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.

During the CARIACO time series program, microbial standing stocks, bacterial production, and acetate turnover were consistently elevated in the redox transition zone (RTZ) of the Cariaco Basin, the depth interval (~240–450 m) of steepest gradient in oxidation‐reduction potential. Anomalously high fluxes of particulate carbon were captured in sediment traps below this zone (455 m) in 16 of 71 observations. Here we present new evidence that bacterial chemoautotrophy, fueled by reduced sulfur species, supports an active secondary microbial food web in the RTZ and is potentially a large midwater source of labile, chemically unique, sedimenting biogenic debris to the basin's interior. Dissolved inorganic carbon assimilation (27–159 mmol C m⁻² d⁻¹) in this zone was equivalent to 10%–333% of contemporaneous primary production, depending on the season. However, vertical diffusion rates to the RTZ of electron donors and electron acceptors were inadequate to support this production. Therefore, significant lateral intrusions of oxic waters, mixing processes, or intensive cycling of C, S, N, Mn, and Fe across the RTZ are necessary to balance electron equivalents. Chemoautotrophic production appears to be decoupled temporally from short‐term surface processes, such as seasonal upwelling and blooms, and potentially is more responsive to long‐term changes in surface productivity and deep‐water ventilation on interannual to decadal timescales. Findings suggest that midwater production of organic carbon may contribute a unique signature to the basin's sediment record, thereby altering its paleoclimatological interpretation.

Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84,inD. Kirchman, ed.,Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75,inR. 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,inD. Kirchman, ed.,Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75,inR. 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).

Microbial community samples were collected from the anoxic zone of the Cariaco Basin at depths of 320, 500, and 1,310 m on a November 1996 cruise and were used to construct 16S ribosomal DNA libraries. Of 60 nonchimeric sequences in the 320-m library, 56 belonged to the ɛ subdivision of the Proteobacteria (ɛ- Proteobacteria ) and 53 were closely related to ectosymbionts of Rimicaris exoculata and Alvinella pompejana , which are referred to here as epsilon symbiont relatives (ESR). The 500-m library contained sequences affiliated with the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the division Verrucomicrobia , the division Proteobacteria , and the OP3 candidate division. The Proteobacteria included members of the γ, δ, ɛ and new candidate subdivisions, and γ-proteobacterial sequences were dominant (25.6%) among the proteobacterial sequences. As in the 320-m library, the majority of the ɛ-proteobacteria belonged to the ESR group. The genus Fibrobacter and its relatives were the second largest group in the library (23.6%), followed by the δ-proteobacteria and the ɛ-proteobacteria. The 1,310-m library had the greatest diversity; 59 nonchimeric clones in the library contained 30 unique sequences belonging to the planctomycetes, the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the Proteobacteria , and the OP3 and OP8 candidate divisions. The proteobacteria included members of new candidate subdivisions and the β, γ, δ, and ɛ-subdivisions. ESR sequences were still present in the 1,310-m library but in a much lower proportion (8.5%). One archaeal sequence was present in the 500-m library (2% of all microorganisms in the library), and eight archaeal sequences were present in the 1,310-m library (13.6%). All archaeal sequences fell into two groups; two clones in the 1,310-m library belonged to the kingdom Crenarchaeota and the remaining sequences in both libraries belonged to the kingdom Euryarchaeota . The latter group appears to be related to the Eel-TA1f2 sequence, which belongs to an archaeon suggested to be able to oxidize methane anaerobically. Based on phylogenetic inferences and measurements of dark CO ₂ fixation, we hypothesized that (i) the ESR are autotrophic anaerobic sulfide oxidizers, (ii) sulfate reduction and fermentative metabolism may be carried out by a large number of bacteria in the 500- and 1,310-m libraries, and (iii) members of the Euryarchaeota found in relatively large numbers in the 1,310-m library may be involved in anaerobic methane oxidation. Overall, the composition of microbial communities from the Cariaco Basin resembles the compositions of communities from several anaerobic sediments, supporting the hypothesis that the Cariaco Basin water column is similar to anaerobic sediments.