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.     

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.     

A multiple PCR-primer approach to access the microeukaryotic diversity in environmental samples

The Cariaco Basin off the Venezuelan coast in the Caribbean Sea is the world's largest truly marine body of anoxic water. The first rRNA survey of microbial eukaryotes in this environment revealed a number of novel lineages, but sampled only a fraction of the entire diversity. The goal of this study was to significantly improve recovery of protistan rRNA from the Basin. This was achieved by a systematic application of multiple PCR primer sets and substantially larger sequencing efforts. We focused on the most diverse habitat in the basin, anoxic waters approximately 100m below the oxic-anoxic interface, and detected novel lineages that escaped the single PCR primer approach. All clones obtained proved unique. A 99% sequence similarity cut-off value combined these clones into operational taxonomic units (OTUs), over 75% of which proved novel. Some of these OTUs form deep branches within established protistan groups. Others signify discovery of novel protistan lineages that appear unrelated to any known microeukaryote. Surprisingly, even this large-scale multi-primer rRNA approach still missed a substantial part of the samples' rRNA diversity. The overlap between the species lists obtained with different primers is low, with only 4% of OTUs shared by all three libraries, and the number of species detected only once is large (55%). This strongly indicates that, at least in anoxic environments, protistan diversity may be much larger than is commonly thought. A single sample appears to contain thousands of largely novel protistan species. Multiple PCR primer combinations may be needed to capture these species.     

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.     

Novel Eukaryotes from the Permanently Anoxic Cariaco Basin (Caribbean Sea)

Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000). Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S. L. Baldauf, Science 300:1703-1706, 2003). The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery. Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast. On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages. These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups. Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches. These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s).     

Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119 000 nearly full-length sequences and 28 000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.     

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.     

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.     

Novel eukaryotes from the permanently anoxic Cariaco Basin (Caribbean Sea)

Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000). Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S. L. Baldauf, Science 300:1703-1706, 2003). The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery. Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast. On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages. These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups. Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches. These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s).     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

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.     

Sediment microbial communities in Great Boiling Spring are controlled by temperature and distinct from water communities

Great Boiling Spring is a large, circumneutral, geothermal spring in the US Great Basin. Twelve samples were collected from water and four different sediment sites on four different dates. Microbial community composition and diversity were assessed by PCR amplification of a portion of the small subunit rRNA gene using a universal primer set followed by pyrosequencing of the V8 region. Analysis of 164 178 quality-filtered pyrotags clearly distinguished sediment and water microbial communities. Water communities were extremely uneven and dominated by the bacterium Thermocrinis. Sediment microbial communities grouped according to temperature and sampling location, with a strong, negative, linear relationship between temperature and richness at all taxonomic levels. Two sediment locations, Site A (87–80 °C) and Site B (79 °C), were predominantly composed of single phylotypes of the bacterial lineage GAL35 ($\widehat{p}$=36.1%), Aeropyrum ($\widehat{p}$=16.6%), the archaeal lineage pSL4 ($\widehat{p}$=15.9%), the archaeal lineage NAG1 ($\widehat{p}$=10.6%) and Thermocrinis ($\widehat{p}$=7.6%). The ammonia-oxidizing archaeon ‘Candidatus Nitrosocaldus'' was relatively abundant in all sediment samples <82 °C ($\widehat{p}$=9.51%), delineating the upper temperature limit for chemolithotrophic ammonia oxidation in this spring. This study underscores the distinctness of water and sediment communities in GBS and the importance of temperature in driving microbial diversity, composition and, ultimately, the functioning of biogeochemical cycles.     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

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.     

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.     

Humboldt's spa: microbial diversity is controlled by temperature in geothermal environments

Over 200 years ago Alexander von Humboldt (1808) observed that plant and animal diversity peaks at tropical latitudes and decreases toward the poles, a trend he attributed to more favorable temperatures in the tropics. Studies to date suggest that this temperature–diversity gradient is weak or nonexistent for Bacteria and Archaea. To test the impacts of temperature as well as pH on bacterial and archaeal diversity, we performed pyrotag sequencing of 16S rRNA genes retrieved from 165 soil, sediment and biomat samples of 36 geothermal areas in Canada and New Zealand, covering a temperature range of 7.5–99 °C and a pH range of 1.8–9.0. This represents the widest ranges of temperature and pH yet examined in a single microbial diversity study. Species richness and diversity indices were strongly correlated to temperature, with R² values up to 0.62 for neutral–alkaline springs. The distributions were unimodal, with peak diversity at 24 °C and decreasing diversity at higher and lower temperature extremes. There was also a significant pH effect on diversity; however, in contrast to previous studies of soil microbial diversity, pH explained less of the variability (13–20%) than temperature in the geothermal samples. No correlation was observed between diversity values and latitude from the equator, and we therefore infer a direct temperature effect in our data set. These results demonstrate that temperature exerts a strong control on microbial diversity when considered over most of the temperature range within which life is possible.     

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.     

Thermophilic anaerobic oxidation of methane by marine microbial consortia

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.     

The Effects of Alignment Quality,Distance Calculation Method,Sequence Filtering,and Region on the Analysis of 16S rRNA Gene-Based Studies

Pyrosequencing of PCR-amplified fragments that target variable regions within the 16S rRNA gene has quickly become a powerful method for analyzing the membership and structure of microbial communities. This approach has revealed and introduced questions that were not fully appreciated by those carrying out traditional Sanger sequencing-based methods. These include the effects of alignment quality, the best method of calculating pairwise genetic distances for 16S rRNA genes, whether it is appropriate to filter variable regions, and how the choice of variable region relates to the genetic diversity observed in full-length sequences. I used a diverse collection of 13,501 high-quality full-length sequences to assess each of these questions. First, alignment quality had a significant impact on distance values and downstream analyses. Specifically, the greengenes alignment, which does a poor job of aligning variable regions, predicted higher genetic diversity, richness, and phylogenetic diversity than the SILVA and RDP-based alignments. Second, the effect of different gap treatments in determining pairwise genetic distances was strongly affected by the variation in sequence length for a region; however, the effect of different calculation methods was subtle when determining the sample''s richness or phylogenetic diversity for a region. Third, applying a sequence mask to remove variable positions had a profound impact on genetic distances by muting the observed richness and phylogenetic diversity. Finally, the genetic distances calculated for each of the variable regions did a poor job of correlating with the full-length gene. Thus, while it is tempting to apply traditional cutoff levels derived for full-length sequences to these shorter sequences, it is not advisable. Analysis of β-diversity metrics showed that each of these factors can have a significant impact on the comparison of community membership and structure. Taken together, these results urge caution in the design and interpretation of analyses using pyrosequencing data.     

Microbial community structures in anoxic freshwater lake sediment along a metal contamination gradient

Contamination, such as by heavy metals, has frequently been implicated in altering microbial community structure. However, this association has not been extensively studied for anaerobic communities, or in freshwater lake sediments. We investigated microbial community structure in the metal-contaminated anoxic sediments of a eutrophic lake that were impacted over the course of 80 years by nearby zinc-smelting activities. Microbial community structure was inferred for bacterial, archaeal and eukaryotic populations by evaluating terminal restriction fragment length polymorphism (TRFLP) patterns in near-surface sediments collected in triplicate from five areas of the lake that had differing levels of metal contamination. The majority of the fragments in the bacterial and eukaryotic profiles showed no evidence of variation in association with metal contamination levels, and diversity revealed by these profiles remained consistent even as metal concentrations varied from 3000 to 27 000 mg kg⁻¹ total Zn, 0.125 to 11.2 μ pore water Zn and 0.023 to 5.40 μ pore water As. Although most archaeal fragments also showed no evidence of variation, the prevalence of a fragment associated with mesophilic Crenarchaeota showed significant positive correlation with total Zn concentrations. This Crenarchaeota fragment dominated the archaeal TRFLP profiles, representing between 35% and 79% of the total measured peak areas. Lake DePue 16S rRNA gene sequences corresponding to this TRFLP fragment clustered with anaerobic and soil mesophilic Crenarchaeota sequences. Although Crenarchaeota have been associated with metal-contaminated groundwater and soils, this is a first report (to our knowledge) documenting potential increased prevalence of Crenarchaeota associated with elevated levels of metal contamination.