SOMATIC CELL FLAGELLAR APPARATUSES IN TWO SPECIES OF VOLVOX (CHLOROPHYCEAE)1

The somatic cell flagellar apparatuses of Volvox carteri f. weismannia (Powers) Iyengar and V. rousseletii G. S. West have parallel or nearly parallel basal bodies which are separated at their proximal ends. The four microtubular rootlets alternate between two and four members, and all are associated with a striated microtubular associated component (SMAC) that runs between the basal bodies. In addition, each half of the flagellar apparatus apparently rotates during development and loses the 180° rotational symmetry characteristic of most unicellular chlorophycean motile cells. All of these features appear necessary for efficient motion of a colony composed of numerous radially arranged cells. However, the structural details of the flagellar apparatuses of these two species differ. The distance between flagella is greater in V. rousseletii than in V. carteri. One distal striated fiber and two proximal striated fibers connect the basal bodies in V. carteri, but both types of fibers are absent from V. rousseletii. In the latter species, a striated fiber wraps around each of the basal bodies and attaches to the rootlets and the SMAC. No such fiber is present in V. carteri. Since the similarities in the flagellar apparatuses can be explained as a result of adaptation for efficient colonial motion in organisms with similar colonial morphology, the differences suggest a wider phylogenetic distance than previously believed.     

The Simplest Integrated Multicellular Organism Unveiled

Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR ORIENTATION IN THE COLONIAL GREEN ALGA GONIUM PECTORALE (VOLVOCALES)1

Vegetative cells of Gonium pectorale have a fine structure similar to that of Chlamydomonas. In addition, three zones comprise an extracellular matrix; a fibrillar sheath and tripartite boundary surround individual cells, and a fragile capsule zone surrounds the entire colony. Cytokinesis is accomplished by a phycoplast and cleavage furrow. The flagellar apparatus of the immature vegetative cell of this colonial alga is similar to that of Chlamydomonas, but the basal bodies are slightly separated at their proximal ends. The four microtubular rootlets alternate between two and four members. During development, the basal bodies become further separated and nearly parallel. The distal fiber is stretched, but it remains attached to both basal bodies. At maturity, the basal bodies of peripheral cells of the colony have rotated in opposite directions on their longitudinal axes resulting in a displacement of the distal fiber to one side, an asymmetrical orientation of the rootlets and loss of 180° rotational symmetry. Central cells remain similar to Chlamydomonas in that basal bodies do not rotate, rootlets are cruciate, the distal fiber remains medially inserted and 180° rotational symmetry is conserved. A “pin-wheel” configuration of flagellar pairs and the orientation of parallel rootlets toward the colony perimeter probably accounts for the rotation of the colonies during forward swimming. In addition, these ultrastructural features support the traditional placement of G. pectorale as an intermediate between the unicellular Chlamydomonas and the more complex colonial volvocalean genera.     

Do Protoplasmic Connections Function in the Phototactic Coordination of the Volvox Colony During Light Stimulation?*

SYNOPSIS. The flagellar behavior of the colonial flagellates Volvox carteri Stein and Volvox perglobator Powers was examined by placing 1.01 μm polystyrene particles in solution with swimming colonies, and photographing these particle movements. When directional light stimulation was administered to individual colonies, a cessation of flagellar activity occurred in the anterior cells of the stimulated side in both species. Since Volvox perglobator possesses prominent intercellular connections and Volvox carteri does not, the results of these experiments suggest that the connections linking colony members in some species do not function in the coordination of flagellar activity associated with light orientation behavior.     

Mitochondrial and Plastid Genomes of the Colonial Green Alga Gonium pectorale Give Insights into the Origins of Organelle DNA Architecture within the Volvocales

Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion.     

Stable nuclear transformation of Gonium pectorale

Background  

Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri.     

THE FLAGELLAR PHOTORESPONSE IN VOLVOX SPECIES (VOLVOCACEAE,CHLOROPHYCEAE)1

Steering their swimming direction toward the light is crucial for the viability of Volvox colonies, the larger members of the volvocine algae. While it is known that this phototactic steering is achieved by a difference in behavior of the flagella on the illuminated and shaded sides, conflicting reports suggest that this asymmetry arises either from a change in beating direction or a change in beating frequency. Here, we report direct observations of the flagellar behavior of various Volvox species with different phyletic origin in response to light intensity changes and thereby resolve this controversy: Volvox barberi W. Shaw from the section Volvox sensu Nozaki (2003) changes the direction of the flagellar beating plane, while species encompassed in the group Eudorina (Volvox carteri F. Stein, Volvox aureus Ehrenb., and Volvox tertius Art. Mey.) decrease the flagellar beating frequency, sometimes down to flagellar arrest.     

ULTRASTRUCTURE OF THE FLAGELLAR APPARATUS OF PYROBOTRYS (CHLOROPHYCEAE)1

The flagellar apparatus of Pyrobotrys has a number of features that are typical of the Chlorophyceae, but others that are unusual for this class. The two flagella are inserted at the apex, but they extend to the side of the cell toward the outside of the colony, here designated as the ventral side. Four basal bodies are present, two of which extend into flagella. Four microtubular rootlets alternate between the functional and accessory basal bodies. In each cell, the two ventral rootlets are nearly parallel, but the dorsal rootlets are more widely divergent. The rootlets alternate between two and four microtubules each. A striated distal fiber connects the two functional basal bodies in the plane of the flagella. Two additional, apparently nonstriated, fibers connect the basal bodies proximal to the distal fiber. Another striated fiber is associated with each four-membered rootlet near its insertion into the flagellar apparatus. A fine periodic component is associated with each two-membered rootlet. A rhizoplast-like structure extends into the cell from each of the functional basal bodies. The arrangement of these components does not reflect the 180° rotational symmetry that is usually present in the Chlorophyceae, but appears to be derived from a more symmetrical ancestor. It is suggested that the form of the flagellar apparatus is associated with the unusual colony structure of Pyrobotrys.     

Ultrastructure of the quadriflagellate zoospores of the filamentous green algaeChaetophora incrassata andPseudoschizomeris caudata (Chaetophorales,chlorophyceae) with emphasis on the flagellar apparatus

Quadriflagellate zoospores ofChaetophora incrassata andPseudoschizomeris caudata have similar features including an appressed membrane between the pyrenoid matrix and the starch sheath, and identical flagellar apparatuses. Components of the flagellar apparatus include: directly opposed upper basal bodies, lower basal bodies in the clockwise absolute orientation, a grooved distal fiber, peripheral and terminal fibers between adjacent basal bodies, proximal fibers connecting the lower basal bodies to the X-membered rootlets two- and X-membered rootlets associated with electron-dense components, and at least one rhizoplast. The X-membered rootlets, are comprised of five microtubules inC. incrassata and four or five inP. caudata. These features of the flagellar apparatus suggest that the two algae are closely related, and together withStigeoclonium, Uronema, Draparnaldia andFritschiella, form a natural group, the Chaetophoraceae, Chaetophorales (sensu Mattox and Stewart).     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR POSITION IN THE COLONIAL GREEN ALGA PLATYDORINA CAUDATA (CHLOROPHYCEAE)1

The ultrastructure of the flagellar apparatus in pre-inversion and inversion stages of Platydorina resembles that of Chlamydomonas in having 180° rotational symmetry and clockwise absolute orientation. Basal bodies are in a “V” configuration and connected by one distal and two proximal fibers. Alternating two- and four-membered microtubular rootlets are cruciately arranged. During maturation, the basal bodies rotate and separate, and 180° rotational symmetry is lost. Simultaneously, each proximal fiber detaches from one of the functional basal bodies, and the distal fiber detaches from both. The mature apparatus has widely separated and nearly parallel basal bodies. Flagellar orientation in Platydorina is completed just after inversion and a flattening of the colony called intercalation, resulting in the pairs of flagella of neighboring cells extending from the colony in opposite directions in an alternating fashion. Flagellar orientation and separated basal bodies minimize the interference between the flagella of neighboring cells. Basal bodies and rootlets of the two intercalated halves of a colony rotate, resulting in the effective strokes of the flagella of every cell being towards the colonial posterior. The flagella of each cell beat with an effective stroke in the direction of the two inner rootlets. The flagella have an asymmetrical ciliary type beat. The rotated, separated, and parallel basal bodies, together with the nearly parallel rootlets probably are adaptations for movement of this colonial volvocalean alga. The flagellar apparatus in immature stages of Platydorina lends support to the suggestion that the alga has evolved from a Chlamydomonas-like ancestor.     

Three‐dimensional organization of flagellar basal apparatus in Ectocarpus gametes

The flagellar basal apparatus of the brown alga Ectocarpus siliculosus was re‐investigated in details using transmission electron microscopy and electron tomography. As a result, three‐dimensional structures with spatial arrangement of bands and microtubular flagellar rootlets were observed. Fibrous structures linking the anterior flagellar basal body to the major anterior rootlet (R3) or the bypassing rootlet was newly discovered in this study. A direct attachment from the minor anterior rootlet (R4) to the anterior and posterior basal bodies was also discovered, as were attachments from the minor posterior rootlet (R1) to the deltoid striated band and from the major posterior rootlet (R2) to the posterior fibrous band. The microtubular flagellar rootlets were connected to the bands and to the anterior or posterior basal body. These bands may have a role in maintaining the spatial arrangement of the anterior and posterior flagellar basal bodies and the microtubular flagellar rootlets. A numbering system of the basal body triplets was established by tracing axonemal doublets in the serial sections. From these observations, the precise position of two flagellar basal bodies, bands, and flagellar rootlets was determined.     

Cleavage, incomplete inversion, and cytoplasmic bridges in Gonium pectorale (Volvocales, Chlorophyta)

Multicellularity arose several times in evolution of eukaryotes. The volvocine algae have full range of colonial organization from unicellular to colonies, and thus these algae are well-known models for examining the evolution and mechanisms of multicellularity. Gonium pectorale is a multicellular species of Volvocales and is thought to be one of the first small colonial organisms among the volvocine algae. In these algae, a cytoplasmic bridge is one of the key traits that arose during the evolution of multicellularity. Here, we observed the inversion process and the cytoplasmic bridges in G. pectorale using time-lapse, fluorescence, and electron microscopy. The cytoplasmic bridges were located in the middle region of the cell in 2-, 4-, 8-, and 16-celled stages and in inversion stages. However, there were no cytoplasmic bridges in the mature adult stage. Cytoplasmic bridges and cortical microtubules in G. pectorale suggest that a mechanism of kinesin-microtubule machinery similar to that in other volvocine algae is responsible for inversion in this species.     

ABSOLUTE CONFIGURATION ANALYSIS OF THE FLAGELLAR APPARATUS IN CLADOPHORA AND CHAETOMORPHA MOTILE CELLS,WITH AN ASSESSMENT OF THE PHYLOGENETIC POSITION OF THE CLADOPHORACEAE (ULVOPHYCEAE,CHLOROPHYTA)

Absolute configurational analyses of flagellar apparatus components were performed on the motile cells produced by three species of Cladophora, Cl. dalmatica Kütz., Cl. flexuosa (Dillw.) Harv., and Cl. glomerata (L.) Kütz., and by Chaetomorpha aerea (Dillw.) Kütz. There was little variation among the species. All of the flagellar apparatuses demonstrated the ulvophyceous features of 180° rotational symmetry, counterclockwise absolute orientation, and basal body overlap, as well as the alignment of the basal bodies perpendicular to the long axis of the cell. Diagnostic features included the nearly complete absence of C tubules from the basal bodies and the presence of a coarsely striated component dorsal to the two-membered rootlets in all cells, as well as, in quadriflagellate cells, a tetralobate distal fiber, the coaxial arrangement of the lowermost pair of basal bodies, and the presence of a characteristic array of basal-body-associated striated bands. The distal fiber architecture, the presence of a “wing” in the X-membered rootlets, and the “flattening” of the flagellar apparatus components suggests a close relationship of the Cladophoraceae to the Dasycladales, and indicates that these two groups may have shared a common ancestor, possibly ancient in terms of the geological time scale but relatively recent in the context of ulvophyceous evolution. A sizeable phylogenetic gap exists between the Cladophoraceae and uninucleate-celled, presumably primitive members of the Ulvophyceae.     

The absolute configuration of the flagellar apparatus in zoospores from two species ofLaminariales (Phaeophyceae

Summary We examined the zoospores produced by the unilocular sporangia ofLaminaria digitata (L.) Lamour. andNereocystis luetkeana Post. & Rupr. by serial sectioning to determine the absolute configuration of their flagellar apparatuses. The basal bodies, which are interconnected by three striated bands, lie parallel to the ventral face of the zoospore, and the posterior basal body always is found to the right of the anterior basal body when the cell is viewed from the ventral face, anterior end up. The four rootlets associated with the basal bodies include a major anterior rootlet of about seven microtubules extending from the anterior basal body along the ventral face towards the apex, a five-membered bypassing rootlet that passes ventral to the basal bodies and is connected to the posterior basal body by a posterior fibrous band, and two short rootlets having a single member each, the minor anterior and posterior rootlets. We consider the configuration observed here to be typical of most phaeophycean motile cells. The flagellar apparatus features suggest a considerable phylogenetic difference between thePhaeophyceae and other classes of chlorophyll c-containing organisms.     

Mitosis and cytokinesis in androgonidia ofVolvox carteri f.weismannia

Summary Reproductive cells (androgonidia) ofVolvox carteri f.weismannia divide to form packets of 64 or 128 sperm cells. The androgonidium morphology, stages of mitosis, and cytokinesis were examined by electron microscopy. The biflagellate androgonidium loses its flagella before mitosis but the flagellar bases at the anterior end of the cell are retained. Two additional basal bodies are formed and the nucleus migrates from its central position to the area of the basal bodies before mitosis begins. A five-layered kinetochore is present on the chromosomes and remnant nucleolar material persists during mitosis. A furrow at the chloroplast end of the cell and the formation of phycoplast microtubules and vesicles signal the beginning of cytokinesis at early telophase. The cells maintain cytoplasmic connections until after the packet of sperm cells completes its development.     

THE FLAGELLAR APPARATUS OF ENTOCLADIA VIRIDIS MOTILE CELLS,AND THE TAXONOMIC POSITION OF THE RESURRECTED FAMILY ULVELLACEAE (ULVALES,CHLOROPHYTA)1

The flagellar apparatuses of the quadriflagellate zoo-spores and biflagellate female gametes of the marine chaetophoracean alga Entocladia viridis Reinke are significantly different from those of algae belonging to Chaetophoraceae sensu stricto, but closely resemble those of ulvacean genera. These differences permit the taxonomic reassignment of certain marine chaetophoracean genera and an evaluation of the flagellar apparatus features used to characterize the class Ulvophyceae. Critical features of the zoospore include arrangement of the four basal bodies into an upper and a lower pair with the proximal ends of the upper basal bodies overlapping, terminal caps, proximal sheaths connected to one another by striated bands, and a cruciate microtubular rootlet system having a 3-2–3-2 alternation pattern and striated microtubule-associated components that accompany the two-membered rootlets. An indistinct distal fiber occurs just anterior to the basal bodies, and is closely associated with the insertion into the flagellar apparatus of the three-membered rootlets. The flagellar apparatus demonstrates 180° rotational symmetry, and its components show counterclockwise absolute orientation when viewed from above. Newly described features include the prominently bilobed structure of the terminal caps on the upper basal body pair, and the presence of both a granular zone and an additional single microtubule anterior to each of the four rootlets, an arrangement termed the “stacked rootlet configuration.” Rhizoplasts were not observed and are presumed to be absent. The gamete is identical, except for the absence of the lower basal body pair and the presence of an electron-dense membrane associated structure that resembles the mating structure found in Ulva gametes. These findings, correlated with life history data, sporangial and gametangial structure and developmental patterns, chloroplast pigment arrays, and vegetative cell ultrastructural features, compel the removal of Entocladia viridis and similar members of the marine Chaetophoraceae to a separate family, the Ulvellaceae. The latter is referred to the order Ulvales of the Ulvophyceae. The counterclockwise absolute orientation of components, and terminal caps, may be the most consistent flagellar apparatus features of ulvophycean green algae, while variations in other features previously considered diagnostic for the Ulvophyceae may serve instead to identify discrete lineages within this class.     

A COMPARATIVE CYTOCHEMICAL STUDY OF VOLVOCACEAN MATRIX POLYSACCHARIDES1

The colonial matrices of the volvocacean algae were examined for the presence of sulfated and carboxylated polysaccharides. These results were compared to a similar examination of the single-celled Chlamydomonas reinhardtii Dang. The colonial algae examined were Pandorina morum Bory, Eudorina elegans Ehr., Platydorina caudata Kofoid, Pleodorina californica Shaw, Pleodorina illinoisensis Kofoid and Volvox carteri var. nagariensis Iyengar. Alcian blue staining of whole colonies at pH 0.5 and 2.5 showed evidence for the presence of both sulfated and carboxylated polysaccharides in the extracellular matrix. Quantitative measurement of alcian blue bound to solubilized matrices supported the in vivo results. There was a trend toward an increase in sulfated polysaccharides in the more evolutionary advanced forms with the exception of Pleodorina. This trend was readily seen in the sulfate: carboxyl ratios: Pandorina morum—0.4, Eudorina elegans—1.0, Platydorina caudata—2.1 and Volvox carteri—2.2. The acidic nature of the Pleodorina matrix with a sulfate: carboxyl ratio of 0.2 appeared to be more like that of Pandorina rather than that of the more advanced Volvox.     

Comparative Analysis of Embryonic Inversion in Algae of the Genus <Emphasis Type="Italic">Volvox</Emphasis> (Volvocales,Chlorophyta)

Recent literary data on inversion (turning inside out) in the embryos of flagellated algae of the genus Volvox are critically analyzed. In this process, active changes in the shape of embryonic cells and the displacement of intercellular cytoplasmic bridges play an important role. After inversion, the flagella appear on the outer side of the young colony and provide its motility. Within the genus Volvox, two main modes of embryo inversion have been recently established during the asexual developmental cycle—inversion of type A and inversion of type B—represented by the two species most thoroughly studied, respectively, Volvox carterif. nagariensis and V. globator. However, the published opinion that the inversion of V. aureus embryos is of the type B seems to be doubtful. Comparative and evolutionary aspects of embryonic inversion in Volvox are discussed with the use of data on other genera of colonial volvocine algae.     

Ultrastructure of Volvox carteri

Summary Somatic cells of mature asexual colonies of Volvox carteri do not possess a true cell wall, but are otherwise similar in ultrastructure to Chlamydomonas. Somatic cells are embedded in multilayered fibrillar material of the colonial matrix. The reproductive cells (gonidia) of Volvox carteri lie internal to the somatic cell layer of the colony matrix in an apparently structureless portion of the colony matrix. Mature gonidia are large vacuolate cells with a central nucleus and parietal chloroplasts and mitochondria. They are non-flagellated at maturity, but each contains a pair of kinetosomes.