ATRACTOMORPHA PORCATA SP. NOV., A NEW MEMBER OF THE SPHAEROPLEACEAE (CHLOROPHYCEAE) FROM CALIFORNIA1

Atractomorpha porcata sp. nov. is described from culture isolates derived in 1981 from zygotes present in a 28 year old, dried soil sample collected from near Lemon-cove, Tulare County, California. Vegetative individuals are coenocytic, spindle-shaped unicells with long, thin-pointed apices. Asexual reproduction is by means of large, biflagellate zoospores or, frequently, by aplanospores. Sexual reproduction is usually monoecious, with a single spindle-shaped gametangial cell producing small, biflagellate male gametes at either end, and larger female gametes in the midportion. Female gametes are often biflagellate, but more commonly they lack flagella and are liberated by squeezing through slit-like openings in the gametangial wall. Sexual reproduction may thus be considered as either oogamous or anisogamous, depending on whether or not a particular female gamete has flagella; most often it is oogamous. Atractomorpha porcata is readily distinguished from A. echinata, the only other known member of the genus, by (1) its greater tendency toward oogamy (versus anisogamy), (2) its bisexual gametangia, (3) its frequent production of aplanospores in asexual reproduction, (4) its unusual primary membranes that frequently bear long, delicate bristles, and (5) its distinctive zygote wall ornamentation.     

Parthenogenesis is rare in the reproduction of a sexual field population of the isogamous brown alga Scytosiphon (Scytosiphonaceae,Ectocarpales)

Parthenogenetic development of unfused gametes is commonly observed in laboratory cultures among various brown algal taxa. There is, however, little information on the contribution of parthenogenesis to the reproduction of field populations. In this study, we investigated whether parthenogenesis is present in a sexual population of the isogamous brown alga Scytosiphon with a 1:1 sex ratio. In culture, both female and male gametes showed higher mortality and slower development compared to zygotes. More than 90% of surviving partheno‐germlings formed parthenosporophytes irrespective of the culture conditions tested. Therefore, if parthenogenesis occurs in the field, most unfused gametes are expected to form parthenosporophytes. Contrary to this expectation, parthenosporophytes were rare in the field population. We collected 126 sporophytic thalli and isolated and cultured a unilocular sporangium from each of them. We confirmed that cultures of 120 unilocular sporangia produced both female and male gametophytes by the observation of zygotes or amplification of PCR‐based sex markers indicating that these sporangia originated from zygotic sporophytes. Only females were detected in cultures from two sporangia and only males from four sporangia suggesting that these sporangia originated from parthenosporophytes. In the Scytosiphon population, although parthenogenesis is observable in culture, our results demonstrate that the contribution of parthenogenesis to reproduction is small (≤4.8%) compared to sexual reproduction. Unfused gametes may not survive to form mature parthenosporophytes in significant numbers in the field partly due to their higher mortality and slower development compared from zygotes.     

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

 The reproductive strategy of a marine alga with a heteromorphic biphasic life cycle was studied by analyzing various sexual reproductive characters in light of the evolution of anisogamy. Gametophytes of Monostroma angicava were dioecious and their gametes were slightly anisogamous. Volume of gametangium, density of gametangia and area of mature gametangial parts on each gametophyte did not differ from male to female. Therefore, the reproductive biomass investment for gamete production was considered to be the same for each sex. Anisogamy in this alga appeared to be derived from the difference in the number of cell divisions during gametogenesis, because the majority of male gametangia each produced 64 (2⁶) gametes and the female produced 32 (2⁵) gametes. This corresponded with measurements of cell size in male and female gametes. Further, the sex ratio was 1:1 for sexually mature plants sampled at Charatsunai. Therefore, it was suggested that in the field twice as many male gametes are released as female gametes. Liberated gametes of both sexes showed positive phototaxis. The swimming velocity of freshly liberated male gametes was a little higher than that of female gametes. Male gametes had the potential to swim for ca. 72 h and female gametes for ca. 84 h. The difference in gamete motility between the two sexes seemed to be related to cell size. Planozygotes were negatively phototactic and swam more rapidly than gametes of either sex. Received: 5 March 1997 / Revision accepted: 18 July 1997     

MORPHOLOGY AND DEVELOPMENT OF AHNFELTIA PLICATA (RHODOPHYTA): PROPOSAL OF AHNFELTIALES ORD. NOV.1

Ahnfeltia plicata (Hudson) Fries, the type species of Ahnfeltia Fries, is currently assigned to the Phyllophoraceae (Gigartinales). Several morphological and biochemical characters distance A. plicata from the Phyllophoraceae but, because sexual reproduction has never been demonstrated, an alternative placement has not been possible. A. plicata now is shown to have a heteromorphic sexual life history. Erect branched gametophytes are dioecious. In male sori, spermatangia are cut off transversely from spermatangial mother cells. Female sori form numerous terminal sessile carpogonia. Following fertilization, several zygotes in each sorus fuse facultatively with undifferentiated intercalary cells of the female sorus and cut off gonimoblast initials obliquely outwards. These initials give rise to branching gonimoblast filaments that fuse with apical and intercalary female sorus cells and with each other, then grow radially outward in the compound external carposporophyte and terminate in carposporangia. Carpospores develop in culture into crustose tetrasporophytes identical to Porphyrodiscus simulans Batters. Field-collected P. simulans tetraspores grew into erect A. plicata axes. Tetrasporangia are formed by division and enlargement of crust apical cells followed by sequential enlargement and maturation of tetrasporocytes in an erosive process. Monosporangia are formed in sori on male gametophytes. Pit plugs of both gametophyte and tetrasporophyte phases consist of naked plug cores without cap layers of membranes. Gametophytes exhibit both cell fusions and secondary pit connections whereas tetrasporophytes form cell fusions but lack secondary pit connections. On the basis of the unique female and postfertilization reproductive development and in conjunction with the pit plug structure which is unique among florideophytes, the order Ahnfeltiales, containing the family Ahnfeltiaceae, is proposed.     

On auxospore formation inCaloneis and the nature ofAmphiraphia (Bacillariophyta)

Amphiraphia Chen & Zhu, together with theAmphiraphiaceae andAmphiraphidales, should be abandoned, sinceAmphiraphia cells are the heterovalvar initial cells ofCaloneis. WhenCaloneis reproduces sexually two cells pair and become surrounded by a two-layered capsule of mucilage. Each cell produces two gametes and these become rearranged within the gametangial frustule before plasmogamy. The gametes are amoeboid and fusion is isogamous. Following plasmogamy the zygote contracts, becomes ellipsoidal, and lays down a primary perizonial band. This is a complete, wide hoop, while subsequent perizonial bands are narrow and open.     

NUCLEAR HISTONE PROTEINS OF GAMETES IN AN OOGAMOUS AND TWO ISOGAMOUS BROWN ALGAE1

Nuclear basic proteins (histones) were studied in male and female gametes of the isogamous brown algae, Colpomenia bullosa (Saunders) Yamada and Analipus japonicus (Harvey) Wynne and sperm of the oogamous Cystoseira hakodatensis (Yendo) Fensholt by using SDS‐ and AUT‐PAGE. Four major core histones and several linker histone H1s were detected by electrophoresis. Each of the core histones was identified by amino acid sequence analysis and peptide mapping. Electrophoresis patterns of histones were the same in male and female gametes and quite similar between the two species. The composition of histone H1s in conspicuously condensed sperm nuclei of C. hakodatensis was different from that in isogamous gametes. Electrophoresis after micrococcal nuclease digestion of chromatin in male and female gamete nuclei of C. bullosa and A. japonicus and sperm of C. hakodatensis resulted in regular ladder patterns of DNA fragments (ca. 200 base pair). The chromatin of the brown algal gametes thus has the typical nucleosome structure. These results showed that chromatin condensation in sperm nuclei of C. hakodatensis was associated with a modification of linker histone H1 but not by change of core histones, replacement by other basic proteins, changes of repeating patterns, or disappearance of nucleosomes.     

Sexual auxosporulation of the marine diatom Navicula directa var. directa

Sexual auxosporulation was observed in a mixed culture of two strains of Navicula directa var. directa. Two gametes did not re‐arrange in their gametangium and each adhered to the inner surfaces of the gametangial theca. Each of the two gametes of one gametangium fused with a gamete of the other gametangium iso‐gamously. As a result, two zygotes and hence two auxo‐spores were produced per paired gametangia. As the gametangial thecae kept close to the gametes during fusion, the zygote became associated with two different thecae. The presence of type IB2a of Geitler's (1973) system was confirmed by the present observations.     

THE ULTRASTRUCTURE OF DERBESIA TENUISSIMA (DE NOTARIS) CROUAN. I. ORGANIZATION OF THE GAMETOPHYTE PROTOPLAST,GAMETANGIUM, AND GAMETANGIAL PORE1,2,3

The fine structure of vegetative and reproductive gametophytes of Derbesia tenuissima is described. Development of the gametangium and release of the gametes progress as follows: (1) In initial stages of gametangium formation, prior to 24 hr before gamete release, there is an accumulation and proliferation of nuclei, chloroplasts, and other organelles. (2) This is followed by separation of the gametangium from the rest of the plant by a gametangial membrane; segregation of organelles into gametes has begun by 12 hr before release and the process is completed by 2.5 hr before release. (3) Enzymatic wall dissolution of the pore area occurs between 2.5 and, 12 hr before normal lights-on time. (4) The release mechanism appears to be an instantaneous light-induced increase in lurgor pressure rupturing the weakened pore area, of the wall and causing a forcible expulsion of the gametes. (5) Following release, the pore is sealed by organellar debris and the gametangial membrane. Additional wall layers are presumed to be laid down internal to the plugged pore by the vegetative protoplasm which migrates into the area.     

EFFECTS OF FIXATIVES ON THE ULTRASTRUCTURE OF PHYSODES IN VEGETATIVE CELLS OF SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYTA)1

The effects of different glutaraldehyde-osmium fixation schedules on the ultrastructure of the vegetative cells from the meristematic regions of Scytosiphon lomentaria (Lyngbye) Link fronds are described. The best overall preservation of cell structure was obtained with a 2 h fixation in 2.5–3.5% glutaraldehyde in 0.1 M cacodylate buffered seawater (pH 7.0), followed after washing by 1 h post fixation in 1% osmium tetroxide. The addition of 1% caffeine to the glutaraldehyde fixative resulted in better retention and spatial localization of the electron dense phenolic deposits within the cells. Particular attention was paid to the effects of the various fixation schedules on the electron-dense material within the cells and the images obtained were compared with previous accounts of brown algal cells. It is proposed that the term physode should be restricted to the discrete electron dense spherical bodies within the vacuoles and not applied to electron dense material in general. Although the organization of Scytosiphon cells was similar to that previously reported in the Scytosiphonaceae, the organization of the plasmodesmata into pit fields is at variance with previous accounts.     

PYRENOID FORMATION ASSOCIATED WITH THE CELL CYCLE IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti‐RUBISCO antibody in vegetative cells of gametophytes. From mid‐S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.     

Light- and electron-microscopic studies of growth and reproduction inCutleria (Phaeophyta)

Summary Differentiation of the female gametangium inCutleria bancockii Dawson is described. Four series of mitoses result in a 16-locule structure (four tiers of four cells each). The organelles in each locule become polarized after partitioning is complete, with the mitochondria lying near the longitudinal axis of the gametangium. The nucleus and plastids are centrally located, with abundant osmiophilic material present in the cytoplasm subjacent to the gametangial surface. Both electron density and Toluidine Blue 0 staining of the material increase. Two flagella are then produced: one becomes tightly appressed to the plasmalemma near its base, and the other is free. A prominent eyespot forms in the plastid nearest the developing flagella. Golgi and endoplasmic reticulum vesicles are prolific in this region and seem to be involved with mastigoneme production and deposition on the free flagellum. Immediately beneath the plasmalemma, flagellar rootlet tubules emanate from amorphous masses near the basal bodies. Some of these tubules are associated with the eyespot. Most of the osmiophilic material is then secreted into the extracytoplasmic spaces while the gametes are rounding up. Granular-cored vesicles may be involved with pore formation and gamete release.     

DISAPPEARANCE OF MALE MITOCHONDRIAL DNA AFTER THE FOUR‐CELL STAGE IN SPOROPHYTES OF THE ISOGAMOUS BROWN ALGA SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYCEAE)1

Mitochondrial DNA (mtDNA) of the isogamous brown alga Scytosiphon lomentaria (Lyngb.) Link is inherited maternally. We used molecular biological and morphological analyses to investigate the fate of male mitochondria. Ultrastructural observations showed that the number of 25 mitochondria in a zygote coincided with the number of mitochondria derived from male and female gametes. This number remained almost constant during the first cell division. Strain‐specific PCR in single germlings suggested that mtDNA derived from the female gamete remained in the germling during development, while the male mtDNA gradually and selectively disappeared after the four‐cell stage. One week after fertilization, male mtDNA had disappeared in sporophytic cells. Using bisulfite DNA modification and methylation mapping assays, we found that the degree of methylation on three analyzed sites of mtDNA was not different between male and female gametes, suggesting that maternal inheritance of mtDNA is not defined by its methylation. This study indicates that the mechanism of selective elimination of male mtDNA is present in each cell of a four‐celled sporophyte and that it does not depend on different degrees of DNA methylation between male and female mtDNA.     

Dissection of conjugants and mating type plus and minus cells in selfing clones of the isogamous green alga Closterium ehrenbergii

In the sexual reproduction of the green alga Closterium ehrenbergii, two sexually competent cells that are morphologically indistinguishable from the vegetative cells first come close to each other to form a sexually interacting pair. Each then divides into two gametangial cells. Isogamous conjugation occurs between nonsister gametangial cells of the two resulting pairs. With unusual selfing clones derived from a certain cross of heterothallic strains, we dissected apart a pair of gametangial cells that had already been united together by a delicate transparent tube, into which each gametangial cell was going to develop its conjugation papilla. In spite of such a degree of differentiation, when each was cultured in fresh medium, individual gametangial cells could dedifferentiate into vegetative cells and form subclones. By crossing such subclones with standard stable heterothallic mating-type strains, we show that each selfing clone of this alga actually produces both stable mt ⁺ and stable mt ^- cells, in addition to unstable mt ^- cells with selfing potency, during its mitotic vegetative growth. Although the selfing in C. ehrenbergii studied here differs in certain points from true homothallism, the results of the present study provide insight into how homothallism might have evolved from heterothallism.     

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.     

SEX‐SPECIFIC CELL SURFACE STRUCTURE OF ANISOGAMETES: MORPHOLOGICAL CHANGES DURING FERTILIZATION OF BRYOPSIS MAXIMA (ULVOPHYCEAE,CHLOROPHYTA) REVEALED BY ULTRA–HIGH‐RESOLUTION FIELD EMISSION SEM1

Cell surfaces of biflagellate gametes and their morphological changes during fertilization of Bryopsis maxima Okamura were observed using a high‐resolution field emission scanning electron microscope. Male gametes have broad and narrow faces, which are divided into at least five morphologically distinct regions: 1) the apical plate is a plate‐like structure that is approximately 380–530 nm long and approximately 190 nm wide, in the center of the papilla and slightly protruded from the plasma membrane; 2) strips are smooth materials on ridges that originate from the basal part of the papilla and extend downward; 3) the lateral belt is a belt‐shaped structure on the center of the narrower faces; 4) the flagellar surface; and 5) the other region of the cell body has a fine‐grained appearance. In contrast, the entire female gamete surface is rough because of many granular or amorphous cell coats on the plasma membrane. When both gametes were mixed together, the initial fusion proceeded between the broader face of the male gamete and the anterior side of the female one near the basal bodies. Morphology of the male gamete's cell surface changed gradually as fusion proceeded and was covered by the granular materials; that surface closely resembled those of female gametes except for the apical plate. It was present until the planozygote attached itself to the substrate by the papilla. It finally disappeared after settlement. Therefore, these results indicate that gametes of B. maxima have sex‐specific surface structures that change their morphology during fertilization and settlement.     

Parthenogenetic female populations in the brown alga Scytosiphon lomentaria (Scytosiphonaceae,Ectocarpales): decay of a sexual trait and acquisition of asexual traits

In isogamous brown algae, the sexuality of populations needs to be tested by laboratory crossing experiments, as the sexes of gametophytes are morphologically indistinguishable. In some cases, gamete fusion is not observed and the precise reproductive mode of the populations is unknown. In the isogamous brown alga Scytosiphon lomentaria in Japan, both asexual (gamete fusion is unobservable) and sexual populations (gamete fusion is observable) have been reported. In order to elucidate the reproductive mode of asexual populations in this species, we used PCR‐based sex markers to investigate the sex ratio of three asexual and two sexual field populations. The markers indicated that the asexual populations consisted only of female individuals, whereas sexual populations are composed of both males and females. In culture, female gametes of most strains from asexual populations were able to fuse with male gametes; however, they had little to no detectable sexual pheromones, significantly larger cell sizes, and more rapid parthenogenetic development compared to female/male gametes from sexual populations. Investigations of sporophytic stages in the field indicated that alternation of gametophytic and parthenosporophytic stages occur in an asexual population. These results indicate that the S. lomentaria asexual populations are female populations that lack sexual reproduction and reproduce parthenogenetically. It is likely that females in the asexual populations have reduced a sexual trait (pheromone production) and have acquired asexual traits (larger gamete sizes and rapid parthenogenetic development).     

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae)

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

     

SEXUAL REPRODUCTION,MATING SYSTEM,AND PROTOPLAST DYNAMICS OF SEMINAVIS (BACILLARIOPHYCEAE)1

Cell division, the mating system, and auxosporulation were studied in the marine epipelic diatom Seminavis cf. robusta Danielidis & D. G. Mann. The interphase protoplast contains two girdle‐appressed chloroplasts, each with an elongate bar‐like pyrenoid, and also a central nucleus, located in a bridge between two vacuoles. Before cell division, the chloroplasts divide transversely and translocate onto the valves. The nucleus relocates to the ventral side for mitosis. After cytokinesis and valve formation, the chloroplasts move back to the girdle, showing a constant clockwise movement relative to the epitheca of the daughter cell. Seminavis cf. robusta is dioecious, and sexual reproduction is possible once cells are less than 50 μm. In crosses of compatible clones, gametangia pair laterally, without the formation of a copulation envelope, and produce two gametes apiece. The intensity of sexualization increases as cells reduce further in size below the 50‐μm threshold. At plasmogamy, the gametangia dehisce fully and the gametes, which were morphologically and behaviorally isogamous, fuse in the space between the gametangial thecae. The auxospore forms a transverse and longitudinal perizonium. After expansion is complete, there is an unequal contraction of the protoplast within the perizonium, creating the asymmetrical shape of the vegetative cell. Apart from this last feature, almost all characteristics exhibited by the live cell and auxospores of Seminavis agree with what is found in Navicula sensu stricto, supporting the classification of both in the Naviculaceae. Haploid parthenogenesis and polyploid auxospores were found, lending support to the view that change in ploidy may be a significant mechanism in diatom evolution.     

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.     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole