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.     

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.     

Parametric Analysis of Alignment and Phylogenetic Uncertainty

To infer a phylogenetic tree from a set of DNA sequences, typically a multiple alignment is first used to obtain homologous bases. The inferred phylogeny can be very sensitive to how the alignment was created. We develop tools for analyzing the robustness of phylogeny to perturbations in alignment parameters in the NW algorithm. Our main tool is parametric alignment, with novel improvements that are of general interest in parametric inference. Using parametric alignment and a Gaussian distribution on alignment parameters, we derive probabilities of optimal alignment summaries and inferred phylogenies. We apply our method to analyze intronic sequences from Drosophila flies. We show that phylogeny estimates can be sensitive to the choice of alignment parameters, and that parametric alignment elucidates the relationship between alignment parameters and reconstructed trees.     

PHYLOGENETIC AFFINITIES OF THE SARCINOCHRYSIDALES AND CHRYSOMERIDALES (HETEROKONTA) BASED ON ANALYSES OF MOLECULAR AND COMBINED DATA1

Small-subunit ribosomal RNA nucleotide sequences were inferred for Giraudyopsis stellifera Dangeard (Chrysomeridales), as well as for Pulvinaria sp. and Sarcinochrysis marina Geitler (Sarcinochrysidales,). Phylogenetic analyses of the molecular data indicate that the former is weakly related to the Phaeophyceae/Xanthophyceae clade, whereas the latter two have affinities to the Pelagophyceae, and the Sarcinochrysidales sensu stricto is transferred to this class. A recent study proposed that the Pelagophyceae belongs to a larger assemblage of chromophytic species characterized by reduced flagellar apparatuses. Although the flagellar apparatus characterizing the Sarcinochrysidales is reduced relative to the Chysomeridaels and some other chromophytes, it is the most complicated to be associated with “the reduced flagellar apparatus” lineage. Cladistic analyses of a traditional data set (largely ultrastructural features of the flagellar apparatus) and a combined traditional/molecular data set were used to assess the evolutionary trends of reduction in the flagellar apparatus within the heterokont chromophytes.     

Molecular Cloning and Antiserum Development of Cyclin Box in the Brown Tide Alga Aureococcus anophagefferens

Cyclins can be useful cell cycle markers for growth rate studies on harmful algal blooms. In this study, a gene fragment corresponding to cyclin box was cloned for the brown tide alga Aureococcus anophagefferens. This algal gene fragment, designated as Btcycl1, was most similar to cyclin B. Oligopeptides based on the deduced amino acid sequence were synthesized and used to raise an antiserum that reacted on Western blots with a protein of about 63 kDa, the same size as cyclin B in other organisms. The cyclin B–like protein recognized by this antiserum, and the messenger RNA amplified using the primers, were more abundant in exponential cultures and decreased markedly in stationary cultures. This protein also appeared to be cell cycle dependent. Immunofluorescence labeling showed that this antiserum specifically stained a protein in Aureococcus cells and had no cross-reaction with bacteria that were present in the algal culture. The Btcycl1 sequence and the antiserum will provide a useful tool for studies on regulation of in situ growth rate for this brown tide alga. Received May 22, 2000; accepted July 13, 2000.     

A Three-Gene Dinoflagellate Phylogeny Suggests Monophyly of Prorocentrales and a Basal Position for <Emphasis Type="Italic">Amphidinium</Emphasis> and <Emphasis Type="Italic">Heterocapsa</Emphasis>

Many outstanding questions about dinoflagellate evolution can potentially be resolved by establishing a robust phylogeny. To do this, we generated a data set of mitochondrial cytochrome b (cob) and mitochondrial cytochrome c oxidase 1 (cox1) from a broad range of dinoflagellates. Maximum likelihood, maximum parsimony, and Bayesian methods were used to infer phylogenies from these genes separately and as a concatenated alignment with and without small subunit (SSU) rDNA sequences. These trees were largely congruent in topology with previously published phylogenies but revealed several unexpected results. Prorocentrum benthic and planktonic species previously placed in different clusters formed a monophyletic group in all trees, suggesting that the Prorocentrales is a monophyletic group. More strikingly, our analyses placed Amphidinium and Heterocapsa as early splits among dinoflagellates that diverged after the emergence of O. marina. This affiliation received strong bootstrap support, but these lineages exhibited relatively long branches. The approximately unbiased (AU-) test was used to assess this result using a three-gene (cob + cox1 + SSU rDNA) DNA data set and the inferred tree. This analysis showed that forcing Amphidinium or Heterocapsa to relatively more derived positions in the phylogeny resulted in significantly lower likelihood scores, consistent with the phylogenies. The position of these lineages needs to be further verified. Reviewing Editor: Dr. Martin Kreitman     

EFFECTS OF NUTRIENT ENRICHMENT ON NATURAL POPULATIONS OF THE BROWN TIDE PHYTOPLANKTON AUREOCOCCUS ANOPHAGEFFERENS (CHRYSOPHYCEAE)1

The brown tide picoalga Aureococcus anophagefferens Hargraves et Sieburth was present in approximately equal numbers in 12 large scale (13,000 L) mesocosms at the start of a nutrient addition experiment in June 1985. Increases in abundance in untreated systems mimicked the pattern of bloom development in Narragansett Bay, Rhode Island, the seawater source for the experiment. Aureococcus increased to maximal values of 2.6 × 10⁹ cells. L^?1 and persisted at high numbers (10⁸ cells·L^?1) for 7–8 weeks. In nutrient addition tanks, the picoalgae bloomed briefly (1–3 weeks) but rapidly declined to the usual level (～10⁷ cells·L^?1) for eukaryotic algae in Narragansett Bay. The decline in picoalgae abundance was followed by an increase in total diatoms in all nutrient treated tanks. Mean picoalgae abundance in the mesocosms and the bay was significantly (P < 0.05) and inversely correlated (r =–0.93) with mean concentration of dissolved inorganic nitrogen. The persistence of the brown tide species in control mesocosms and Narragansett Bay appears related to its ability to grow at very low concentrations of dissolved inorganic nitrogen, levels previously shown to limit diatom growth.     

α‐ and β‐Tubulin Phylogenies Support a Close Relationship Between the Microsporidia Brachiola algerae and Antonospora locustae

ABSTRACT. Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced α‐ and β‐tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein‐based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.     

基于ITS和trnL-F以及rps4基因片段的 粗疣藓属分类学地位研究

选择粗疣藓属及相关属样本作为内群,油藓属样本作为外群,选取核基因ITS片段以及叶绿体基因trnL-F和rps4片段进行PCR扩增、测序,运用贝叶斯推论(BI)、最大似然法(ML)和最大简约法(MP)构建分子系统发育树,以明确粗疣藓属的分类学地位。结果表明:(1)用于分子系统分析的基因序列共有1 744个位点,其中747个位点属于核基因片段,997个位点属于叶绿体基因片段。(2)3种系统发育树拓扑结构基本一致,都显示粗疣藓属与毛锦藓科成员聚成一支,支持粗疣藓属应隶属于毛锦藓科的观点。(3)小粗疣藓和粗疣藓的种间界限模糊。(4)小鼠尾藓属的3个种与棉藓属成员聚成一支,支持将其归入棉藓科的观点。     

Ultrastructure and taxonomy of a marine photosynthetic stramenopile Phaeomonas parva gen. et sp. nov. (Pinguiophyceae) with emphasis on the flagellar apparatus architecture

Phaeomonas parva gen. et sp. nov., a marine photosynthetic stramenopile from oceanic water near the Caroline Islands, is described. Cells are naked and spherical to ovoid. The alga is motile with two laterally inserted flagella during the light period, whereas during the dark period, it absorbs the flagella and rounds up. The anterior (immature, No. 2) long flagellum possesses tubular tripartite mastigonemes. The posterior (mature, No. 1) short flagellum is smooth and has autofluorescence at the base. The cupshaped, yellowish‐brown chloroplast occupies the posterior half of the cell, and a pyrenoid occurs in the inner cavity of the cup‐shaped chloroplast. The flagellar apparatus has several unusual features. Two basal plates and a two‐gyred proximal helix in the flagellar transitional region may suggest that P. parva is related to the Pelagophyceae, Dictyochophyceae and Sulcochrysis biplastida, a photosynthetic stramenopile of uncertain taxonomic position. The R3 and R4 roots form a loop that resembles phagotrophic chrysophytes. However, this resemblance is superficial because Phaeomonas is not phagotrophic, its R3 root has a different number of microtubules and its R3 root does not split to form a food‐uptake mouth. Phaeomonas has a ‘bypassing root’, which is found only with the Phaeophyceae, Giraudyopsis stellifera (Chrysomerophyceae), and Ankylochrysis lutea (probably a member of the Pelagophyceae). The taxonomic position of P. parva could not be determined solely from ultrastructural features. However, molecular phylogeny and biochemical analyses (published separately) strongly supported a relationship between P. parva and four other monotypic strameno‐piles, Glossomastix, Pinguiochrysis, Pinguiococcus and Polypodochrysis. Although these algae are morphologically distinct, they have unusually high percentages of polyunsaturated fatty acids, especially eicosapentoic acid. This unusual assemblage of stramenopiles is classified in a new class, the Pinguiophyceae (published separately), and P. parva is its only biflagellate member.     

Regeneration process of coniferous forests in northern Hokkaido I.Abies sachalinensis forest andPicea glehnii forest

Regeneration of natural forests was studied in the Nakagawa Experiment Forest of Hokkaido University using age distribution surveys made by the clear felling method. In Plot 1 (30 m × 65 m),Abies sachalinensis dominated the canopy layer but there were also a fewBetula ermanii trees.Sasa senanensis densely covered the forest floor. Most of the canopy trees were from 122 to 195 years old. Seedlings younger than 50 years old ofA. sachalinensis were found on fallen logs and root bases. There were, however, few trees from 50 to 120 years old. The present canopy trees seemed to have regenerated after competitive pressure from old canopy andSasa disappeared 180 years ago. Plot 2 (50 m × 100 m) on serpentinite soil was dominated byPicea glehnii. Sasa kulirensis covered the floor but not as densely asS. senanensis in Plot 1. The ages ofP. glehnii ranged from 1 to 586 years old, and the age distribution ofA. sachalinensis was L-shaped. A small gap in the canopy formed about 290 years ago, and it gradually extended. Conifers regenerated continuously in the extending gap butB. ermanii did not. One hundred thirty years ago, part of Plot 2 was again destroyed andA. sachalinensis andB. ermanii regenerated. Thus, two types of regeneration were found. One regenerated both conifers andBetula after a sudden disturbance of canopy layer or death ofSasa, and the other, under an extending gap, regenerated only conifers.     

Phylogenetic relationships among eukaryotic kingdoms inferred from ribosomal RNA sequences

Summary Phylogenetic trees among eukaryotic kingdoms were inferred for large- and small-subunit rRNAs by using a maximum-likelihood method developed by Felsenstein. Although Felsenstein's method assumes equal evolutionary rates for transitions and transversions, this is apparently not the case for these data. Therefore, only transversiontype substitutions were taken into account. The molecules used were large-subunit rRNAs fromXenopus laevis (Animalia), rice (Plantae),Saccharomyces cerevisiae (Fungi),Dictyostelium discoideum (Protista), andPhysarum polycephalum (Protista); and small-subunit rRNAs from maize (Plantae),S. cerevisiae, X. laevis, rat (Animalia), andD. discoideum. Only conservative regions of the nucleotide sequences were considered for this study. In the maximum-likelihood trees for both large- and small-subunit rRNAs, Animalia and Fungi were the most closely related eukaryotic kingdoms, and Plantae is the next most closely related kingdom, although other branching orders among Plantae, Animalia, and Fungi were not excluded by this work. These three eukaryotic kingdoms apparently shared a common ancestor after the divergence of the two species of Protista,D. discoideum andP. polycephalum. These two species of Protista do not form a clade, andP. polycephalum diverged first andD. discoideum second from the line leading to the common ancestor of Plantae, Animalia, and Fungi. The sequence data indicate that a drastic change occurred in the nucleotide sequences of rRNAs during the evolutionary separation between prokaryote and eukaryote.     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Molecular evolution and secondary structural conservation in the B-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products

The nature of the bcl-2 family of protooncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. Phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for BCL-2 and BCL-X, human MCL1, murine A1, the nematode Caenorhabditis elegans and Caenorhabditis briggsiae ced-9 proteins, and the sequences BHRF1 from Epstein-Barr and LMW5-HL from African swine fever viruses. Both sequence alignment and secondary structure prediction techniques supported the conservation of both the overall secondary structure and the carboxy-terminal transmembrane domain in all members of the family. All the treeing methods employed (distance matrix, maximum likelihood, and parsimony) supported a tree in which the proapoptotic proteins BCL-2 and BCL-X represent the most recent additions to the group. All the trees also indicated that the viral proteins BHRF1 and LMW-HL arose from a common ancestor, an ancestor they shared in common with the pro-apoptotic control protein BAX, indicating that this function of BAX evolved only recently. The most ancient branches are represented by the nematode ced-9 protein and by the control genes MCL1 and A1, which in the treeing methods employed represent separate lineages within the most ancient grouping. These results demonstrate the evolution of a highly conserved family of developmental control genes from nematode to man—genes that encode proteins essential for normal development but which are highly conserved in terms of predicted structure and possible cellular localization. The evolutionary analysis also indicates that the family may be even larger than originally predicted and that other members are waiting to be discovered. Correspondence to: D. Lloyd Evans     

Branch sacrifice: cavitation-associated drought adaptation of riparian cottonwoods

In their native riparian zones (floodplains), Populus deltoides (prairie cottonwood) and P. fremontii (Fremont cottonwood) commonly experience substantial branch die-back. These trees occur in semi-arid areas of North America and unexpectedly given the dry regions, they are exceptionally vulnerable to xylem cavitation, drought-induced air embolism of xylem vessels. We propose that the vulnerability to cavitation and branch die-back are physiologically linked; drought-induced cavitation underlies branch die-back that reduces transpirational demand enabling the remaining shoot to maintain a favorable water balance. This proposal follows field observation along various western North American rivers as precocious branch senescence, the yellowing and death of leaves on particular branches during mid- to late summer, was common for P. deltoides and P. fremontii during hot and dry periods of low stream-flow. Branches displaying precocious senescence were subsequently dead the following year. The proposed association between cavitation, precocious senescence and branch die-back is also supported by experiments involving external pressurization of branches to about 2.5 MPa with a branch collar or through an adjacent cut-branch. The treatments induced xylem cavitation and increased leaf diffusive resistance (stomatal closure) that was followed by leaf senescence and branch death of P. deltoides. P. trichocarpa (black cottonwood) appeared to be less affected by the pressurization treatment and this species as well P. angustifolia (narrowleaf cottonwood) and P. balsamifera (balsam poplar) seldom display the patchy summer branch senescence typical of P. deltoides and P. fremontii. ’Branch sacrifice’ describes this cavitation-associated senescence and branch die-back that may provide a drought adaptation for the prairie and Fremont cottonwoods. Received: 13 May 1999 / Accepted: 4 November 1999     

Two new species ofPichia isolated from slime fluxes of deciduous trees

Two new homothallic species ofPichia, associated with exudates ofPopulus trichocarpa andSalix sp. have been described.Pichia trehalophila sp.n. was isolated from slime exudates in two widely separatedPopulus trichocarpa trees andPichia salictaria sp.n. was isolated from two exudates ofSalix sp. The latter species was previously present as an unidentified strain ofPichia in the culture collection of the Centraalbureau voor Schimmelcultures and had been isolated from a patient in Germany. The natural habitat ofP. salictaria is considered to be in association with willow trees.     

Reproductive allocation and fruit-set pattern in the euhalophyte Suaeda salsa in controlled and field conditions

Reproductive allocation and fruit-set pattern are important characteristics of halophytes, and these characteristics were investigated in euhalophyte Suaeda salsa (S. salsa). The plants comprised of those treated with 1 mM (control) or 500 mM (high salinity) of NaCl for 120 days in a glasshouse and those collected in the field. In controlled conditions, high salinity increased reproductive allocation and the number of fruits and seeds in each node of the branches. In field conditions, reproductive allocation and the number of fruits and brown seeds in S. salsa at the upper tide line were higher than at the lower tide line. There were more fruits and seeds distributed in the central part than those in the base or top of branches in both controlled and field conditions. Soil organic matter, total nitrogen and total phosphorus content at the upper tide line were lower than at the lower tide line. An opposite trend could be observed in the total soluble salt, Na⁺ and Cl^? content in soil. In conclusion, the fruit-set pattern of S. salsa may be affected by both resource constraints and resource allocation. Reproductive allocation and fruit-set pattern may ensure the establishment of S. salsa in high saline and low nutrient environments.     

Antimicrobial activity of berberine--a constituent of Mahonia aquifolium

The antimicrobial activity of the protoberberine, alkaloid, berberine, isolated fromMahonia aquifolium, was evaluated against 17 microorganisms including two Gram-negative bacteria—Pseudomonas aeruginosa andEscherichia coli (both resistant and sensitive), two Gram-positive bacteria—Bacillus subtilis andStaphylococcus aureus, Zoogloea ramigera, six filamentous fungi—Penicilium chrysogenum, Aspergillus niger, Aureobasidium pullulans (black and white strain),Trichoderma viride (original green strain and brown mutant),Fusarium nivale, Mycrosporum gypseum, and two yeasts—Candida, albicans andSaccharomyces cerevisiae. The IC₅₀, minimum inhibitory concentration (MIC), minimum microbicidal concentration (MMC) and minimum microbistatic concentration (MMS) varied considerably depending on the microorganism tested, the sensitivity decreasing as follows:S. aureus >P. aeruginosa S (sensitive) >E. coli S>P. aeruginosa R (resistant) >E. coli R>B. subtilis>Z. ramigera>C. albicans>S. cerevisiae>A. pullulans B (black)>A. pullulans W (white)>T. viride Br (brown)>M. gypseum>A niger>F. nivale>P. chrysogenum>T. viride G (green).     

Molecular phylogeny in the Lardizabalaceae

Eleven species belonging to seven genera in the Lardizabalaceae were analyzed in terms of restriction fragment length polymorphism (RFLP) of chloroplast DNA and the sequence of the chloroplast gene,rbcL, of Lardizabalaceae and its related families. Phylogenetic trees inferred from parsimony, neighbor joining and maximum likelihood methods based on RFLP data showed that two South American genera,Boquila andLardizabala, and three East Asian genera,Akebia, Holboellia andStauntonia are closely related to each other, respectively. On the other hand, the parsimony, neighbor joining and maximum likelihood trees constructed using sequence data of therbcL gene showed thatAkebia, Stauntonia, Boquila andLardizabala clustered as(((Akebia, Stauntonia), Boquila), Lardizabala). This difference may be attributable to fewer informative sites inrbcL genes than in RFLP in this family.Decaisnea diverges at the very base of the Lardizabalaceae.