Diversification of unicellular eukaryotes: cryptomonad colonizations of marine and fresh waters inferred from revised 18S rRNA phylogeny

The cryptomonads is a well-defined lineage of unicellular eukaryotes, composed of several marine and freshwater groups. However, the evolutionary relationships among these groups are unclear due to conflicting inferences between morphological and molecular phylogenies. Here, we have inferred the evolutionary relationships among marine and freshwater species in order to better understand the importance of the marine-freshwater boundary on the historical diversification patterns of cryptomonads. We have constructed improved molecular phylogenies by taking into account rate variation both across sites and across sequences (covarion substitutions), and by analysing the vast majority of publicly available cryptomonad 18S rRNA sequences and related environmental phylotypes. The resulting phylogenies included 55 sequences, and revealed two novel freshwater cryptomonad clades (CRY1 and CRY2) and a large hidden diversity of cryptomonads. CRY1 was placed deeply within the cryptomonad phylogeny together with all the major freshwater lineages (i.e. Goniomonas and Cryptomonas), while CRY2 was placed within a lineage of marine species identified as Plagioselmis-like with the aid of a new sequence generated from a cultured species. The inferred phylogenies suggest only few successful marine-freshwater transitions over the history of cryptomonads. Most of the transitions seem to have occurred from marine to fresh waters, but re-colonizations of marine habitats have also taken place. This implies that the differences in the biogeophysical conditions between marine and fresh waters constitute a substantial barrier for the cross-colonization of these environments by cryptomonads.     

Diversity of the cadmium-containing carbonic anhydrase in marine diatoms and natural waters

A recent report of a novel carbonic anhydrase (CDCA1) with Cd as its metal centre in the coastal diatom Thalassiosira weissflogii has led us to search for the occurrence of this Cd enzyme (CDCA) in other marine phytoplankton and in the environment. Using degenerate primers designed from the published sequences from T. weissflogii and a putative sequence in the genome of Thalassiosira pseudonana, we show that CDCA is widespread in diatom species and ubiquitous in the environment. All detected genes share more than 64% amino acid identity with the CDCA of T. pseudonana. Analysis of the amino acid sequence of CDCA shows that the putative Cd binding site resembles that of beta-class carbonic anhydrases (CAs). The prevalence of CAs in diatoms that presumably contain Cd at their active site probably reflects the very low concentration of Zn in the marine environment and the difficulty in acquiring inorganic carbon for photosynthesis. The cdca primers developed in this study should be useful for detecting cdca genes in the field, and studying the conditions under which they are expressed.     

Lysogens and free viruses in fresh, brackish, and marine waters: a Bayesian analysis

A yearlong study was conducted to determine factors that affect the abundance and distribution of lysogens and free viruses at fresh-, brackish-, and saltwater stations in Newport Bay, CA. The viral and bacterial abundance were highest in the freshwater (average 1.1 × 10⁸ and 1.1 × 10⁷ mL⁻¹, respectively) and lowest in the marine water (average 0.4 × 10⁸ and 0.5 × 10⁷ mL⁻¹, respectively). Bacterial and viral counts were also several times higher during the summer than in winter. Approximately, 35% of the 141 samples were inducible in the presence of mitomycin C. The highest percentage of inducible lysogens was observed in marine waters (42%), while the lowest percentage was observed in the warmer freshwater (23%). A statistical model for the joint occurrence of lysogens and free viruses was formulated and estimated using Bayesian techniques to understand the key environmental determinants of viruses and lysogens. Our results support the existence of significant heterogeneity between the saltwater and freshwater sites. A parsimonious model that combines the two saltwater sites performs best among the specifications that were considered. Bacteria and water temperature were significant determinants of virus counts, whereas lysogen relationships are unclear. Importantly, conditional on the covariates, viruses and lysogen fractions exhibit robust negative correlation.     

Survival of Candida albicans in tropical marine and fresh waters

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

Description of diatoms from the Southwest to West Greenland coastal and open marine waters

Detailed studies on sub-Arctic and Arctic marine diatom assemblages contribute to the understanding of spatial distribution patterns and their physical drivers. In this study, diatom species were analyzed from water samples collected with a Niskin bottle rosette combined with a CTD along the West Greenland coast (63°58′N–71°08′N and 50°49′W–59°06′W) during summer 2007. Diatom community was represented mainly by three genera Thalassiosira, Fragilariopsis, and Chaetoceros and linked to observed hydrographic and environmental conditions. Thalassiosira spp. were common in coastal waters (particularly Godthåbsfjord) and linked to increased surface water temperature, typical of summer water stratification in West Greenland. Fragilariopsis spp., along with other dominant species associated with higher geographic latitudes, dominated in Arctic fjords (Uummannaq Fjord-Qaumarujuk Fjord). These species generally characterized coastal waters influenced by melting sea ice and/or glacial ice. Chaetoceros spp. were linked to more saline open marine waters, particularly in the Davis Strait south of 70°N, probably corresponding to weaker water stratification and the influence of the West Greenland Current. The present paper provides new knowledge on diatom assemblages along a south–north climate gradient in West Greenland, which is necessary in order to understand how observed ocean-climate changes influence Arctic marine ecosystems. This study provides a reference for palaeoenvironmental reconstructions using diatom microfossils deposited in the West Greenland marine sediments.     

Survival of Candida albicans in tropical marine and fresh waters.

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

Phyloproteomics: what phylogenetic analysis reveals about serum proteomics

Phyloproteomics is a novel analytical tool that solves the issue of comparability between proteomic analyses, utilizes a total spectrum-parsing algorithm, and produces biologically meaningful classification of specimens. Phyloproteomics employs two algorithms: a new parsing algorithm (UNIPAL) and a phylogenetic algorithm (MIX). By outgroup comparison, the parsing algorithm identifies novel or vanished MS peaks and peaks signifying up or down regulated proteins and scores them as derived or ancestral. The phylogenetic algorithm uses the latter scores to produce a biologically meaningful classification of the specimens.     

Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.

BACKGROUND:The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. METHODS AND RESULTS: Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CONCLUSIONS:CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.     

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments

Apoptin, a protein derived from chicken anemia virus (CAV), induces apoptosis selectively in human tumor cells as compared with normal cells. This activity depends on phosphorylation and relocation of apoptin to the nucleus of cancer cells. Here, we describe an in vitro kinase assay that allows the biochemical characterization of apoptin kinase activity in tumor cells. The kinase phosphorylates apoptin in a strictly ATP-dependent fashion and in a broad salt range. The kinase activity is present constitutively in both cytoplasm and nucleus of various human tumor cells. Q-column chromatography showed that both cytoplasmic and nuclear fractions have identical fractionation characteristics, suggesting that the same kinase is present in both cellular compartments. Kinase activity derived from positive Q-column fractions bound to amylose-maltose-binding protein (MBP)-apoptin and could be eluted with ATP only in the presence of the cofactor Mg(2+). Apparently, unphosphorylated apoptin interacts with the kinase and is released only after phosphorylation has occurred, proving that our assay recognizes the genuine apoptin kinase. This is further corroborated by the finding that apoptin is phosphorylated in vitro at positions Thr108 and Thr107, in concert with earlier in vivo observations. Our assay excludes cyclin-dependent kinase 2 (CDK2) and protein kinase C beta (PKC-β), previously nominated by two separate studies as being the genuine apoptin kinase.     

Viable bacterial biomass and functional diversity in fresh and marine waters in the Canadian Arctic

Bacterial biomass and functional diversity in four marine and four freshwater samples, collected from Resolute Bay, Nunavut, Canada, were studied using fluorescent nucleic-acid staining and sole-carbon-source utilization. Viable microbial counts using the LIVE/DEAD BacLight Viability Kit estimated viable marine bacterial numbers from 0.7 to 1.8᎒⁶ cells/l, which were lower than viable bacterial numbers in freshwater samples (2.1-9.9᎒⁶ cells/l) (RCBD-ANOVA). Calculations of the Shannon-Wiener diversity index and average well colour development were based on substrate utilization in ECO-Biolog plates incubated at 4°C and 20°C for 38 and 24 days, respectively. The Shannon-Wiener diversity of the marine water samples was significantly greater ( x _H'=2.40ǂ.08, P <0.005; RCBD-ANOVA) than that of freshwater samples ( x _H'=1.20ǂ.00, P <0.005; RCBD-ANOVA). Differences in microbial diversity between fresh and marine water samples at 4°C ( x _4°C =2.01) and 20°C (x_20°C =2.31) were also detected by RCBD-ANOVA analysis. Interactions between water type and incubation temperature were not significant ( F =1.926, F _c=5.12). Principal component analysis revealed differences in metabolic substrate utilization patterns and, consequently, the microbial diversity between water types and samples.     

Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms

The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor expression of a gene encoding a fucoxanthin, chlorophyll a/c-binding protein (FCP) in the centric planktonic diatom Thalassiosira weissflogii. Analysis of FCP gene expression in dark-adapted diatom cultures revealed that mRNA levels increase 5- to 6-fold in response to white light irradiation and peak around 6 to 8 h. To determine the photoreceptors involved in this response action spectra of FCP gene expression were determined using the Okazaki large spectrograph. Responses consistent with the presence of cryptochrome-, rhodopsin- and phytochrome-type receptors could be detected. The apparent presence of phytochrome-mediated responses is of particular interest given the low fluences of red and far-red light wavelengths in the marine environment.     

Global phylogenetic structure of the hyperdiverse ant genus Pheidole reveals the repeated evolution of macroecological patterns

Adaptive radiations are of particular interest owing to what they reveal about the ecological and evolutionary regulation of biodiversity. This applies to localized island radiations such as Darwin''s finches, and also to rapid radiations occurring on a global scale. Here we analyse the macroevolution and macroecology of Pheidole, a famously hyperdiverse and ecologically dominant ant genus. We generate and analyse four novel datasets: (i) a robust global phylogeny including 285 Pheidole species, (ii) a global database on regional Pheidole richness in 365 political areas summarizing over 97 000 individual records from more than 6500 studies, (iii) a global database of Pheidole richness from 3796 local communities and (iv) a database of Pheidole body sizes across species. Analysis of the potential climate drivers of richness revealed that the patterns are statistically very similar across different biogeographic regions, with both regional and local richness associated with the same coefficients of temperature and precipitation. This similarity occurs even though phylogenetic analysis shows that Pheidole reached dominance in communities through serial localized radiations into different biomes within different continents and islands. Pheidole body size distributions have likewise converged across geographical regions. We propose these cases of convergence indicate that the global radiation of Pheidole is structured by deterministic factors regulating diversification and diversity.     

Survey of human enterovirus occurrence in fresh and marine surface waters on Long Island.

A variety of surface water systems, including a lake, a creek, and two marine embayments, were analyzed on a monthly basis for indigenous human enteroviruses and coliform bacteria. Findings are discussed in terms of the probable pollution sources to each system and their relationship to data from previous studies.     

Survey of human enterovirus occurrence in fresh and marine surface waters on Long Island.

A variety of surface water systems, including a lake, a creek, and two marine embayments, were analyzed on a monthly basis for indigenous human enteroviruses and coliform bacteria. Findings are discussed in terms of the probable pollution sources to each system and their relationship to data from previous studies.     

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.     

Index of the Venezuelan marine microflora: diatoms, dinoflagellates and cocolithophorids

The marine phytoplankton of Venezuela has been studied on a regular basis since the mid 20th century. However, a species checklist that can be used as a framework for taxonomic studies is lacking. In this paper, an index of the marine microflora of Venezuela is presented for the first time. The index includes only those diatoms (89 centric and 186 pennate species), dinoflagellates (eight naked and 154 thecate species) and coccolithophores (24 species) for which formal diagnosis and illustrations (drawings and/or photographs) have been reported in the scientific literature (journals and/or first degree or master's theses). It is ordered alphabetically according to class, order, family, and species. It includes the author (s) of the taxa.     

Effect of chlorination on the development of marine biofilms dominated by diatoms

This study addressed the antifouling efficiency of commercially available chlorine at different concentrations (0.5%, 1%, and 2%) and exposure times (0.5?min, 1?min, 5?min, and 15 min). The rapid and non-destructive FIRe (fluorescence induction and relaxation) technique was used to evaluate the effects of the biocide on diatom dominated biofilms. The efficiency of chlorine in removing diatoms from the developed biofilms increased with an increase in concentration and exposure time. The fluorescence measurements revealed low F(v)/F(m) and high σ(PSII) values for chlorine-treated Navicula and Amphora biofilms indicating that chlorination was efficient in damaging the photosystem-II reaction centers. Chlorination also caused mortality of diatom cells by damaging the cell body. In natural biofilms, the biocidal effect of chlorine was species specific; species of Amphiphrora, Navicula, Cylindrotheca, and Coscinodiscus showed an increase in the density of the population, but species of Pleurosigma, Amphora, and Thalassionema did not increase in density after chlorine treatment. It was also demonstrated that diatoms can colonize, grow and photosynthesize on chlorine-treated surfaces. Under pulse chlorination (treatment every 6 h), irrespective of chlorine concentration, the development of biofouling decreased with an increase in exposure time. Differences between exposure times of 1 to 15?min were not significant. Additionally, transmission levels of the control (non-chlorine-treated) fouled coupons reduced significantly (～20%) compared to the chlorine-treated fouled coupons (<2%). These results suggest that chlorine can be used as a biocide to control the development of diatom biofilms.     

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton.

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.     

Proteome-wide analysis of protein function composition reveals the clustering and phylogenetic properties of organisms

A 17-dimensional vector named the proteome vector is defined to represent an organism. The components of the vector reflect the relative contents of protein-encoding genes of the 17 cluster of orthologous groups of proteins (COGs) classes in the whole genome of the relevant organism. Based on the definition of this proteome vector, the fuzzy clustering of 36 completely sequenced organisms (8 archaea, 24 bacteria, and 4 eukarya) was performed and a proteome tree was constructed. Our results show that (1) the 36 organisms can be 100% correctly classified into three clusters corresponding to the three primary kingdoms, (2) our proteome tree is remarkably similar to that derived from 16S rRNA, and (3) the chromosomes and/or plasmids belonging to the same organism have very similar gene composition. Based on these results, we argue that the 17-dimensional proteome vector could be a good criterion for clustering approaches and to a large extent reveals the phylogenetic properties of organisms; the Three Primary Kingdoms Hypothesis is trustworthy although the existence of lateral gene transfer (LGT) brings controversy to the construction of the "universal tree of life."