PHYTOPLANKTON COMMUNITY COMPOSITION AND GENE EXPRESSION OF FUNCTIONAL GENES INVOLVED IN CARBON AND NITROGEN ASSIMILATION1

A functional gene microarray was developed and used to investigate phytoplankton community composition and gene expression in the English Channel. Genes encoding the CO₂‐fixation enzyme RUBISCO (rbcL) and the nitrate assimilation enzyme nitrate reductase (NR) representing several major groups of phytoplankton were included as oligonucleotide probes on the “phytoarray.” Five major groups of eukaryotic phytoplankton that possess the Type 1D rbcL gene were detected, both in terms of presence (DNA) and activity (rbcL gene expression). Changes in relative signal intensity among the Type 1D rbcL probes indicated a shift from diatom dominance in the spring bloom to dominance by haptophytes and flagellates later in the summer. Because of the limitations of a smaller database, NR probes detected fewer groups, but due to the greater diversity among known NR sequences, NR probes provided higher phylogenetic resolution than did rbcL probes and identified two uncultivated diatom phylotypes as the most abundant (DNA) and active (NR gene expression) in field samples. Unidentified chlorophytes and the diatom Phaeodactylum tricornutum Bohlin were detected at both the DNA and cDNA (gene expression) levels. The reproducibility of the array was evaluated in several ways, and future directions for further improvement of probe development and sensitivity are outlined. The phytoarray provides a relatively high‐resolution, high‐throughput approach to assessing phytoplankton community composition in marine environments.     

LIGHT BOUNDARIES AND THE COUPLED EFFECTS OF SURFACE HYDROPHOBICITY AND LIGHT ON SPORE SETTLEMENT IN THE BROWN ALGA HINCKSIA IRREGULARIS (PHAEOPHYCEAE) 1

We examined the effects of light and surface hydrophobicity individually and in tandem on Hincksia irregularis (Kützing) Amsler spore settlement. Hincksia irregularis spores were determined to be negatively phototactic by the use of computer‐assisted motion analysis. Spore settlement was significantly influenced by surface hydrophobicity and by light, individually and in tandem. Experiments conducted using culture plates modified to reduce well edge artifacts revealed significantly higher settlement on hydrophobic surfaces and in dark environments when compared with negatively charged surfaces and lighted environments. Experimental light/dark boundaries elicited distinct spore settlement responses, with spores displaying dissimilar settlement patterns on plates with different surface hydrophobicities. The results of this study indicate H. irregularis spores possess the capacity for complex responses to their environment. These complex responses may influence dispersal and aid spores in the detection of suitable settlement locations in marine microenvironments.     

Circadian rhythmicity in the expression of a novel light-regulated rice gene

We have identified and analyzed cDNAs corresponding to a single-copy gene from rice, designated lir1, whose expression exhibits dramatic diurnal fluctuations. The cDNAs encode a putative protein of 128 amino acids with no homology to known proteins. Lir1 mRNA accumulates in the light, reaching maximum and minimum steady-state levels at the end of the light and dark period, respectively. The oscillations of lir1 mRNA abundance persist after the plants have been transferred to continuous light or darkness. Plants germinated in the dark have very low levels of lir1 mRNA, whereas plants germinated in continuous light express lir1 at an intermediate but constant level. These results indicate that lir1 expression is controlled by light and a circadian clock.     

PHYLOGENETIC ANALYSIS OF 32 STRAINS OF VAUCHERIA (XANTHOPHYCEAE) USING THE rbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA‐rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.     

NUCLEOTIDE SEQUENCE OF THE GENES FOR RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT AND RIBOSOMAL PROTEIN S14 FROM A CHLORELLA-LIKE GREEN ALGA1

Two chloroplast genes were sequenced from an exsymbiotic strain of a eukaryotic, Chlorella-like green alga. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the ribosomal protein S14 (rps14) were oriented in the same direction and were separated by 402 bp. The rbcLs of the exsymbiont and a free living Chlorella ellipsoidea were compared with other reported rbcL sequences. The rbcL gene of the exsymbiont is closely related to that of free-living Chlorella ellipsoidea. This is the first published report of an rps 14 gene sequence from an alga.     

Ultrastructure of the ascospores of some species of the Torulaspora group

Development and germination of the ascospores in species of the Torulaspora group of yeasts have been described. Most species had warty spores which, in sections, showed a dark outer layer consisting of the outer unit membrane of the prospore wall and a layer underneath formed at an early stage of development of the spores. In mature spores the light inner layer of the wall was delimited at the outside by a thin dark layer. The warts often contained dark material. The ascospores of two Pichia and three Debaryomyces species were studied for comparison; they differed in sections from the Torulaspora spores. The taxonomic implications of the ultrastructural observations have been discussed.     

MOLECULAR PHYLOGENY OF THE GREEN ALGAL ORDER PRASIOLALES (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The systematics of the Prasiolales was investigated by phylogenetic inference based on analyses of the rbcL and 18S rRNA genes for representatives of all four genera currently attributed to this order (Prasiococcus, Prasiola, Prasiolopsis, Rosenvingiella), including all type species. The rbcL gene had higher sequence divergence than the 18S rRNA gene and was more useful for phylogenetic inference at the ranks of genus and species. In the rbcL gene phylogeny, three main clades were observed, corresponding to Prasiola, Prasiolopsis, and Rosenvingiella. Prasiococcus was nested among species of Prasiola occurring in subaerial and supralittoral habitats. Trichophilus welckeri Weber Bosse, a subaerial alga occurring in the fur of sloths in Amazonia, was closely related to Prasiolopsis ramosa Vischer. The species of Prasiola were grouped into three well‐supported clades comprising (i) marine species, (ii) freshwater and terrestrial species with linear blades, and (iii) terrestrial species with rounded or fan‐shaped blades. Sequence divergence was unexpectedly low in the marine group, which included species with different morphologies. For the 18S rRNA gene, the phylogenetic analyses produced several clades observed for the rbcL gene sequence analysis, but, due to very little sequence variation, it showed considerably lower resolution for inference at the species and genus levels. Due to the low support of some internal branches, the results of the analyses did not allow an unambiguous clarification of the origin and the early evolution of the Prasiolales.     

OBSERVATIONS ON THE SURVIVAL AND GERMINATION OF RESTING SPORES OF THREE CHAETOCEROS (BACILLARIOPHYCEAE) SPECIES1,2

Resting spores (hypnospores) of Chaetoceros diadema (Ehrenberg) Gran, Chaetoceros vanheurckii Gran, and Chaetoceros didymus Ehrenberg were collected from a large plastic enclosure moored in Saanich Inlet, B.C., Canada. The effects of combinations of temperature and irradiance on the germination of these resting spores were investigated. Nutrient uptake, carbon fixation, and changes in the photosynthetic capacity of the germinating spores were also examined. Resting spores germinated optimally at combinations of temperature and irradiance similar to those in the environment during sporulation. They did not germinate at irradiances 1.3 μEin m^?2 s^?1 or temperatures >25.3° C. Nitrate, phosphate and silicate were taken up after the resting spores had germinated and resumed vegetative growth. Chlorophyll a fluorescence in vivo, and the DCMU-induced increase in in vivo fluorescence also increased after the resting spores had germinated. Resting spores began to fix carbon as soon as they were placed in light. Spores remained viable for at least 645 d. The length of time between first exposure to light and germination did not change during this period; however, the percentage of viable resting spores decreased markedly. None of the Chaetoceros spores germinated after 737 d of storage at 2–4° C in darkness.     

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE,VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold‐tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL–18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.‐A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.‐D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.‐ANT1, and Chloromonas sp.‐ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex.     

GLOBAL SYSTEMATIC AND PHYLOGENETIC ANALYSIS OF SARGASSUM IN THE GULF OF MEXICO,CARIBBEAN AND PACIFIC BASIN

Sargassum is one of the most species‐rich genera in the brown algae with over 400 described species worldwide. The bulk of these species occurs in Pacific‐Indian ocean waters with only a small portion found on the Atlantic side of the Isthmus of Panama. Sargassum also has one of the most subdivided and complex taxonomic systems used within the algae. Systematic distinctions within the genus are further complicated by high rates of phenotypic variability in several key morphological characters. Molecular analyses in such systems should allow testing of systematic concepts while providing insights into speciation and evolutionary patterns. Global molecular phylogenetic analyses using both conserved and variable regions of the Rubisco operon (rbcL and rbcL‐IGS‐rbcS) were performed with species from the Gulf of Mexico, Caribbean, and Pacific basin. Results confirm earlier analyses based on rbcL‐IGS‐rbcS from Pacific species at the subgeneric and sectional level while providing additional insights into the systematics and phylogenetics on a global scale. For example, species east of the Isthmus of Panama form a distinct well‐resolved clade within the tropical subgenus. This result in sharp contrast to traditional systematic treatments but provides a window into the evolutionary history of this genus in the Pacific and Atlantic Ocean basins and a possible means to time speciation events.