THE INABILITY OF THE TEXAS “BROWN TIDE” ALGA TO USE NITRATE AND THE ROLE OF NITROGEN IN THE INITIATION OF A PERSISTENT BLOOM OF THIS ORGANISM1

A planktonic alga similar in general morphology and pigments to Aureococcus anophagefferens Hargraves and Sieburth has caused persistent and ecologically damaging blooms along the south Texas coast. Experiments using 100 μM NO₃^?, NO₂^?, and NH₄⁺ demonstrated that the alga could not use NO₃^? for growth but could use NO₂^? and NH₄⁺. Doubling iron or trace metal concentrations did not permit growth on NO₃^?. Chemical composition data for cultures grown in excess NO₃^? or NH₄⁺, respectively, were as follows: N·cell^?1 (0.88 vs. 1.3 pg), C:N ratio (25:1 vs. 6.4:1), C:chlorophyll a (chl a) (560:1 vs. 44:1), and chl a·cell^?1 (0.033 vs. 0.16 pg). These data imply that cells supplied with NO₃^? were N-starved. Culture addition of 10 mM final concentration chlorate (a nitrate analog) did not affect the Texas isolate while NO₃^? utilizing A. anophagefferens was lysed, suggesting that the NO₃^? reductase of the Texas isolate is nonfunctional. Rates of primary productivity determined during a dense bloom indicated that light-saturated growth rates were ca. 0.45 d^?1, which is similar to maximum rates determined in laboratory experiments (0.58 d^?1± 0.16). However, chemical composition data were consistent with the growth rate of these cells being limited by N availability (C:N 28, C:chl a 176, chl a·cell^?1 0.019). Calculations based on a mass balance for nitrogen suggest that the bloom was triggered by an input of ca. 69 μM NH₄⁺ that resulted from an extensive die-off of benthos and fish.     

PHYSIOLOGICAL RESPONSES DURING DARK SURVIVAL AND RECOVERY IN AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE)1

The harmful bloom alga Aureococcus anophagefferens Hargraves et Sieburth can survive prolonged darkness, which could facilitate overwinter survival and dispersal by anthropogenic vectors such as ballast water. Experiments were conducted to examine the biochemical and photosynthetic changes in cells during dark storage. Cells were stored in the dark for periods from 2 to 14 days and were sampled at days 0, 1, 2, 4, 7, 10, and 14. Samples from days 2, 7, and 14 were monitored during a recovery period of 4–5 days. Physiological and photosynthetic parameters were measured during the dark storage and recovery periods. Cultures resumed growth quickly when returned to light, and bacterial counts remained constant during the dark storage period but increased rapidly during recovery periods. Cellular protein, carbohydrate, and lipid concentrations declined slightly during the dark period. There were no changes in chl a per cell or in RUBISCO per cell during 14 days of darkness. The data therefore suggest that A. anophagefferens is able to maintain its photosynthetic apparatus during dark storage periods of at least 2 weeks and relies on cellular reserves until it is returned to light to resume photosynthesis. During the recovery period in the light, the cells are able to acclimate rapidly to current light levels and resume growth.     

ANALYSIS OF CLONAL CULTURES OF THE BROWN TIDE ALGAE AUREOCOCCUS AND AUREOUMBRA (PELAGOPHYCEAE) USING 18S rRNA, rbcL, AND RUBISCO SPACER SEQUENCES

Variability among clonal cultures of the brown tide algae Aureococcus anophagefferens Hargraves et Sieburth and Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson was examined by DNA sequence comparisons. Nuclear-encoded 18S rRNA and plastid-encoded rbc L gene sequences were determined for six Aureococcus strains. RUBISCO spacer sequences were determined for 14 strains of Aureococcus. No differences among Aureococcus strains were found in the DNA regions examined. The rbc L and RUBISCO spacer sequences for three Aureoumbra strains were identical but differed from those of Aureococcus. These data indicate that blooms of these species are comprised of cells that are very similar and also imply that Aureococcus and Aureoumbra do not contain varieties or cryptic species. Separate and combined phylogenetic analyses of the 18S rRNA and rbc L gene sequences were performed. Results confirm that the brown-tide-causing algae of Long Island Sound, New York ( Aureococcus ), and Laguna Madre, Texas ( Aureoumbra ), are best classified in separate genera within the Pelagophyceae. Phylogenetic trees place Aureococcus and Aureoumbra within the Pelagomonadales and Sarcinochrysidales, respectively.     

CHARACTERIZATION OF NITROGEN UPTAKE BY NATURAL POPULATIONS OF AUREOCOCCUS ANOPHAGEFFERENS (CHRYSOPHYCEAE) AS A FUNCTION OF INCUBATION DURATION,SUBSTRATE CONCENTRATION,LIGHT, AND TEMPERATURE1

Nitrogen uptake studies were conducted during an aestival “brown tide” bloom in Shinnecock Bay, Long Island, New York. The same station was sampled in late July and mid-August 1995 when Aureococcus anophagefferens composed >90% and 30–40% of the total cell density, respectively. Experiments were designed to examine the effect of incubation duration on the uptake kinetics, and the effect of light and temperature dependencies of NH₄⁺, urea, and NO₃^? uptake. Maximum specific uptake rates (V'_max) decreased in the order NH₄⁺, urea, NO₃^? and were nonlinear with time for NH₄⁺ and urea, both of which exhibited an exponential decline between 1 and 10 min and then did nut significantly change for 60 min. Nitrogen uptake kinetic experiments exhibited a typical hyperbolic response for urea and NO₃^?. Half-saturation constants. (K_s) were calculated to he 0.03 and 0.12 μmol · L^?1 for urea and NO₃^?; respectively, but could not be calculated for NH₄⁺ under these experimental conditions. Nutrient uptake rate versus, irradiance (NI) experiments showed that maximum uptake rates occurred at ≤% of incident irradiance on both sampling dates and that values of V′_max-cell (NH₄⁺) were on average 30% greater than V′_max-cell (urea). A7°–9°C temperature decrease in incubation temperature between the two NI experiments in August resulted in a 30% decrease in V′_max-cell(NH₄⁺), no change in V′_max-cell(urea), and a 3–4-fold decrease in calculated K_lt values for both NH₄⁺ and urea. The results from these experiments demonstrate that A. anophagefferens has a higher affinity for NH₄⁺ and urea than for NO₃^? and that this particular species is adapted to use these substrates at low irradiances and concentrations. The data presented in this study are also consistent with the hypothesis that A. anophagefferens may be an oceanic clone that was displaced by an anomalous oceanographic event.     

DESCRIPTION AND CHARACTERIZATION OF THE ALGAL SPECIES AUREOUMBRA LAGUNENSIS GEN. ET SP. NOV. AND REFERRAL OF AUREOUMBRA AND AUREOCOCCUS TO THE PELAGOPHYCEAE1

The Texas brown tide alga (strain TBA-2) is described as Aureoumbra lagunensis Stockwell, DeYoe, Hargraves, et Johnson, gen. et sp. nov. Pigment composition, chloroplast structure, and 18s ribosomal RNA gene sequence data indicate that A. lagunensis and the east coast brown tide alga Aureococcus anophagefferens (originally placed in the Chrysophyceae) belong in the class Pelagophyceae. The new genus Aureoumbra with A. lagunensis as the type species differs from Aureococcus in 18s ribosomal RNA gene sequence, pyrenoid form, nitrogen physiology, and possession of basal bodies. The genus Aureococcus is placed in the order Pelagomonadates and family Pelagomonadaceae while ordinal placement of Aureoumbra is deferred.     

PHYLOGENY OF THE SUBFAMILY SCOTIELLOCYSTOIDEAE (CHLOROPHYCEAE, CHLOROPHYTA) AND RELATED TAXA INFERRED FROM 18S RIBOSOMAL RNA GENE SEQUENCE DATA

Nuclear-encoded small subunit ribosomal RNA gene (18SrDNA) sequences were determined for Coelastrella multistriata (Trenkwalder) Kalina et Punčochářová, two species of Scotiellopsis ( S. oocystiformis (Lund) Kalina et Punčochářová and S. terrestris (Reisigl) Kalina et Punčochářová) and two species of Muriella ( M. aurantiaca Vischer and M. terrestris Boye- Petersen). Coelastrella and Scotiellopsis are members of the subfamily Scotiellocystoideae, and Muriella is a member of the subfamily Chlorelloideae in the family Chlorellaceae. Phylogenetic trees were constructed based on these sequence data and on previously known 18SrDNA sequences of 25 taxa. Coelastrella and Scotiellopsis were closely related to each other and formed a cluster with Scenedesmus vacuolatus (Shihira et Krauss) Kessler et al. This cluster shared a monophyletic ancestry with other Scenedesmus species. Muriella aurantiaca formed a sister relationship with the monophyletic lineage of Scenedesmus. However, another species, M. terrestris, was placed in the Trebouxiophyceae and was strongly related to Chlorella. The genus Mychonastes belonging to the Scotiellocystoideae was also not monophyletic. This study suggests that the subfamily Scotiellocystoideae should be removed from the Chlorellaceae.     

血卵涡鞭虫核糖体18SrDNA和ITS1-5.8SrDNA-ITS2序列测定与系统发育分析

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ULTRASTRUCTURE AND 18S RRNA GENE SEQUENCE FOR PELAGOMONAS CALCEOLATA GEN. ET SP. NOV. AND THE DESCRIPTION OF A NEW ALGAL CLASS,THE PELAGOPHYCEAE CLASSIS NOV.1

Pelagomonas calceolata gen. et sp. nov., an ultra-planktonic marine alga, is described using electron microscopy and the cytoplasmic small subunit (18S) ribosomal RNA (rRNA) gene sequence. Cells are uniflagellate, about 1.5 × 3 μm in size. The flagellium has two rows of bipartite hairs, the paraxonemal rod has a dentate appearance, and a two-gyred transitional helix is present between two transitional plates. Microtubular roots, striated roots, and a second basal body are absent. A thin organic theca surrounds most of the cell. There is a single chloroplast with a girdle lamella and a single, dense mitochondrion with tubular cristae. A single Golgi body with swelled cisternae lies beneath the flagellum, and each cell has an ejectile organelle that putatwely releases a cylindrical structure. A vacuole, or cluster of vacuoles, contains the putative carbohydrate storage product. The 18S rRNA gene was sequenced completely in both directions, excluding three primer regions. When compared to the same gene sequence from other organisms, Pelagomonas calceolata gen. et sp. nov. occupies an unresolved position among other chromophyte algae and is distinct from members of any of these classes. Based on morphological, ultrastructural, and molecular data, we describe this alga as a new species, and we place this highly unusual new species in a new genus, family, order, and class.     

INFERRING TAXONOMIC POSITIONS AND TESTING GENUS LEVEL ASSIGNMENTS IN COCCOID GREEN LICHEN ALGAE: A PHYLOGENETIC ANALYSIS OF 18S RIBOSOMAL RNA SEQUENCES FROM DICTYOCHLOROPSIS RETICULATA AND FROM MEMBERS OF THE GENUS MYRMECIA (CHLOROPHYTA, TREBOUXIOPHYCEAE CL. NOV.)1

Complete nuclear-encoded small-subunit ribosomal RNA (18S rRNA) coding sequences were determined for the coccoid green algae Dictyochloropsis reticulata (Tschermak-Woess) Tschermak-Woess , Myrmecia astigmatica Vinatzer, and M. bisecta Reisigl, to investigate the taxonomic position of Dictyochloropsis Geitler and of the genus Myrmecia Printz. Phylogenies inferred from these data revealed a sister-group relationship between D. reticulata and certain coccoid green algae that lack motile stages (autosporic coccoids) within the order Microthamniales. The monophyletic origin of the Microthamniales, including autosporic coccoids previously classified in the Chlorophyceae, is clearly resolved by the rRNA sequence data. This finding. shows the considerable taxonomic breadth of that order, whose taxonomic position has been unclear so far. A new class, Trebouxiophyceae, is proposed for this group of green algae. Phylogenetic inferences from the rRNA sequences show paraphyly of the genus Myrmecia. The 18S rRNA sequence data suggest that, among taxa that share similar vegetative cell morphologies, the zoospore characters resolve better the actual genus and species boundaries. Within identical zoospore types, the rRNA data allow further resolution of taxonomic relationships. On the basis of the.se findings, I propose that the genus Friedmannia Chantanachat ± Bold be merged into Myrmecia and that only those species be left in the genus Myrmecia that are identical in particular zoospore characters (i.e. those described in detail for M. israeliensis ( Chantanachat ± Bold) comb, nov.), namely M. astigmatica, M. biatorellae (Tschermak-Woess ± Ptesst) Petersen, and M. israeliensis. Myrmecia bisecta has to be excluded from Myrmecia; its taxonomic position within the Trebouxiophyceae is unclear .