We investigated the origin of dissolved free amino acids (DFAA) in stream water focusing on biofilms in the river bed by means of incubation experiments in the laboratory. Stones were placed in the Toyohira River, Hokkaido, Japan, for 3 months, allowing formation of biofilms, and then incubated for 24 h in the laboratory at stream water temperature. After incubation, the composition and concentrations of DFAA in the incubation solution and total hydrolyzed amino acids (THAA) in biofilms were measured by high-performance liquid chromatography. The amount of chlorophyll a (Chl. a) and the number of bacteria were also measured. The DFAA concentration increased greatly in the biofilm incubation solution, but the DFAA composition (mol %) did not change relative to the inception of incubation, where it was similar to stream water. There was no correlation between the increase in DFAA concentration and the THAA concentration, Chl. a amount, or the number of bacteria in biofilms. These results suggest that biofilms are one of the major sources of DFAA in stream water.

Culture strains of the Closterium moniliferum-ehrenbergii species complex were classified on the basis of three types of dormant spores: zygospores formed by homothallic and heterothallic conjugation and parthenospores formed without conjugation. Two parthenosporic strains of C. moniliferum from Hiroshima, Japan, and Ho Chi Minh, Vietnam, and two parthenosporic strains of C. ehrenbergii from Okinawa, Japan, and Ho Chi Minh, Vietnam, were used in the present study. Morphological observations on the process of parthenospore formation and germination were made using light and fluorescence microscopy. Parthenosporic strains of the two species essentially shared the same manner of parthenospore formation. Germination of parthenospores in C. monilferum produced one germling without any meiotic event. The phylogenetic relationship between parthenosporic strains of the two species and among homothallic, heterothallic and parthenosporic strains were analysed on the basis of joint sequences of the 1506 group I intron and internal transcribed spacer-2 (ITS-2) regions of nuclear ribosomal DNA, using maximum likelihood. Phylogenetic analysis showed that parthenosporic strains of C. monoliferum and C. ehrenbergii were not monophyletic. Parthenosporic strains of C. ehrenbergii were in the same clade with heterothallic mating group K of the same species, whereas parthenosporic strains of C. moniliferum were sister to heterothallic mating group M of C. ehrenbergii. Inferred secondary-structure models of ITS-2 showed some common features among the three reproductive systems. The present study confirmed the hypothesis that parthenosporic populations have been derived from heterothallic populations in the C. moniliferum-ehrenbergii complex.

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.

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. calosproum 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 VI 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.

The nuclear ribosomal DNA internal transcribed spacer (ITS-1 and ITS-2) sequences were determined for 10 of 12 Japanese non-digitate Laminaria species, Kjellmaniella gyrata (Kjellman) Miyabe, Costaria costata (Turner) Saunders, Alaria praelonga Kjellman and Chorda filum (L.) Stackhouse collected at Hokkaido. Phylogenetic analyses (maximum parsimony and distance matrix) of these sequences, including published data for L. saccharina (L.) Lamouroux from Canada, showed strong nucleotide conservation among these species of Laminaria, but two phylogenetically distinct species groups were recognized. A L. japonica group encompassing L. japonica Areschoug, L. religiosa Miyabe, L. ochotensis Miyabe, L. diabolica Miyabe, L. longipedalis Okamura, L. angustata Kjellman and L. longissima Miyabe and a L. saccharina group including L. coriacea Miyabe, L. saccharina, L. cichorioides Miyabe and L. yendoana Miyabe. As to other laminarialean genera, Kjellmaniella gyrata was most closely related to the genus Laminaria, being related to the second Laminaria species group based on both parsimony and distant tree values.

We investigated whether allelochemicals that could suppress other algae were released from the coralline red algae, Corallina pilulifera and Pneophyllum zostericolum, Bialgal cultures were composed of either of the two coralline species and Laminaria sporelings in a millicell-CM insert. Higher culture temperatures tended to increase suppressive activities of both coralline algae against female gametophyte maturation and normal sporophyte formation of Laminaria. Even if dissolved iron, which is known to be required for the gametophyte maturation, was amply supplied to PES medium, the suppressive activity was still recognized. In bialgal cultures, when C. pilulifera and L. japonica sporelings were horizontally placed, the suppressive activity decreased compared to those vertically placed. Suppressive effects against the female gametophyte maturation in the conditioned medium of C. pilulifera was apparently lower than those in bialgal cultures. These results led us to assume that allelochemicals released from the coralline algae suppressed development of Laminaria sporelings and that they were volatile and/or labile substances. Three kinds of volatile halomethanes known to be released from C. pilulifera: bromoform, dibromochloromethane, and dibromomethane, were examined for suppressive effects on the development of L. angustata sporelings. Bromoform at a concentration as low as 3 ppm apparently reduced the normal sporphyte formation. Dibromochloromethane and dibromomethane showed weaker toxicities; both halomethanes contain less bromine atoms than bromoform. However, since we could detect only extremely low concentrations of bromoform in the cultures of C. pilulifera, we came to the conclusion that the actual allelochemicals in the bialgal cultures could not be the above halomethanes.

An allelopathic substance, which displays growth-inhibitory activity and spore-settlement suppressive activity, was isolated from some red algae and identified as (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid on the basis of spectroscopic and chemical evidence. Copyright (C) 1996 Elsevier Science Ltd

An antiserum against gibberellin A4 (GA4) raised in rabbits and its partially purified antibodies were used to develop radioimmunoassay (RIA) and indirect enzyme-linked immunosorbent assays (ELISAs) for GA4. Of three immunoassays tested, an ELISA based on the NAD-dependent redox cycle (enzyme-amplified ELISA) had highest sensitivity. Levels of methylated GA4 detected by this most sensitive method ranged from 0.1 fmol/assay (3.5 fg/assay) to 0.1 pmol/assay (3.5 pg/assay) suggesting applicability of this method to the detection of gibberellins in purified plant extracts.