STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Disappearance of the heteroplasmic state for chloroplast markers in zygospores of Chlamydomonas reinhardtii

In the investigations reported here, the length of zygospore incubation or “maturation” prior to the induction of meiosis was found to affect the inheritance pattern of chloroplast genes. The frequency of zygospores transmitting chloroplast alleles from both parents drops with increasing zygospore age following mating, while the frequencies of zygospores homoplasmic for maternal or paternal chloroplast alleles increase correspondingly. Since there is a negligible reduction in viability, zygospores which are initially biparental appear to become pure for the chloroplast genes from one or the other parent prior to the occurrence of cell division. These results are amplified in crosses of mt⁺ cells which have been irradiated with ultraviolet (uv) light or grown in the presence of the base analog, 5-fluorodeoxyuridine, which also perturbs maternal inheritance. Low doses of uv irradiation, applied to zygospores derived from crosses in which the maternal parent was also irradiated prior to mating, increase the biparental zygospore frequency while reducing the proportion of maternal zygospores. This indicates that at least some maternal zygospore clones are actually derived from zygospores which still contain both parental chloroplast genomes prior to the induction of germination. Thus, a subclass of zygospores must contain paternal chloroplast genomes which are either eliminated upon germination or are not expressed in the resulting zygospore clone. Tetrad analysis of biparental zygospores derived from uv-irradiated mt⁺ gametes demonstrates that the frequency of maternal chloroplast alleles in biparental zygospores decreases as they age. One result is an increase in the proportion of meiotic products homoplasmic for all paternal markers. The increased segregation of homoplasmic daughter cells during the meiotic divisions may result from a reduction in chloroplast ploidy by elimination of maternal genomes. Alternatively, it may reflect an altered ratio of maternal:paternal genomes due to continuous rounds of pairing and gene conversion between heterologous chloroplast DNAs leading to genetic drift within the DNA population of the organelle.     

ALTERED ZYGOSPORE WALL ULTRASTRUCTURE CORRELATES WITH REDUCED ABIOTIC STRESS RESISTANCE IN A MUTANT STRAIN OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

The zygospore of Chlamydomonas is a diploid resting stage that provides protection from environmental extremes. The remarkable abiotic stress resistance of the zygospore can be explained, in part, by the presence of a massive wall that includes a sporopollenin‐containing surface layer ( Van Winkle‐Swift and Rickoll 1997 ). A Chlamydomonas monoica Strehlow zygospore‐specific mutant strain (D19) was obtained previously by screening for loss of chloroform resistance in zygospore populations derived from self‐mating of post‐mutagenesis clones. Exposure of D19 zygospores to solar UV radiation or germicidal radiation also resulted in a pronounced decrease in survival of D19 zygospores relative to wildtype zygospore survival. Similarly, resistance to NaCl‐induced osmotic shock was reduced in D19 zygospores, especially when exposed to very high (e.g., 20% w/v) salt concentrations. Mature zygospores of C. monoica exhibit a UV‐induced blue surface autofluorescence that may indicate the presence of phenolic wall components. The intensity of zygospore autofluorescence was significantly reduced in D19 zygospores. As revealed by TEM, the surface layer of mature homozygous D19 zygospores was disrupted, suggesting a defect in wall assembly. Zygospore‐specific chloroform sensitivity, UV sensitivity, and reduced autofluorescence cosegregated in tetrads derived from D19 heterozygotes (i.e., if a progeny clone from a cross involving D19 and a normal strain was found to be chloroform sensitive, it was always also UV sensitive and showed reduced autofluorescence), indicating that all three characteristics were the consequence of the same Mendelian mutation.     

Natural populations of the Closterium ehrenbergii (Desmidiales, Chlorophyta) species complex in Nepal

Several natural populations of the Closterium ehrenbergii Meneghini ex Ralfs species complex were collected in Nepal, in October–December 1982. Water temperature and pH were also recorded. Clonal isolates from these populations were identified to one of four mating groups (H, I, J and M) by test crossing with standard mating-type strains of known mating groups. Groups H and M have smooth walled zygospores, while Groups I and J have scrobiculated zygospore walls. Several undetermined isolates were found in some population samples. In contrast to the previously reported population samples from Nepal, especially from dried soil samples, some of these populations appeared to be rather heavily loaded with mutations that are deleterious to the sexual cycle (i.e. sexual compatibility, zygospore formation and germination). By genetic analysis, a zygote maturation-defective mutation (zym) was detected. One reason for such a heavy genetic load was suggested to be that most population samples had been maintained exclusively by asexual reproduction for a long period in large lakes and nearby ponds, or left-over vegetative populations in paddy fields after other members entered into dormancy through sexual reproduction. The significance of studying such mutations at sexual gene loci is discussed in the light of speciation problems in microalgae.     

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.     

ENVIRONMENTAL FACTORS AND SEXUAL EXPRESSION IN CHLOROCOCCUM ECHINOZYGOTUM (CHLOROPHYCEAE)1

Several environmental factors affected total growth and zygospore production in Chlorococcum echinozygotum Starr. The temperature range at which zygospore production occurred was more restricted than the range that supported vegetative growth. Light intensity had little effect upon zygospore formation: gamete production and gamete pairing occurred in darkness. Zygospore production occurred over a wide pH range; bicarbonate had a minor effect upon zygospore formation. Nitrogen concentration was the factor of primary importance. As the level of nitrogen supplied as nitrate, ammonia, urea and asparagine in the medium was increased, zygospore production first increased (over no nitrogen) at low levels and then decreased at high levels. All levels of glutamine supplied reduced zygospore production. A possible way in which nitrogen concentration in the medium and sexual expression are linked is discussed.     

Zygospore formation inMortierella capitata

A novel type of zygospore formation is described in the heterothallic speciesMortierella capitata, which was repeatedly isolated from soils inhabited by pillbugs (Armadillidium vulgare, Isopoda). Zygospore formation was induced on media containing sterilized arthropods. Anisogamy and colorless zygospore walls are shared with other zygosporic species ofMortierella, but a unique feature ofM. capitata is the production of zygospores on elongated macrosuspensors which are covered by branches of the microsuspensors. This kind of zygosporogenesis is termed “capitata-type” here. The taxonomic position ofM. capitata is discussed based on the zygospore characteristics.     

Variations of the Surface Sculpture and Cell Wall Ultrastructure of the Zygospores in Chlamydomonas geitleri (Chlorophyta)

The superficial cell wall ornamentation in the zygospores of the alga Chlamydomonas geitleri Ettl (Chlorophyta) is formed by thickenings of the cell wall which are shaped into a network of anastomosing ribs, sometimes with local wart-like protuberances. Clearly different sculpture patterns (given by presence, arrangement and/or morphological modification of sculpture elements) were accompanied by many transient forms. Sculpture variations occurred even in clonal cultures. In the zygospore cell wall of C. geitleri, the inner, outer and middle layer can be distinguished from the morphological point of view. The relatively thin outer (sporopollenin) layer covers the whole surface of the zygospore wall. The thicker inner layer adhering to the zygospore protoplast forms, either solely or together with the middle layer (possessing a fine meshwork substructure), variously shaped thickening of the zygospore cell wall. Discussed are the ultrastructural morphology of the cell wall in Chlamydomonas zygospores, the striking similarity of the cell wall ultrastructure of zygospores in C. geitleri to the ultrastructure of the cell wall of vegetative cells in some green algae (subfamily Scotiellocystoideae), as well as the extensive morphological variability of the zygospore wall sculpture in C geitleri and its species specificity.     

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.     

Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

CULTURE STUDY OF REPRODUCTIVE CYCLES AND SYSTEMATICA IN MOUGEOTIA TRANSEAUI (CHLOROPHYTA)1,2

Asexual and sexual reproductive cyries are described and illustrated for a cultured homothallic strain of Mougeotia transeaui Collins. Reproduction is by aplanospores and zygospores. Aplanospore formation precedes zygospore formation and continues longer with regreening of older cultures occurring regularly from precocious germination of aplanospores. Aplanospores typically form when most of the protoplast moves into the central swollen region of a cell and two new cross walls form to delimit an aplanosporangium. Conjugation is scalariform without the movement and fusion of well-organized gametes. During zygospore maturation, three new cross walls form in the receptive gametangium and conjugation tube to produce a zygosporangium.     

SELF-STERILE AND MATURATION-DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA,CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

Homothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform-resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly-outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation-defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self-sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully-matured, chloroform-resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.     

SEXUAL REPRODUCTION IN PANDORINA UNICOCCA1

The effects of nutrients and light on sexual reproduction were investigated in 2 mating pairs of Pandorina unicoca. Strains 101–104 required nitrogen deficiency for sexual differentiation. Sulfur deficiency also appeared to be effective in evoking sexual differentiation. The time required for sexual differentiation of strains 103–104 after removal of KNO₃ and MgSO from the medium was 24 hr. Addition of KNO₃ and MgSO₄ after 24hr did not reverse sexual differentiation. Light was required during the first 10–12 hr. Strains 105–106 did not require a period of sexual differentiation and, exhibited mating activity in the presence of nitrogen and sulfur in the medium. Both mating pairs required light for the mating response and calcium was necessary for zygote formation. The pH range for mating activity was determined.     

The genetics of two homothallic species of the Mucoraceae

Summary Auxotrophic strains of Mucor genevensis and Zygorhynchus exponens were crossed and the resulting zygospores germinated. The presence of a true sexual cycle in both species was demonstrated by the recovery of recombinant genotypes. Expected Mendelian ratios were not realized, however. The presence of selfed zygospores among those isolated makes this observation understandable. It was possible to demonstrate nutritional complementation when young mating mycelium was transferred to minimal medium and forced heterokaryons were recovered.     

Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses ofPhycomyces blakesleeanus

Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages. Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors.     

MATURATION OF ALGAL ZYGOTES: ALTERNATIVE EXPERIMENTAL APPROACHES FOR CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Young zygotes from crosses of Chlamydomonas reinhardtii Dang. mutant and wild-type strains were incubated, in the presence or absence of light and/or nitrogen to determine whether continuation of conditions inducing gamete formation permits zygospore formation without loss of viability. Different culture media, continuous illumination vs. dark incubation and various durations of the maturation period were tested, for effect on zygospore germination efficiency, zygospore “burst size” and zoospore viability. Following either the routine maturation procedure of dark incubation on standard minimal medium, or following a new procedure of incubation under continuous illumination on N-free medium, zygospore formation can be ensured and high germination efficiencies obtained within 3 days after mating. Tetrad analysis indicates meiosis occurs normally whether zygotes have been matured in the presence or absence of light or nitrogen. Preliminary data suggest an effect of increased maturation time on the transmission of cytoplasmic genes, if a N-free continuous illumination maturation protocol is followed. Two experimental approaches for the maturation of C. reinhardtii zygotes are suggested and advantages of each are discussed.     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

Lipid content during sexual development in the homothallic mucor Zygorhynchus moelleri

In Zygorhynchus moelleri, a homothallic Mucor, triglycendes are the main components of chloroform/methanol extractable lipids. The triglycerides accumulate in the aerial hyphae, particularly in the developing zygospores and in the lateral suspensors, but only after zygospore maturation. They are probably transported from the submerged mycelium to the aerial hyphae. Most of the fatty acid synthetase activity is found in the submerged mycelium. The fatty acid composition of the triglycerides does not change appreciably during sexual development. No influence of trisporic acids has been found on triglyceride synthesis or transport.     

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.