Next-Generation Sequencing of Microbial Communities in the Athabasca River and Its Tributaries in Relation to Oil Sands Mining Activities

The Athabasca oil sands deposit is the largest reservoir of crude bitumen in the world. Recently, the soaring demand for oil and the availability of modern bitumen extraction technology have heightened exploitation of this reservoir and the potential unintended consequences of pollution in the Athabasca River. The main objective of the present study was to evaluate the potential impacts of oil sands mining on neighboring aquatic microbial community structure. Microbial communities were sampled from sediments in the Athabasca River and its tributaries as well as in oil sands tailings ponds. Bacterial and archaeal 16S rRNA genes were amplified and sequenced using next-generation sequencing technology (454 and Ion Torrent). Sediments were also analyzed for a variety of chemical and physical characteristics. Microbial communities in the fine tailings of the tailings ponds were strikingly distinct from those in the Athabasca River and tributary sediments. Microbial communities in sediments taken close to tailings ponds were more similar to those in the fine tailings of the tailings ponds than to the ones from sediments further away. Additionally, bacterial diversity was significantly lower in tailings pond sediments. Several taxonomic groups of Bacteria and Archaea showed significant correlations with the concentrations of different contaminants, highlighting their potential as bioindicators. We also extensively validated Ion Torrent sequencing in the context of environmental studies by comparing Ion Torrent and 454 data sets and by analyzing control samples.     

Phylogenetic Estimation of Community Composition and Novel Eukaryotic Lineages in Base Mine Lake: An Oil Sands Tailings Reclamation Site in Northern Alberta

Reclamation of anthropogenically impacted environments is a critical issue worldwide. In the oil sands extraction industry of Alberta, reclamation of mining‐impacted areas, especially areas affected by tailings waste, is an important aspect of the mining life cycle. A reclamation technique currently under study is water‐capping, where tailings are capped by water to create an end‐pit lake (EPL). Base Mine Lake (BML) is the first full‐scale end‐pit lake in the Alberta oil sands region. In this study, we sequenced eukaryotic 18S rRNA genes recovered from 92 samples of Base Mine Lake water in a comprehensive sampling programme covering the ice‐free period of 2015. The 565 operational taxonomic units (OTUs) generated revealed a dynamic and diverse community including abundant Microsporidia, Ciliata and Cercozoa, though 41% of OTUs were not classifiable below the phylum level by comparison to 18S rRNA databases. Phylogenetic analysis of five heterotrophic phyla (Cercozoa, Fungi, Ciliata, Amoebozoa and Excavata) revealed substantial novel diversity, with many clusters of OTUs that were more similar to each other than to any reference sequence. All of these groups are entirely or mostly heterotrophic, as a relatively small number of definitively photosynthetic clades were amplified from the BML samples.     

Quantifying long‐term recurrence in planktonic microbial eukaryotes

How much temporal recurrence is present in microbial assemblages is still an unanswered ecological question. Even though marked seasonal changes have been reported for whole microbial communities, less is known on the dynamics and seasonality of individual taxa. Here, we aim at understanding microbial recurrence at three different levels: community, taxonomic group and operational taxonomic units (OTUs). For that, we focused on a model microbial eukaryotic community populating a long‐term marine microbial observatory using 18S rRNA gene data from two organismal size fractions: the picoplankton (0.2–3 µm) and the nanoplankton (3–20 µm). We have developed an index to quantify recurrence in particular taxa. We found that community structure oscillated systematically between two main configurations corresponding to winter and summer over the 10 years studied. A few taxonomic groups such as Mamiellophyceae or MALV‐III presented clear recurrence (i.e., seasonality), whereas 13%–19% of the OTUs in both size fractions, accounting for ~40% of the relative abundance, featured recurrent dynamics. Altogether, our work links long‐term whole community dynamics with that of individual OTUs and taxonomic groups, indicating that recurrent and non‐recurrent changes characterize the dynamics of microbial assemblages.     

Bioprocessing-Based Approach for Bitumen/Water/Fines Separation and Hydrocarbon Recovery from Oil Sands Tailings

Oil sands are a major source of oil, but their industrial processing generates tailings ponds that are an environmental hazard. The main concerns are mature fine tailings (MFT) composed of residual hydrocarbons, water, and fine clay. Tailings ponds include toxic contaminants such as heavy metals, and toxic organics including naphthenics. Naphthenic acids and polyaromatic hydrocarbons (PAHs) degrade very slowly and pose a long-term threat to surface and groundwater, as they can be transported in the MFT. Research into improved technologies that would enable densification and settling of the suspended particles is ongoing. In batch tests, BioTiger?, a microbial consortium that can metabolize PAHs, demonstrated improved oil sands tailings settling from a Canadian tailings pond. Results also showed, depending on the timing of the measurements, lower suspended solids and turbidity. Elevated total organic carbon was observed in the first 48 hours in the BioTiger?-treated columns and then decreased in overlying water. Oil sands tailings mixed with BioTiger? showed a two-fold reduction in suspended solids within 24 hours as compared to abiotic controls. The tailings treated with BioTiger? increased in microbial densities three orders of magnitude from 8.5 × 105 CFU/mL to 1.2 × 108 CFU/mL without any other carbon or energy source added, indicating metabolism of hydrocarbons and other available nutrients. Results demonstrated that bioaugmentation of BioTiger? increased separation of organic carbon from particles in oil sands and enhanced settling with tailings with improved water quality. Journal style is for Abstract to be less than 200 words, and contain no citations to other sources; please edit as needed     

Bioreactor studies predict whole microbial population dynamics in oil sands tailings ponds

Microorganisms in oil sands fluid fine tailings (FFT) are critical to biogeochemical elemental cycling as well as to the degradation of residual hydrocarbon constituents and subsequent methane and CO₂ production. Microbial activity enhances particulate matter sedimentation rates and the dewatering of FFT materials, allowing water to be recycled back into bitumen extraction. A bulk of this evidence comes from bioreactor studies and has implications for engineering and environmental management of the FFT ponds. Yet, it is largely uncertain whether such laboratory populations are representative of whole field scale microbial communities. By using population ecology tools, we compared whole microbial communities present in FFT bioreactors to reference populations existing in Syncrude's West In Pit (WIP) tailings pond. Bacteria were found to be persistent in a sulfidic zone in both the oxic and anoxic bioreactors at all occasions tested. In contrast to the WIP, archaea only became predominant in bioreactors after 300 days, at which point analysis of similarity (global R statistic p?<?0.5) revealed no significant dissimilarities between the populations present in either system. A whole community succession pattern from bacterial dominated prevalence to a new assemblage predominated by archaea was suggested. These results have implications for the stepwise development of microbial model systems for predictive management of field scale FFT basins.     

Effects of long‐term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach

To understand the fine‐scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long‐term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae‐Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88 706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic‐ (farmyard manure), mineral‐ and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co‐occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.     

Mock communities highlight the diversity of host‐associated eukaryotes

Host‐associated microbes are ubiquitous. Every multicellular eukaryote, and even many unicellular eukaryotes (protists), hosts a diverse community of microbes. High‐throughput sequencing (HTS) tools have illuminated the vast diversity of host‐associated microbes and shown that they have widespread influence on host biology, ecology and evolution (McFall‐Ngai et al. 2013 ). Bacteria receive most of the attention, but protists are also important components of microbial communities associated with humans (Parfrey et al. 2011 ) and other hosts. As HTS tools are increasingly used to study eukaryotes, the presence of numerous and diverse host‐associated eukaryotes is emerging as a common theme across ecosystems. Indeed, HTS studies demonstrate that host‐associated lineages account for between 2 and 12% of overall eukaryotic sequences detected in soil, marine and freshwater data sets, with much higher relative abundances observed in some samples (Ramirez et al. 2014 ; Simon et al. 2015 ; de Vargas et al. 2015 ). Previous studies in soil detected large numbers of predominantly parasitic lineages such as Apicomplexa, but did not delve into their origin [e.g. (Ramirez et al. 2014 )]. In this issue of Molecular Ecology, Geisen et al. ( 2015 ) use mock communities to show that many of the eukaryotic organisms detected by environmental sequencing in soils are potentially associated with animal hosts rather than free‐living. By isolating the host‐associated fraction of soil microbial communities, Geisen and colleagues help explain the surprisingly high diversity of parasitic eukaryotic lineages often detected in soil/terrestrial studies using high‐throughput sequencing (HTS) and reinforce the ubiquity of these host‐associated microbes. It is clear that we can no longer assume that organisms detected in bulk environmental sequencing are free‐living, but instead need to design studies that specifically enumerate the diversity and function of host‐associated eukaryotes. Doing so will allow the field to determine the role host‐associated eukaryotes play in soils and other environments and to evaluate hypotheses on assembly of host‐associated communities, disease ecology and more.     

Marked seasonality and high spatial variability of protist communities in shallow freshwater systems

Small eukaryotes have key roles in aquatic ecosystems, influencing their local environment, global biogeochemical cycles and climate. Their impact depends on community structure, which varies along time. However, very few studies take into account temporal variation. This is especially true for small, shallow freshwater systems, which remain largely understudied despite their wide variety, global surface and intense microbial activity. We have monthly followed changes in the community structure of small microbial eukaryotes (0.2–5 μm cell diameter) for 2 years in four ponds and one brook located in North-Western France based on massive 18S rDNA amplicon 454 pyrosequencing. We detected a total of 3742 stringently defined operational taxonomic units (OTUs) encompassing all recognized eukaryotic supergroups and lineages of uncertain affiliation. Although geographically close, protist communities in the five ecosystems were contrasting, with very few shared OTUs, suggesting that environmental selection mainly drives community structure. The temporal dynamics of different high-rank taxa appeared complex and rapid at monthly scales. Despite this, a clear and reproducible seasonality was observed. As expected, low-abundance OTUs dominated the community. Although some of them appeared sporadically or remained at low frequencies during the survey, others occasionally reached relatively high abundances, sometimes recurrently. This shows that at least a fraction of low-abundance eukaryotes constitutes a seed bank. The annual proportion of primary producers, free-living heterotrophs and parasites appeared remarkably constant among the different ecosystems, suggesting underlying trends of ecosystem carrying capacity for these functional groups.     

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.     

DNA metabarcoding reveals that 200‐μm‐size‐fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes

Microeukaryotic plankton (0.2–200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High‐throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size‐fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore‐size sieve –by using SSU rDNA‐based high‐throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore‐size sieve. We further found that both >0.2 μm and 0.2–200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size‐fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.     

Abrupt burial imparts persistent changes to the bacterial diversity of turbidite‐associated sediment profiles

The emplacement of subaqueous gravity‐driven sediment flows imposes a significant physical and geochemical impact on underlying sediment and microbial communities. Although previous studies have established lasting mineralogical and biological signatures of turbidite deposition, the response of bacteria and archaea within and beneath debris flows remains poorly constrained. Both bacterial cells associated with the underlying sediment and those attached to allochthonous material must respond to substantially altered environmental conditions and selective pressures. As a consequence, turbidites and underlying sediments provide an exceptional opportunity to examine (i) the microbial community response to rapid sedimentation and (ii) the preservation and identification of displaced micro‐organisms. We collected Illumina MiSeq sequence libraries across turbidite boundaries at ~26 cm sediment depth in La Jolla Canyon off the coast of California, and at ~50 cm depth in meromictic Twin Lake, Hennepin County, MN. 16S rRNA gene signatures of relict and active bacterial populations exhibit persistent differences attributable to turbidite deposition. In particular, both the marine and lacustrine turbidite boundaries are sharply demarcated by the abundance and diversity of Chloroflexi, suggesting a characteristic sensitivity to sediment disturbance history or to differences in organic substrates across turbidite profiles. Variations in the abundance of putative dissimilatory sulfate‐reducing Deltaproteobacteria across the buried La Jolla Canyon sediment–water interface reflect turbidite‐induced changes to the geochemical environment. Species‐level distinctions within the Deltaproteobacteria clearly conform to the sedimentological boundary, suggesting a continuing impact of genetic inheritance distinguishable from broader trends attributable to selective pressure. Abrupt, <1‐cm scale changes in bacterial diversity across the Twin Lake turbidite contact are consistent with previous studies showing that relict DNA signatures attributable to sediment transport may be more easily preserved in low‐energy, anoxic environments. This work raises the possibility that deep subsurface microbial communities may inherit variations in microbial diversity from sediment flow and deformation events.     

Evaluating the Metal Tolerance Capacity of Microbial Communities Isolated from Alberta Oil Sands Process Water

Anthropogenic activities have resulted in the intensified use of water resources. For example, open pit bitumen extraction by Canada’s oil sands operations uses an estimated volume of three barrels of water for every barrel of oil produced. The waste tailings–oil sands process water (OSPW)–are stored in holding ponds, and present an environmental concern as they are comprised of residual hydrocarbons and metals. Following the hypothesis that endogenous OSPW microbial communities have an enhanced tolerance to heavy metals, we tested the capacity of planktonic and biofilm populations from OSPW to withstand metal ion challenges, using Cupriavidus metallidurans, a known metal-resistant organism, for comparison. The toxicity of the metals toward biofilm and planktonic bacterial populations was determined by measuring the minimum biofilm inhibitory concentrations (MBICs) and planktonic minimum inhibitory concentrations (MICs) using the MBEC ™ assay. We observed that the OSPW community and C. metallidurans had similar tolerances to 22 different metals. While thiophillic elements (Te, Ag, Cd, Ni) were found to be most toxic, the OSPW consortia demonstrated higher tolerance to metals reported in tailings ponds (Al, Fe, Mo, Pb). Metal toxicity correlated with a number of physicochemical characteristics of the metals. Parameters reflecting metal-ligand affinities showed fewer and weaker correlations for the community compared to C. metallidurans, suggesting that the OSPW consortia may have developed tolerance mechanisms toward metals present in their environment.     

Absence of biomarker evidence for early eukaryotic life from the Mesoproterozoic Roper Group: Searching across a marine redox gradient in mid‐Proterozoic habitability

By about 2.0 billion years ago (Ga), there is evidence for a period best known for its extended, apparent geochemical stability expressed famously in the carbonate–carbon isotope data. Despite the first appearance and early innovation among eukaryotic organisms, this period is also known for a rarity of eukaryotic fossils and an absence of organic biomarker fingerprints for those organisms, suggesting low diversity and relatively small populations compared to the Neoproterozoic era. Nevertheless, the search for diagnostic biomarkers has not been performed with guidance from paleoenvironmental redox constrains from inorganic geochemistry that should reveal the facies that were most likely hospitable to these organisms. Siltstones and shales obtained from drill core of the ca. 1.3–1.4 Ga Roper Group from the McArthur Basin of northern Australia provide one of our best windows into the mid‐Proterozoic redox landscape. The group is well dated and minimally metamorphosed (of oil window maturity), and previous geochemical data suggest a relatively strong connection to the open ocean compared to other mid‐Proterozoic records. Here, we present one of the first integrated investigations of Mesoproterozoic biomarker records performed in parallel with established inorganic redox proxy indicators. Results reveal a temporally variable paleoredox structure through the Velkerri Formation as gauged from iron mineral speciation and trace‐metal geochemistry, vacillating between oxic and anoxic. Our combined lipid biomarker and inorganic geochemical records indicate at least episodic euxinic conditions sustained predominantly below the photic zone during the deposition of organic‐rich shales found in the middle Velkerri Formation. The most striking result is an absence of eukaryotic steranes (4‐desmethylsteranes) and only traces of gammacerane in some samples—despite our search across oxic, as well as anoxic, facies that should favor eukaryotic habitability and in low maturity rocks that allow the preservation of biomarker alkanes. The dearth of Mesoproterozoic eukaryotic sterane biomarkers, even within the more oxic facies, is somewhat surprising but suggests that controls such as the long‐term nutrient balance and other environmental factors may have throttled the abundances and diversity of early eukaryotic life relative to bacteria within marine microbial communities. Given that molecular clocks predict that sterol synthesis evolved early in eukaryotic history, and (bacterial) fossil steroids have been found previously in 1.64 Ga rocks, then a very low environmental abundance of eukaryotes relative to bacteria is our preferred explanation for the lack of regular steranes and only traces of gammacerane in a few samples. It is also possible that early eukaryotes adapted to Mesoproterozoic marine environments did not make abundant steroid lipids or tetrahymanol in their cell membranes.     

Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny

Recent culture-independent molecular analyses have shown the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In the present study we directly extracted DNA from anoxic sediment near active fumaroles on a submarine caldera floor at a depth of 200 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit (SSU) rDNA. By sequencing cloned SSU rDNA of the libraries and their phylogenetic analyses, it was shown that most sequences have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, stramenopiles and Opisthokonta). In particular, some sequences were closely related to those of representatives of eukaryotic parasites, such as Phagomyxa and Cryothecomonas of Cercozoa, Pirsonia of stramenopiles and Ichthyosporea of Opisthokonta, although it is not clear whether the organisms occur in free-living or parasitic forms. In addition, other sequences did not seem to be related to any described eukaryotic lineages suggesting the existence of novel eukaryotes at a high-taxonomic level in the sediment. The community composition of microbial eukaryotes in the sediment we surveyed was different overall from those of other anoxic marine environments previously investigated.     

Time series are critical to understand microbial plankton diversity and ecology

How diverse are marine planktonic protist communities? How much seasonality do they exhibit? For a very long time, these two old and challenging questions in the field of plankton ecology could be addressed only for large‐size protist species, based on cell counting under the microscope. The recent application of molecular techniques, notably massive marker‐gene amplicon sequencing approaches (metabarcoding), has allowed investigating with unprecedented level of resolution the small‐sized (<20 µm) planktonic eukaryotes too. An amazing diversity of these tiny organisms has been unveiled but details about their temporal dynamics remain much more elusive. In a From the Cover article in this issue of Molecular Ecology, Giner et al. (2019) introduce a new Recurrence Index (RI) to specifically look for seasonality in time‐series metabarcoding data. They inspected the temporal dynamics of all operational taxonomic units (OTUs) in a rich sequence data set of pico‐ and nanoplanktonic eukaryotes in samples collected monthly during 10 years. Although most OTUs did not show seasonality, some abundant ones did, which explains why some averaging methods can find seasonality at the less detailed level of whole planktonic communities. Not surprisingly, the very complex small‐sized eukaryotic plankton communities are composed of organisms with miscellaneous temporal dynamics.     

Microbial community diversity and composition varies with habitat characteristics and biofilm function in macrophyte‐rich streams

Biofilms in streams play an integral role in ecosystem processes and function yet few studies have investigated the broad diversity of these complex prokaryotic and eukaryotic microbial communities. Physical habitat characteristics can affect the composition and abundance of microorganisms in these biofilms by creating microhabitats. Here we describe the prokaryotic and eukaryotic microbial diversity of biofilms in sand and macrophyte habitats (i.e. epipsammon and epiphyton, respectively) in five macrophyte‐rich streams in Jutland, Denmark. The macrophyte species varied in growth morphology, C:N stoichiometry, and preferred stream habitat, providing a range in environmental conditions for the epiphyton. Among all habitats and streams, the prokaryotic communities were dominated by common phyla, including Alphaproteobacteria, Bacteriodetes, and Gammaproteobacteria, while the eukaryotic communities were dominated by Stramenopiles (i.e. diatoms). For both the prokaryotes and eukaryotes, the epipsammon were consistently the most diverse communities and the epiphytic communities were generally similar among the four macrophyte species. However, the communities on the least complex macrophyte, Sparganium emersum, had the lowest richness and evenness and fewest unique OTUs, whereas the macrophyte with the most morphological complexity, Callitriche spp., had the highest number of unique OTUs. In general, the microbial taxa were ubiquitously distributed across the relatively homogeneous Danish landscape as determined by measuring the similarity among communities (i.e. Sørensen similarity index). Furthermore, we found significant correlations between microbial diversity (i.e. Chao1 rarefied richness and Pielou's evenness) and biofilm structure and function (i.e. C:N ratio and ammonium uptake efficiency, respectively); communities with higher richness and evenness had higher C:N ratios and lower uptake efficiency. In addition to describing the prokaryotic and eukaryotic community composition in stream biofilms, our study indicates that 1) physical habitat characteristics influence microbial diversity and 2) the variation in microbial diversity may dictate the structural and functional characteristics of stream biofilm communities.     

Microbial diversity of an anoxic zone of a hydroelectric power station reservoir in Brazilian Amazonia

Microbial diversity was evaluated in an anoxic zone of Tucuruí Hydroelectric Power Station reservoir in Brazilian Amazonia using a culture-independent approach by amplifying and sequencing fragments of the 16S rRNA gene using metagenomic DNA as a template. Samples obtained from the photic, aphotic (40 m) and sediment (60 m) layers were used to construct six 16S rDNA libraries containing a total of 1,152 clones. The sediment, aphotic and photic layers presented 64, 33 and 35 unique archaeal operational taxonomic units (OTUs). The estimated richness of these layers was evaluated to be 153, 106 and 79 archaeal OTUs, respectively, using the abundance-based coverage estimator (ACE) and 114, 83 and 77 OTUs using the Chao1 estimator. For bacterial sequences, 114, 69 and 57 OTUs were found in the sediment, aphotic and photic layers, which presented estimated richnesses of 1,414, 522 and 197 OTUs (ACE) and 1,059, 1,014 and 148 OTUs (Chao1), respectively. Phylogenetic analyses of the sequences obtained revealed a high richness of microorganisms which participate in the carbon cycle, namely, methanogenic archaea and methanotrophic proteobacteria. Most sequences obtained belong to non-culturable prokaryotes. The present study offers the first glimpse of the huge microbial diversity of an anoxic area of a man-made lacustrine environment in the tropics.     

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).     

Distribution and Diversity of Microbial Eukaryotes in Bathypelagic Waters of the South China Sea

Little is known about the biodiversity of microbial eukaryotes in the South China Sea, especially in waters at bathyal depths. Here, we employed SSU rDNA gene sequencing to reveal the diversity and community structure across depth and distance gradients in the South China Sea. Vertically, the highest alpha diversity was found at 75‐m depth. The communities of microbial eukaryotes were clustered into shallow‐, middle‐, and deep‐water groups according to the depth from which they were collected, indicating a depth‐related diversity and distribution pattern. Rhizaria sequences dominated the microeukaryote community and occurred in all samples except those from less than 50‐m deep, being most abundant near the sea floor where they contributed ca. 64–97% and 40–74% of the total sequences and OTUs recovered, respectively. A large portion of rhizarian OTUs has neither a nearest named neighbor nor a nearest neighbor in the GenBank database which indicated the presence of new phylotypes in the South China Sea. Given their overwhelming abundance and richness, further phylogenetic analysis of rhizarians were performed and three new genetic clusters were revealed containing sequences retrieved from the deep waters of the South China Sea. Our results shed light on the diversity and community structure of microbial eukaryotes in this not yet fully explored area.