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.     

Chemical signals and habitat selection by three zooplankters in Andean Patagonian ponds

1. Zooplankton may react differently to chemical signals produced by macrophytes in shallow systems. They may be attracted by macrophytes, as the plants may be used as a refuge against predators, or the plants may have a repellent effect (e.g. when the plants are a habitat for numerous invertebrate predators or fish). In fishless Patagonian ponds, the structural complexity provided by macrophytes modulates the rate of predation on zooplankton by the invertebrate predator Mesostoma ehrenbergii (Turbellaria). 2. We performed a field study to analyse the coexistence of M. ehrenbergii and three of its prey (two copepods, the calanoid Boeckella gracilis and the cyclopoid Acanthocyclops robustus, and the cladoceran Ceriodaphnia dubia) in four ponds. In two of the ponds, we carried out day and night sampling to evaluate the influence of macrophytes on the distribution of these zooplankters. 3. In laboratory experiments, we analysed the response of the zooplankters to the chemical signals produced by macrophytes (the emergent Juncus pallescens and the submerged Myriophyllum quitense), the predator M. ehrenbergii and the ‘alarm signal’ provided by a homogenate of conspecifics. 4. Our field studies demonstrated the coexistence of M. ehrenbergii and the selected prey in different seasons and that A. robustus and C. dubia choose the vegetated area (a mixed bed of J. pallescens and M. quitense) over the non‐vegetated area. The habitat choice experiments indicated that the presence of M. ehrenbergii may directly affect the habitat selection of B. gracilis, because this zooplankter swam away from the predator. In addition, Mesostoma may indirectly affect the habitat selection of the cyclopoid copepod A. robustus and the cladoceran C. dubia as both zooplankters exhibited a negative response to the alarm signal produced by crushed conspecifics. 5. The presence of the submerged M. quitense did not affect the horizontal movements of any of the zooplankters studied. In contrast, the emergent macrophyte J. pallescens elicited a positive response of B. gracilis, suggesting that this aquatic plant may act as a predation refuge. 6. Our results suggest that predator avoidance behaviour can occur in fishless environments in response to a tactile invertebrate predator like Mesostoma. In addition, the refuge effect of emergent macrophytes, enhancing the survival of pelagic zooplankters, may act as a key factor in stabilizing predator–prey interactions in fishless Patagonian ponds, as has been widely recorded in northern temperate lakes with fish.     

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.     

PLASMOLYSIS,HECHTIAN STRAND FORMATION,AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID,CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a “lattice”‐like arrangement with interstrand spacing of 120–130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis‐deplasmolysis. Application of microtubule‐ or microfilament‐affecting agents or various proteolytic/polysaccharide‐degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.     

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.     

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.     

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.     

Predator–prey instability: individual-level mechanisms for population-level results

1. In previous work we established that increasing temperature led to a destabilization of the population dynamics of the invertebrate carnivore Mesostoma ehrenbergii and its prey Daphnia pulex , which ultimately resulted in the local extinction of Daphnia at higher temperatures. Two mechanisms are proposed to explain the population-level phenomena: (1) quantitative changes in carnivore vital rates with increasing temperature led to stronger functional and numerical response and (2) qualitative changes in the dynamic allocation of energy to reproduction by the predator with increasing temperature introduces inverse density dependence in the predator's response.
2. The growth of individual M. ehrenbergii was monitored under various food conditions to determine the effect of two temperatures (18 and 24 °C) and five food levels on rates of growth, prey consumption and reproduction and on reproductive allocation patterns.
3. The first mechanism was supported by both higher consumption rates (stronger functional response) and faster growth rates with earlier age at maturity and shorter generation time (stronger numerical response).
4. Evidence for mechanism two was also provided by an alteration of the reproductive allocation pattern with temperature. Viviparous (subitaneous) eggs were more likely to be produced by this carnivore at low food levels at 24 °C, while at 18 °C, high food levels were required before individuals made this switch. This shift actually introduces inverse density dependence in the predator's numerical response which is highly destabilizing.
5. Based on the results of this study, the differential effect of M. ehrenbergii on the dynamics and structure of its D. pulex prey populations can be attributed to changes in both physiological rates and reproductive allocation patterns with temperature.     

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.