Mitsunobu Kamiya

A rapid method to score plastid haplotypes in red seaweeds and its use in determining parental inheritance of plastids in the red alga Bostrychia (Ceramiales)

A method for the rapid identification of plastid haplotypes in red algae is presented. This method is based on single-stranded conformation polymorphism (SSCP) to detect variation in DNA sequence of PCR-amplified plastid-encoded ribulose bisphosphate carboxylase/oxygenase large and small subunit intergenic spacer. The method detects variation within a population of the mangrove red alga Caloglossa leprieurii from New South Wales, Australia. Three haplotypes (plastid variants) were easily scored in our samples, and a spatial variation of haplotypes within the intertidal is suggested. This method also reveals maternal inheritance of plastids in crosses between isolates of Bostrychia radicans and in multiple crosses between isolates of B. moritziana. Fluorescence microscopy shows that plastids are contained within the spermatia of these species and that bi- or paternal inheritance of plastids is possible. SSCP is a simple and rapid method for the detection of plastid variation (haplotypes) within populations of red algae that should also be applicable to brown seaweeds.

ISOLATION AND CHARACTERIZATION OF PARMALES (HETEROKONTA/HETEROKONTOPHYTA/STRAMENOPILES) FROM THE OYASHIO REGION, WESTERN NORTH PACIFIC1

A small siliceous species of marine phytoplankton, order Parmales (Heterokonta), was isolated and characterized for the first time with the aid of a fluorescent silicon tracer 2‐(4‐pyridyl)‐5‐([4‐(2‐dimethylaminoethylaminocarbamoyl)‐methoxy]phenyl)oxazole (PDMPO). This dye was easily detected by clear fluorescence in newly produced silica cell plates. Our isolate was surrounded by eight smooth plates without any ornamentation, suggesting a similarity to Triparma laevis B. C. Booth. TEM observation showed the typical ultrastructure of photosynthetic heterokontophytes; with two chloroplast endoplasmic reticulate membranes, a girdle lamella, three thylakoid lamellae, and mitochondrion with tubular cristae. Molecular phylogenetic analyses of SSU rDNA and rbcL genes showed that the parmalean alga was within the bolidophycean clade of autotrophic naked flagellates and a sister group of diatoms. HPLC analysis detected chl a, c1 + c2, and c3; fucoxanthin; and diadinoxanthin as major photosynthetic pigments, and a composition that is shared with Bolidophyceae and diatoms. Together, these data indicate a close evolutionary relationship between Parmales, Bolidophyceae, and diatoms. The PDMPO‐staining procedure should accelerate isolation of other Parmales species, helping to establish their diversity and aiding quantitative study of their role in oceanic processes.

EVOLUTIONARY DIVERGENCE IN THE RED ALGAE CALOGLOSSA LEPRIEURII AND C. APOMEIOTICA

Morphological comparisons, hybridization experiments, and molecular phylogenetic analyses using the RUBISCO spacer region were undertaken on 12 populations of Caloglossa leprieurii (Montagne) J. Agardh in order to clarify their relationships. In addition, data from one population of the morphologically similar but asexual species, C. apomeiotica (West et Zuccarello), were included in the assessment. Three morphological types were recognized on the basis of the number of cell rows at nodes of the main axis opposite to the lateral branch and blade width: single/ broad (with three mating groups), multiple/broad (three mating groups) and multiple/slender (one mating group). In the molecular analyses, C. leprieurii was resolved as two clusters that correspond phenetically to the single and multiple cell row types. Both the morphological and molecular data indicate that the asexual species was derived from sexual plants of the multiple cell row type. The reproductive compatibility correlates with genetic distance rather than geographical distance. Sympatric mating groups are completely incompatible and have 10–21 nucleotide changes in the examined region, whereas mating groups that produce abnormal progeny or pseudocystocarps are allopatrically distributed with 5–7 nucleotide changes. The present data suggest that the two populations, one with single and the other with multiple cell rows, which are sympatrically distributed in southeastern Japan, have probably evolved by allopatric speciation. The single/broad type that is restricted to the western Pacific, may have diverged genetically between eastern and western Australia, with subsequent dispersal from the western population as far as Japan.

Reproductive patterns of Caloglossa species (Delesseriaceae, Rhodophyta) from Australia and New Zealand: multiple origins of asexuality in C. leprieurii. Literature review on apomixis, mixed-phase, bisexuality and sexual compatibility

Reproduction and life history patterns in culture of five Caloglossa speaes from Australia and New Zealand are compared. Caloglossa adhaerens King et Puttock and Caloglossa bengalensis (Martens) King et Puttock have a Polyslphonla‐type sexual life history (P‐type, isomorphic alternation of generations). Caloglossa monosticha Kamiya occurs only in Western Australia (WA) and is a P‐type. Caloglossa ogasawaraensis Okamura occurs in WA, Northern Territory (NT), Queensland (QLD), New South Wales (NSW), Victoria (VIC) and South Australia (SA) and is for the most part a P‐type in culture. A few isolates have bisexual gametophytes that are self‐compatible, while most are unisexual. Caloglossa ogasawaraensis from Adelaide, SA and from Wilsons Promontory, VIC are new records for these States. In Australia, Caloglossa postiae (King et Puttock) Kamiya et King occurs in NSW, NT and QLD. All nine isolates are P‐type. Isolates of Caloglossa leprieurii (Montagne) G. Martens from NSW, NT, QLD, Tasmania, VIC and New Zealand are P‐type except for the freshwater isolates in which tetraspore germlings do not reproduce. In some isolates mixed‐phase reproduction is seen with male gametophytes producing both viable spermatia and tetrasporangia and female gametophytes producing procarps and sori with non‐dividing sporangia. All isolates of C. leprieurii irom Spencer Gulf and Gulf of St Vincent, SA and one isolate from QLD give rise to successive asexual generations of tetrasporophytes. Based on RuBisCO spacer DNA data an asexual life history has arisen several times in the C. leprieurii complex. The literature on apomixis, mixed‐phase reproduction, bisexuality and sexual compatibility in red algae is surveyed.

Seasonal variation of phlorotannin in sargassacean species from the coast of the Sea of Japan

Variations in phlorotannin concentrations among the developmental stages of brown algae have been reported; however, the phlorotannin concentration plasticity associated with fluctuations in environmental factors make it difficult to determine the essential ontogenetic variation. The phlorotannin concentrations in five perennial sargassacean species where newly sprouted branches appear in summer and become fertile the following spring were examined every month during a year; and correlation with the developmental or seasonal environmental factors was determined. Although the phlorotannin fluctuated greatly throughout the year, the fluctuation patterns were relatively similar among the five species: phlorotannin showed a peak during July and August; gradually decreased in the winter; and increased in April. Performing a multiple regression analysis, the phlorotannin concentration did not correlate with thallus size in all species; and phlorotannin amounts were significantly affected by ambient abiotic factors in some species. The phlorotannin contents in newly sprouted branches were always higher than those in the long main branches during all seasons. When the phlorotannin contents were determined monthly for S. fulvellum (Turner) C. Agardh where the thalli were cultured from embryos in outdoor tanks, the phlorotannin concentrations were 3–4% of the dry matter (DM) in the juveniles and decreased to less than 1% of the DM in thalli >7.5 cm in length. However, the phlorotannin in these cultured thalli suddenly increased to 5.3% DM after being transplanted to the inshore coast; and then the concentration gradually decreased. The data show higher phlorotannin concentrations in younger sargassacean algae thalli and fluctuation of the phlorotannin amounts with extrinsic environmental factors.

Evolutionary relationships of the genus Caloglossa (Delesseriaceae, Rhodophyta) inferred from large-subunit ribosomal RNA gene sequences, morphological evidence and reproductive compatibility, with description of a new species from Guatemala

Abstract Systematic studies on the genus Caloglossa were performed based on morphology, reproductive crossability and the 26S ribosomal RNA gene sequences of 47 specimens of 12 species. The molecular phylogenetic data suggest that Caloglossa is divided into three major clades, which are distinguishable by the secondary branching method (endogenous or adventitious). A new species from Guatemala, C. rotundata sp. nov., is characterized by a unique arrangement of rhizoids and the production of both endogenous and adventitious secondary branches, and this species makes a sister clade with the adventitiously branched group. Although the distinction between C. monosticha and C. saigonensis had been debated, their heterogeneity was demonstrated by consistent morphological difference under similar culture conditions and reproductive incompatibility. Caloglossa continua ssp. axillaris is characterized by having monosiphonous hyphae along the axis and wide blades, but neither feature is stable under culture conditions. This subspecies should be combined with C. monosticha due to their reproductive compatibility and close evolutionary relationship. Caloglossa apomeiotica, which was described as an asexual species derived from C. leprieurii, should be taxonomically reduced because the molecular phylogeny demonstrates that asexualization happened more than once in the C. leprieurii lineage. Caloglossa leprieurii is divided into two lineages based on the number of cell rows at the nodes (single or multiple), and we propose to resurrect C. vieillardii for the single-cell row type.

ACCUMULATION OF SULFURIC ACID IN DICTYOTALES (PHAEOPHYCEAE): TAXONOMIC DISTRIBUTION AND ION CHROMATOGRAPHY OF CELL EXTRACTS

Four species in the order Dictyotales (Dictyopteris latiuscula (Okamura) Okamura, D. prolifera (Okamura) Okamura, D. repens (Okamura) Børgesen, and Spatoglossum crassum J. Tanaka) were found to be highly acidic as in some species of the order Desmarestiales (Phaeophyceae). The pH within their cells, presumably that of the vacuole, was estimated to be 0.5 to 0.9 by pH measurements of their cell extracts in distilled water. However, other species of these genera (D. divaricata (Okamura) Okamura, D. undulata Holmes, and S. pacificum Yendo) did not show high acidity. Ion chromatography of the cell extracts showed that those species contained high concentrations of SO within their cells, up to 10 times that in seawater but relatively low Cl−. The sum of cations examined (Na+, NH, K+, Mg2+, Ca2+) was significantly lower than that of anions (Cl−, Br−, NO, SO), and the difference is presumed to represent protons (H+), causing the extremely low cell sap pH. Estimated cellular proton concentrations calculated from the pH data roughly agreed with those calculated from differences between the sum of cations and anions and that of anions. Although certain other, nonacidic, dictyotalean species also contained high concentrations of SO, these species contained high concentrations of Mg2+, and the sums of cations and anions were balanced.

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE1

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form‐genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form‐genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form‐genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form‐genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form‐genus Cyanothece were observed to be in a close relationship with our isolates.

Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms

Bolidomonas is a genus of picoplanktonic flagellated algae that is closely related to diatoms. Triparma laevis, a species belonging to the Parmales, which are small cells with a siliceous covering, has been shown to form a monophyletic group with Bolidomonas. We isolated several novel strains of Bolidophyceae that have permitted further exploration of the diversity of this group using nuclear, plastidial and mitochondrial genes. The resulting phylogenetic data led us to formally emend the taxonomy of this group to include the Parmales within the Bolidophyceae, to combine Bolidomonas within Triparma and to define a novel species, Triparma eleuthera sp. nov. The global distribution of Bolidophyceae was then assessed using environmental sequences available in public databases, as well as a large 18S rRNA V9 metabarcode data set from the Tara Oceans expedition. Bolidophyceans appear ubiquitous throughout the sampled oceans but always constitute a minor component of the phytoplankton community, corresponding to at most ~4% of the metabarcodes from photosynthetic groups (excluding dinoflagellates). They are ~10 times more abundant in the small size fraction (0.8–5 μm) than in larger size fractions. T. eleuthera sp. nov. constitutes the most abundant and most widespread operational taxonomic unit (OTU) followed by T. pacifica, T. mediterranea and the T. laevis clade. The T. mediterranea OTU is characteristic of Mediterranean Sea surface waters and the T. laevis clade OTU is most prevalent in colder waters, in particular off Antarctica.

Effects of silicon-limitation on growth and morphology of Triparma laevis NIES-2565 (Parmales, Heterokontophyta).

The order Parmales (Heterokontophyta) is a group of small-sized unicellular marine phytoplankton, which is distributed widely from tropical to polar waters. The cells of Parmales are surrounded by a distinctive cell wall, which consists of several siliceous plates fitting edge to edge. Phylogenetic and morphological analyses suggest that Parmales is one of the key organisms for elucidating the evolutionary origin of Bacillariophyceae (diatoms), the most successful heterokontophyta. The effects of silicon-limitation on growth and morphogenesis of plates were studied using a strain of Triparma laevis NIES-2565, which was cultured for the first time in artificial sea water. The cells of T. laevis were surrounded by eight plates when grown with sufficient silicon. However, plate formation became incomplete when cells were cultured in a medium containing low silicate (ca. <10 µM). Cells finally lost almost all plates in a medium containing silicate concentrations lower than ca. 1 µM. However, silicon-limitation did not affect growth rate; cells continued to divide without changing their growth rate, even after all plates were lost. Loss of plates was reversible; when cells without plates were transferred to a medium containing sufficient silicate, regeneration of shield and ventral plates was followed by the formation of girdle and triradiate plates. The results indicate that the response to silicon-limitation of T. laevis is different from that of diatoms, where cell division becomes inhibited under such conditions.