correspond to the junction of the individual cell walls (Figs. 29,31). The wall is not greatly modified in the area of the Junction (Figs* 29,31). The outer layers of the wall are not In direct contact at the junction. There is, however, a very fine fibrillar component that extends from cell to cell at this point
(Fig. 31). The cell wall, and especially the cell to cell connections of P. casinoensis. are not generally elongated as in
Chlorcorona. However, the walls form short extensions at the point of contact (Fig. 31). In addition, the fibrous component connecting the cells is similar to that of Chlorcorona (cf., Figs. 30,31).
Flagella exit the apex of each cell, but extend outwards, away from the colony (Figs. 27,28). Although the cell wall lacks a papilla, the flagellar apparatus is located in an elevation of the protoplast, that is created by the slight withdrawal of the protoplast from the wall in the region surrounding the flagellar apparatus. The flagellar appparatus is very similar to that observed for Pyrobotrys, therefore, a complete account of the details of
Chlorcorona need not be presented. However, to substantiate the s im ila r ity of the two f la g e lla r ap p aratu ses, a summary is p resen ted .
Flagella are Inserted into the cell in the typical V-shaped configuration (Fig. 28). The two basal bodies are connected by a striated fiber that Is located in the middle of the V, thus to the side of the cell towards the outside of the colony and not towards the anterior of the cell (Fig. 32). In addition, two non-striated fibers connect the basal bodies at a more proximal level (not shown). 29 The two functional basal bodies end In amorphous appearing material
(Figs. 29,32). Two accessory basal bodies are also present, one of which is associated with the amorphous material (not shown)• The
four rootlets are arranged with the two rootlets between the
functional basal bodies nearly parallel (Fig. 32), while the other
two are more widely divergent. The rootlets alternate between four and two microtubules (Fig. 32). Each four membered rootlet Is present In the 3 over 1 configuration near the flagellar apparatus
(Fig. 32). There Is a striated component associated with the larger two rootlets (not shown). Unlike Pyrobotrys a rhlzoplast-like structure was not observed. In view of the reduced nature of the apparent rhlzoplast In Pyrobotrys, the apparent absence of a similar structure could be a result of oblique sectioning. This is supported by the fact that in some cells of Pyrobotrys, serial sections did not clearly reveal thiB structure.
DISCUSSION
Chlorcorona bohemlca has been of uncertain taxomomic affinities
(Fott, 1950; Bourrelly, 1972; Starr, 1980; Silva, In press). This condition has probably been maintained because of the rarity of the alga, as well as Its unusual colony construction* Therefore, we utilized the opportunity provided by Its local occurrence to attempt to Increase the information about the morphology and taxonomy of this organism . The morphology of this alga corresponds closely with that reported by Fott (1950). The presence of connections between cells, as well as the structure of the colony, were the primary features used to establish the new family* As demonstrated here, the structure of the connections is unlike those of the Volvocaceae
(Blsalputra and Stein, 1966; Ikushlma and Maruyama, 1968;
Plckett-Heaps, 1970; Merchant, 1977; Blrchem and Kochert, 1979), but similar to Pyrobotrys. In both Chlorcorona and Pyrobotrys the connections consist of unspeclallzed outgrowths of the wall, connected to corresponding structures from the adjacent cell by a fine fibrous component* In addition, the components of the wall itself seem to be identical in the two algae. Thus the key difference between the connections of Pyrobotrys and Chlorcorona is in length. Although Fott considered the connections in Chlorcorona to be nearly the same length In all colonies, some variation was found in our sample. There was also some variation in the length of the connections in P. caslnoensls. Additionally, at least one species of Pyrobotrys (P. lncurva Arnoldi; see Bourrelly, 1972) has connections that are elongated, although not to the extent of
Chlorcorona.
The flagellar apparatus is also very similar in these two algae
In each alga, flagella extend from the apex of the cell, but to the side. This arrangement of flagella is associated with an unusual arrangement of mlcrotubule rootlets and striated fibers, and is o strikingly different from the 180 rotational symmetry that is characteristic of the flagellar apparatus of most members of the Chlorophyceae (Floyd et al. 1980). All structures present In the
flagellar apparatus of Pyrobotrys, with the possible exception of the
rhlzoplast-llke structure, appear to be present In Chlorcorona,
including a striated connecting fiber, striated fibers associated with the four membered microtubule rootlets, two non-strlated
connecting fibers, two accessory basal bodies and the amorphous material Into which one of the accessory basal bodies and two of the
functional basal bodies Insert.
These features suggest that Chlorcorona Is closely related to
Pyrobotrys. Although recognized species of Pyrobotrys may have eight cells, the cell connections are never as long as they are in
Chlorcorona. In addition, no species of Pyrobotrys has the cell
arrangement present in Chlorcorona. Although Chlorcorona should be
retained as a genus, the taxon should be positioned in the
Spondylomoraceae along with Pyrobotrys. 32
Fig. 24. Chlorcorona bohemlca colony. In this light microscopic view the cells are arranged in alternating rhomboids, (x 500).
Fig. 25. Chlorcorona side view. The anterior ends of the cells in a colony all face the same direction, (x 650).
Fig. 26. Squashed colony of Chlorcorona. Note the connections between cells. These connections break at the line in the middle of the connection (arrow). (x 525).
Fig. 27. SEM of Chlorcorona. Although the flagella have broken during preparation, the flagellar stubs in each cell point away from the colony, (x 3,500).
Fig. 28. SEM of individual cell of Chlorcorona. Note that the flagella extend to the same side of the cell, (x 12,600). Fig. 29. TEM of Chlorcorona cell. The cell to cell connections are outgrowths of the cell wall. Note the location of the flagellar apparatus (FA); CV - contractile vacuole, CH ■ chloroplast, N “ nucleus, (x 19,700).
Fig. 30. Cell to cell connections in Pyrobotrys. Note that these connections are outgrowths of undifferentiated wall material connected by a fibrous component (arrow), (x 38,800).
Fig. 31. Cell to cell connections in Chlorcorona. Note that these connections are very similar to those of Pyrobotrys, except that the wall outgrowths are longer. (x 38,800).
Fig. 32. Flagellar apparatus of Chlorcorona. The two functional flagella insert into amorphous material (AM), and are connected by a striated fiber (DF). (x 81,000).
Fig. 33. Microtubule rootlets in Chlorcorona. Ventral rootlets are nearly parallel and alternate between 3 over 1 and 2 microtubules. (x 81,000).
33
CHAPTER 3
ULTRASTRUCTURE OF THE FLAGELLAR APPARATUS AND
FLAGELLAR MOTION IN ASTREPHOMENE GUBERNACULIFERA
The colonial Volvocales are a diverse group of organisms demonstrating considerable variation of colonial morphology. They are regarded as evolutionarily derived from unicellular Volvocalean
representatives (Crow, 1918; Frltsch, 1935; Lang, 1963;
Pickett-Heaps, 1975a; E ttl, 1976), such as Chlamydomonas, for which the structure of the flagellar apparatus and many aspects of
flagellar motion have been described (Ringo, 1967; Hyams and Borisy,
1975; 1978; Schmidt and Eckert, 1976; Moestrup, 1978; Goodenough and
Weiss, 1978; Katz and McLean, 1979). The transition from unicellular to colonial habit Imposed a new set of opportunities and constraints on flagellar motion which is presumably reflected in the construction of the flagellar apparatus. For example, a spherical colony with radially arranged cells could not move efficiently, if all of the cells composing the colony used the breaststroke-like motion characteristic of Chlamydomonas. It has long been noted that the
flagella of cells in this type of colony are Inserted into the cell separately, and not together in the V-shaped arrangement of
Chlamydomonas. In this chapter the ultrastructure of the flagellar
35 36 apparatus of the colonial green alga Astrephomene gubernacullfera will be described and related to the structure of flagellar motion as determined by flash clnephotomiorography. The results will be
Interpreted with respect to the form of the colony and colonial m otion.
MATERIALS AND METHODS
Astrephomene gubernacullfera Pocock (LB 1068) was obtained from the Culture Collection of Algae at the University of Texas.
Non-axenic stocks were maintained in sol1-water medium to which pea cotyledon had been added (Stein, 1958). These cultures grew slowly, but contained large colonies with both the somatic rudder cells as well as vegetative cells. Axenic cultures were obtained by the spray plate method, grown in Modified Volvocalean Medium (Brooks, 1966) and transferred every 2 to 3 weeks. The axenic cultures grew rapidly, resulting in smaller cells and colonies. The rudder cells did not differentiate In the young colonies from these cultures. Axenic cultures 1 to 3 days old or non-axenic cultures 2 weeks old were fix ed in IX g lu tarald eh y d e In c u ltu re medium for 10-15 min and transferred directly to IX glutaraldehyde in 0.1 to 0.2 M sodium calcodylate, pH 7.2 for about two hours. After rinsing in buffer the cells were collected on a milllpore filter, embedded in IX agar and postfixed in IX 0 b04 In b u ffer for 1 ho u r. The sample was washed in water, poststained overnight in IX aqueous uranyl acetate, dehydrated 37 In acetone and embedded In either Spurrs or Epon-Araldite> In some
cases the cells were not embedded in the agar before processing, but
a loose pellet was fixed, en bloc stained and dehydrated as above,
and embedded in Spurrs between teflon coated glass slides* Serials were picked up on formvar coated slot grids, stained with lead
citrate, and viewed on an Hitachi H300 transmission electron microscope* The clnephotomlcrography was conducted in Dr* George
Witman's laboratory, Princeton University, using phase contrast on a
Zeiss Universal microscope, with Tri-X reversal movie film in a
Redlake Locam model 51, 16 mm high-speed motion picture camera
synchronized with a Chadwlck-Helmlth Strobex power supply and lamp*
The fram ing ra te was e ith e r 128 o r 256 frames per second. To allow observation of the flagellar motion, the colonies were held by gentle suction utilizing a micropipet and held away from both the coverslip and the slide (Ray and Witman, unpublished).
RESULTS
Ultrastructure of the flagellar apparatus
This report will deal only with the nature of the flagellar apparatus of the mature cell * The structure of the vegetative cell and a description of cell division will be presented elsewhere
(Chapter 5). All drawings and serial sections are in the correct absolute orientation (Floyd et al. 1980). The details of the
flagellar apparatus are different from any algal flagellar apparatus 38 previously reported. Therefore, we have Included a reconstruction
(Fig. 34) that may assist In interpreting the various micrographs.
In Astrephomene the flagella are Inserted separately. As cells
In a culture age there is a tendency for the flagella separate. As the distance between the flagella Increases, it becomes more difficult to obtain good serial sections that contain both flagella in an appropiate plane. Therefore, most of the flagellar apparatuses examined were of young cells from actively growing cultures.
Sections of older vegetative cells from the slowly growing, non-axenic cultures (not shown), Indicate that there are few, if any, differences between the flagellar apparatus of mature cells of these two cell types, except for the distance separating the flagella and the length of the structures that run between them. The specalized rudder cells were not differentiated in the younger colonies used here, so their structure will not be considered in this report.
Flagella are Inserted in a small depression in the surface of the cell (Figs. 35-38). Although the flagella are nearly parallel, usually only basal body is in good cross section at a time
(Figs. 36-38), indicating that the two flagella are not perfectly parallel* Since the flagella are not always synchronized, this may reflect the differing stages in the beat cycle. The structure of the flagella and basal bodies is similar to those reported for related organisms Buch as Chlamydomonas (Rlngo, 1967), Volvox (Olson and
Kochert, 1970) and Eudorlna (Hobbs, 1971) and will not be detailed here* However, the components that are associated with each of the flagella (basal bodies) are arranged differently (Fig. 34). For 39 convenience we have arbitrarily labeled the flagellum (basal body) to the right In Figure 34 as number 1, and the second as number 2. An electron dense structure, here termed the "strut", extends past the basal bodies on each side of the flagellar apparatus (Figs. 33-38).
In cross section, this structure Is semicircular (Figs * 42-44)■
Where it passes each of the functional basal bodies, It connects to them by a series of thin shelf-like projections that arise from the flat side of the strut (Figs. 41,44). These projections are present on the strut between the basal bodies (Fig. 41), but are not associated with the strut when it extends beyond the basal bodies
(Fig. 42). The plasma membrane is pulled down towards this structure leaving a cleft just to the outside of the basal bodies
(Figs. 42-45), however, there does not appear to be a direct connection of the strut to the plasma membrane. The presence of the strut on the same side of each functional basal body, means that the flagellar apparatus possesses a definite polarity. As shown with light microscope observations (see below), the flagella of each cell in the colony beat in nearly the same direction, thus giving the colony a functional polarity. In addition, the rudder cells, if present, are conslstantly located at the posterior end of the colony.
We have determined that the strut is on the side of the flagellar apparatus toward the anterior end of the colony by sectioning cells from the slowly growing, non-axenic cultures in which rudder cells could be unamblgously identified (thus allowing the determination of the colony polarity). 40 Four microtubule rootlets extend from the flagellar apparatus just under the cell surface for an undetermined distance. The two
Inner rootlets, each of which Inserts on the Inside of one of the functional basal bodies, are parallel (Figs. 36-40). These inner rootlets alternate between 2 and 3 over 1 members (Figs. 39,40). A second 3 over 1 rootlet is located on the outside of basal body number X, and is nearly parallel to the Inner rootlets (Figs. 37,39).
The fourth rootlet Is nearly perpendicular to the other three as viewed from above. This rootlet, composed of two microtubules
(Fig. 42) rises toward the cell surface at a steep angle (Fig. 41), so it is not obvious in the plane that shows the longitudinal views of the other rootlets (i.e., Fig. 35-38). This rootlet runs in nearly the same direction as the strut although it appears to attach to the strut only at the Insertion of the rootlet into the basal body
(Fig. 42). A periodic component runs under the 3 over 1 rootlet that is inserted into basal body number 2 (Fig. 37). There does not appear to be a similar component associated with the second 3 over 1 rootlet (Figs. 37-38).
An accessory basal body is near each of the two membered rootlets (Figs. 37,39,41). These accessory basal bodies are at the level of the bottom of the functional basal body or lower and always in the same position relative to the rest of the flagellar apparatus.
The accessory basal body associated with basal body number 1 is lo cated between the two fla g e lla w ith i t s axis p o in tin g away from the functional basal body (Fig. 39; the position of this accessory basal body has been slightly altered in Fig. 34 to allow details of the 41 proximal area of basal body number 1 to be seen). The accessory basal body associated with the second flagellum Is on the outside of that basal body, again with its axis pointing in roughly the same direction (Fig. 41). A large striated fiber is found on the same side of each of the flagella as the accessory basal body. From above, this striated fiber appears to attach directly to the electron dense strut (Figs. 36-38). Sections in other planes, however, indicate that the focus of the fiber is below the strut, although the side of the fiber appears to touch it (Fig. 43). The fiber is triangular in shape and has a characteristic strlatlon pattern. Fine filaments run perpendicular to the strlations. The fiber does not attach to the basal body along the entire base of the fiber; approximately half goes under the proximal portion of the basal body and ends at a "spade-like*' structure that also extends below the basal body (Fig. 43). At the other (pointed) end of each striated fiber there is Invariably a membrane enclosed vesicle of varying size
(F ig . 4 3 ).
A thin electron dense structure extends from basal body number 1 towards, but does not reach, the other basal body (Fig. 40). This structure attaches to the baBal body of the functional flagellum on the side opposite the rootlets. It touches the electron dense strut, but it is not a part of that structure.
A spade-like structure is associated with each of the functional basal bodies. This structure Is present on the side of the striated fiber that is associated with the proximal end of each of the basal bodies. The spade-like structures are located roughly perpendicular 42 to the line between flagella but, as viewed from above, to the
opposite side of the electron dense strut (Fig. 38). They are
associated with the amorphous material that partially occludes the
proximal region of each of the functional basal bodies* Fine,
fibrous material runs from the outside of the spade-like structure of
basal body 1 to the amorphous material associated with the Inside of
the spade—like structure on basal body 2 (Fig. 39,46). This
fibrillar structure is associated with membranes that may look like
endoplasmic reticulum, or irregular vesicles (Figs. 39,40,46).
Because of the Irregular appearance of the membranes, it Is difficult to determine their precise arrangement, but they do not appear to completely enclose the fibrous material (serial sections not shown).
A ribosome free region is present between the nucleus and each of the functional basal bodies (Fig. 45). Such areas are occupied by a collection of very fine filaments. The nucleus appears to be pulled up towards the flagellar apparatus at this point (Fig. 45).
Analysis of flagellar motion
The flagella of Astrephomene beat In a tonsate or ciliary manner (Fig. 47). This figure was obtained from tracings of film
taken at 128 frames per second, thus the Intervals between the stages shown here are about 7.8 milliseconds apart. Unlike the case in many cilia, the effective stroke takes about as long, or in some cases slightly longer, than the recovery stroke. A flagellum may stop or pause near the beglnlng of the effective stroke, sometimes for a 43 considerable period. If both flagella of a given cell are In the
same portion of the beat cycle (I.e., are synchronized) they are
nearly parallel (Fig. 48). The direction of the effective stroke Is
towards the posterior end of the colony, and varies little for a
given flagellum over the period in which it was observed. Both of
the flagella of a given cell beat in parallel planes in the same
direction. The plane of the effective stroke approaches the
perpendicular of an imaginary line running between the two separate
basal bodies, but consistantly does not reach that position. The
difference presumably represents the component of flagellar motion
which is responsible for colony rotation rather than forward
swimming. The flagella of each cell are not necessarily
synchronized; frequently one flagellum is in the middle of the
effective stroke while the second is in the middle of the recovery
stroke (Fig. 48). At times both flagella may pause, then one
flagellum will start the effective stroke, followed almost
immediately by the second.
The flagella of different cells in a colony beat towards the
posterior of the colony. However, all flagella do not always beat in
exactly the same direction (Fig. 48), although the three dimentlonal component of the flagellar motion makes determination of the exact direction of beat difficult. The significance of this is not known. DISCUSSION
Light m icroscopists have noted that the flag ella of Astrephomene
(Pocock, 1953; Stein, 1958), as well as many other colonial members of the Volvocaceae (e .g ., Fritch, 1935; Smith, 1950; Bourrelly, 1972) are Inserted separately, a situation uncommon but not unique in the
Chlorophyceae. In ABtrephomene the details of the flagellar apparatus are different from those commonly associated with
Chlorophycean motile cells. Unlike the situation in most
Chlorophycean cells, including Gloeomonas (Schnepf et al. 1976) and
C arterla type I I (Lerabi,1975), two u n icellu lar Chlorophyceae in which the basal bodies are also separated, the entire flagellar apparatus o of Astrephomene does not show 180 rotational symmetry* This gives the flagellar apparatus an additional component of directionality, which is correlated with the directionality of flagellar motion.
The strut extends past both the functional basal bodies, and presumably functions as a structural support. In addition, the fine, fibrous component that extends from one spade-like structure to the other, might also serve a structural function. The fibrous component is located more proximally in respect to the flagellar apparatus than the electron dense strut, but runs from basal body to basal body on the opposite side of the cell (i.e., the side of the cell that is towards the posterior of the colony). The flagella will continue to separate as the cel1 b in the colony grow older. Presumably, growth of either the fibrous component, or of the portion of the strut between the basal bodies, or both, Is responsible for this movement. 45 The four mlcrotubular rootlets alternate between four membered
rootlets In the 3 over 1 configuration at their origin near the basal bodies, and 2 membered rootlets. This configuration is common in diverse members of the Chlorophyceae (see Moestrup, 1978; Melkonian,
1980a). In Astrephomene, however, the rootlets are not arranged in a cruciate manner, but are spaced asymmetrically, with two rootlets in association with each of the functional basal bodies. Two of the rootlets are present at right angles to the strut, while a third rootlet runs nearly parallel to these two. The fourth rootlet, however, extends away from the flagellar apparatus, near to, but not exactly parallel to the strut, thus perpendicular to the two parallel rootlets. This rootlet is inserted at the basal body near the presumed attachment of the strut to that basal body. Away from the
flagellum, the rootlet and strut diverge slightly, but the two structures remain close to one another. This reflects the role that this mlcrotubular rootlet plays in the generation of the strut (see next chapter).
The triangular striated fibers are parallel to each other, but on the same side of their respective basal bodies• Each fiber has a small vesicle at the apex, and attaches to the strut on the proxipal side. Striated fibers also attach the proximal fibers of Carteria, type I I , to the "electron dense rods" of the "B complex" (Lembl,
1975). However, the relationship between the rods in Carteria and the strut in Astrephomene is uncertain, as is the relationship between the striated fibers of these two algae. A connecting band Is present In Gloeomonas (Schnepf et al. 1976), but in this case the 46 connecting band appears to be branched, with extensions that run past
each of the basal bodies on both sides, a situation that Is different
from that found in Astrephomene.
Of the colonial members of the Volvocales with radially arranged
cells, the flagellar apparatuses of the vegetative cells have been partially characterized for Eudorlna lllinolensis (Hobbs, 1971) and
Volvox carter! (Olson and Kochert, 1970). In Eudorlna a single
"proximal band" uas found connecting the separated basal bodies, while in V. carter! both proximal and distal "kinetosome bridges" were observed. Our work in progress on V. rouselettli and V. carterl f. weismannla Indicates that the nature of the flagellar apparatus components, with the exception of the basal bodies and axonemes, is different from that of Astrephomene. Pyrobotrys and
Chlorcorona, two colonial Volvocales with a different colonial morphology, have a V-shaped arrangement of the functional basal bodies, with the flagella extending outward from the apex of each cell; and thus are different from the colonial members with radially arranged cells (Chapters 1 and 2).
Analysis of the form of flagellar beat shows that the flagella of Astrephomene beat with a c ilia ry type motion, but unlike
Chlamydomonas, the effective stroke of each flagellum is in the same direction. A transition to the "flagellar" type motion as occurs in
Chlamydomonas (Rlngo, 1967; Schmidt and Ekert, 1976; Hyams and
Borlsy, 1978) was never observed. Both flagella of a given cell, and the flagella of all the cells In a colony have the effective stroke toward the posterior of the colony, therefore generating effective 47 colonial motion. In Astrephomene the flagella did not beat continously, nor did both flagella from a given cell necessarily beat synchronously. In Volvox, high light intensity is thought to prevent or slow flagellar beating in positive phototaxis (Huth, 1970; Hand and Haupt, 1971; Sakaguchi and Twada, 1977; Sakaguchi and Iwasat
1979), and a sim ilar effect may be present In Astrephomene , which also has a well developed phototaxic response* Due to the high intensity flash used in filming, the rate of flagellar beating, and periods between the flagellar strokes may not represent the situation typically found In the swimming colony. This may account for the unusual length of time that a given flagellum is in the effective stroke compared to the recovery stroke. It is assumed however, that the form of the flagellar beat observed is unchanged. Therefore, the flagella beat in a plane that is perpendicular to the electron dense strut and ItB associated small rootlet, but nearly parallel to the three other rootlets. The effective stroke is towards the 2 parallel rootlets and away from the position of the electron dense strut.
Presumably both the rootlets and the electron dense strut are
Involved in anchoring the basal bodies as postulated for many types of flagellar roots and rootlets (Ringo, 1967; Petelka, 1974;
Stephans, 1975; Goodenough and Weiss, 1978; Gardiner, H iller and
Marsh, 1981).
It is noteworthy that the striated fibers are parallel to the plane of flagellar beat. If these fibers are contractile as some other algal striated fibers (Salisbury and Floyd, 1978; Melkonian,
1980b), they may play a role in the flagellar beat initiation. The 48 vesicle present at the apex of these fibers would not appear to give
these fibers sufficient anchoring, however, such anchoring may be
accomplished by the association of these fibers with the electron dense strut.
Note that both the construction of the flagellar apparatus, and
the form and direction of fla g e lla r motion Is unlike forward swimming
Chlamydomonas, where the V-shaped flag ella beat with a breastroke motion (Rlngo, 1967; Hyams & Borlsy, 1975; 1978), as well as the
reverse swimming where the flag ella beat with a fla g e lla r type motion
(Rlngo, 1967; Hyams and Borlsy, 1978; Schmidt and E kert, 1976). The combination of separate, nearly parallel flagella and the ciliary motion of both flagella In parallel planes is admirably suited for movement in this colonial organism. First, both flagella of a given
cell and all cells of the colony beat in the same direction, that is,
towards the posterior of the colony. This Is necessary for the
efficient motion of the colony. Secondly the nearly parallel arrangement of the flagella minimizes detrimental interference of the
flagella from a given cell and those of its neighbors. Thirdly the
flagellar separation would minimize detrimental interference between
the two flagella of the same cell, which otherwise could arise as a result of the nearly parallel flagellar arrangement. 49
BB1 BB2
Fig. 34. Diagrammatic representation of the flagellar apparatus of the mature cell of Astrephomene. In this view basal body 1 (BB1) is on the le ft of basal body 2 (BB2). The s tru t (S) extends past both basal bodies. The striated fibers (SF) extend under the strut. Both four membered (R4) and two membered (R2) microtubule rootlets extend from the basal bodies. A fibrous component (FC) connects the spade-like structure (SS) attached to basal body 1 to the amorphous material associated with basal body 2* A flap-like structure (FS) is attached to basal body 1. An accessory basal body (ABB) is near each of the smaller rootlets. Fig. 35. Top view of Astrephomene flag ellar apparatus, f ir s t section. The flagella are separate and nearly parallel. The strut (S) extends past the basal body.
Fig. 36. Top view of Astrephomene flagellar apparatus, second section. One flagellum is in good cross section while the other is slightly oblique. Note one four membered rootlet (R4).
Fig. 37. Top view of Astrephomene flagellar apparatus, third section. The strut extends past the basal body on both sides. The second four membered rootlet (R4) is present, and a periodic component underlies the other large rootlet (arrow). Note the striated fiber (SF) attached to basal body number two and the accessory basal body nearby.
Fig. 38. Top view of Astrephomene fla g ellar apparatus, fourth section. The striated fiber is associated with the spade-like structure (SS) and amorphous m aterial (AM). A two membered rootlet (R2) extends from Inside of basal body number 1. (Figs. 34-38 x55,000)•
50 51 Fig. 39. L ateral view of Astrephomene fla g ellar apparatus* f ir s t section. The mlcrotubular rootlets alternate between four and two members (R2* R4). Note the fibrous component running from the spade-llke structure of basal body 1 to the amorphous m aterial associated with basal body 2 (straight arrow)* and the accessory basal body near basal body 1 (curved arrow).
Fig. 40. Lateral view of Astrephomene flag ellar apparatus, second section. The dense flap (DF) extends from basal body 1 (out of the plane of section In this figure) toward the second basal body. One of the s tria te d fibers (SF) is In cross section.
Fig. 41. Laterlal view of Astrephomene flagellar apparatus* third section. The inside of the strut has thin shelf-llke projections (arrow) on the side toward the basal bodies. The fourth rootlet (R2) is approximately perpendicular to the other three. Also note the second accessory basal body (ABB). (Figs. 39-41 x68*00Q).
52 53 Fig* 42. Astrephomene; cross section of the accessory basal body associated with functional basal body 2. Note the fourth rootlet (R2) in association with the strut. (xl00,000).
Fig.43. Striated fiber of Astrephomene. This fiber is associated with the spade-like structure (SS) and the strut (S). Avesicle (V) is present at the apex of the fiber* (xl0,000).
Fig. 44. Astrephomene; strut near the vicinity of the basal body. Note the shelf-like structures that extend from thestrut (S). Also note the microtubule rootlet (R4). (xlO^OQO).
Fig. 45. Region proximal to the basal body in Astrephomene * Note the ribosome free area traversed by fine filamemts (arrow). (xl05,000).
54 55 56
Fig. 46. Basal body region of Astrephomene. A fibrous component (FC) runs between the spade-like structure (SS) attached to one basal body and the amorphous m aterial (AM) associated with the other. Note the membranes around the filaments• (x71,000). 57
10
Fig. 47. Astrephomene flagellar motion. From a tracing of cine film taken at 128 frames per second. Numbers Indicate order from beginning of effective stroke to end of recovery stroke. 58
Fig. 48. Single frame of Astrephomene flag ellar motion from cine film. In one cell both flagella are in the parallel position at the beggining of the effective stroke. Note the lack of synchrony in the other cells which have one flagellum in the effective stroke and the other in the recovery stroke. CHAPTER 4
THE DEVELOPMENT OF THE
FLAGELLAR APPARATUS IN ASTREPHOMENE
In the preceding chapter the unusual nature of the flagellar
apparatus of the mature cell of Astrephomene gubernacullfera was
described. The flagellar apparatus Is apparently adapted to the
constraints on flagellar motion Imposed by the nature of the colonial
habit. In this report the development of the flagellar apparatus is
traced from Its early stages which are similar to those of the
chlamydomonad-type (Melkonian, 1980a), to the situation found in the mature cell. These changes give further insight into
structure-functlon relationships of the mature flagellar apparatus, as well as suggesting certain evolutionary relationships.
MATERIALS AND METHODS
Astrephomene gubernacullfera Pocock was grown axenically (Chapter 3).
One to three day old cultures at 1-3 hrs before the onset of the
light period were fixed as described previously (Chapter 3).
Examination was by an Hitachi H300 TEM.
59 RESULTS
Like other Volvocalean algae, each of the reproductive cells of
Astrephomene will divide a number of times to yield a new daughter colony. It Is difficult to determine if a cell would have divided again if the sample had not been fixed, because the cell number varies In colonies grown under the conditions used here. Also it is difficult to determine the cell number of an entire colony in thin section. Therefore, it is uncertain whether the initial stage In the development of the flagellar apparatus represents the condition of the flagellar apparatus shortly before, or after, the last division.
Nevertheless the appearance of this first stage is quite consistent and frequently encountered.
In the earliest stage observed, the flagellar apparatus consists of four basal bodies, none of which extend into flag ella
(Figs. 49-54). Two opposite basal bodies are connected by a broad striated fiber (Fig. 50). The other two basal bodies are further apart than the connected pair (Figs* 51-53). Four cruciate rootlets descend from the region of the basal bodies (Figs. 51-53). The rootlets are more closely associated with the basal bodies that are connected by the s tria te d fiber than the other two. These rootlets consist of alternating 3 over 1 and 2 microtubules each. In addition to the ro o tlets, numerous cytoplasmic microtubules lie just under the plasmalemma and extend towards the posterior of the cell
(Figs. 49-54). Connections from the basal bodies not attached by the 61 striated fiber to either the rootlets or the other basal bodies are present (Figs. 51,52). Two striated fibers also connect the proximal ends of the two basal bodies that are connected by the striated distal fiber (Fig. 53). Lastly, there is a disorganized meshwork of thin filaments that extends downward from the region below the fla g e lla r apparatus (Fig. 5A).
As the cells enlarge the flagellar apparatus undergoes many changes. The basal bodies that are connected by the striated distal fiber give rise to flagella (Fig. 55-58). The flagellar apparatus s t i l l has cruciate microtubular rootlets (Figs. 56,57), which alternate between 2 and A members with each larger rootlet present in the 3 over 1 configuration near *"he basal bodies (Figs. 59,60). In addition, the two striated proximal fibers are still present
(Fig. 57,58). The additional pair of full sized basal bodies is no longer present, rather two short accessory basal bodies are located to the side of the functional ones (Fig. 58). The accessory basal bodies are lower In the cell than the functional pair, and they are not arranged in the V-shape characteristic of the functional basal bodies or the second pair present in the initial stage. It is not certain If this condition is due to the rearrangement and shortening of the original second pair, or If the accessory basal bodies are formed after the redistribution of the original two pairs to daughter cells following division.
Cells at this stage, sectioned in a plane perpendicular to the cell In Figs. 55-58, reveal further information about the flagellar apparatus. Each accessory basal body has a complete set of triplets In addition to the cartweel structure (Fig. 61). The functional basal bodies are arranged In a V-shaped configuration about 90° degrees apart (Fig. 62-64). The proximal fibers are triangular and possess a distinctive striatlon pattern (Figs. 62,64). The apex of the trian g le is located to the outside of the basal body, but toward the anterior of the cell. The base of the triangle is associated with the other basal body, however, much of the base extends off the proximal side (Fig. 62). The apex of the second striated fiber attaches to the same basal body that is associated with the base of the first striated fiber. Also associated with the proximal region of each basal body is a region of amorphous electron dense m aterial
(Figs. 63,64). This material is found In association with the posterior side of the basal body and p a rtia lly occludes the lumen. A ribosome free region surrounds the base of the flagellar apparatus
(Fig. 63).
When viewed with the light microscope cells of this stage have two flagella capable of motion (Pocock, 1953). The structure of the flagellar apparatus is similar to certain other motile Chlorophycean algal cells (see discussion), but very different from the adult cell of Astrephomene• In view of the Importance of the structure of the flagellar apparatus at this stage, a reconstruction (Fig. 65) is provided to aid in Interpretation, and for comparison with published information on the flagellar apparatus of other Chlorophycean cells.
The next step in the development of the flagellar apparatus
Involves several changes that occur concurrently. One of the first changes noted is the appearance of an electron dense strut which 63 forms near one of the 2 membered rootlets (Fig. 67). This structure
Is not appressed to the mlcrotubular rootlet near which It forms. At this stage the strut does not extend past the second functional basal body (Figs. 67,66). The striated distal fiber detaches from one corner of each functional basal body (Fig. 67). The rootlets are no longer cruclately arranged, but are now asymmetrical. One four membered rootlet and one two membered rootlet are closer together, while the other two and four membered rootlets diverge (Figs. 67-69).
The position of the accessory basal bodies has also changed, so that one is between the pair of rootlets that are close together
(Fig. 67), while the second accesssory basal body appears under the 2 membered ro o tlet and the forming stru t (Fig. 70). These changes suggest that each half of the flagellar apparatus is rotating, the two halves rotating in opposite directions.
Another change involves the proximal striated fibers. Each fiber dissociates at Its pointed end from one of the two basal bodies
(Figs. 69-71). Electron dense material remains at the former site of attachment (Fig. 69). Initially, these fibers do not reattach to any particular structure, although in Figs. 66-71, one striated fiber underlies the developing strut while the second terminates near that structure. A narrow spade-like structure extends from one (Fig. 71), or both (not shown) of the basal bodies at this stage.
Later, the functional basal bodies continue to become more nearly parallel, and begin to separate (Figs. 72-75). At this stage a spade-like structure is associated with each basal body (Fig. 75).
A fibrous component extends from the outside (relative to its 64 associated basal body) of one spade-like structure to the amorphous matertlal associated with the Inside of the second spade-like structure* At this stage two rootlets become nearly parallel, while the other two diverge still further. The strut extends past both of the functional basal bodies and remains to the same side of each
(Fig. 75). The strut Is striated at a periodicity of approximately 5 to 6 nm* Although similar striations are present in the strut of the mature cell (not shown), they are most evident In the early stages of development. The distal fiber remains attached at two corners to the two basal bodies (Fig. 73), and extends laterally In the direction of the elongating strut. The fiber appears to be stretched, presumably as a result of the separating basal bodies.
After the basal bodies become parallel, further separation results in the disjunction of the former distal fiber from one of the basal bodies. Thereafter the former distal fiber remains as a dense flap associated with the strut and one of the functional basal bodies
(see previous paper). Both the fine fibrous material that extends between the spade-llke structures and the strut elongate during this time. Although the flagella will continue to move apart until they are widely separated, there does not appear to be further rearrangement of the flag ellar apparatus components a fte r the disjunction of the distal fiber. 65 DISCUSSION
In the previous chapter It was demonstrated that the flagellar apparatus of the mature cell of Astrephomene gubernacullfera Is unusual among the green algae for its deviation from the typical symmetry, and how the arrangement of the components Is advantageous to a colony possessing radially arranged cells with isokont flagella.
This chapter describes the development of the flagellar apparatus from the early stages to the mature form.
During early stages of development the flagellar apparatus supports 2 flag ella in a V-shaped arrangement, which Pocock (1953) has reported to be capable of movement. At this stage the flagellar apparatus resembles the chlamydomonad-type (Melkonian, 1980a). The
Astrephomene flagellar apparatus at this stage resembles the flagellar apparatus of Chlamydomonas (Ringo, 1967, Moestrup, 1978;
Goodenough and Weiss, 1978; Katz and McLean, 1979) in possessing two functional basal bodies in a V-shaped configuration, two proximal and one distal striated connecting fibers, and 4 cruciately arranged microtubular rootlets. Initially, Chlamydomonas was reported to have four rootlets with 4 microtubules in each (Ringo, 1967), but alternating 2 and 4 membered rootlets have since been demonstrated
(Moestrup, 1978; Goodenough & Weiss, 1978; Katz and McLean, 1979), like Astrephomene at this early stage. In both genera the four membered microtubule rootlets are present in the 3 over 1 configuration in the region of the basal bodies. Although the vegetative cells of Chlamydomonas relnhardti do not possess fully formed basal bodies during most of Interphase (Cavalier-Smith, 1974;
Gould, 1975), gametes In this species have accessory basal bodies In a position similar to that of the flagellar apparatus In the Immature c e ll of Astrephomene (Friedmann, Colwln and Colwln, 1968)* Both the flagellar apparatus of Chlamydomonas. and Astrephomene at this stage, have 180° ro tatio n al symmetry. While a number of these features are present in a variety of green algae (Moestrup, 1978; Melkonian 1980a;
Floyd et al> 1980), taken together, the similarities suggest a close phylogenetic relationship between Astrephomene and the unicellular algae of the chlamydomonad-type. The sperm cells of Volvox carter!
(Birchem and Kochert, 1979b), V. areus (Peason et a l. 1969, Deason and Darden, 1971) and V. tertlus (Plckett-Heaps, 1975b) also resemble the chlamydomonad-type of flagellar apparatus in having the two functional basal bodies in a V-shaped arrangement and connected by a distal fiber and in possessing cruciate microtubular rootlets. This is in contrast to the mature vegetative cells of Volvox. where the flagella are inserted separately. Work in progress Indicates that the ultrastructure of the mature vegetative cells of V. carter! and
V. ro u e sele tii is sig n ifican tly d ifferen t from Chlamydomonas, but the flagellar apparatuses of the embryonic cells are the chlamydomonad-type.
It has repeatedly been suggested that the colonial Volvocales evolved from u n icellu lar members of the order (Crow, 1918; F ritsch,
1935; Lang, 1963; Pickett-Heaps, 1975a; E ttl, 1976). The sim ilarity of the fla g e lla r apparatus of the immature cells of Astrephomene, as well as the sperm c ells of Volvox, to the algae with the 67 chlamydomonad-type flagellar apparatus supports this view. However, some of the d e tails of the fla g e lla r apparatus of Astrephomene at this stage do differ from those noted in algae of this type. In particular, the proximal striated fibers of Astrephomene appear unusual in their triangular shape and in that much of the striated fiber Is proximal to the base of the basal body. The similarity between flagellar apparatuses of certain stages of the colonial
Volvocales and Chlamydomonas should not be taken as evidence that the ancestor was a present day Chlamydomonas. Careful Investigation of other unicellular algae may reveal species that have additional features in common with the immature flag ellar apparatus of
Astrephomene and the sperm c ells of Volvox.
The flagellar apparatus does not remain in the chlamydomonad configuration, but rather undergoes a major reorganization involving the separation, rotation, and rejoining of certain structures.
Others are newly developed. This is dlagrammatically illustrated in
Figure 28. The strut forms in association with one of the two membered rootlets and remains close to this structure during development to the mature state • The strut elongates during development* However, at present, the mechanism for this elongation is not understood. The association with the smaller rootlet, as well as the fine periodicity, indicates that it may be a derived form of the SMAC (striated microtubular-associated component, Floyd et a l.f
1980), which is present in association with the smaller microtubular ro o tle ts in a variety of algae, including Chlamydomonas (Goodenough and Weiss, 1978). 68 Another modification In the flagellar apparatus Includes the
formation of the spade-like structures and the associated filaments
that run between them* The spade-like structures are not developed when the flagellar apparatus is in the chlamydomonad-type stage,
although there Is amorphous material In association with the proximal
end of the basal bodies. This material Is present in association with the spade-llke strutures in the mature flagellar apparatus. The
spade—like structures form during the early stages of reorganization, when the basal bodies are rotating and becoming more nearly p a ra lle l.
Both the time of formation and the position of these structures
suggest that they are involved In the reorganization movement•
In addition to the rearrangement that causes basal bodies to
become separate and parallel, there is also rotation of the two
halves of the flagellar apparatus about 90 in opposite directions.
Although no internal markers were found associated with each basal
body, the position of the s tria te d fiber and its former attachment,
as well as the position and attachments of the distal fiber during
this process, suggest that the basal body itself rotates during this
period. In addition to the basal body and its attached flagellum,
each rotating half includes an accessory basal body, one small and
one large rootlet, amorphous material at the proximal end of the
basal body, the developing spade-like structure, and one striated
fiber (formerly one of the proximal striated fibers).
Flash cinephotomicrographic analysis (Chapter 3) of the
flagellar motion of Astrephomeme is consistent with the development
described here. Figure 29 diagrammatlcally compares fla g e lla r motion 69 In Chlamydomonas and Astrephomene* The face view of Chlamydomonas is defined here as the view in which fla g e lla r motion is in the plane of
the page, while in side view, the plane of flagellar motion is at
right angles to the paper* Cell orientation of Astrephomene in the
chlamydomonad-stage is determined by comparison with Chlamydomonas, and does not change during flagellar reorientation. Note that each o flagellum of Astrephomene beats in a plane that is rotated about 90
from that of Chlamydomonas. Since each half of the flag ellar apparatus has rotated a comparable amount, this Implies that a
structural component determines the plane of flagellar beat.
Precisely what stru c tu ra l component is responsible continues to
remain an important question. The striated proximal fiber is a
possible candidate. This fiber appears to be in the right plane to
initiate flagellar motion in both the chlamydomonad and mature
stages. Some algal striated fibers are contractile (Salisbury and
Floyd, 1978; Melkonian, 1980b), and it has been suggested that these or similar fibers might function in the initiation or control of
fla g e lla r motion (Salisbury and Floyd, 1978; Hyams and Borisy, 1975;
1978). The distal striated fiber cannot be responsible since this
structure does not rotate fully during development and moreover it does not connect both functional basal bodies in the mature flagellar apparatus.
A lternatively, the stru ctu ral component that determines the direction of the effective stroke might be located in the axoneme or basal body. Under certain conditions, isolated axonomes of
Chlamydomonas beat with an asymmetric form, presumably equivalent to 70 the ciliary type motion characteristic of forward swimming (Bessen,
Fay and Witman, 1980) and sim ilar to the motion in Astrephomene* The
axonemes of many organisms possess a bridge between doublets 5 and 6
(Warner, 1975)* Although these have not been reported for any alga,
including Chlamydomonas and Astrephomene, such a bridge would disrupt
the radial symmetry of the axoneme and might be a structural
component responsible for some aspect of directionality* Some green
algae have one, two, or three septatlons or beaks in the flagellar
axoneme (see Hoops et al* in press; Witman, Carlson, B erliner, and
Rosenbaum, 1972)* When three such stru ctu res are present they are in
two adjacent doublets, and the doublet directly across from this
pair. Although it is difficult to envision how these structures would directly influence the directionality of flagellar beat, their presence might reflect a structural polarity, that in Itself may be difficult to directly observe* A further possibility involves the orientation of the central pair. These microtubules are thought to have a particular relationship to the direction of flagellar beat
(Satir, 1963; Tamm and Horridge, 1970; Satir, 1975). In a given organism, however, the orientation of the central pair, is not necessarily fixed. In the dilate Opalina, the plane of the central pair varies with the flagellar beat (Tamm and Horrige, 1970). There appears to be rotation during flagellar motion in Paramecium (Omoto and Kung, 1979; 1980) and in the green alga Micromonas (Omoto and
Witman, 1981). It is notable that the flagellar motion in all of these organisms is strongly three dlmentional. In contrast, the central pair orientation does not change during forward or reverse swimming In the ptenophore Pleurobrachla (Tamm and Tamm, 1981).
Since the position of the central pair of microtubules Is not always
fixed, it seems unlikely that the rotation of each half of the
flagellar apparatus would be necessary to reorient the central pair in Astrephomene.
It is also possible that the structural component that determines the plane of the effective stroke might be axonemal, but
the component responsible for the in itia tio n of the stroke might be external to the axoneme. This theory is similar to that suggested by
Tamm and Horridge (1970). In the case of Opalina the effective stroke is probably initiated by viscous-coupling. In the case of
Astrephomene, where flagella are considerably further apart, initiation may be accomplished by other means, such as contraction of the striated fiber. This would explain why not only the flagellum, but associated components, in particular the proximal fibers, underwent rotation during development. Fig* 49. Very early stage in flagellar apparatus development in Astrephomene. first section. None of the basal bodies extend into fla g e lla .
Fig. 50. Very early stage in flagellar apparatus development in Astrephomene. second section. Two of the basal bodies are connected by a striated distal fiber (DF).
Fig. 51. Very early stage In flagellar apparatus development in Astrephomene. third section. Four mlcrotubular rootlets (R2, R4) are cruclately arranged.
Fig. 52. Very early stage in flagellar apparatus development in Astrephomene, fourth section. The mlcrotubular rootlets run toward the p osterior of the c e ll ju st under the plasma membrane.
Fig. 53. Very early stage in flagellar apparatus development in Astrephomene, fifth section. The two basal bodies connected by the distal fiber are also connected by two proximal striated fibers (SF).
Fig. 54. Very early stage in flagellar apparatus development in Astrephomene» sixth section. Numerous fine filaments are present under the flagellar apparatus. (Figs. 49-54 x55,000).
72 73
V * ' i \ ■ flRb ■Xvk'^j. Fig. 53. Chlamydomonad-stage of flagellar apparatus development In Astrephomene, first section. Two of the basal bodies extend into flagella* These basal bodies are connected by the distal striated fiber (DF).
Fig. 56. Chlamydomonad-stage of flagellar apparatus development in Astrephomene, second section. Mlcrotubular rootlets (R2, R4) descend from the region of the basal bodies.
Fig. 57. Chlamydomonad-stage of flagellar apparatus development in Astrephomene. third section. Two basal bodies connected by the distal fiber are also connected by 2 striated proximal fibers (SF).
Fig. 58. Chlamydomonad-stage of flagellar apparatus development in Astrephomene, fourth section. Two accessory basal bodies (ABB) are below the Functional basal bodies. (Figs. 55-58 x59,Q00).
Fig. 59. Larger rootlet of the chlamydomonad-stage of Astrephomene. This rootlet is in the 3 over 1 configuration near the basal bodies. (x68,000).
Fig. 60. Smaller rootlet of the chlamydomonad-stage of Astrephomene. This rootlet is 2 membered. (x68,000).
74 75
y e H . V* K S X*i Fig. 61. Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, first section. The accessory basal body has the normal complement of microtubule triplets and the cartwheel s t r u c tu r e .
Fig. 62. Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, second section. Note the triangular proximal striated fiber. Half of this fiber extends off the proximal end of the basal body.
Fig. 63. Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, third section. The distal fiber connects the basal bodies which are arranged in a V. Note the amorphous material associated with the underside and proximal end of the basal body (curved arrow).
Fig. 64. Lateral view of the chlamydomonad-stage of Astrephomene flagellar apparatus, fourth section. Note that the second proximal striated fiber points in the direction opposite the first. (Figs. 61-64 x65,000).
76 77 Fig. 65. Reconstruction of the chlamydomonas-stage of flagellar development in Astrephomene. The two functional basal bodies are connected by a s tr ia te d d i s t a l fib e r (DF) and two s t r i a t e d proximal fibers (SF). The rootlets alternate between A (RA) and 2 (R2) membered rootlets. Note also the amorphous material (AM) and the accesso ry b a sa l bodies (ABB). Fig. 66. Developing flagellar apparatus of Astrephomene, first section. The two flagella are nearly parallel.
Fig. 67. Developing flagellar apparatus of Astrephomene, second section. The distal fiber is detached from the flagellum at one corner* Two of the rootlets (R2, R4) are now nearly parallel. The strut (S) is associated with one of the two membered rootlets (R2), Note the accessory basal body (ABB).
Fig. 68. Developing flagellar apparatus of Astrephomene. third section. The strut extends to the other basal body, but does not extend beyond i t •
Fig. 69. Developing flagellar apparatus of Astrephomene. fourth section. Each striated proximal fiber (SF) has detached at one point, leaving an electron dense remnant at the former attachment point (unlabeled arrow). Note the 4 membered mlcrotubular rootlet (R4) and the other accessory basal body (ABB).
Fig. 70. Developing flagellar apparaus of Astrephomene, fifth section. Note attachment of the striated fibers at the base of each functional basal body.
Fig. 71. Developing flagellar apparatus of Astrephomene. sixth section. A spade-like structure (SS) descends below the proximal end of the basal body* Note the ribosome free region under the basal bodies. (Figs. 66-71 x49,000).
79 80
«
4 *
• * fsm,- ■.*.»- **•'. * ■
M m r Fig. 72. Nearly mature flagellar apparatus of Astrephomene, first section. The flagella are nearly parallel to each other. The fibrous component (arrow) extends from the spade-like structure attached to basal body number 1 to the amorphous material near basal body 2.
Fig. 73. Nearly mature flagellar apparatus of Astrephomene, second section. The two inner rootlets (R2, R4) are nearly parallel, and the other four membered rootlet (arrow) is in about the same position as in the fully mature cell. Note that the accessory basal body (ABB) is also near the position it will occupy in the mature cell.
Fig. 74. Nearly mature flagellar apparatus of Astrephomene. third s e c tio n . The d i s t a l fib e r (DF) s tre tc h e s between the two f la g e lla . The two striated fibers, here in cross section, are parallel.
Fig. 75. Nearly mature flagellar apparatus of Astrephomene. fourth section. The electron dense strut, is finely striated (arrow),and now extends past both basal bodies. (Figs. 7 2-75 x71,000).
81 > 1 * W A 82 Fig* 76. Diagrammatic representation of flagellar apparatus development in Astrephomene. In the earliest stage (A), four full sized basal bodies are present, 2 of which are connected by the striated distal fiber (DF) and two striated proximal fibers (PF). The mlcrotubular rootlets (R2,R4) are cruciately arranged. Later the cells enter the chlamydomonad-stage (B) as two basal bodies grow flagella, while two smaller accessory basal bodies (ABB) are also p re s e n t. During development the basal bodies become more nearly parallel, the proximal fibers detach at one end, and each half of the flagellar apparatus begins to rotate (C). This rotation is reflected in the position of the mlcrotubular rootlets, the striated fibers (SF; detached proximal fibers), and the accessory basal bodies. In addition, an electron dense strut (S) forms near one of the two rootlets and extends past one of the basal bodies. For simplicity, the fibrous component that extends between the basal bodies is not shown. In the mature stage (D), the strut extends past both basal bodies and rotation of each half of the flagellar apparatus ceases. The striated fibers attach to the underside of the strut. The distal fiber has detached from one basal body and becomes the stage. Again, the fibrous component between the basal bodies was omitted for clarity. Although the basal bodies will continue to separate there is no further rearrangement of components.
83 84
B A
DF i 1
“ ^TT~ PF 1------1 ABB
R 4 85
Fig. 77. Diagrammatic comparison of flagellar motion in Chlamydomonas and Astrephomene. The top two rows show the f la g e lla r motion of Chlamydomonas in two different views* In the top row the flagellar motion is in the plane of the page* while the cells in the second row are views of the same stages* but viewed at right angles to the plane of flagellar motion* The bottom row represents flagellar motion in Astrephomene. In this case cell orientation is the same as the second row of Chlamydomonas. For simplicity the flagella of Astrephomene are shown as synchronous. Note that each flagellum of Astrephomene beats in a plane about 90v different from the Chlamydomonas cell with the same orientation. This appears to be correlated with the development of the flagellar apparatus in Astrephomene. The flagellar motion in Chlamydomonas is after Ringo, 1967; Hyams and Borisy, 1974; while the motion in Astrephomene is taken from Chapter 3. CHAPTER 5
MITOSIS, CYTOKINESIS AND COLONY
FORMATION IN ASTREPHOMENE GUBERNACULIFERA
Although the colonies of Astrephomene are spherical with radially arranged cells like many members of the Volvocaceae, the colonies do not undergo Inversion as do all Investigated members of that family (Pocock, 1953; Stein, 1958). Pocock (1953) suggested that this feature was important enough to serve as the basis for a new family, the Astrephomenaceae• Other unusual features of the alga
Include the lack of a common envelope and the presence of specllized
"rudder" cells at the posterior of the colony* Most subsequent authors have followed this classification (i.e., Starr, 1980; Stein,
1958; Lang 1963). This family is not recognized, however, by
Bourrelly (1972), who includes this genus in the Volvocaceae.
The morphology and sexual cycle of Astrephomene has been extensively studied at the light microscope level (Pocock, 1953;
Stein, 1958; Brooks, 1966), but the alga has only been superficially examined at the ultrastructural level (Lang, 1963). In view of the uncertain relationship of this alga to members of the Volvocaceae, and the paucity of information on both mitosis and colony development
In this family, the ultrastructural study of the vegetative
86 87 structure, mitosis, cytokinesis and colony formation In Astrephomene was undertaken.
MATERIALS AND METHODS
Astrephomene gubernacullfera Pocock (LB 1968) was obtained from the Culture Collection of Algae at the University of Texas.
Non-axenlc stock cultures were maintained in soil-water tubes fortified with a portion of a pea cotyledon (Stein, 1958). Axenic cultures were grown in Modified Volvocalean Medium (MV medium;
Brooks, 1966), and tra n s fe rre d every two weeks* The alg a was grown on a 16:8 light dark regime under 1800 lux at 20* C. Cells usually divided 1“3 hrs before onset of the dark period. Axenic cultures 1 to 3 days old were fixed in IX g lu tarald eh y d e in c u ltu re medium for
10-15 min, and transferred directly to IX glutaraldehyde in 0.1 or
0.2 M sodium cacodylate buffer, pH 7.2 for two hours. After rinsing in buffer the cells were collected on a millipore filter, embedded in
2% agar and postfixed in IX 0s04 in buffer for 1 hour. Samples were washed in water, poststained overnight with 1% aqueous uranyl acetate, dehydrated in acetone and embedded in either Spurrs or
Epon-Araldlte. Sections were picked up on formvar-coated slot grids, stained with lead citrate or potassium permanganate followed by lead citrate (Bray and Wagenaar, 1978), and viewed on a Hitachi H300 transmission electron microscope. 88 RESULTS
Each cell of the colony is surrounded by a separate cell wall
(Fig. 78). In the mature colony the cells are round to kidney-shaped with two flagella arising out of a shallow depression or groove
(Fig. 79). The flagella are inserted separately and are nearly parallel. The flagellar apparatus Is highly unusual and is described elsewhere (Chapters 3 and 4). Mitochondria are located near the periphery of the cell, and a many-lobed chloroplast Is internal to the mitochondria The chloroplast haB a variable number of internal membranes; the thylakoids are sparse In cells recently Inoculated in to the organic medium (F ig . 7 8 ), but they are more numerous In older cultures (Fig. 79). A single multilayered eyespot is present in one lobe of the chloroplast (Fig. 78). Golgl bodies are located internal to the chloroplast (Fig. 78). The central nucleus has a large nucleolus. In interphase, most of the nucleolus stains densely, although less dense regions are present and the rest of the nucleus stains only lightly (Fig. 18).
Mitosis and cytokinesis.
In Astrephomene, any cell of the mature colony, except the rudder cells, may divide several times to yield a daughter colony.
The stag es shown here rep re se n t developm ental events subsequent to the first division of the reproductive cells. 89 In the period between divisions, the nuclei appear similar to those of the adult interphase cell (cf. Figs. 80 and 95). Each cell has centrloles located at its apex and near the nucleus
(Figs. 61,95). In every case examined by serial sectioning each cell had four centrloles (basal bodies; Fig. 81), therefore the two additional centrloles are presumably formed very early in the cell cycle. The fate of the parental flagella was not examined in detail, but each flagellum remains attached to a daughter cell throughout development* These flagella have been observed to beat under the light microscope (Pocock, 1953). Each flagellum at this stage lacks a basal body (Fig. 82).
Prophase. As prophase proceeds the chromatin begins to condense
(Fig. 83). During early stages of condensation the chromatin appears to be associated with the nuclear envelope. At this point numerous virus-like particles appear in association with the chromatin
(Fig. 84). These structures have a polygonal, lightly staining region about 90 nm in diameter surrounding a darker, circular layer, which in turn surrounds a moderately electron dense inner core. They are occasionally observed in cells that possess a nucleolus
(Fig. 95), but are uncommon in cells without condensed chromatin.
The next stage Involves the partial breakdown of the nucleolus as microtubles appear to the Inside of the nuclear envelope (Fig. 85).
Metaphase. At metaphase the chromosomes are in the typical metaphase plate alignment (Fig. 86). The nuclear envelope is closed except at the fenestrate poles. The spindle is crescent-shaped, consequently a medial section does not normally Intersect both poles. 90 Flagellar stubs are located at the cell surface and sometimes project slightly above It (Figs. 86,90,91). Microtubules radiate from a ribosome free region around the centrloles at each pole. Some of these microtubules enter the fenestrae and become part of the spindle while some remain to the outside of the nuclear envelope (Fig. 87).
The centrloles are located lateral to the focus of the Bplndle
(Fig. 87). During this stage multilayered kinetochores are present
(see below).
Early anaphase. Individual chromosomes are evident just after separation as they move towards the poles (Fig. 88). Each chromosome has a trllayered kinetochore (Figs. 88,89) that stains strongly with permanganate-lead citrate. Kinetochores are difficult to discern with the standard uranyl acetate, lead-cltrate staining procedure.
Micrographs of material stained with the latter method do, however, reveal faintly stained kinetochores (not shown). Serial sections of the curved spindle show each pole to have a pair of centrloles
(Figs. 88,90,91), with each pair of centrloles bridged by a connecting fiber (Fig. 91). The two centrloles of one spindle pole are oriented perpendicular to the pair at the opposite pole. At least two microtubular rootlets are present in association with each of the centriolar complexes (Figs. 87,90).
Late anaphaBe-early telophase. At this stage the chromosomes have traversed to the poles. The Interzonal spindle remains completely surrounded by the nuclear envelope (Fig. 92). The virus-like particles have accompanied the chromatin to the poles and are not found in the Interzonal region. 91 Telophase. At telophase the Interzonal spindle becomes narrow and presumably pinches off. Serial sections Indicate that the remains of the nuclear membrane and Interzonal spindle connect daughter nuclei such as those Illustrated in Fig* 93. Chromosomes begin to disperse at this stage*
Cytokinesis. Cytokinesis begins with Infurrowlng at the anterior end of the cell and progresses towards the posterior. Infurrowlng also occurs In the posterior region, but at a slower rate. The plane of cleavage Is predicted by a phycoplast which developes between daughter nuclei (Figs. 93,94). Many of the phycoplast microtubules appear to originate from the region of the centrloles (Fig. 93).
Initially cytokinesis Is incomplete leaving numerous bridges between daughter cells (Fig. 94). At a later stage these bridges are less numerous (Fig. 95). After a number of cell divisions a curved plate
Is formed with each cell of the newly formed plate possessing a nucleus and the centrloles towards the convex side (Fig. 95).
Division continues until a hollow sphere Is formed. Unlike the case
In the Volvocaceae, the centrloles are on the side of the cell towards the outside of the colony. Therefore, it is not necessary for the colony to invert to allow flagellar formation toward the o u ts id e .
DISCUSSION
With the exception of the flagellar apparatus, the ultrastructure of the interphase cell of Astrephomene appears similar 92 to that of members of the Volvocaceae (Lang, 1963; Deason et al.,
1969; 1971; Kochert and Olson, 1970; Plckett-Heaps, 1970; Hobbs,
1971; Gottlieb and Goldstein, 1977; Marchant, 1977; Birchem and
Kochert, 1979), as well as unicellular members of the Volvocales
Including Chlamydomonas (e.g., E ttl, 1976). Although the chloroplasts are almost devoid of thylakolds when the alga is first
placed into organic growth media, they develop internal membranes as
the culture ages. This activity is presumably correlated with the
greater contribution of photosynthesis to the energy balance of the cell which occurs due to depletion of organic nutrients.
The adult cell has a pit or groove from which the flagella
extend. This groove was not mentioned by Pocock (1953), although she did state that the cells become flattened at their anterior end prior
to division. The pit is clearly visible, however, in the micrographs of Stein (1958). This pit is not always present, as the flagellar apparatus of very young cells Is located in a pointed apex* A pit has been cited as a characteristic of the class Prasinophyceae
(Peterfl and Manton, 1968; Round, 1971), but has not been considered a normal occurrence in the Chlorophyceae. However, Hafniomonas, an alga previously thought to be a species of Pyramimonas, but
apparently more closely related to the Volvocales (Ettl and Moestrup,
1980), also possesses a pit. Although the presence of a pit In
Astrephomene and Hafniomonas may reflect an independent evolution of
that character, the finding of flagellar pits in cells that otherwise are clearly Chlorophycean, suggests that this feature should probably not be used In the delineation of a class. 93 To our knowledge, virus-like particles have not been reported for any other colonial Volvocalean alga* It is not certain if these structures are present in the chromatin of other Isolates of
Astrephomene, nor has it been determined that they are infective*
Kinetochores are present in some members of the Volvocales
(i.e ., Chlamydomonas moewusii, Triemer and Brown, 1974; Asteromonas,
Floyd, 1978; Volvox carterl. Birchem and Kochert, 1979) but they were not shown for Volvox aureus (Deason and Darden, 1971), or Eudorlna elegans (Gottlieb and Goldstein, 1977; Marchant 1977). In
Astrephomene the kinetochores are only faintly visible when stained with uranyl acetate and lead citrate, but are clearly Been when stained with potassium permanganate followed by lead citrate*
Perhaps the latter staining method would reveal kinetochores in the
Volvocales for which none were reported.
The presence of a closed mitotic spindle in the colonial
Volvocales was doubted before electron microscopic examination (Cave and Pocock, 1951), but has since been described for divisions during sperm packet formation in two species of Volvox (Deason and Darden,
1971; Birchem and Kochert, 1979), the reproductive cells of Eudorlna
(Gottlieb and Goldstein, 1977; Marchant, 1977), and the unicellular
Volvocales, Chlamydomonas (Triemer and Brown, 1974; Johnson and
Porter, 1968), Dunallella (Marano, 1976) and Asteromonas (Floyd,
1978). Volvox carter! has been reported to have large gaps over much of the nuclear envelope (Birchem and Kochert, 1979), but this unusual situation should be reexamined to rule out possible explanations such as oblique sectioning of the membrane. Species of Volvox, Eudorlna, 94 Dunallella and Chlamydomonas have been shown to possess fenestrate poles. It has been suggested that both Asteromonas and Chlamydomonas moewusli (Floyd 1978; Triemer and Brown, 1974) also have polar fenestre, but these openings are present only briefly during the division process*
Another feature common to the Volvocalean algae examined to date is the lateral position of the centrloles near the spindle poles.
The combination of the closed nuclear envelope and centrloles in this position is also found in certain green algae with persistent spindles, sometimes placed in the class Prasinophyceae (Mantoniella,
Barlow and Cattollco, 1981 and Heteromastix (Mattox and Stewart,
1977). It is possible that closed mitosis with fenestrate poles and lateral centrloles represents a primitive condition in the unicellular green algae which haB been retained in the otherwise more advanced Volvocales.
The presence of cytoplasmic bridges between cells of the developing colonies is of considerable Interest since similar bridges are found in many of the Volvocaceae (Blsalputra and Stein, 1966;
Ikushima and Maruyama, 1968; Deason, Darden and E ly,1969;
Pickett-Heaps, 1970; Deason and Darden, 1971; Gottlieb and Goldstein,
1977; Marchant, 1977; Birchem and Kochert, 1979). In some members of the Volvocaceae these bridges remain in the adult colony, while in others, as in Astrephomene, the bridges disappear during development.
The cytoplasmic bridges in Astrephomene, like those of the
Volvocaceae, have their origin in Incomplete cytokinesis, and like the Volvocaceae, the number of bridges diminish as development 95 continues. Cytoplasmic bridges, along with changes In cell shape, are thought to play an Important role In the process of Inversion In the Volvocaceae (Pickett-Heaps, 1970, Gottlieb and Goldstein, 1977;
Kelland, 1977; Marchant, 1977; Viamontes and Kirk; 1977, Viamontes,
Fochtmann and Kirk, 1979). The absence of inversion Is the primary reason that Astrephomene is placed in its own family (Pocock, 1953).
Perhaps Astrephomene, unlike members of the Volvocaceae, successfully achieves the proper cellular orientation by integrating the processes of division and cell shape changes. The presence of cytoplasmic bridges in Astrephomene Buggests that it shares a common ancestor with the Volvocaceae which may have inverted during development. Fig. 78. Vegetative ultrastructure of Astrephomene gubernaculifera. The chloroplasts have very few thylakoids and are located mainly Internal to the mitochondria. A three-layered eyespot is present in one chloroplast lobe. The nucleus has a large nucleolus* xl0,300.
Fig* 79. Section through the flagellar pit of Astrephomene. The flagella are inserted separately into the cell in a pit or groove. Note that they are nearly parallel. xl4,700.
96 f t Fig. 80. Interphase nucleus of Astrephomene. Most of the nucleolus stains densely* although leBS dense regions are also present. Note the lack of virus-like particles. x21,100.
Fig. 81. Centrioles of interphase cell in Astrephomene. Four full-sized centrioles are present. x65*600.
Fig. 82. Parental flagellum of Astrephomene. The flagellum lacks a basal body. x51,600.
98 99 Fig. 83. Prophase nucleus of Astrephomene. Although the nucleolus is still intact, chromatin has started to condense, especially near the envelope, and numerous virus-like particles are present. x35,200.
Fig. 84. Intranuclear virus-like particles in Astrephomene« These particles consist of lightly staining polygon (arrow) surrounding a darker layer which in turn contains a lighter core. x82,400.
Fig. 85. Late prophase nucleus in Astrephomene * Numerous virus-like partlcleB are associated with the condensed chromatin. The nucleolar material is partially broken down and microtubules are found inside the nuclear envelope. x30,200.
100 m sam Fig. 86. Metaphase spindle of Astrephomene. The crescent-shaped spindle is closed except at the poles. One basal body stub is lateral to the pole. The parental wall (U) is also present. x27,000.
Fig. 87. Spindle pole of Astrephomene. Some of the microtubules that arise near the basal bocfy are on the outside of the nuclear envelope) while others enter the nucleus through polar fenestrae (arrow). A microtubular rootlet (MR) extends from the region of the centriole. x51,800.
102 103 Fig. 88. Early anaphase In Astrephomene. The chromosomes have started to separate and move toward the poles. Two pairB of sister chromosomes are v isib le and the kinetochores of each member of one pair are present In the section (arrows). The centrioles are lateral to the poles of the spindle. x31,700.
Fig. 89. Kinetochore of anaphase chromosome in Astrephomene. Three layers are evident. The kinetochore microtubules are Inserted in an outer dense layer, while a clear layer, and a thin dense Inner layer are also present• CH*chromatin. x82,900.
Fig. 90. First pair of polar centrloles of Astrephomene. This micrograph is of a section serial to Fig. 11, and represents the pair of centrioles to the left in that figure. Note 2 microtubule rootlets. x53,600.
Fig. 91. Second pair of polar centrioles of Astrephomene. This micrograph is of a section serial to the right side of Fig. 11, and from the same negative as Fig. 13. Note the connections between them (arrow). This pair of centrloles has an orientation at nearly, right angles to the pair at the other pole. Also note the connection between them (arrow). x53,600.
104 105 Fig. 92. Late anaphase-early telophase in Astrephomene. The chromosomes have separated to opposite poles. Microtubules remain between the chromatin, x 27,500.
106 107 Fig. 93. Telophase In Astrephomene. Note that the chromatin has begun to disperse. The daughter nuclei In this Figure are connected by the remnants of the interzonal spindle (serial not shown). Also note the phycoplast mlcrotubles (P) which come from the direction of both pairs of centrloles (arrows), x 21,600.
Fig. 94. Phycoplast of Astrephomene. Note the numerous microtubules which line the cleavage furrow (P). Also note the small cytoplasmic bridges (arrows), x 36,600.
108 109 110
Fig. 95. Developing daughter colony of Astrephomene. The anterior end of each cell containing the nucleus and the centrioles (basal bodies; arrow) is on the convex side. Cytoplasmic bridges (CB) connect adjacent cells. x13,200. CHAPTER 6
OVERVIEW
The green algae encompass a diverse assemblage of organisms.
However, the limited number of character states visible with the unaided eye, or even the light microscope, have lead to a taxonomic system that may contain many unnatural groupings. With the utilization of electron microscopy, many aspects of green algal systematics have been refined. Especially noteworthy are the phylogenetic implications based on the ultrastructure of the mitotic and cytokinetic processes (see Pickett-Heaps, 1975; Stewart and
Mattox, 1975; Stewart, Mattox and Floyd, 1973; Mattox and Stewart,
1977), as well as the detailed ultrastructure of the flagellar apparatus Including the microtubule rootlets and striated components
(see Floyd, Hoops and Swanson, 1980; Hoops, Floyd and Swanson, in press; Stewart and Mattox, 1978; Moestrup, 1978). Newly formulated classification systems have differed from the more classical taxonomies in a number of respects. For example, certain algae traditionally placed in the Ulotrichales have a mitotic and cytokinetic apparatus like those of the land plants and unlike other members of the Ulotrichales and Chaetophorales (Floyd, Stewart and
Mattox, 1972a, 1972b; Merchant and Pickett-Heaps 1973; Pickett-Heaps
111 112 1972, 1974, 1976). In addition, the motile cells of one of these algae, Klebsormldlum (Merchant, Pickett-Heaps and Jacobs, 1973), resemble the motile cells of Coleochaete (Graham and McBride, 1979),
Chara (Pickett-Heaps, 1968), Chaetospheridlum (Moestrup, 1974), and archegoniate plants (e .g ., Robbins and Carothers, 1978). Also,
Chlorokvbus. a green alga with a sarcinold growth habit, has motile cells of similar structure (Rogers, Mattox and Stewart, 1980). Such
Information is beginning to accumulate on evolutionary relationships among members of other orders that are considered closely related by both traditional and recent criteria (see Pickett-Heaps, 1973;
Stewart and Mattox, 1975).
As an extension to the new and Improved classific atio n and phylogenetic schemes, the author has studied a number of colonial
Volvocalean algae with the light and electron microscopes for aspects of vegetative cell structure, the detailed structure of the flagellar apparatus, mode of colony movement and features of mitosis and cytokinesis.
It is believed that the colonial Volvocalean algae have descended from unicellular Chlorophyta, which were thought to resemble the present day Chlamydomonas or a probable close relative,
Haematococcus (Crow, 1918; F ritsch, 1935; Lang, 1963; Pickett-Heaps,
1975; E ttl, 1976). Recently, this has been p a rtia lly supported by the finding that mitosis in a number of these organisms, particularly during sperm formation in two species of Volvox (Deason and Darden,
1971; Blrchem and Kochert, 1979a), is sim ilar to that of
Chlamydomonas (Johnson and Porter, 1968; Cross, 1974; Triemer and 113
Brown, 1974). The author has demonstrated in these studies that the division of reproductive cells of Astrephomene resembles mitosis in both Volvox and the unicellular Volvocales*
To date, confirming evidence on other aspects of ultrastructure is lacking. Many phycologlsts seem to consider that cells of colonial members of this group are similar to each other and to
Chlamydomonas. For example in an early survey Lang (1963) stated:
"Although gross morphological differences among the
colonial flagellates have resulted in the establishment of
various genera, the ultrastructure of the individual cells
of these different genera is basically Blmllar and thus
the establishment of any taxonomic criteria based on the
ultrastructural differences is precluded...... In
Internal structure the vegetative cells of Volvox cannot
be distinguished from those of Gonium or of
Chlamydomonas."
Although there have been many ultrastructural studies on the colonial forms since then (Pickett-Heaps, 1970; Kochert and Olson, 1970; Olson and Kochert, 1970; Deason and Darden, 1971; Hobbs, 1971; Gottlieb and
Goldstein, 1977; 1979; Vlamontes and Kirk, 1977; Viamontes, Fochmann and Kirk, 1979; Blrchem and Kochert, 1979), the flagellar apparatus of the vegetative cells or sperm cells has not been c ritic a lly examined. Blrchem and Kochert (1979) have demonstrated that the sperm cells of Volvox have features sim ilar to Chlamydomonas, however detailed comparisons were not made. For example, the alternation of
4 and 2 membered microtubule rootlets is also present in a number of other green algae belonging to the Chlorophyceae and the Ulvaphyceae
(see Moestrup, 1978; Melkonlan, 1980a; Hoops et a l. In p ress). The similarities between the flagellar apparatuses of the vegetative cell of Volvox c a rte rl and Chlamydomonas were described as "impressive" by
Olson and Kochert, (1971); however this conclusion was based almost exclusively on the flag ella and basal bodies which are known to be nearly lnvarient in the green algae, and not on the other flagellar associated structures such as rootlets and striated structures» now widely used (see Moestrup, 1978; Melkonlan, 1978; Floyd et a l, 1980;
Hoops et al, in press).
In this study the flagellar apparatuses of Chlorcorona,
Pyrobotrys, and Astrephomene have been examined in d e ta il. In no case was the flagellar apparatus indistinguishable from that of
Chlamydomonas. In fact, in every case, the flag ellar apparatus of the mature alga possessed unusual features, particularly in lacking e the 180 rotational symmetry that is present in the majority of
Chlorophyceae (Floyd et al. 1980). Further, there was considerable variation among the two major types of flagellar apparatuses examined. Therefore, flagellar apparatus ultrastructure may provide useful characters for phylogenetic analyis of colonial green algae.
The similarity of the flagellar apparatuses of Pyrobotrys and
Chlorcorona, as well as the similarity of wall structure and cell to cell connections, indicate that these two algae are closely related and allows the placement of Chlorcorona, formerly of uncertain taxonomic position, into the same family as Pyrobotrys, the
Spondylomoraceae. This relationship is strengthened by several 115 flagellar apparatus features shared by these two species which are
unique among those algae studied to date*
Taxonomlcally, the situation for Astrephomene Is more difficult.
The flagellar apparatus Is very complicated, and In the mature
configuration, It Is appreciably differen t from Chlamydomonas.
Astrephomene is often (Pocock, 1953; Stein, 1958; Bold and Wynne,
1978; Starr, 1980), but not always (Bourrelly, 1976), placed in its
own family, because, unlike other spherical colonies with radially
arranged cells, it does not undergo the process of inversion (Pocock,
1953; Stein, 1958). Work is nearly complete on the flagellar
apparatuses of Volvox ro u seletii and V. c a r te r i, two algae which do
undergo Inversion and thus are placed In the Volvocaceae. The
flagellar apparatuses of these two algae are quite different from
Astrephomeme. This would appear to suggest a separate evolutionary
history for Astrephomene and the members of the Volvocaceae; however,
the fla g e lla r apparatuses of the two species of Volvox d iffe r between
themselves about as much as they d iffer from Astrephomeme. Further,
neither bears a very close resemblance to Chlamydomonas. It appears,
therefore, that the limitation of using flagellar apparatus ultrastructure for taxonomic problems within the colonial Volvocales,
is not the lack of variability, but too much variability to allow unequivical use of such information to imply a separate origin of the
colonial habit. In this regard, studies underway on Stephanosphaera
show the flagellar apparatus of this organism to be yet another different form. 116 These studies have value In addition to the taxomonlc and phylogenetic aspects* In particular, detailed fine structural studies of the flagellar apparatus provides Information about structure-function relationships* The colonial flagellates show considerable promise In this regard because they have numerous colony
types which Impose differing constraints on flagellar and colony motion* In addition* the presumed close phylogenetic relationships among the members of this group make the comparisons of differences
in the structure of the flagellar apparatus more likely to reflect differing functlonial constraints than would be the case with unrelated forms.
Cinephotomicrographic analysis is a powerful tool that can be used to correlate flagellar motion with the flagellar apparatus ultrastructure of the colonial green algae. This type of analysis has shown that the flagellar apparatus of Pyrobotrys is modified* presumably to conform to the unusual constraints placed on flagellar and colony motion by the form of the colony. In Pyrobotrys, the cells are arranged either parallel to one another, or with their anterior ends convergent. If these cells swam with a motion similar to the breastroke motion of Chlamydomonas (Ringo, 1967; Hyams and
Borisy, 1975, 1978; Schmidt and Ekert, 1976) the flag ella either would become tangled with the flag ella of the neighboring c e l l , or if the cell had an orientation that would minimize this interaction, one flagellum from each cell would project to the inside of the colony.
Some species of Pyrobotrys swim very rapidly which suggests that neither of the above conditions apply. This is confirmed by both 117 clnephotomlcrographic analysis and the detailed analysis of the structure of the flagellar apparatus.
The flagellar apparatus of this alga has apparently lost the e ancestral 180 rotational symmetry while evolutlonarlly tilting Its flagellar apparatus so the flagella extend to the side of the cell.
The flagellar apparatus doest however, retain its apical position.
Also each of the basal bodies (flagella) appears to have rotated so the anterior non-strlated fiber and the posterior non-strlated fiber are of different lengths. Clnephotomlcrographic analysis confirms that the flagella of Pyrobotrys beat in a ciliary manner, but unlike
Chlamydomonas, they beat to the side of the cell (and thus the outside of the colony). This basal body rotation reflects a change in the plane of flagellar beat. While the flagella of Chlamydomonas beat In the same plane, but in different directions (Ringo, 1967,
Hyams and Borisy, 1975), the flagella of Pyrobotrys beat in two different planes on the same side of the cell.
Clnephotomlcrographic and ultrastructural analysis have also been done on Astrephomene, here used as a representative of the spherical colonies with radially arranged cells. The flagella of the mature cells of Astrephomene are separate and nearly parallel.
Unlike Chlamydomonas, the flag ella beat In separate, p a ra llel planes, with the effective stroke of each flagellum In the same direction.
The combination of separate, nearly parallel flagella and the ciliary motion of both flagella in parallel planes Is admirably suited for movement In this organism. First, both flagella of a cell and the flagella of all cells of the colony beat in the same direction, that 118 is, towards the posterior end of the colony. Secondly, the parallel arrangement of the flagella minimizes detrimental Interference of the flagella from adjacent cells. Thirdly, the flagellar separation would minimize detrim ental interference between the two flagella of the same cell, that otherwise would occur as a result of the nearly parallel flagellar arrangement. Interestingly, other algae with a similar colony construction, in particular Volvox, share some of these features, although the detailed construction of the flagellar apparatus is different. This is not necessarily a sign of a close phylogenetic relationship; these features may be the most efficient arrangement of fla g e lla r components for any colony of sim ilar construction that Is composed of cells with isokont flagella.
The development of the fla g e lla r apparatus in Astrephomene has been examined to determine additional aspects of structure-functlon relatio n sh ip s. The immature fla g ellar apparatus is sim ilar to the flagellar apparatus of Chlamydomonas« In addition to supporting the idea that the colonial Volvocales, including Astrephomene, may be derived from a chlamydomonad ancestor, this information indicates that the changes in the flagellar apparatus during maturation are likely correlated with the colonial habit. Further, some of the changes that take place clearly implicate certain structural components as being responsible for certain aspects of m o tility . For example, the striated distal fiber does not persist in the mature stage and thus cannot be responsible for the initiation of flagellar activity. However, striated proximal fibers are present in both the chlamydomonad-stage and in the mature stage. Further, these fibers run In the same direction as the effective stroke In each of the two stages, so they are prime candidates to initiate flagellar motion.
Since each half of the flagellar apparatus In Astrephomene rotates c about 90 In development, and the direction of the effective stroke changes a comparable amount from that exiblted by Chlamydomonas (and therefore presumably the Chlamydomonad stage of Astrephomene) . there appears to be a stru c tu ra l component present in the fla g e lla r apparatus that determines flagellar beat. This is in contrast to the situation In the dilates where the direction of the flagellar beat does not appear to be determined by a stru c tu ra l component of the
fla g e lla r apparatus (Tamm and Horridge, 1970; Machmer, 1972; 1974).
Thus, the colonial Volvocales are not simply polymers of
Chlamydomonas-like cells as previously thought by many phycologists.
There Is variability in the flagellar apparatuses of members of this group which allows flagellar apparatus ultrastructure to be used for taxonomic and phylogenetic analysis. In addition the wide range of colony types and colonial motion allow certain structure-function relationships to be determined. LIST OF REFERENCES
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