Synthetic Statistical Approach Reveals a High Degree of Richness of Microbial Eukaryotes in an Anoxic Water Column

Molecular surveys suggest that communities of microbial eukaryotes are remarkably rich, because even large clone libraries seem to capture only a minority of species. This provides a qualitative picture of protistan richness but does not measure its real extent either locally or globally. Statistical analysis can estimate a community's richness, but the specific methods used to date are not always well grounded in statistical theory. Here we study a large protistan molecular survey from an anoxic water column in the Cariaco Basin (Caribbean Sea). We group individual 18S rRNA gene sequences into operational taxonomic units (OTUs) using different cutoff values for sequence similarity (99 to 50%) and systematically apply parametric models and nonparametric estimators to the OTU frequency data to estimate the total protistan diversity. The parametric models provided statistically sound estimates of protistan richness, with biologically meaningful standard errors, maximal data usage, and extensive model diagnostics and were preferable to the available nonparametric tools. Our clone library exceeded 700 clones but still covered only a minority of species and less than half of the larger protistan clades. Our estimates of total protistan richness portray the target community as very rich at all OTU levels, with hundreds of different populations apparently co-occurring in the small (3-liter) volume of our sample, as well as dozens of clades of the highest taxonomic order. These estimates are among the first for microbial eukaryotes that are obtained using state-of-the-art statistical methods and can serve as benchmark numbers for the local diversity of protists.     

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.     

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.     

Diversity estimates of microeukaryotes below the chemocline of the anoxic Mariager Fjord, Denmark

Microbial communities of extreme environments have often been assumed to have low species richness. We analysed 18S rRNA gene signatures in a sample collected below the chemocline of the anoxic Mariager Fjord in Denmark, and from these data we computed novel parametric and standard nonparametric estimates of protistan phylotype richness. Our results indicate unexpectedly high richness in this environment: at the 99.5% phylotype definition, our most conservative estimate was 568 phylotypes (+/-114, standard error). Phylogenetic analyses revealed that the sequences collected cover the majority of described lineages in the eukaryotic domain. Out of 384 sequences analysed, 307 were identified as protistan targets, none of which was identical to known sequences. However, based on what is known about species that are phylogenetically related to the Mariager sequences, most of the latter seem to belong to strictly or facultative anaerobe organisms. We also found signatures that together with other environmental 18S rRNA gene sequences represent environmental clades of possibly high taxonomic levels (class to kingdom level). One of these clades, consisting exclusively of sequences from anoxic sampling sites, branches at the base of the eukaryotic evolutionary tree among the earliest eukaryotic lineages. Assuming eukaryotic evolution under oxygen-depleted conditions, these sequences may represent immediate descendants of early eukaryotic ancestors.     

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.??     

“Missing” protists: a molecular prospective

Molecular ecology methods based on 18S rRNA amplification and sequencing have revealed an astounding diversity of microbial eukaryotes in every environment sampled so far. This is certainly true of new species and genera, as essentially every new survey discovers a wealth of novel diversity at this level. This is almost certain for taxa that are higher in taxonomic hierarchy, as many molecular surveys reported novel clades within established protistan phyla, with some of these clades repeatedly confirmed by subsequent studies. It may also be that the molecular approaches discovered several lineages of the highest taxonomic order, but this claim has not been vigorously verified as yet. Overall, the field of protistan diversity remains in its infancy. The true scale of this diversity is unknown, and so are the distribution of this diversity, its patterns, spatial and temporal dynamics, and ecological role. The sampled diversity appears to be just the tip of the iceberg, and this offers outstanding opportunities for microbial discovery for the purposes of both basic and applied research. Special Issue: Protist diversity and geographic distribution. Guest editor: W. Foissner.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. II. Habitat specialization

This is the second paper in a series of three that investigates eukaryotic microbial diversity and taxon distribution in the Cariaco Basin, Venezuela, the ocean''s largest anoxic marine basin. Here, we use phylogenetic information, multivariate community analyses and statistical richness predictions to test whether protists exhibit habitat specialization within defined geochemical layers of the water column. We also analyze spatio-temporal distributions of protists across two seasons and two geographic sites within the basin. Non-metric multidimensional scaling indicates that these two basin sites are inhabited by distinct protistan assemblages, an observation that is supported by the minimal overlap in observed and predicted richness of sampled sites. A comparison of parametric richness estimations indicates that protistan communities in closely spaced—but geochemically different—habitats are very dissimilar, and may share as few as 5% of total operational taxonomic units (OTUs). This is supported by a canonical correspondence analysis, indicating that the empirically observed OTUs are organized along opposing gradients in oxidants and reductants. Our phylogenetic analyses identify many new clades at species to class levels, some of which appear restricted to specific layers of the water column and have a significantly nonrandom distribution. These findings suggest many pelagic protists are restricted to specific habitats, and likely diversify, at least in part due to separation by geochemical barriers.     

Toward a census of bacteria in soil

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome.     

Locality or habitat? Exploring predictors of biodiversity in Amazonia

Amazonia is an environmentally heterogeneous and biologically megadiverse region, and its biodiversity varies considerably over space. However, existing knowledge on Amazonian biodiversity and its environmental determinants stems almost exclusively from studies of macroscopic above‐ground organisms, notably vertebrates and trees. In contrast, diversity patterns of most other organisms remain elusive, although some of them, for instance microorganisms, constitute the overwhelming majority of taxa in any given location, both in terms of diversity and abundance. Here, we use DNA metabarcoding to estimate prokaryote and eukaryote diversity in environmental soil and litter samples from 39 survey plots in a longitudinal transect across Brazilian Amazonia using 16S and 18S gene sequences, respectively. We characterize richness and community composition based on operational taxonomic units (OTUs) and test their correlation with longitude and habitat. We find that prokaryote and eukaryote OTU richness and community composition differ significantly among localities and habitats, and that prokaryotes are more strongly structured by locality and habitat type than eukaryotes. Our results 1) provide a first large‐scale mapping of Amazonian soil biodiversity, suggesting that OTU richness patterns might follow substantially different patterns from those observed for macro‐organisms; and 2) indicate that locality and habitat factors interact in determining OTU richness patterns and community composition. This study shows the potential of DNA metabarcoding in unveiling Amazonia's outstanding diversity, despite the lack of complete reference sequence databases for the organisms sequenced.     

Statistical approaches for estimating actinobacterial diversity in marine sediments

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >/= 99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. F(ST) and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.     

Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Introducing SONS, a Tool for Operational Taxonomic Unit-Based Comparisons of Microbial Community Memberships and Structures

The recent advent of tools enabling statistical inferences to be drawn from comparisons of microbial communities has enabled the focus of microbial ecology to move from characterizing biodiversity to describing the distribution of that biodiversity. Although statistical tools have been developed to compare community structures across a phylogenetic tree, we lack tools to compare the memberships and structures of two communities at a particular operational taxonomic unit (OTU) definition. Furthermore, current tests of community structure do not indicate the similarity of the communities but only report the probability of a statistical hypothesis. Here we present a computer program, SONS, which implements nonparametric estimators for the fraction and richness of OTUs shared between two communities.     

Introducing SONS, a tool for operational taxonomic unit-based comparisons of microbial community memberships and structures

The recent advent of tools enabling statistical inferences to be drawn from comparisons of microbial communities has enabled the focus of microbial ecology to move from characterizing biodiversity to describing the distribution of that biodiversity. Although statistical tools have been developed to compare community structures across a phylogenetic tree, we lack tools to compare the memberships and structures of two communities at a particular operational taxonomic unit (OTU) definition. Furthermore, current tests of community structure do not indicate the similarity of the communities but only report the probability of a statistical hypothesis. Here we present a computer program, SONS, which implements nonparametric estimators for the fraction and richness of OTUs shared between two communities.     

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.     

Global patterns of diversity and community structure in marine bacterioplankton

Because of their small size, great abundance and easy dispersal, it is often assumed that marine planktonic microorganisms have a ubiquitous distribution that prevents any structured assembly into local communities. To challenge this view, marine bacterioplankton communities from coastal waters at nine locations distributed world-wide were examined through the use of comprehensive clone libraries of 16S ribosomal RNA genes, used as operational taxonomic units (OTU). Our survey and analyses show that there were marked differences in the composition and richness of OTUs between locations. Remarkably, the global marine bacterioplankton community showed a high degree of endemism, and conversely included few cosmopolitan OTUs. Our data were consistent with a latitudinal gradient of OTU richness. We observed a positive relationship between the relative OTU abundances and their range of occupation, i.e. cosmopolitans had the largest population sizes. Although OTU richness differed among locations, the distributions of the major taxonomic groups represented in the communities were analogous, and all local communities were similarly structured and dominated by a few OTUs showing variable taxonomic affiliations. The observed patterns of OTU richness indicate that similar evolutionary and ecological processes structured the communities. We conclude that marine bacterioplankton share many of the biogeographical and macroecological features of macroscopic organisms. The general processes behind those patterns are likely to be comparable across taxa and major global biomes.     

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

Background  

Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

Dose-response curve estimation: a semiparametric mixture approach

In the estimation of a dose-response curve, parametric models are straightforward and efficient but subject to model misspecifications; nonparametric methods are robust but less efficient. As a compromise, we propose a semiparametric approach that combines the advantages of parametric and nonparametric curve estimates. In a mixture form, our estimator takes a weighted average of the parametric and nonparametric curve estimates, in which a higher weight is assigned to the estimate with a better model fit. When the parametric model assumption holds, the semiparametric curve estimate converges to the parametric estimate and thus achieves high efficiency; when the parametric model is misspecified, the semiparametric estimate converges to the nonparametric estimate and remains consistent. We also consider an adaptive weighting scheme to allow the weight to vary according to the local fit of the models. We conduct extensive simulation studies to investigate the performance of the proposed methods and illustrate them with two real examples.     

Comparison of wintertime eukaryotic community from sea ice and open water in the Baltic Sea, based on sequencing of the 18S rRNA gene

The Baltic Sea is one of the world’s largest brackish water basins and is traditionally considered to be species poor. Here, we assessed the diversity of the nano-sized eukaryotic microbial wintertime community, using molecular ecological methods based on sequencing of small-subunit ribosomal RNA gene clone libraries. The results demonstrate that a rich community of small eukaryotes inhabits the Baltic Sea ice and water during winter. The community was dominated by alveolates and stramenopiles. Ciliates and cercozoans were the richest groups present, while in contrast to previous studies, diatoms showed a lower richness than expected. Furthermore, fungi and parasitic Syndiniales were present both in the water and in the sea ice. Some of the organisms in the sea-ice community were active, based on the RNA data, but a number of organisms were inactive or remnants from the freezing process. The results demonstrate that the sea-ice communities in the Baltic Sea are highly diverse and that water and ice of different ages include different protistan assemblages. Our study emphasizes the potential loss in biodiversity through diminishing ice cover as a result of climate change.