Rapid shifts in the structure and composition of a protistan assemblage during bottle incubations affect estimates of total protistan species richness

Changes in the structure and composition of a protistan community were characterized through the analysis of small-subunit ribosomal RNA gene (18S) sequences for a 3-day bottle incubation using a single sample collected in the western North Atlantic. Cloning and sequencing was used to investigate changes in perceived species richness and diversity as a consequence of environmental perturbation. The treatments included a control (unamended seawater), inorganic nutrient enrichment, and enrichment with a complex organic mixture. Five clone libraries were constructed and analyzed at the time of collection (t-0?h) and after 24 (t-24?h) and 72 (t-72?h) h for the control, and at t-72?h for the inorganic and organic enrichments, resulting in an analysis of 1,626 partial 18S rDNA sequences that clustered into 238 operational taxonomic units (OTUs). Analysis of the clone libraries revealed that protistan assemblages were highly dynamic and changed substantially at both the OTU level and higher taxonomic classifications during time frames consistent with many oceanographic methods used for measuring biological rates. Changes were most dramatic in enrichments, which yielded community compositions that were strongly dominated by one or a few taxa. Changes in community structure during incubation dramatically influenced estimates of species richness, which were substantially lower with longer incubation and especially with amendment, even though all incubated samples originated from the same aliquot of seawater. Containment and enrichment of the seawater sample led to the detection of otherwise undetected protistan taxa, suggesting that characterization of protistan diversity in a sample only at the time of collection could lead to an underrepresentation of unique taxa. Additionally, the rapid increase in the relative abundance of some members of the ??rare biosphere?? in our results implies an ecological importance of at least some of the taxa comprising the ??rare biosphere.??     

The growing contributions of molecular biology and immunology to protistan ecology: molecular signatures as ecological tools

Modern genetic and immunological techniques have become important tools for assessing protistan species diversity for both the identification and quantification of specific taxa in natural microbial communities. Although these methods are still gaining use among ecologists, the new approaches have already had a significant impact on our understanding of protistan diversity and biogeography. For example, genetic studies of environmental samples have uncovered many protistan phylotypes that do not match the DNA sequences of any cultured organisms, and whose morphological identities are unknown at the present time. Additionally, rapid and sensitive methods for detecting and enumerating taxa of special importance (e.g. bloom-forming algae, parasitic protists) have enabled much more detailed distributional and experimental studies than have been possible using traditional methods. Nevertheless, while the application of molecular approaches has advanced some aspects of aquatic protistan ecology, significant issues still thwart the widespread adoption of these approaches. These issues include the highly technical nature of some of the molecular methods, the reconciliation of morphology-based and sequence-based species identifications, and the species concept itself.     

Protistan community patterns within the brine and halocline of deep hypersaline anoxic basins in the eastern Mediterranean Sea

Environmental factors restrict the distribution of microbial eukaryotes but the exact boundaries for eukaryotic life are not known. Here, we examine protistan communities at the extremes of salinity and osmotic pressure, and report rich assemblages inhabiting Bannock and Discovery, two deep-sea superhaline anoxic basins in the Mediterranean. Using a rRNA-based approach, we detected 1,538 protistan rRNA gene sequences from water samples with total salinity ranging from 39 to 280 g/Kg, and obtained evidence that this DNA was endogenous to the extreme habitat sampled. Statistical analyses indicate that the discovered phylotypes represent only a fraction of species actually inhabiting both the brine and the brine-seawater interface, with as much as 82% of the actual richness missed by our survey. Jaccard indices (e.g., for a comparison of community membership) suggest that the brine/interface protistan communities are unique to Bannock and Discovery basins, and share little (0.8–2.8%) in species composition with overlying waters with typical marine salinity and oxygen tension. The protistan communities from the basins’ brine and brine/seawater interface appear to be particularly enriched with dinoflagellates, ciliates and other alveolates, as well as fungi, and are conspicuously poor in stramenopiles. The uniqueness and diversity of brine and brine-interface protistan communities make them promising targets for protistan discovery.     

Microbial eukaryotes in the hypersaline anoxic L'Atalante deep-sea basin

The frontiers of eukaryote life in nature are still unidentified. In this study, we analysed protistan communities in the hypersaline (up to 365 g l⁻¹ NaCl) anoxic L'Atalante deep-sea basin located in the eastern Mediterranean Sea. Targeting 18S ribosomal RNA retrieved from the basin's lower halocline (3501 m depth) we detected 279 protistan sequences that grouped into 42 unique phylotypes (99% sequence similarity). Statistical analyses revealed that these phylotypes account only for a proportion of the protists inhabiting this harsh environment with as much as 50% missed by this survey. Most phylotypes were affiliated with ciliates (45%), dinoflagellates (21%), choanoflagelates (10%) and uncultured marine alveolates (6%). Sequences from other taxonomic groups like stramenopiles, Polycystinea , Acantharea and Euglenozoa , all of which are typically found in non-hypersaline deep-sea systems, are either missing or very rare in our cDNA clone library. Although many DHAB sequences fell within previously identified environmental clades, a large number branched relatively deeply. Phylotype richness, community membership and community structure differ significantly from a deep seawater reference community (3499 m depth). Also, the protistan community in the L'Atalante basin is distinctively different from any previously described hypersaline community. In conclusion, we hypothesize that extreme environments may exert a high selection pressure possibly resulting in the evolution of an exceptional and distinctive assemblage of protists. The deep hypersaline anoxic basins in the Mediterranean Sea provide an ideal platform to test for this hypothesis and are promising targets for the discovery of undescribed protists with unknown physiological capabilities.     

A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates

A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150 ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750-800 base pairs (bp) encompassing hypervariable regions in the 5' portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram + high GC content, Gram + low GC content). The 2273 microorganisms surveyed encompass 834 alpha-Proteobacteria (representing 60 different phylotypes), 25 beta-Proteobacteria (3 phylotypes), 767 gamma-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram + high GC content (43 phylotypes), and 198 Gram + low GC content isolates (24 phylotypes). Notably, 11 phylotypes were < or =93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (approximately 1400 bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported "uncultured" microbes, such as sponge-specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html).     

High level of bacterial diversity and novel taxa in continental shelf sediment

The bacterial diversity of the continental shelf sediment in the Yellow Sea was investigated by the cloning and sequencing of PCR-amplified 16S rRNA genes. The majority of the cloned sequences were distinct phylotypes that were novel at the species level. The richness estimator indicated that the sediment sample might harbor up to 32 phylum-level taxa. A large number of low-abundance, phylum-level taxa accounted for most of the observed phylogenetic diversity at our study site, suggesting that these low-abundance taxa might play crucial roles in the shelf sediment ecosystem.     

Bacterial Diversity in Three Different Antarctic Cold Desert Mineral Soils

A bacterial phylogenetic survey of three environmentally distinct Antarctic Dry Valley soil biotopes showed a high proportion of so-called “uncultured” phylotypes, with a relatively low diversity of identifiable phylotypes. Cyanobacterial phylotypic signals were restricted to the high-altitude sample, whereas many of the identifiable phylotypes, such as the members of the Actinobacteria, were found at all sample sites. Although the presence of Cyanobacteria and Actinobacteria is consistent with previous culture-dependent studies of microbial diversity in Antarctic Dry Valley mineral soils, many phylotypes identified by 16S rDNA analysis were of groups that have not hitherto been cultured from Antarctic soils. The general belief that such “extreme” environments harbor a relatively low species diversity was supported by the calculation of diversity indices. The detection of a substantial number of uncultured bacterial phylotypes showing low BLAST identities (<95%) suggests that Antarctic Dry Valley mineral soils harbor a pool of novel psychrotrophic taxa.     

Microeukaryote community patterns along an O2/H2S gradient in a supersulfidic anoxic fjord (Framvaren, Norway)

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O(2)/H(2)S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O(2)/H(2)S gradient.     

Global biogeography of highly diverse protistan communities in soil

Protists are ubiquitous members of soil microbial communities, but the structure of these communities, and the factors that influence their diversity, are poorly understood. We used barcoded pyrosequencing to survey comprehensively the diversity of soil protists from 40 sites across a broad geographic range that represent a variety of biome types, from tropical forests to deserts. In addition to taxa known to be dominant in soil, including Cercozoa and Ciliophora, we found high relative abundances of groups such as Apicomplexa and Dinophyceae that have not previously been recognized as being important components of soil microbial communities. Soil protistan communities were highly diverse, approaching the extreme diversity of their bacterial counterparts across the same sites. Like bacterial taxa, protistan taxa were not globally distributed, and the composition of these communities diverged considerably across large geographic distances. However, soil protistan and bacterial communities exhibit very different global-scale biogeographical patterns, with protistan communities strongly structured by climatic conditions that regulate annual soil moisture availability.     

Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic Fjord (Framvaren, Norway)

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O₂/H₂S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O₂/H₂S gradient.     

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil

In spite of the techniques based on the amplification of 16S rRNA genes (16S rDNA) to compare bacterial communities that are now widely in use in microbial ecology, little is known about the composition of the soybean continuous cropping (CC) and rotational cropping (RC) soil microbial community. To address this, we compared the levels of bacterial community diversity in RC and 5-year CC rhizosphere soil samples. We selected 407 clones in RC and 490 clones in CC for restriction fragment length polymorphism analysis. A total of 123 phylotypes were identified among the 16S rDNA clones, while 78 unique and 21 common phylotypes were identified among the CC soil isolates. Analysis of sequences from a subset of the phylotypes showed that at least 11 bacterial divisions were represented in the clone libraries. The phylotype richness, frequency distribution (evenness), and composition of the two clone libraries were investigated using a variety of diversity indices. Although the analysis of diversity indices and LIBSHUFF comparisons revealed that the compared libraries were not significantly different ( P =0.05) between the RC vs. CC soils, some differences could be observed in terms of specific phyla and groups. We concluded that the group variance was not determined immediately by the cropping system's induction, but was a long-term and slow process.     

Diversity of aquatic prokaryotic communities in the Cuatro Cienegas basin

The Cuatro Cienegas basin (Coahuila, México) is a composite of different water systems in the middle of the desert with unusually high levels of endemism and diversity in different taxa. Although the diversity of macrobiota has been well described, little is known about the diversity and distribution of microorganisms in the oligotrophic ponds. Here we describe the extent and distribution of diversity found in aquatic prokaryotic communities by analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes and phylogenetic analysis of cloned genes. Twelve locations within the basin were sampled. Among all the samples, we found a total of 117 operational taxonomic units (OTUs) using T-RFLPs, which ranged in any single sample from four to 49. OTU richness and Shannon diversity indices for different sites varied, but none were particularly high. 16S rRNA gene sequence data showed 68 different phylotypes among 198 clones. The most abundant phylotypes were Gamma- and Betaproteobacteria, and extreme halophiles. The differences among sites were significant; 45 TRFs were found only once, and 37% of the total diversity was represented by differences between sites, suggesting high beta-diversity. Further studies are needed to test whether this is a direct consequence of environmental heterogeneity in the basin.     

Diversity and distribution of marine microbial eukaryotes in the Arctic Ocean and adjacent seas

We analyzed microbial eukaryote diversity in perennially cold arctic marine waters by using 18S rRNA gene clone libraries. Samples were collected during concurrent oceanographic missions to opposite sides of the Arctic Ocean Basin and encompassed five distinct water masses. Two deep water Arctic Ocean sites and the convergence of the Greenland, Norwegian, and Barents Seas were sampled from 28 August to 2 September 2002. An additional sample was obtained from the Beaufort Sea (Canada) in early October 2002. The ribotypes were diverse, with different communities among sites and between the upper mixed layer and just below the halocline. Eukaryotes from the remote Canada Basin contained new phylotypes belonging to the radiolarian orders Acantharea, Polycystinea, and Taxopodida. A novel group within the photosynthetic stramenopiles was also identified. One sample closest to the interior of the Canada Basin yielded only four major taxa, and all but two of the sequences recovered belonged to the polar diatom Fragilariopsis and a radiolarian. Overall, 42% of the sequences were <98% similar to any sequences in GenBank. Moreover, 15% of these were <95% similar to previously recovered sequences, which is indicative of endemic or undersampled taxa in the North Polar environment. The cold, stable Arctic Ocean is a threatened environment, and climate change could result in significant loss of global microbial biodiversity.     

Past President's Address: Protistan Biogeography: Why All The Fuss?1

ABSTRACT. Conflicting hypotheses regarding the distribution of protistan species on our planet have led to rather impassioned arguments in the recent literature, and heated discussions at scientific meetings. Both sides of this debate have mounted seemingly credible arguments for endemism on the one hand, or ubiquitous dispersal of protists on the other. At present, the controversy appears unending, perhaps because it is fueled by a convergence of unresolvable issues involving the enormous phylogenetic breadth of species presently encompassed by the term “protist,” the application of multiple species concepts to these taxa, the inability of extant techniques and technology to plumb the depths of microbial diversity in natural ecosystems, and a lack of knowledge regarding the relationship between dispersal rates and rates of evolution of protists. These issues have made it difficult to erect and test hypotheses concerning the distribution of protists. In the absence of definitive experimental or observational information, preconceived attitudes regarding protistan distributions have dominated the interpretation of the available data. On the positive side, the debate has led to the development and application of new approaches to the study of protistan diversity, and stimulated discussions involving how (and why) we define protistan species.     

Broadly sampled multigene trees of eukaryotes

Background  

Our understanding of the eukaryotic tree of life and the tremendous diversity of microbial eukaryotes is in flux as additional genes and diverse taxa are sampled for molecular analyses. Despite instability in many analyses, there is an increasing trend to classify eukaryotic diversity into six major supergroups: the 'Amoebozoa', 'Chromalveolata', 'Excavata', 'Opisthokonta', 'Plantae', and 'Rhizaria'. Previous molecular analyses have often suffered from either a broad taxon sampling using only single-gene data or have used multigene data with a limited sample of taxa. This study has two major aims: (1) to place taxa represented by 72 sequences, 61 of which have not been characterized previously, onto a well-sampled multigene genealogy, and (2) to evaluate the support for the six putative supergroups using two taxon-rich data sets and a variety of phylogenetic approaches.     

Characterization of the prokaryotic diversity through a stratigraphic permafrost core profile from the Qinghai-Tibet Plateau

Permafrost on the Qinghai-Tibet Plateau is one of the most sensitive regions to climate warming, thus characterizing its microbial diversity and community composition may be important for understanding their potential responses to climate changes. Here, we investigated the prokaryotic diversity in a 10-m-long permafrost core from the Qinghai-Tibet Plateau by restriction fragment length polymorphism analysis targeting the 16S rRNA gene. We detected 191 and 17 bacterial and archaeal phylotypes representing 14 and 2 distinct phyla, respectively. Proteobacteria was the dominant bacterial phylum, while archaeal communities were characterized by a preponderance of Thaumarchaeota. Some of prokaryotic phylotypes were closely related to characterized species involved in carbon and nitrogen cycles, including nitrogen fixation, methane oxidation and nitrification. However, the majority of the phylotypes were only distantly related to known taxa at order or species level, suggesting the potential of novel diversity. Additionally, both bacterial α diversity and community composition changed significantly with sampling depth, where these communities mainly distributed according to core horizons. Arthrobacter-related phylotypes presented at high relative abundance in two active layer soils, while the deeper permafrost soils were dominated by Psychrobacter-related clones. Changes in bacterial community composition were correlated with most measured soil variables, such as carbon and nitrogen contents, pH, and conductivity.     

Diversity and Distribution of Marine Microbial Eukaryotes in the Arctic Ocean and Adjacent Seas

We analyzed microbial eukaryote diversity in perennially cold arctic marine waters by using 18S rRNA gene clone libraries. Samples were collected during concurrent oceanographic missions to opposite sides of the Arctic Ocean Basin and encompassed five distinct water masses. Two deep water Arctic Ocean sites and the convergence of the Greenland, Norwegian, and Barents Seas were sampled from 28 August to 2 September 2002. An additional sample was obtained from the Beaufort Sea (Canada) in early October 2002. The ribotypes were diverse, with different communities among sites and between the upper mixed layer and just below the halocline. Eukaryotes from the remote Canada Basin contained new phylotypes belonging to the radiolarian orders Acantharea, Polycystinea, and Taxopodida. A novel group within the photosynthetic stramenopiles was also identified. One sample closest to the interior of the Canada Basin yielded only four major taxa, and all but two of the sequences recovered belonged to the polar diatom Fragilariopsis and a radiolarian. Overall, 42% of the sequences were <98% similar to any sequences in GenBank. Moreover, 15% of these were <95% similar to previously recovered sequences, which is indicative of endemic or undersampled taxa in the North Polar environment. The cold, stable Arctic Ocean is a threatened environment, and climate change could result in significant loss of global microbial biodiversity.     

SSU‐rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep‐sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high‐throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full‐length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.     

Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.