DISTRIBUTION OF THE XANTHOPHYLL LOROXANTHIN IN DESMIDS (CHAROPHYCEAE,CHLOROPHYTA)1

Forty-seven species of desmids, representing all four families, were examined for the presence of the xanthophyll loroxanthin by reverse-phase high-performance liquid chromatography. In the Desmidiaceae 28 of the 35 species examined possessed loroxanthin, and in the Mesotaeniaceae two of the six examined had loroxanthin present. All six species of the families Peniaceae and Closteriaceae examined possessed loroxanthin. Although the distribution of loroxanthin appears to be disjunct in the desmids and does not have strict taxonomic significance, it does follow a coherent pattern consistent with current ideas on desmid phylogeny. This pattern suggests that loroxanthin synthesis probably evolved once in the desmid lineage, with one or more subsequent reversals.     

LOROXANTHIN, A PHYLOGENETICALLY USEFUL CHARACTER IN CHLAMYDOMONAS AND OTHER CHLOROPHYCEAN FLAGELLATES1

The distribution of loroxanthin in chlorophycean flagellates is consistent with their phylogeny as inferred from molecular and organismal characters. Although polarity of this character cannot yet be determined, if the absence of loroxanthin is plesiomorphic for chlorophycean flagellates, the presence of loroxanthin defines a clade that includes the Euchlamydomonas morphological group, two other species of Chlamydomonas, C. asymmetrica and C. oblonga, and Gonium pectorale. A subsequent reversal to loroxanthin absence is apomorphic for the more complex volvocacean taxa. These results suggest that loroxanthin distribution will be useful for resolving phylogenetic questions in green algae .     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

STRUCTURE OF THE ANISOGAMETES OF THE GREEN SIPHON PSEUDOBRYOPSIS SP. (CHLOROPHYTA)1

The fine structure of the male and female gametes of Pseudobryopsis, particularly that of the flagellar apparatus, is compared with that of swarmers of other green algae. There is general similarity, with differences in detail, to the Ulvales and other green siphons that have been studied. The similarities include overlapping basal bodies, the capping plate type of connective between basal bodies, terminal caps, and system II fibrous roots (rhizoplasts). The capping plate of the female gamete differs from that in other green siphons and the Ulvales in form and in the presence of a faint striation. A diagram illustrating the actual arrangement of the components of the flagellar apparatus is given, along with a discussion of the fact that the mirror image of the true arrangement has been given in some reports on ulvaphycean algae.     

TRACKING DISPERSAL ROUTES: PHYLOGEOGRAPHY OF THE ARCTIC-ANTARCTIC DISJUNCT SEAWEED ACROSIPHONIA ARCTA (CHLOROPHYTA)1

Phylogenetic relationships in the Arctic-Antarctic disjunct seaweed species Acrosiphonia arcta (Dillwyn) J. G. Agardh (Acrosiphoniales, Chlorophyta) were examined using restriction fragment-length polymorphism analysis of the fast-evolving nuclear ribosomal intergenic spacer (IGS) region and random amplified polymorphic DNA (RAPD) markers. Twenty-two isolates collected from 10 different locations in both hemispheres were compared. Five IGS length classes were identified among the 10 locations. Throughout the North Atlantic, IGS regions were found to be extremely homogeneous whereas RAPD patterns revealed subdivided populations that suggest founder effects. Acrosiphonia arcta populations found in the Arctic and North Atlantic oceans are hypothesized to be of Pacific origin. Extensive differences found between Arctic Greenland populations and those in the North Atlantic suggest that colonization of Arctic Greenland occurred as an independent event. Recolonization of the Antarctic peninsula from Southern Chile is favored, whereas the directionality of transequatorial passage along the western coast of the Americas could be in either direction.     

THE OPTIMUM PH OF FOUR STRAINS OF ACIDOPHILIC SNOW ALGAE IN THE GENUS CHLOROMONAS (CHLOROPHYTA) AND POSSIBLE EFFECTS OF ACID PRECIPITATION1

Four axenic strains of snow algae were examined for optimum pH under laboratory conditions using M-1 growth medium. Growth was measured using cell counts, cell measurements and absorbance readings at 440 nm. Strains C204 and C479A of Chloromonas sp. from the Adirondack Mountains, New York, grew optimally at pH 4.0 to 5.0. Strains C381F and C381G, Chloromonas polyptera (Fritsch) Hoh., Mull. & Roem. from the White Mountains, Arizona, grew optimally at pH 4.5 to 5.0. Growth was significantly higher at pH 4.0 in the northeastern species (Chloromonas sp.), but no significant difference was observed in final growth at pH 4.5, 5.0 and 5.5 between species. It is postulated that the more acidic precipitation in the northeastern United States may be selecting for strains of snow algae with greater tolerance to acidity than in strains from the southwestern United States or that the different pH optima reported are simply species differences. New York strain C204 was also grown in heavily buffered AM medium where it had an optimum pH of 5.0, but cells became irregularly shaped and tended to clump at pH 6.0 to 7.0. Growth of C204 in AM medium was significantly lower than in M-1 medium for snow algae. These findings justify the use of M-1 medium for this type of experimentation.     

PHOTOSYNTHETIC PIGMENTS OF PSEUDOSCOURFIELDIA MARINA AND SELECT GREEN FLAGELLATES AND COCCOID ULTRAPHYTOPLANKTON: IMPLICATIONS FOR THE SYSTEMATICS OF THE MICROMONADOPHYCEAE (CHLOROPHYTA)1

Photosynthetic pigments of the green flagellate Pseudoscourfieldia marina (Throndsen) Manton (Micromonadophyceae) are similar to those of the coccoid Pycnococcus provasolii Guillard; prasinoxanthin is the predominant carotenoid. Other organisms that possess prasinoxanthin also possess additional pigments not found in either P. marina or P. provasolii. Uriolide, a xanthophyll previously described from the coccoid done URI 266G, was also found in Mantoniella squamata (Manton et Parke) Desikachary, Micromonas pusilla Manton et Parke and Mamiella gilva (Parks et Rayns) Moestrup, all flagellate members of the Mamiellales, and the coccoid clone IV E5G. Other unidentified carotenoids were also present in M. squamata, M. pusilla, and M. gilva. These results suggest that P. marina and the coccoid organisms URI 266G and IV E5G may be related to the Mamiellales, and that P. provasolii may be more closely related to P. marina than to M. squamata, M. pusilla, and M. gilva.     

DIVISION APPARATUS OF THE CHLOROPLAST IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA)1

Chloroplast division in Nannochloris bacillaris Naumann (Chlorophyta) was examined by electron microscopy after preparation of samples by freeze-substitution. A pair of belts appeared on the surface of the outer and inner envelope membranes at the middle of the chloroplast. These belts seemed to be constructed of thin fibrils that run parallel to the longitudinal direction of the belts. The outer fibrillar belt increased in width as the constriction of the chloroplast advanced. It appears that the fibrillar belt is the division apparatus of the chloroplast. It encircles the chloroplast and finally divides the chloroplast in two as the diameter of the belt decreases.     

PIGMENT COMPOSITION OF THE SCALY GREEN FLAGELLATE MESOSTIGMA VIRIDE (MICROMONADOPHYCEAE) IS SIMILAR TO THAT OF THE SIPHONOUS GREEN ALGA BRYOPIS PLUMOSA (ULVOPHYCEAE)1

Pigments were isolated from Mesostigma viride Lauterborn by reversed-phase high-performance liquid chromatography and compared to standards from Chlamydomonas reinhardtii Dang. and Bryopsis plumose (Huds.) Ag. M. viride possesses chlorophylls a and b, α and β-carotenes, and the xanthophylls siphonaxanthis, siphonein, neoxanthin, violaxanthin and echinenone. In addition, three unidentified xanthophylla were detected. Neither lutein nor zeaxanthin were detected. The pigment composition of M. viride was similar to that of B. plumosa which had chlorophylls a and b, ?- and α-carotenes, siphonaxanthin, siphonein, neoxanthin, violaxanthin, and two of the unidentified xanthophylls found in M. viride. The similarities in the pigments of Mesostigma and Bryopsis and other characters suggest that Mesostigma may be related to a flagellate ancestor of the Ulvophyceae.     

FREEZE-DRYING OF ALGAE: CHLOROPHYTA AND CHRYSOPHYTA1

Thirty-nine of 106 algal strains tested were successfully lyophilized using at least one of the following as suspending agents: 20% (w/v) skim milk, 12% (w/v) sucrose, 100% lamb serum and 100% horse serum. The majority of the Chlorella and Scenedesmusstrains tested (27/40) were amenable to freeze-drying. Much less success (3/18) occurred with the Chlamydomonas strains tested. At least one strain of Ankistrodesmus, Bracteacoccus, Characium, Trebouxia, Haematococcus, Coccomyxa, Interfilum, Hormidium and Nephrodiella were also recovered. More strains were recovered with 20% skim milk as the protective agent than with 12% sucrose; however, 12% sucrose generally provided higher yields. Freeze-drying appears to be an effective method of preservation far some algal strains.     

IMMUNOLOGICAL IDENTIFICATION OF THE ALTERNATIVE OXIDASE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Using a monoclonal antibody to the alternative oxidase from voodoo lily, we provide evidence that the green alga Chlamydomonas reinhardtii Dang, possesses a protein that is immunologically related to the higher plant alternative oxidase. Mitochondria were isolated from a cell wall-less mutant strain (CW-15), and the presence of cyanide-resistant oxygen consumption was confirmed in these mitochondria. The voodoo lily antibody was used as a probe for immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of mitochondrial proteins of C. reinhardtii. The antibody reacted with a protein from C. reinhardtii with the same molecular mass (36 kDa) as the alternative oxidase from voodoo lily and tobacco mitochondria. These results suggest that cyanide-resistant respiration in C. reinhardtii is mediated by a higher plant-type alternative oxidase.     

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA,SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.     

OCCURRENCE OF PRASIOLA FLUVIATILIS (CHLOROPHYTA) ON ELLESMERE ISLAND IN THE CANADIAN ARCTIC1

Three occurrences of the macroalga Prasiola fluviatilis (Sommerf.) Aresch. were recorded after the examination of 32 stream segments from the Ellesmere Island National Park Reserve (81°23′ N) in the High Arctic of Canada. The collection sites were small (second-third order) glacial melt streams characterized by pH 7.3–8.2, specific conductance of 52–159 μS·cm^?1, low ion levels, extremely low nutrient levels, and the lack of other prominent macroalgae. These occurrences confirm the reported circumpolar distribution of the species.     

PHYLOGENETIC RELATIONSHIPS AMONG ISOLATES OF EUDORINA SPECIES (VOLVOCALES, CHLOROPHYTA) INFERRED FROM MOLECULAR AND BIOCHEMICAL DATA

Phylogenetic analyses of 19 strains representing five species of Eudorina, one strain of Pleodorina indica, and seven strains of Yamagishiella unicocca were carried out by sequencing the internal transcribed spacer region (ITS 1 and ITS 2) of the nuclear ribosomal DNA (rDNA) repeats. The sequence data resolved five phylogenetic groups, one consisting of Y. unicocca and the other four encompassing all the Eudorina species. Two isolates, Eudorina sp. (ASW 05157) and Pleodorina indica (ASW 05153), were of uncertain affiliation. Whereas one monophyletic group included strains of E. elegans only, the other strains of E. elegans appeared alongside E. cylindrica, E. illinoisensis, and E. unicocca var. unicocca in the other Eudorina clades. The distribution pattern of the carotenoid loroxanthin ([3R,3'R,6'R]-β,ε-carotene-3,19,3'-triol), a systematically useful biochemical marker within chlorophycean flagellates, was shown to match the evaluated molecular data. Whereas it was either totally absent or universally present in six of the deduced phylogenetic lines, it occurred randomly in the E. elegans clade containing only E. elegans isolates. The results substantiated the current hypothesis that the unique vegetative morphology of E. elegans has independently arisen at various times during evolution and that it is not a marker of a monophyletic group.     

POLYAMINE TRANSPORT IN THE SEAWEED ULVA RIGIDA (CHLOROPHYTA)1

The excessive growth of Ulva rigida C. Agardh, a green seaweed present in the Northern Adriatic Sea, is a problem for the inhabitants and the economy of the region. As information about hormonal control of growth in seaweeds is scarce, our aim was to investigate the presence of endogenous polyamines and their absorption by algal cells and to correlate the findings with terrestrial plants. Free polyamines (putrescine, spermidine, and spermine) were present endogenously in the algal thallus at concentrations ranging from 4 to 134 μM. Putrescine and spermidine were also present in the seawater in which the alga usually grows at concentrations between 0 and 0.9 μM. Uptake of labeled polyamines occurred, but it was inhibited by cations present in the seawater. Uptake was investigated also by incubation in distilled water. In this case, uptake displayed characteristics similar to those observed in higher plant systems. Uptake studies in seawater showed that polyamine accumulation in algal cells occurred and that it followed a concentration gradient and displayed linear kinetics. The mechanism proposed that of a passive uptake, as indicated also by the inability of metabolic inhibitors to block transport. There was evidence for polyamine binding to external cell sites, but polyamine uptake by protoplasts as well as polyamine translocation and secretion by the whole thallus was also demonstrated. Since cultured and actively growing thallus discs displayed a higher uptake ability than freshly collected ones, a role for polyamines in sustaining growth is discussed.     

ABUNDANT PLASMID-LIKE DNA IN VARIOUS MEMBERS OF THE ORDERS SIPHONOCLADALES AND CLADOPHORALES (CHLOROPHYTA)1

In all seven genera belonging to the order Siphonocladales and in two genera of the Cladophorales (Chlorophyta) that were examined, extracts of total DNA contained abundant low molecular weight (LMW) molecules. This DNA typically ranged in size from 1.5 to 3.0 kb among the different genera studied. The complexity of the populations of molecules found in different genera also varied with regard to sizes and numbers of bands evident in electrophoresis gels. Our data suggest that the LMW DNA is ubiquitous among these algae since it occurred in each of our six isolates of Ernodesmis verticillata (Kützing) Børgesen collected in both the Caribbean and the Gulf of California. The LMW DNA from Ernodesmis and Ventricaria ventricosa (J. Agardh) Olsen et J. West was denser than high molecular weight (HMW) DNA in CsCl/bisbenzimide gradients. Ultrastructurally, the LMW DNA molecules were linear, averaging 0.65 μm (Ernodesmis) and 1.0 μm (Ventricaria) in length. Approximately 2%–5% of the former possessed an apparent loop or lariat at one end, as visualized in the electron microscope after rotary shadowing. The LMW DNA molecules appeared to be predominantly double-stranded DNA based upon staining with acridine orange. The 2.2-kb DNA from Ernodesmis has features typical of plasmids in that it rapidly reannealed after heat denaturation, and it was selectively enriched in an alkaline-lysis extraction procedure. In denaturing gels, this DNA migrated at >4.0 kb, indicating that the molecules may actually be single-stranded DNA species with intramolecular base pairing, each containing a long inverted repeat folded back on itself in a hairpin conformation in the native state. Digestion with mung bean nuclease revealed that additional single-stranded regions may occur in many of the LMW molecules, in addition to the hairpin loop. Although the endonuclease DNase I rapidly digested the LMW DNA, neither exonuclease III nor lambda exonuclease digested it. This suggests that the ends of these DNA molecules are protected. Collectively, the data indicate that members of the Siphonocladales and Cladophorales contain linear plasmid-like DNA molecules that appear to have a novel combination of features.     

TAXONOMIC SIGNIFICANCE OF SECONDARY CAROTENOID FORMATION IN NEOSPONGIOCOCCUM (CHLOROCOCCALES,CHLOROPHYTA)1

The pigmentation of 23 species of Neospongiococcum Deason was studied in the stationary phase of nitrogen-limited cultures on agar with or without glucose or in liquid medium. Fourteen species gradually developed an orange or brown color, and in all, secondary carotenoids were detected by thin-layer chromatography. Among these, the keto-carotenoids echinenone, canthaxanthin and esterified astaxanthin were identified, although the last named was not always present. When aging on glucose agar, the remaining nine species developed only a yellow-green or yellow color and a pronounced tendency to bleach. In these, the aforementioned keto-carotenoids were lacking, and β-carotene and lutein lucre dominant. From this study it is not clear whether secondary carotenoid formation is species-specific in Neospongiococcum.     

MICROSENSOR STUDIES OF OXYGEN AND LIGHT DISTRIBUTION IN THE GREEN MACROALGA CODIUM FRAGILE1

Scalar irradiance, oxygen concentration, and oxygenic photosynthesis were measured at 0.1 mm spatial resolution within the tissue of the siphonous green macroalga Codium fragile subsp. tomentosoides (van Goor) Silva by fiber-optic scalar irradiance microsensors and oxygen microelectrodes. The scalar irradiance of visible light was strongly attenuated in the outer 0.2 mm of the tissue but was nearly constant for the subsequent 1.0 mm of photo-synthetic tissue. Far-red scalar irradiance at 750 nm increased below the tissue surface to a maximum of 200% of incident irradiance at 1.2 mm depth due to multiple scattering in the medullary tissue. The constant intensity of visible light below 0.2 mm was thus a result of the combined effects of absorption and backscattering from the medulla. The oxygen exchange between the alga and the surrounding water was diffusion-limited with a steep O₂gradient inside and around the alga. In darkness, the tissue below 0.6 mm became anoxic, and endophytic extracellular space provided an environment where anoxygenic microbial processes may occur. When illuminated at 160 nmol photons·^?2·^?1, O₂ concentrations exceeded ambient levels throughout the thallus, with a maximum of 250% of air saturation just below the surface. The amplitude of oxygen variation was buffered by gas bubbles formed in the medullary tissue.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.     

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.