PHYLOGENY OF THE ULVOPHYCEAE (CHLOROPHYTA): CLADISTIC ANALYSIS OF NUCLEAR-ENCODED rRNA SEQUENCE DATA1

Cladistic analysis of nuclear-encoded rRNA sequence data provided us with the basis for some new hypotheses of relationships within the green algal class Ulvophyceae. The orders Ulotrichales and Ulvales are separated from the clade formed by the remaining orders of siphonous and siphonocladous Ulvophyceae (Caulerpales, Siphonocladales /Cladophorales [S/C] complex, and the Dasycladales), by the Chlorophyceae and Pleurastrophyceae. Our results suggest that the Ulvophyceae is not a monophyletic group. Examination of inter- and intra-ordinal relationships within the siphonous and siphonocladous ulvophycean algae revealed that Cladophora, Chaetomorpha, Anadyomene, Microdictyon, Cladophoropsis and Dictyosphaeria form a clade. Thus the hypothesis, based on ultrastructural features, that the Siphonocladales and Cladophorales are closely related is supported. Also, the Caulerpales is a monophyletic group with two lineages; Caulerpa, Halimeda, and Udotea comprise one, and Bryopsis and Codium comprise the other. The Dasycladales (Cymopolia and Batophora) also forms a clade, but this clade is not inferred to be the sister group to the S/C complex as has been proposed. Instead, it is either the sister taxon to the Caulerpales or basal to the Caulerpales and S/C clade The Trentepohliales is also included at the base of the siphonous and siphonocladous ulvophycean clade. The Pleurastrophyceae, which, like the Ulvophyceae, posses a counter-clockwise arrangement of flagellar basal bodies, are more closely related to the Chlorophyceae than to the Ulvophyceae based on rRNA sequences. Thus, the arrangement of basal bodies does not diagnose a monophyletic group. Previously reported hypotheses of phylogenetic relationships of ulvophycean algae were tested. In each case, additional evolutionary steps were required to obtain the proposed relationships. Relationships of ulvophycean algae to other classes of green algae are discussed.     

A STUDY OF THE SPERMATOZOIDS OF DICHOTOMOSIPHON TUBEROSUS (CHLOROPHYCEAE)1

Spermatozoids of the siphonous green alga Dichotomosiphon tuberosus (A. Br.) Ernst are specialized gametes which differ in many respects from other green algal motile cells, but whose microanatomy nevertheless indicates its chlorophycean affinities. Each cell is anteriorly biflagellate and contains an irregularly shaped nucleus attached to the flagellar bases by a complex support apparatus. There is a single reduced chloroplast in each spermatozoid and numerous (50–100) minute spherical mitochondria, only 0.3 μm diam. These move vigorously in the living cell and when viewed with the light microscope they bear a striking resemblance to bacteria. Rather unexpectedly, no contractile vacuoles could be detected, even though the gametes are naked freshwater cells. Daring spermatogenesis the nucleoli of the vegetative cells disperse and are replaced by a large dense body presumably formed from either nucleolar material or condensed chromatin. The flagellar apparatus includes a cruciate flagellar root system, a feature now known to be characteristic of most green algae, exceptions being those putative ancestors of the higher plants and bryophytes. Discharge of spermatozoids from the antheridia is extremely rapid and the whole process may be finished in 30 sec. The antheridium lacks a pore apparatus, but at maturity bursts open explosively at the apex. Phyletic affinities are discussed and it is concluded that the ultrastructure of the motile cells does not, at this time, support the separation of the siphonous green algae from other green algae into a separate class.     

IMMUNOLOCALIZATION OF NITRATE REDUCTASE IN THE MARINE DINOFLAGELLATE GONYAULAX POLYEDRA (PYRROPHYTA)1

A polyclonal antibody, raised against nitrate reductase (NR) purified from the photosynthetic dinoflagellate Gonyaulax polyedra Stein, was used as a probe in immunogold-labeling experiments on thin sections prepared from cells harvested both during day and night phases. Previous experiments have shown that both NR activity and the amount of immunoreactive NR in cell extracts is greater when day-phase cells are examined, and this property was exploited as an internal control for the cytochemical labeling. We observed that in day-phase cells, chloroplasts contained approximately three times more gold particles than night-phase cells (highly significant difference; P < 0.0001), whereas cytoplasmic labeling levels remained relatively level between day and night. We conclude from the diurnal difference in labeling that our antibody faithfully reflects the distribution of NR in Gonyaulax cells. Thus, as in to some other higher plants and green algae, Gonyaulax compartmentalizes active NR in its chloroplasts.     

STRUCTURE AND ABSOLUTE CONFIGURATION OF THE FLAGELLAR APPARATUS IN THE ISOGAMETES OF BATOPHORA (DASYCLADALES,CHLOROPHYTA)1

The overall appearance of the flagellar apparatus in the isogametes of Batophora oerstedii. J. Ag. is most like that which occurs in motile cells of the Ulvophyceae. Like other Ulvophyceae, the basal bodies overlap and are arranged in the 11/5 configuration, microtubular roots are arranged in a cruciate pattern and system II striated fibers are present. The basal body connective which generally lacks striation in the Ulvophyceae is clearly different in Batophora, being composed of two large non-striated halves which connect to the anterior surface of each basal body and are then connected to one another by a distinctly fibrous centrally striated region. This variation in the basal body connective and the presence of two posteriorly directed system II striated fibers is clearly different from homologous structures reported in siphonous green algae of the Caulerpales. Based upon these variations and similarities among flagellar apparatus components in siphonous green algae, it is suggested that the Dasycladales and Siphonodadales are more closely related to one another than to the Caulerpales.     

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE MALI GAMETES OF BIDDULPHIA LEVIS EHR.1

The marine centric diatom Biddulphia levis produced uniflagellate fusiform male gametes completely within the parent cell frustule. These gametes lacked both a central pair of microtubules in the flagellar axoneme and chloroplasts but did contain a cone of microtubules which passed posteriorly from the base of the kinetosome along the nuclear envelope. The gametes were released through a specialized pore in the girdle band leaving behind a cytoplasmic mass which contained chloroplasts and other cytoplasmic components. Tubules which resembled the flimmer hairs on the gamete flagellum occurred in cisternae of the cytoplasmic reticulum in the residual cytoplasm and in the nuclear envelope of the gametes. Gametogenesis in B. levis is compared with similar processes in other centric diatoms.     

CYTOPLASMIC BIOSYNTHESIS OF LIGHT-HARVESTING POLYPEPTIDES IN THE GREEN FLAGELLATE MANTONIELLA SQUAMATA (MICROMONADOPHYCEAE)1

The biosynthesis of the light-harvesting complex (LHC) polypeptides of the green flagellate Mantoniella squamata (Manton et Parke) Desikachary (Micromonadophyceae, Chlorophyta) was examined by in vivo polypeptide labeling and immunoprecipitation of in vitro translation products. Using protein synthesis inhibitors, the LHC polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes and not in the chloroplasts of cells. Poly (A)+ RNA was isolated and proteins were synthesized by a rabbit reticulocyte lysate system, with antisera raised against M. squamata LHC used for immunoprecipitation from the translation products. One polypeptide 3-5 kDa larger than mature LHC polypeptides was immunoprecipitated. These studies indicate that although the LHC of M. squamata is quite different from the LHC of most green plants, the LHC polypeptides are synthesized as precursors in the cytoplasm of the cell and suggest that the genes encoding these polypeptides are located in the nucleus.     

THE QUANTITATIVE ULTRASTRUCTURE OF THE MERISTEMATIC CELLS OF XANTHIUM STRUMARIUM DURING THE TRANSITION TO FLOWERING

Ultrastructural changes occurring in the central part of the apical meristem of the SDP Xanthium strumarium, induced to flower by a single 16-hr long night, were quantitatively investigated using stereological methods and compared to the changes previously reported in other species, particularly the LDP Sinapis alba. Changes detected in Xanthium, which are also found in other species, included: increase in cellular, cytoplasmic, cytoplasmic matrix and nucleolar sizes, change in nucleolar structure; increase in mitochondrial number and chondriome size, increase in dictyosome number. These changes are believed to be essential for floral evocation because of their universality. Other changes were specific to Xanthium and not detected in Sinapis. Accordingly, they were thought to be accompanying nonessential events of floral evocation in Xanthium. These changes included an increase in the number of plastid profiles and in plastidome size. The size of the nucleus, chromatin and vacuolar apparatus, as well as the number of vacuolar profiles, did not change in Xanthium, contrary to what was observed in other plants.     

CHLOROPLAST SPECIALIZATION IN DICOTYLEDONS POSSESSING THE C4-DICARBOXYLIC ACID PATHWAY OF PHOTOSYNTHETIC CO2 FIXATION

The C₄-dicarboxylic-acid pathway of photosynthetic CO₂ fixation found in tropical grasses has recently been demonstrated in members of the Amaranthaceae and Chenopodiaceae. In the tropical grasses this CO₂-fixation pathway is correlated with specialized leaf anatomy and chloroplast structure. This investigation was undertaken to determine if leaf cells of some representatives of these other families had structural features similar to those of tropical grasses. The leaf anatomy of Amaranthus edulis and a variety of Atriplex species is very similar and it resembles that of grasses such as sugar cane. Prominent bundle sheaths are surrounded by a layer of palisade cells. Bundle-sheath cells of Am. edulis have large chloroplasts containing much starch, but the chloroplasts have grana. The palisade cells have much smaller chloroplasts containing very little starch. The bundle-sheath cell chloroplasts of At. lentiformis have grana, their profiles tend to be ovoid, and they contain abundant starch grains. The palisade cell chloroplasts contain little starch and their profiles are discoid. The bundle-sheath cells of both species contain mitochondria which are much larger than those in the palisade cells. The chloroplasts in both types of cells in both species have a highly developed peripheral reticulum. This reticulum is composed of anastomosing tubules which are contiguous with the inner plastid membrane. The leaf anatomy and cell ultrastructure of these dicots are similar to those of the tropical grasses possessing this new photosynthetic carbon-fixation pathway. These morphological features are interpreted as adaptations for the rapid transport of precursors and end products of photosynthesis. A hypothesis is presented stating that the unique morphological and biochemical characters of these plants represent adaptations for efficient and rapid carbon fixation in environments where water stress frequently limits photosynthesis.     

THE RPS12 GENE IN SPROGYRA MAXIMA (CHLOROPHYTA) AND ITS EVOLUTIONARY SINGNIFICANCE1

The str operon consists fo four genes in eubacteria. Portions of his operon are conserved in the chloroplast genomes of green algae and land plants. In land plant chloroplasts, the str operon comprises only two genes, rps12 and rps7, and is arranged in a trans-spliced state. Since no other previously studied chloroplast genome contains this arrangement, and because the charophyte lineage is the sister group of land plants, we chose to look for this arrangement in the Charophyceae. The two str genes, rps12 and rps7, present in the chloroplasts of Spirogyra maxima Hanssall, were identified by hybridization of a Southern blot and requenced. The results indicate that Spirogyra contains a str operon almost identical to that of land plant chloroplasts. Based upon the structure of the operon in other chloroplasts and eubacterial genomes, the trans-spliced state most likely evolved early within the charophyte lineage.     

THE DEVELOPMENT AND POTENTIAL ROLES OF CELL‐WALL TRABECULAE IN CAULERPA MEXICANA (CHLOROPHYTA)1

Siphonous plants represent an alternate scheme to the way most macroscopic plants are constructed. They are single, often large (1–2 m), sometimes morphologically complex, multinucleate (coenocytic) cells where the whole of the cytoplasm is a continuum. Caulerpa mexicana Sond. ex Kütz. is a siphonous tropical marine green alga characterized by four morphologically distinct regions and, as with other members of the genus, by the presence of a dense network of anastomosing cylindrical cell wall in growths called trabeculae. Based on the results of this study, we propose several roles for trabeculae: (i) They are structural components, which likely add some small amount of support in compression but add considerable strength in tension. (ii) As extensions of the cell wall and plasma membrane, they act as diffusion channels from the cell exterior to the interior cytoplasm. It is possible that trabeculae also play a role in determining cell shape through developmental positioning and placement patterns, thus facilitating the diverse shapes found in the morphologically distinct regions of Caulerpa sp.     

Electrophysiology of Turgor Regulation in Marine Siphonous Green Algae

We review electrophysiological measures of turgor regulation in some siphonous green algae, primarily the giant-celled marine algae, Valonia and Ventricaria, with particular comparison to the well studied charophyte algae Chara and Lamprothamnium. The siphonous green algae have a less negative plasma membrane potential, and are unlikely to have a proton-based chemiosmotic transport system, dominated by active electrogenic K⁺ uptake. We also make note of the unusual cellular structure of the siphonous green algae. Hypertonic stress, due to increased external osmotic pressure, is accompanied by positive-going potential difference (PD), increase in conductance, and slow turgor regulation. The relationship between these is not yet resolved, but may involve changes in K⁺ conductance (G _K) or active K⁺ transport at both membranes. Hypotonic turgor regulation, in response to decreased external osmotic pressure, is ∼3 times faster than hypertonic turgor regulation. It is accompanied by a negative-going PD, although conductance also increases. The conductance increase and the magnitude of the PD change are strongly correlated with the magnitude of hypotonic stress.     

EVIDENCE THAT THE NUCLEOMORPHS OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYCEAE) ARE VESTIGIAL NUCLEI: MORPHOLOGY,DIVISION AND DNA-DAPI FLUORESCENCE1

Chlorarachnion reptans Geitler shows affinities to both the Chlorophyceae and the chloroplast endoplasmic reticulum-containing chromophyte algae in possessing chlorophyll b and chloroplasts which are limited by four membranes, respectively. In the periplastidal compartment surrounding each of the four to eight chloroplasts of a C. reptans cell are putative eukaryotic-sized ribosomes, scattered tubules and vesicles, and a small double-membrane-limited nucleus-like organelle named the nucleomorph. The nucleomorphs display 4′-6-diamidino-2-phenylindole (DAPI)fluorescence which is sensitive to DNase digestion, but not to treatment with RNase. The nucleomorphs also contain a fibrillogranular body which resembles a nucleolus. Nucleomorph division occurs by the sequential infolding of the inner and outer envelope membranes and subsequent constriction in two, with no involvement of microtubules. In all these characteristics, the nucleomorphs of C. reptans are similar to the cryptomonad nucleomorph which has been hypothesized to be the vestigial nucleus of an ancestral red alga which gave rise to the chloroplasts of the Cryptophyceae. The presence of chlorophyll b and the contents and morphology of C. reptans chloroplast compartments suggest a green algal origin for the chloroplasts of these cells. The discovery of a second organism with a DNA-containing, nucleus-like organelle in its chloroplast compartment lends strong support to the hypothesis that the chloroplasts of many algae have evolved from eukaryotic endosymbionts.     

TAXONOMIC SIGNIFICANCE OF NUCLEUS-MICROBODY ASSOCIATIONS,SEGREGATED NUCLEOLI AND OTHER NUCLEAR FEATURES IN SIPHONOUS GREEN ALGAE1

On the basis of fine-structural features of nuclei the “true” siphonous green algae (Eusiphoniidae) are divided into three groups. In the Codium group nuclei are generally similar to those in most other algae. The Penicillus group is characterized by the association of microbodies with nuclei. In the Avrainvillea group nucleoli are segregated into a granular and a fibrillar component. This condition, known as macrosegregation, persists throughout the vegetative phase of the life history and may indicate a peculiar ribosomal RNA cycle. The Penicillus group corresponds to the order Caulerpales Feldmann. Avrainvillea and Cladocephalus probably constitute a new order. Blastophysa, although having a type of segregated nucleoli, is probably more closely related to Siphonocladales than to any order in Eusiphoniidae.     

An interconnected plastidom inAcetabularia: Implications for the mechanism of chloroplast motility

Summary In the unicellular green algaAcetabularia, the vital fluorochrome 3,3′-dihexyloxacarbocyanine (DiOC₆) readily accumulates in chloroplasts and mitochondria at low concentrations, suboptimal for the visualization of the endoplasmic reticulum (ER). These organelles align along motility tracks and partially obscure each other, resulting in the loss of image information in conventional fluorescence microscopy. However, superior imaging of organelles was achieved by confocal laser scanning microscopy, which was particularly evident in areas where mitochondrial profiles overlap with chloroplasts. In addition to the tubular mitochondria, a new type of tubular membrane profiles was discovered inAcetabularia which connects the chloroplasts with each other. These tubules may either form short bridges or may stretch over hundreds of micrometers before connecting to the next chloroplast. Because staining intensity, size and overall shape of mitochondria and the connecting membrane tubules were very similar, pharmacological treatments have been applied to differentiate more clearly between the two compartments. Inhibitors of mitochondrial function are shown here to affect mitochondrial shape but not that of the chloroplast tubules. Finally, electron microscopic analysis of thin sectioned materials revealed long tubular emanations from the chloroplasts proving their plastidal origin. The function of these hitherto unknown plastidal membrane tubules is not known, but their behaviour suggests that they interact with the cytoskeleton and effectively modify chloroplast behaviour.     

Studies on Halimeda IV. An endogenous rhythm of chloroplast migration in the siphonous green alga,H. distorta 1

Abstract

Halimeda has been found particularly suitable for studies of long‐distance chloroplast migration by virtue of its coenocytic structure and calcium carbonate skeleton. A circadian rhythm of chloroplast migration in Halimeda distorta was monitored by videography of segment surface pigmentation. In normal 12 h light/12 h dark treatments synchronised with dawn and dusk, the segments were green all day, began to become pale immediately the light was turned off, and then remained almost white for most of the night until beginning to re‐green a few hours before dawn. As a result of that, they were already quite green by the time the light was turned on. In continuous darkness a similar cycle, albeit with reducing amplitude and a period of about 23 hours, was maintained for at least 7 days. However, this cycle differed significantly from the normal one in that the segments did not remain green after the light was not switched on at dawn, but rather began to pale immediately thereafter. Conversely, in continuous light the segments did not become pale at any time. Thus, the rhythmical re‐emergence of the chloroplasts before dawn and their subsequent withdrawal appears to be controlled by an endogenous rhythm which is independent of light. However, light does completely, but reversibly, inhibit the chloroplast withdrawal component of the cycle. This behaviour of the chloroplasts in Halimeda is very similar to that in the related alga, Caulerpa, but it is quite different from that in another extensively Studied but unrelated siphonous green alga, Acetabularia, in which the circadian rhythm of chloroplast migration is maintained in continuous light.     

FURTHER EVIDENCE FOR OSMOREGULATION IN EPIDERMAL LEAF CELLS OF SEAGRASSES

A comparison of the epidermal leaf cell ultrastructure of three seagrasses, Thalassia testudinum (tropical, high salinity), Zostera marina (North temperate, moderate salinity), and Ruppia maritima (North temperate, brackish) provides confirmation for the theory that an invaginated plasmalemma-mitochondrial transport system is developed at least in part as a response to salt concentration. Cytochemical localization of presumed Cl⁻ ion provides further evidence for the presence of a salt secretion or exclusion mechanism. Immature epidermal leaf cells communicate with each other and with mesophyll cells through numerous plasmodesmata, but during cell maturation these cytoplasmic connections are lost and the apoplastic transport system develops to replace the symplastic one. The two North temperate region seagrasses contain cytoplasmic lipids which are absent in the tropical species. Thalassia and Zostera have chloroplasts which lack starch, but stain densely for polysaccharides with thiocarbohydrazide. The polysaccharide staining is essentially negative in the chloroplasts of Ruppia, but mesophyll chloroplasts of this brackish water species contain starch. These and other cytological findings are compared with other seagrasses.     

PHOTOSYNTHETIC PIGMENT COMPOSITION IN THE PRIMITIVE GREEN ALGA MESOSTIGMA VIRIDE (PRASINOPHYCEAE): PHYLOGENETIC AND EVOLUTIONARY IMPLICATIONS1

The photosynthetic pigment composition of Mesostigma viride Lauterborn, a primitive green alga, was determined. This alga contained chl a and b, lycopene, lutein, siphonaxanthin, γ‐carotene, β‐carotene, antheraxanthin, violaxanthin, neoxanthin, and two novel carotenoid fatty acid esters, siphonaxanthin C12:0 ester and siphonaxanthin C14:0 ester. The esters were saturated, whereas all previously identified siphonaxanthin and loroxanthin esters have been mono‐unsaturated (trans‐Δ2). Neoxanthin was the all‐trans form. This is the first such case detected in the chloroplasts of green plants. The 9′‐cis form of neoxanthin is believed to be universally present in the chloroplasts of green plants (Streptophyta and Chlorophyta) and is a precursor of abscisic acid. However, the 9′‐cis form was not found in M. viride. Based on these results, we discuss the phylogenetic implications and early evolution of the antenna pigment system in green plants.     

THE ASSOCIATION OF A SINGLE B-CHROMOSOME WITH MALE STERILITY IN PLANTAGO CORONOPUS

Paliwal , Ripsudan L. (B. R. College, Agra, India.), and Beal B. Hyde . The association of a single B-chromosome with male sterility in Plantago coronopus. Amer. Jour. Bot. 46(6): 460–466. Illus. 1959.—Two species of Plantago showing male sterility have been studied cytogenetically. In P. ovata (n=4) the sterility appears to be cytoplasmic. In P. coronopus (n=5) all male-sterile plants contain a single extra chromosome which is largely heterochromatic, shorter, and not homologous with any of the other chromosomes. No male-fertile plants contain this B-chromosome. Meiosis is regular in the male-sterile lines. The accessory chromosome usually does not divide and moves to one pole at the first division of meiosis and divides regularly in the second division. Degeneration of all microspores occurs before pollen mitosis. Male-sterile plants are apomictic, but whether or not male-fertile plants are also apomictic has not yet been determined.     

PHYLOGENY OF LOBOCHARACIUM (CHLOROPHYCEAE) AND ALLIES: A STUDY OF 18S AND 26S rDNA DATA1

The phylogenetic affinities of Lobocharacium coloradoense were investigated by analysis of combined 18S and 26S rDNA data. Results from both parsimony and likelihood methods supported a close alliance among Lobocharacium, Characiosiphon, and Characiochloris. These three taxa formed a clade near the base of the “Dunaliella” group within the chlamydomonad lineage. Protosiphon, which exhibits a siphonous habit similar to Characiosiphon and Lobocharacium, was not resolved as a close ally of the latter two taxa. The Lobocharacium alliance was characterized by the presence of an attachment pad associated with the nonmotile vegetative stage and pyrenoids that possess cytoplasmic invaginations. The pyrenoid feature is an ultrastructural trait that has now been observed in five different chlorophycean lineages. The Lobocharacium–Characiosiphon–Characiochloris clade is not predicted by any classifications of green algae. Additional taxon and data sampling need to be completed to resolve inconsistencies between the molecular phylogenetic evidence and at least some of the current family‐level taxa.     

Co-Localization of Particle Transport with Microtubules in Cytoplasmic Exudates of the Siphonous Green Alga Bryopsis

Organelles in the cortical cytoplasm of the siphonous green alga Bryopsis display various types of motile activities. One of them, saltatory movement along axially oriented linear tracks is typical for mitochondria and other small particles. A method is described which allows in vitro observation of such movements in thin layers of cytoplasm extruded from the alga and attached to a poly-l -lysine coated glass surface. By comparing video recordings of motile activities with the position of cytoskeletal elements visualized by immunofluorescence in the same area of a cytoplasmic exudate, it can be shown that tracks along which particles have moved in vitro are identical with microtubules (MTs). Depolymerization of MTs in the cytoplasmic exudates by MT-specific inhibitors stops particle movement, whereas depolymerization of actin filaments with cytochalasin D disrupts actin bundles but has little effect on particle motility. These data are consistent with the model of MT guided particle transport.