Terunobu Ichimura

Little divergence in ribosomal DNA internal transcribed spacer-1 and -2 sequences among non-digitate species of Laminaria (Phaeophyceae) from Hokkaido, Japan

The nuclear ribosomal DNA internal transcribed spacer (ITS‐1 and ITS‐2) sequences were determined for 10 of 12 Japanese non‐digitate Laminaria species, Kjell‐maniella gyrata (Kjellman) Miyabe, Costaria costata (Turner) Saunders, Alaria praelonga Kjellman and Chorda filum (L.) Stackhouse collected at Hokkaido. Phyloge‐netic analyses (maximum parsimony and distance matrix) of these sequences, including published data for L. sac‐charina (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. yapon/ca 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. sac‐charina, 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.

Pathogenic bacteria associated with lesions and thallus bleaching symptoms in the Japanese kelp Laminaria religiosa Miyabe (Laminariales, Phaeophyceae)

During early Spring (April–May) when the seawater salinity drops suddenly and the seawater temperature increases drastically, severe lesions and thallus bleaching were observed in the Laminaria religiosa population at Oshoro Bay, Otaru, Hokkaido, Japan. The healthy and diseased kelp blades were collected and subjected to enumeration of total number of culturable bacteria and bacterial species. Bacterial enumerations were done using 3 different media formulations; high-nutrient media (Media 1), low-nutrient media (Media 2) and modified low-nutrient media with 5% kelp extract (Media 3). Seven bacterial species were isolated from the healthy kelp. These were Alcaligenes aquamarinas, Alteromonas sp., Azomonas agilis, Azotobacter beijerinckii, Escherichia coli, Halobacterium sp. and Halococcus sp. All 7 bacterial species were isolated on Media 2 and Media 3, but only 5 species were isolated using Media 1 with the absence of Halobacterium sp. and Halococcus sp. Highest total number of culturable bacteria was 2050 CFU/cm2 on Media 3. Eight species of bacteria were isolated from the diseased kelp thallus with the addition of Erwinia amylovora. All 8 bacteria grew on Media 2 and Media 3, but only 6 species were isolated using Media 1 with the absence of Halobacterium sp. and Halococcus sp. Highest total number of culturable bacteria was 5830 CFU/cm2 on Media 3. However, only 3 species were isolated from the lesioned area. The most abundant species was Alteromonas sp. followed by Halococcus sp. and Alcaligenes aquamarinas. The surface bacteria showed best growth on Media 3. Scanning Electron Microscopic images of the healthy and diseased thallus gave distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population. In an effort to identify the symptoms causative organism, the isolated bacterial species were cultured and used to test Kochs postulates. Out of the 8 species, only Alteromonas sp. induced lesions on the axenic kelp blades. The inoculated bacteria were also re-isolated without any significant contamination. Hence, Alteromonas sp. is suggested as the possible disease causing organism.

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.

Gametic behavior in a marine green alga, Monostroma angicava: an effect of phototaxis on mating efficiency

Abstract. The role of phototactic behavior of gametes was tested experimentally in the slightly anisogamous marine green alga Monostroma angicava Kjellman, and the effect of phototaxis on mating efficiency was discovered. Both male and female gametes showed positive phototaxis in response to a white light source. In contrast, they did not respond to a red light source. Their swimming velocity did not differ between these two illuminating light sources. It was, therefore, suggested that the search ability of the gamete itself might not vary between phototactic and non-phototactic conditions. The number of zygotes formed during the mating process may be expressed as the product of the number of encounters between male and female gametes and the fraction of encounters that result in sexual fusion. In this study, with high densities of male and female gametes mixed in test tubes, almost all minor (fewer in number) gametes fused sexually within 10 min. After dilution of the gamete suspensions by half, mating efficiency in test tubes illuminated by white light from above was higher than that in dark controls. This suggests that male and female gametes gathered at the water surface through their positive phototaxis, thus increasing the rate of encounters. Mating efficiency also decreased if the test tubes were illuminated from above by white light and also shaken. Since negative phototaxis is clearly shown in planozygotes, we suggest that positive phototaxis of male and female gametes in M. angicava is an adaptive trait for increasing the rate of gametic encounters rather than for the dispersal of zygotes as previously reported for zoospores of some marine algae.

Biosystematic studies of theClosterium peracerosum-strigosum-littorale complex

Nine representative pairs of heterothallic (=self-sterile, cross-fertile) strains of theClosterium peracerosum-strigosum-littorale complex from the northern Kanto area in Japan have been studied under the defined standard culture conditions. Since a wall thickening at cell apices was observed in vegetative cells of all the strains, these strains turned out to belong to the morphological group II (Ichimura and Watanabe, 1976). The result of statistical analyses of their cell size variations corresponded well with the result of intercrossing experiments between them. It was shown that these strains are virtually composed of three biologically different groups which are morphologically distinct and reproductively isolated completely or at least partially from each other. For convenience, these three groups have been designated as II-A, II-B, and II-C in the order of from smaller to larger cell size. In intra-group crossings, a large number of zygospores were formed, and they germinated well to yield healthy populations of their progenies in all the three groups. In inter-group crossings, no sign of sexual reproduction was observed between Group II-A and Group II-C or Group II-B and Group II-C, and a marked decrease of zygospore formation was observed between Group II-A and Group II-B, especially between Group II-A minus and Group II-B plus. It was concluded that the distinctions between the three groups are biologically sound and that each represents an evolutionary unit.

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.

Nucleotide sequence diversity of the 5S rDNA spacer in the simple blade kelp genera Laminaria, Cymathaere and Kjellmaniella (Laminariales, Phaeophyceae) from northern Japan

Tandem repeats of the 5S ribosomal RNA gene (rDNA) were confirmed for almost all laminarian, cymathaerean and kjellmaniellan species distributed in northern Japan. The nucleotide sequence of the spacer region between tandemly repeated 5S rDNA was investigated for 79 samples from 31 sites. Phylogenetic analysis of the 29 different sequences detected revealed two lineages: (1) Laminaria coriacea group, including Laminaria coriacea Miyabe, Laminaria cichorioides Miyabe, Laminaria sachalinensis (Miyabe) Miyabe, Laminaria yendoana Miyabe, Cymathaere japonica Miyabe et Nagai, Kjellmaniella gyrata (Kjellman) Miyabe and Kjellmaniella crassifolia Miyabe; (2) Laminaria japonica group including Laminaria japonica Areschoug, Laminaria religiosa Miyabe, Laminaria ochotensis Miyabe, Laminaria diabolica Miyabe, Laminaria longipedalis Okamura, Laminaria angustata Kjellman and Laminaria longissima Miyabe. In addition, the latter group was divided into two: subgroup (2a) including L. angustata and L. longissima and subgroup (2b) including L. japonica, L. religiosa, L. ochotensis, L. diabolica and L. longipedalis. Members of the three groups differ from each other in the appearance of ornaments (bullation, gyration and folds) on the surface of the blade. These are absent in group (2a), only present in the early stages of the lifespan of group (2b), and present for the duration of the lifespan in group (1). Genetic distances among samples were extremely small within group (2a). Together with previous crossing studies and data on ocean currents and distribution, these findings suggest that gene flow occurs within group (2b).

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

Abstract 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.

GAMETE DIMORPHISM IN BRYOPSIS PLUMOSA PHOTOTAXIS, GAMETE MOTILITY AND PHEROMONAL ATTRACTION

An anisogamous marine green alga, Bryopsis plumosa (Hudson) C. Agardh from Muroran, Hokkaido, Japan was studied in light of the theories of the evolution of anisogamy. Male gametes were 5.04 ± 0.86 (means ± SB) μιη long and 2.36 ± 0.45 μηη wide without an eye-spot. Female gametes were 8.96 ± 1.38 μιη long and 4.83 ± 0.78 μιη wide with an eye-spot. Gametes of both sexes were biflagellated and they were simultaneously liberated by light stimulation. Male gametes showed no phototaxis. In contrast, almost all female gametes initially showed positive phototaxis but some changed to negative particularly 12 h after liberation. The swimming velocity of freshly liberated male gametes as much higher than that of female gametes. Male gametes increased their swimming velocity remarkably was water temperatures were raised, although the swimming velocity of female gametes was not affected significantly. Gametes of both sexes changed their swimming velocities only slightly as intensities of irradiance changed. Male gametes had a potential of swimming for ca. 6 h and female ones for ca. 36 h. Planozygotes were negatively phototactic and swam still more rapidly than gametes of both sexes. The differences in the cell motility between male gametes, female gametes, and planozygotes seemed to be related largely to their cell sizes. Male gametes were attracted around mature female gametangia and female gametes. A cell-free bioassay clearly showed the existence of a sexual pheromone from female gametes.