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).     

Viral elements and their potential influence on microbial processes along the permanently stratified Cariaco Basin redoxcline

Little is known about viruses in oxygen-deficient water columns (ODWCs). In surface ocean waters, viruses are known to act as gene vectors among susceptible hosts. Some of these genes may have metabolic functions and are thus termed auxiliary metabolic genes (AMGs). AMGs introduced to new hosts by viruses can enhance viral replication and/or potentially affect biogeochemical cycles by modulating key microbial pathways. Here we identify 748 viral populations that cluster into 94 genera along a vertical geochemical gradient in the Cariaco Basin, a permanently stratified and euxinic ocean basin. The viral communities in this ODWC appear to be relatively novel as 80 of these viral genera contained no reference viral sequences, likely due to the isolation and unique features of this system. We identify viral elements that encode AMGs implicated in distinctive processes, such as sulfur cycling, acetate fermentation, signal transduction, [Fe–S] formation, and N-glycosylation. These AMG-encoding viruses include two putative Mu-like viruses, and viral-like regions that may constitute degraded prophages that have been modified by transposable elements. Our results provide an insight into the ecological and biogeochemical impact of viruses oxygen-depleted and euxinic habitats.Subject terms: Microbial ecology, Metagenomics     

Phylogenetic diversity of bacterial and archaeal communities in the anoxic zone of the Cariaco Basin

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 epsilon subdivision of the Proteobacteria (epsilon-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 gamma, delta, epsilon and new candidate subdivisions, and gamma-proteobacterial sequences were dominant (25.6%) among the proteobacterial sequences. As in the 320-m library, the majority of the epsilon-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 delta-proteobacteria and the epsilon-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 beta, gamma, delta, and epsilon-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.     

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.     

Comparison of vertical distributions of prokaryotic assemblages in the anoxic Cariaco Basin and Black Sea by use of fluorescence in situ hybridization

Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria, Crenarchaeota, and Euryarchaeota were more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances of Archaea in both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group I Crenarchaeota (median, 5.5%) to prokaryotic communities were significantly higher (P < 0.001; n = 20) than those of group II Euryarchaeota (median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4',6'-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select.     

Euphausiids (Crustacea: Melacostraca) from the southern Mexican Caribbean Sea

The composition, abundance and distribution of euphausiids from the southern part of the Mexican Caribbean Sea (August 1986) were sampled during the ARCOMM I cruise on board the R/V "Justo Sierra" of UNAM. Sampling was done using a Bongo-net (0.5 mm mesh size) performing oblique hauls from 200 m to the surface at 28 stations. Of the total euphausiid numbers, the most abundant species was Stylocheiron carinatum (49%), followed by Euphausia americana (9.8%) and E. tenera with (7.8%). The highest total density and that of the three most abundant species occurred during the night sampling, and probably was related to vertical migration patterns. The Bray-Curtis index revealed three station assemblages, related to the day-night variations of the euphausiid community. Species were distributed mainly in the oceanic area, and were absent in the neritic zones. The local fauna shows a strong affinity for the euphausiid community of the Gulf of Mexico and other areas of the Caribbean Sea. Four species are considered to be new records for the western Caribbean Sea.     

Spatial structure of the Caribbean lobster (Metanephrops binghami) in the Colombian Caribbean Sea

Crustaceans of the genus Metanephrops are of great commercial value in some tropical and subtropical regions. With the potential development of a new deep lobster fishery in the Colombian Caribbean Sea, the objective of this work is to describe for first time the patterns of spatial and bathymetric distribution, and diel migratory periodicity of the Caribbean lobster (M. binghami). Data were collected by trawling in depths between 200 and 550 m (100 m strata intervals) in the Colombian Caribbean Sea. Higher biomass and size of these crustaceans were found between 250 and 350 m, with a maximum at about 300 m. The study offers diel patterns of M. binghami, which suggests nocturnal activity and burrowing during daylight hours.     

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.     

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

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

Leptepsilonema gen. n. (Nematoda, Epsilonematidae) from Chile and the Caribbean Sea

The new genus Leptepsilonema is erected. L. procerum sp.n from intertidal sandy beaches of the Caribbean sea and L. macrum sp.n. and L. exile sp.n. from Chile are described. Postembryonal development of L. procerum , covering the juvenile stages II-IV, is presented. Some ecological data are added.     

New fatty acids from Colombian Caribbean Sea sponges

The fatty acid composition of whole phospholipids from marine sponges collected from the Colombian Caribbean Sea was determined as part of our studies on the order Halichondrida: Halichondria magniconulosa, Halichondria lutea, Petromica ciocalyptoides, Axinyssa ambrosia, Didiscus oxeata and Dragmaxia undata. Structure elucidation was accomplished by means of gas chromatography retention parameters and GCMS. Eight new fatty acids were identified by their methyl esters and N-acylpyrrolidide derivatives (i.e. 5-methyl-6-octadecenoic, 16-methyl-11-heptadecenoic, 8-methyl-4-tetracosenoic, 8,17-dimethyl-5-octadecenoic, 23-methyl-8-tetracosenoic, 20-methyl-18-tetracosenoic, 4,17-tetracosadienoic and 22,27-dimethyl-5,9-octacosadienoic acids). These findings establish alternative fatty acid biosynthetic possibilities for these organisms.     

Hermit crabs (Anomura: Paguroidea) distribution patterns in the Colombian Caribbean Sea

Hermit crabs represent the marine life in the Colombian Caribbean, and are important for the dynamic equilibrium maintenance in ecosystems, the ecological interactions and their impact on food web stability. Generally, in order to come up with some conservation strategies, strong bio-geographical information is needed for poll cies definition. With this aim, this study analyzed the distribution patterns of hermit crabs in the Colombian Caribbean Sea. through classification and spatial ordination multivariate analyses, using historical records from years 1916 to 2006. Besides, the world distribution of Colombian species and their geographic affinity in the Caribbean and Western Atlantic were identified. The results show deep differences between coastal and continental slope faunas, and latitudinal differences in the assemblages, with the identification of three groups: Northeast. Center and Southwest. The differences in faunal composition that support these three groups were determined. Based on maps of the Colombian marine ecosystems, it was found that the main factors affecting the distribution of hermit crabs were the Caribaná slope (depth), water-mass temperature, Guajira sea-grass beds, and particular conditions of "Coralline Archipelagos" and "Darién" eco-regions. Colombian hermit crab fauna is more related to the North Atlantic and the Antilles, than to the South Atlantic and the Gulf of Mexico. Additionally, geographical sub-provinces in which Colombia is included, these were found as transition zones among Northern and Austral subprovinces of the Greater Caribbean.     

Halichrysis corallinarius sp. nov. (Rhodymeniaceae, Rhodophyta) from Puerto Rico, Caribbean Sea

A new species, Halichrysis corallinarius sp. nov. is described from coral reef habitats in southwest Puerto Rico as well as from Grand Cayman Island. The new species produces strap‐shaped thalli supported above the substrata by abundant peg‐like stipes. The lobed branches are frequently anastomosed with colonies measuring to 4.0 cm broad with individual axes measuring to 7.0 mm across. Algae possess up to five layers of medullary cells with frequent open spaces in the medulla. Tetrasporangia, measure to 19 × 25 μm, and are borne in sori limited to ventral surfaces. Gametophytes are monoecious and hemispherical carposporophytes are produced dorsally, measuring to 900 μm in diameter and 600 μm high.     

Species of dinoflagellates of the genus Gambierdiscus (Dinophyceae) in the Mexican Caribbean Sea

Some dinoflagellates with benthic habits are related to ciguatera intoxication by fish consumption, especially in tropical areas. In the Mexican Caribbean, ciguatera is relatively common, but only one paper seems to have been published on the subject, and there are very few publicactions on phytoplankton and benthic microalgae. Material collected along the coast of the State of Quintana Roo with phytoplankton net (54 mm) and directly from sediment and epiphytes of macroscopic plants, was searched for toxic and other associated dinoflagellates. Samples were studied by light and scanning electron microscopy. Morphological characters were useful for species identification, but eventually physiological, ecological and molecular characters could also be used. Three species of Gambierdiscus, related to the production of ciguatera toxins, were identified: G. belizeanus, G. toxicus and G. yasumotoi. They are distributed in shallow coastal areas, including coastal lagoons.     

Inclusion bodies in several species of Ceratium Schrank (Dinophyceae) from the Caribbean Sea examined with DNA-specific staining

Several dinoflagellate species in the genus Ceraiium Schrankwere examined, with the help of a DNA-specific dye, for thepresence of a nucleus, chloroplasts and inclusion bodies. Theabundance of Ceratium in the Caribbean Sea in April 1990 wasbetween 1.3 and 4.9 x 10⁵ cells m^–3 Most Ceratium teresKofoid contained 5–10 small chloroplasts and among 529C.teres cells. 8% possessed an inclusion body. The shape andsize of inclusion bodies varied, and 7% of the inclusions hada DNA-containing particle inside. Similar inclusion bodies werealso observed in Ceratium declinatum f. normale Jörgensen,Ceratium furca (Ehrenberg) Claparede and Lachmann, and Ceratiumfusus (Ehrenberg) Dujardin. Our observations indicated thatinclusion bodies are common in oceanic Ceratium. ¹Present address: Institute of Marine Biology, National TaiwanOcean University, Keelung 20224, Taiwan, ROC     

Cruoriella rhodoliths from shallow-water back reef environments in La Parguera, Puerto Rico (Caribbean Sea)

The occurrence of shallow-water (0.9 to 1.3 m) rhodoliths in back reef environments in southwest Puerto Rico is reported. The rhodoliths were generally cylindrical, discoidal or irregular in shape with an average longest dimension of 7.2 cm. They occurred at a maximum density of 524 m⁻². The rhodoliths were composed of mostly coral nuclei with concentric laminations of aragonite-producing Cruoriella armorica (Peyssonneliaceae, Rhodophyta). Maximum Cruoriella accretion around coral nuclei was 30 mm although accretions of 1 to 20 mm were more common. Based on measurements of Cruoriella accretion, these shallow water rhodoliths are estimated to have minimum ages of 12 to 24 years. It is further estimated that approximately 2% of the rhodoliths are turned over daily. Accepted: 1 October 1999     

Unexpected and novel putative viruses in the sediments of a deep-dark permanently anoxic freshwater habitat

Morphological diversity, abundance and community structure of viruses were examined in the deep and anoxic sediments of the volcanic Lake Pavin (France). The sediment core, encompassing 130 years of sedimentation, was subsampled every centimeter. High viral abundances were recorded and correlated to prokaryotic densities. Abundances of viruses and prokaryotes decreased with the depth, contrasting the pattern of virus-to-prokaryote ratio. According to fingerprint analyses, the community structure of viruses, bacteria and archaea gradually changed, and communities of the surface (0–10 cm) could be discriminated from those of the intermediate (11–27 cm) and deep (28–40 cm) sediment layers. Viral morphotypes similar to virions of ubiquitous dsDNA viruses of bacteria were observed. Exceptional morphotypes, previously never reported in freshwater systems, were also detected. Some of these resembled dsDNA viruses of hyperthermophilic and hyperhalophilic archaea. Moreover, unusual types of spherical and cubic virus-like particles (VLPs) were observed. Infected prokaryotic cells were detected in the whole sediment core, and their vertical distribution correlated with both viral and prokaryotic abundances. Pleomorphic ellipsoid VLPs were visible in filamentous cells tentatively identified as representatives of the archaeal genus Methanosaeta, a major group of methane producers on earth.     

Comparison of Vertical Distributions of Prokaryotic Assemblages in the Anoxic Cariaco Basin and Black Sea by Use of Fluorescence In Situ Hybridization

Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria, Crenarchaeota, and Euryarchaeota were more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances of Archaea in both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group I Crenarchaeota (median, 5.5%) to prokaryotic communities were significantly higher (P < 0.001; n = 20) than those of group II Euryarchaeota (median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4′,6′-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select.