TIME LAPSE ANALYSES OF SEXUAL REPRODUCTION IN CLOSTERIUM EHRENBERGII (CONJUGATOPHYCEAE)1

The process of sexual differentiation was studied using heterothallic clones of Closterium ehrenbergii Meneghini. The first visible sign of sexual reproduction was agglutination of two or more cells in a group and this was followed by gametangiogenic division and conjugation of gametangial cells. Movements of gametangial cells were carefully studied. Gametangial cells occasionally participated again in gametangiogenesis instead of proceeding directly to the formation of conjugation papilla. The whole process of sexual differentiation from vegetative cell to zygospore was considered to be basically similar in both of the two closely related mating groups, A and B, of C. ehrenbergii. Nevertheless, there were some differences between the two groups in patterns of the sexual differentiation. In Group A, vegetative cell division was completely suppressed by mixing the two complementary mating type clones together into the same medium with high light illumination. This suppression was not caused by the nitrogen depletion in the medium, but by the presence of cells of opposite mating type. In Group B, vegetative cell division and sexual reproduction occurred side by side repeatedly for several days.     

A SEX DETERMINING MECHANISM IN THE CLOSTERIUM EHRENBERGII (CHLOROPHYTA) SPECIES COMPLEX1

Homozygous mt^?/mt^? diploid clones of the Closterium ehrenbergii Menegh. ex Ralfs species complex were obtained by hypertonic treatment from minus vegetative cells, and mating type segregation ratios in the F₁ progeny of “triploid” zygospores between wild type mt⁺ haploid and mt^?/mt^? homozygous diploui were analyzed. The ratio of plus to minus individuals was 1:4.8, and the ratio of the pairs of opposite mating types to those of minus mating type was 1:2.1. The results clearly show that mt^? is dominant to mt⁺ and that the mating type inheritance in these zygospores follows the triploid-like pattern. The validity of our assumption that the two mating types are determined by one genetic factor (mt^? allele dominant) was confirmed in B₁ progeny analyses as well. The results suggest that this sex determining mechanism is working effectively in the C. ehrenbergii species complex, in which several biological species have evolved through polyploidization.     

MORPHOLOGICAL VARIABILITIES OF THREE CLOSELY RELATED MATING GROUPS OF CLOSTERIUM EHRENBERGII MENEGHINI (CHLOROPHYTA)1

The ranges of morphological variabilities in vegetative cells of three closely related mating groups of Closterium ehrenbergii Meneghini were statistically analyzed, having been grown under standard and uniform culture conditions, using Group A clones from Japan and Australia, Group B clones from Japan and Taiwan and Group H clones from Nepal. Significant differences in the morphological characters were not recognized between the two complementary mating types in any of the three groups. It has been shown that cells of Group A are smallest (mean width 50 μm and mean length 250 μm) and cells of Group B are largest (mean width 67 μm and mean length 404 μm), while cells of Group H are intermediate (mean width 57 μm and mean length 333 μm). There are considerable differences in the mean cell size between the three mating groups, although some intergrading clones were recognized. Degrees of the intergrading overlap were shown to be small between the sympatric groups (A and B) and large between the two pairs of allopatric groups (A and H & B and H). It has been shown that cells of Group A are shorter and much more curved than cells of the other two mating groups. Cells of Groups B and H are slender and less curved. It has also been shown that the ranges in cell size of each mating group are smaller than those currently accepted for C. ehrenbergii.     

Morphological and cytogenetic characteristics of intergroup hybrids between closely related mating groups of the Closterium ehrenbergii species complex (Desmidiales, Chlorophyta)

Five F₁ hybrid strains were established from rare survivors in intergroup crosses between three closely related mating groups (A, B and H) of the Closterium ehrenbergii Meneghini ex Ralfs species complex. Cell sizes of these five strains studied under our standard culture conditions were compared to those of their parental stains and also to the total range of cell-size variation in each mating group. All five F₁ strains were larger in mean cell width than their parental strains. In cell length, three of them were larger than, one was the same as, and the other was intermediate between their parental strains. Their cell sizes were always larger than the range of their respective smaller parental mating group and three of them were larger than the range of their respective larger parental mating group.     

DETECTION AND EVALUATION OF A NOVEL SEXUAL PHEROMONE THAT INDUCES SEXUAL CELL DMSION OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

Mating type-plus (mt⁺; NIES-228) cells of Closterium ehrenbergii undergo a division to form gamete-shaped cells. This cell division is induced by a substance produced by mating type-minus (mt^?; NIES-229) cells. Light and the presence of mt⁺ cells enhanced production of the substance. The active substance is heat labile and has an apparent molecular mass of 20 kDa. From these results, we conclude that the substance is a novel, proteinaceous sexual pheromone involved in reproduction of Closterium ehrenbergii.     

TIME-LAPSE ANALYSES OF SEXUAL ISOLATION BETWEEN TWO CLOSELY RELATED MATING GROUPS OF THE CLOSTERIUM EHRENBERG SPECIES COMPLEX (CHLOROPHYTA)1

Sexual isolation between Groups A and B of Closterium ehrenbergii, two closely related species, was studied by a multiple-choice mating method, as well as the nochoice mating method which has been used in previous work on microalgae. Time lapse photomicrographs and the difference in cell shape and size between the two mating groups allowed identification of a given cell in the mixture as either Group A or B, even when certain morphological changes occurred during the several day culture required for sexual induction. When plus and minus mating types of Group A were mixed with those of Group B (multiplechoice mating), no intergroup hybrid zygospores were formed. However, many intragroup zygospores of either Group A or B were formed. When one plus strain of Group A was mixed with one minus strain of Group B or when one plus strain of Group B was mixed with one minus strain of Group A (no-choice mating), intergroup sexual interactions took place resulting in a small number of hybrid zygospores; however, the process took much longer than intragroup sexual interactions. It was also shown that cell size difference itself hardly affects sexual interactions between haploid and autodiploid strains of Group A. It is suggested that sexual isolation between Groups A and B would be complete in nature, although they may interact sexually in the laboratory.     

DIPLOIDY IN DNA CONTENT IN VEGETATIVE CELLS OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

DNA content of the nucleus in the placoderm desmid, Closterium ehrenbergii Meneghini was measured throughout the life cycle by epifluorescence microspectrophotometry after DNA specific dye [4′,6-diamidino-2-phenylindol (DAPI)] staining. Postulating a mean DNA content of gamete nuclei as 1C, the nucleus of a newly divided vegetative cell was 2C. Most vegetative cells in the stage of exponential growth had a DNA content from 2C to 4C, while most in stationary phase, with the highest frequency of zygote formation, were 2C. They became pre-gametes (2C) upon mixing two heterothallic strains. Four gametes were made by a DNA reduction division of each pre-gamete cell. Therefore, there was a nonmeiotic DNA reduction stage by one half. During germination, the zygote underwent meiosis to produce two gones, each of which contained one surviving nucleus (large nucleus) and one degenerating nucleus (small nucleus). The DNA content of these four nuclei was 1C basically. The DNA of the surviving nucleus duplicated to 2C and further quadruplicated to 4C without cell or nuclear division. These two 4C gones had different cell morphology from ordinary vegetative cells. After the first cell division following meiosis, each gone produced two vegetative cells in which the DNA content became 2C to 4C again.     

COMPOSITION AND SYNTHESIS OF THE PECTIN AND PROTEIN COMPONENTS OF THE CELL WALL OF CLOSTERIUM ACEROSUM (CHLOROPHYTA)

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na₂CO₃-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na₂CO₃ extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans- Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.     

CLOSTERIUM MONILIFERUM-EHRENBERGII (CHAROPHYCEAE, CHLOROPHYTA) SPECIES COMPLEX VIEWED FROM THE 1506 GROUP I INTRON AND ITS2 OF NUCLEAR rDNA

To assess phylogenetic relationships and speciation modes in Closterium , we sequenced two noncoding regions of the nuclear ribosomal cistron, the 1506 group I intron in small subunit and the internal transcribed spacer 2, for a total of 58 strains of the Closterium moniliferum-ehrenbergii species complex. These include both homothallic and heterothallic C. moniliferum Erenberg ex Ralfs v. moniliferum , heterothallic C. moniliferum v. submoniliferum (Woronichin) Krieger, and heterothallic C. ehrenbergii Meneghini ex Ralfs that can be divided into several mating groups. We found no or very little sequence divergence within single mating groups of C. ehrenbergii and among all heterothallic strains of C. moniliferum v. moniliferum or C. moniliferum v. submoniliferum. Nevertheless, sequence divergence was much greater between those mating groups of C. ehrenbergii and also among the three traditional taxa . Maximum parsimony and maximum likelihood analyses showed that the taxon C. ehrenbergii was not monophyletic. The two varieties of C. moniliferum appeared as a sister clade to certain mating groups of C. ehrenbergii . Among the clades that were recovered in different trees by maximum parsimony and maximum likelihood analyses, we consistently found two large conspicuous clades: clade I consisted of mating groups A, B, C, H, K, and L of C. ehrenbergii whose zygospores have smooth-walls, and clade II contained the mating groups D, E, I, J, and S whose zygospores are scrobiculate. Phylogenetic incongruences observed are discussed from the viewpoints of the different molecular nature of the group I intron and internal transcribed spacer 2, as well as putative rapid diversification of the mating groups and probable ancient ancestral hybridization.     

ANALYSIS OF GAMETIC PROTOPLAST RELEASE IN THE CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX (CHLOROPHYTA)1

A biologically active glycoprotein (protoplast-release-inducing protein; PR-IP), which induces the release of gametic protoplasts from mating type minus (mt^-) cells of the Closterium peracerosum-strigosum-littorale complex, was prepared from a medium in which mt^- and mt⁺ cells had been previously incubated together. The process of PR-IP-inducing protoplast release was analyzed. Induction of protoplast release was dependent upon the duration of both PR-IP treatment and preincubation in nitrogen-deficient mating medium before PR-IP treatment. Low cell density in the preculture stage had a significant stimulative effect upon the induction of protoplast release. Light was necessary for protoplast release, especially just before PR-IP treatment. Chloramphenicol and 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) exerted inhibitory effects on protoplast release, especially when they were applied to the preculture stage but not when they were applied to the protoplast-releasing stage after the PR-IP treatment. We suggest that preculture at a low cell density under continuous light conditions that may cause metabolic changes in the chloroplast is a very important stage for gametic protoplast release in this Closterium.     

MONOPHYLY OF THE GENUS CLOSTERIUM AND THE ORDER DESMIDIALES (CHAROPHYCEAE, CHLOROPHYTA) INFERRED FROM NUCLEAR SMALL SUBUNIT rDNA DATA

We newly sequenced the nuclear-encoded small subunit (SSU) rDNA coding region for 21 taxa of the genus Closterium. The new sequences were integrated into an alignment with 13 known sequences of conjugating green algae representing six traditional families (i.e. Zygnemataceae, Mesotaeniaceae, Gonatozygaceae, Peniaceae, Closteriaceae, and Desmidiaceae) and five known charophycean sequences as outgroups. Both maximum likelihood and maximum parsimony analyses supported with high bootstrap values one large clade containing all placoderm desmids (Desmidiales). All the Closterium taxa formed one clade with 100% bootstrap support, indicating their monophyly, but not paraphyly, as suggested earlier. As to the taxa within the genus Closterium , we found two clades of morphologically closely related taxa in both maximum likelihood and maximum parsimony trees. They corresponded to the C. calosporum species complex and the C. moniliferum-ehrenbergii species complex. It is of particular interest that the homothallic entity of C. moniliferum v. moniliferum was distinguished from and ancestral to all other entities of the C. moniliferum-ehrenbergii species complex. Superimposing all 50 charophycean sequences on the higher order SSU rRNA structure model of Closterium , we investigated degrees of nucleotide conservation at a given position in the nucleotide sequence. A characteristic "signature" structure to the genus Closterium was found as an additional helix at the tip of V1 region. In addition, eight base deletions at the tip of helix 10 were found to be characteristic of the C. calosporum species complex, C. gracile , C. incurvum , C. pleurodermatum , and C. pusillum v. maius. These taxa formed one clade with an 82% bootstrap value in maximum parsimony analysis.     

SEXUAL PROCESSES AND PHYLOGENETIC RELATIONSHIPS OF A HOMOTHALLIC STRAIN IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

Members of the Closterium peracerosum–strigosum–littorale (C. psl.) complex are unicellular charophycean algae in which there are two modes of zygospore formation, heterothallic and homothallic. A homothallic strain of Closterium (designation, kodama20) was isolated from a Japanese rice paddy field. Based on alignment of the 1506 group‐I introns, which interrupt nuclear SSU rDNAs, homothallic kodama20 is most closely related to the heterothallic mating group II‐B, which is partially sexually isolated from group II‐A. Time‐lapse photography of the conjugation process in kodama20 revealed that most of the observed zygospores originated from one vegetative cell. The sexual conjugation process consisted of five stages: (1) cell division resulting in the formation of two sister gametangial cells from one vegetative cell, (2) formation of a sexual pair between the two sister gametangial cells (or between gametangial cells of another adjoined individual), (3) formation of conjugation papillae, (4) release of gametic protoplasts from both members of a pair, and (5) formation of the zygospore by protoplast fusion. For conjugation to progress, the cell density and light condition in the culture was critical. We suggested the presence of a conjugation promotion factor.     

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)1

Closterium strains obtained from Japan ( NIES-64 and -65 ) and Nepal ( NIES-67 and -68 ) have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES-64 and -65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS-67 or -68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES-68, called the protoplast-release-inducing protein ( PR-IP ), had no effect on the release of protoplasts from cells of strains NIES-64 or -65. When the media obtained from the culture of NIES-64 and -65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunits of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not structurally similar to PR-IP. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IP inducer ) which is released by NIES-68 cells. In contrast, no protoplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES-64/ NIES-65 and strains NIES-67 /NIES-68 differ, and we propose a possible mechanism of sexual isolation between these biological species .     

ULTRASTRUCTURE OF MALE GAMETOGENSIS IN FUCUS SERRATUS (PHAEOPHYCEAE)1

The main structural features of male gametogenesis in Fucus serratus L. are described. The antheridial parent cells exhibit meristematic characteristics: large nucleus, dense cytoplasm, chloroplasts with few thylakoids. Synaptonemal complexes are frequently observed in the nuclei at the same time that microtubules are very abundant around the centrioles. Meiotic spindles are intranuclear, as are the mitotic spindles of the subsequent synchronous karyokineses. A nuclear membrane is present in all stages observed. Antheridial maturation is accompanied by multiplication of mitochondria and plastids as well as development of two major kinds of “vacuoles.” Cytokinesis proceeds with the progressive fusion of one of these types of vacuoles: this mode of cytoplasmic cleavage is unique among algae. Formation and differentiation of characteristic spermatozoid organelles (flagella, eyespot, proboscis) are also described.     

CORRELATED EVOLUTION OF SELF-FERTILIZATION AND INBREEDING DEPRESSION: AN EXPERIMENTAL STUDY OF NINE POPULATIONS OF AMSINCKIA (BORAGINACEAE)

The relation between inbreeding depression and rate of self-fertilization was studied in nine natural populations of the annual genus Amsinckia. The study included two clades (phylogenetic lineages) in which small-flowered, homostylous populations or species are believed to have evolved from large-flowered, heterostylous, self-compatible ones. In one lineage the small-flowered species is tetraploid with disomic inheritance. Rates of self-fertilization were 25% to 55% in the four large-flowered, heterostylous populations; 72% in a large-flowered but homostylous population; and greater than 99.5% in the four small-flowered, homostylous populations, which produce seed autonomously. When present, inbreeding depression occurred in the fertility but not the survival components of fitness. Using a cumulative fitness measure incorporating both survival and fertility (flower number), we found inbreeding depression to be lower in the four very highly self-fertilizing populations than in the five intermediate ones. The Spearman rank correlation between inbreeding depression and selfing rate for the nine populations was –0.50, but was not statistically significant (P = 0.12). Inbreeding depression was greater in the two tetraploid populations than in the very highly self-fertilizing, diploid ones. Phenotypic stability of progeny from self-fertilization tended to be higher in populations with lower inbreeding depression. We conclude that levels of self-fertilization and inbreeding depression in Amsinckia are determined more by other factors than by each other. Estimates of mutation rates and dominance coefficients of deleterious alleles, obtained from a companion study of the four highly self-fertilizing populations, suggest that a strong relationship may not be expected. We discuss the relationship of the present results to current theory of the coevolution of self-fertilization and inbreeding depression.     

IDENTIFICATION OF THE MINUS MATING‐TYPE SPECIFIC GENE MTD1 FROM GONIUM PECTORALE (VOLVOCALES,CHLOROPHYTA)1

Gonium pectorale O. F. Müll. (Volvocales, Chlorophyta), a colonial 8‐ or 16‐cellular alga, is phylogenetically important as an intermediate form between isogametic unicellular Chlamydomonas and oogamous Volvox. We identified the mating‐type specific gene GpMTD1, from G. pectorale, the first homologue of Chlamydomonas reinhardtii MTD1 (CrMTD1). The GpMTD1 gene was found to be present only in the minus mating‐type locus and was expressed specifically in the gametic phase as is the case for CrMTD1, suggested to participate in development of the minus gametes. This gene is useful as a probe in analyzing the bacterial artificial chromosome (BAC) library for resolving genomic structures of the mating‐type loci in isogamous and oogamous colonial volvocaleans.     

EVIDENCE FOR TUBULAR MATING STRUCTURES INDUCED IN EACH MATING TYPE OF HETEROTHALLIC GONIUM PECTORALE (VOLVOCALES,CHLOROPHYTA)1

Gametes were induced separately in cultures of each mating type of the heterothallic, isogamous colonial volvocalean Gonium pectorale O. F. Müll. to examine the tubular mating structure (TMS) of both mating types plus and minus (plus and minus), referred to as “bilateral mating papillae.” Addition of dibutyryl cyclic adenosine monophosphate (DcAMP or db‐cAMP) and 3‐isobutyl‐1‐methylxanthine (IBMX) to approximately 3‐week‐old cultures of each mating type induced immediate release of naked gametes from the cell walls. Both plus and minus gametes formed a TMS in the anterior region of the protoplasts. Accumulation of actin was visualized by antibody staining in the TMS of both mating types as occurs in the TMS (fertilization tubule) of the plus gametes of the unicellular volvocalean Chlamydomonas reinhardtii P. A. Dang. Induction of naked gametes with a TMS in each mating type will be useful for future cell biological and evolutionary studies of the isogametes of colonial volvocalean algae.     

PHOTOTACTIC MOTILITY OF SYNECHOCYSTIS SP. UNIWG (CYANOBACTERIA) FROM BRACKISH ENVIRONMENT1

Although type IV pilus has been implicated in the phototactic motility of some unicellular cyanobacteria, its regulatory mechanism and the effect of environmental factors on motility are still unknown. Equally important is the ability of cyanobacterial cells to anchor themselves to an environment that is conducive for survival. We compared the motility of a newly isolated unicellular brackish cyanobacterium, Synechocystis sp. UNIWG, with the morphologically and phylogenetically similar freshwater cyanobacterium Synechocystis sp. PCC6803 under different environmental conditions. The phototactic motility of Synechocystis sp. UNIWG on semisolid BG‐11 medium with various concentrations of nitrogen source was significantly faster than that of Synechocystis PCC6803. Interestingly, the cell surface of Synechocystis sp. UNIWG showed the presence of rigid spicules when grown in liquid BG‐11, a phenomenon that was absent in Synechocystis PCC6803. Negative staining of Synechocystis sp. UNIWG revealed the presence of two distinct pilus morphotypes, which resembled type IV pili and thin pili of Synechocystis PCC6803. This finding suggested a similar pattern of phototactic motility in both strains. However, the rigid spicules on Synechocystis sp. UNIWG seem to be more of a hindrance during type IV motility. It was determined that the spicules were degraded when the cells moved, such as under prolonged darkness and/or depletion of nitrogen source, indicating that the function of the spicules is to attach the cell to an environment that is conducive for its survival. Thus, Synechocystis sp. UNIWG shows phototaxis regulation that is more complex than Synechocystis PCC6803.     

EFFECTS OF LIGHT INTENSITY AND TEMPERATURE INTERACTIONS ON GROWTH CHARACTERISTICS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cell division rate, carbon fixation per cell, cell width and chloroplast length of Phaeodactylum tricornutum Bohlin were determined at 30 different combinations of light intensity and temperature. Division rate peaked at 23° C or less depending on light intensity. For each light intensity studied, carbon fixation increased directly with growth temperature from 14 to 25° C. The slope of this relationship was modified by light intensity. Cells grown at 23–25° C tended to be larger than those grown at lower temperatures, possibly due to increased carbon fixation per cell coupled with lower division rates. Chloroplasts were largest at a combination of temperatures above 21° C and low light intensities. This effect could cause cells to sink at a higher than normal rate due to reduced vacuole size and is presented as a possible mechanism affecting the distribution of P. tricornutum.