Biodiversity, Community Structural Shifts, and Biogeography of Prokaryotes within Antarctic Continental Shelf Sediment

16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region. Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions). The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs). At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes. At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria. Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core. Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.     

Statistical approaches for estimating actinobacterial diversity in marine sediments

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

Analysis of the Rumen Bacterial Diversity under two Different Diet Conditions using Denaturing Gradient Gel Electrophoresis,Random Sequencing,and Statistical Ecology Approaches

Bacterial community structure and diversity in the rumen of steers in conditions of hay and corn diets was assessed by in vitro retrieval and analysis of the variable region (V3) of 16S rDNA. Two types of libraries were generated in this study: DGGE libraries, which further were analysed by excising, reamplification, and sequencing, and random shotgun sequence libraries. Phylogenetic and sequence similarity analyses of the resultant 68 clone sequences in DGGE libraries revealed the presence of 42 operational taxonomic units (OTUs) or phylotypes defined as having more than 97% of sequence similarity. One hundred and thirty four clone sequences in shotgun libraries were clustered into 72 phylotypes. The phylotype similarity, diversity, richness, and evenness in these libraries were estimated using a variety of diversity indices. In relation to diet, the corn-fed animals displayed more diverse and rich bacterial populations, which were mostly contributed by CFB-related phylotypes. Proteobacteria were also numerically prevalent on this diet (27%) but were represented by a few phylotypes thus diminishing the overall diversity and species richness values. On hay diet, the principal contributors to general diversity and species richness appeared to be low-G + C gram-positives. Although the ruminal Treponemaes were encountered only in hay-fed animals, their impact on species diversity on hay diet was low because of the limited number of phylotypes.     

Diversity and Spatial Distribution of Prokaryotic Communities Along A Sediment Vertical Profile of A Deep-Sea Mud Volcano

We investigated the top 30-cm sediment prokaryotic community structure in 5-cm spatial resolution, at an active site of the Amsterdam mud volcano, East Mediterranean Sea, based on the 16S rRNA gene diversity. A total of 339 and 526 sequences were retrieved, corresponding to 25 and 213 unique (≥98% similarity) phylotypes of Archaea and Bacteria, respectively, in all depths. The Shannon–Wiener diversity index H was higher for Bacteria (1.92–4.03) than for Archaea (0.99–1.91) and varied differently between the two groups. Archaea were dominated by anaerobic methanotrophs ANME-1, -2 and -3 groups and were related to phylotypes involved in anaerobic oxidation of methane from similar habitats. The much more complex Bacteria community consisted of 20 phylogenetic groups at the phylum/candidate division level. Proteobacteria, in particular δ-Proteobacteria, was the dominant group. In most sediment layers, the dominant phylotypes of both the Archaea and Bacteria communities were found in neighbouring layers, suggesting some overlap in species richness. The similarity of certain prokaryotic communities was also depicted by using four different similarity indices. The direct comparison of the retrieved phylotypes with those from the Kazan mud volcano of the same field revealed that 40.0% of the Archaea and 16.9% of the Bacteria phylotypes are common between the two systems. The majority of these phylotypes are closely related to phylotypes originating from other mud volcanoes, implying a degree of endemicity in these systems.     

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.     

Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures

Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the “hidden” microeukaryotic diversity in the environment examined. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of > 0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G + C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria ( Desulfosarcina group, Syntrophus and Geobacter / Pelobacter / Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.     

Spatial heterogeneity of crenarchaeal assemblages within mesophilic soil ecosystems as revealed by PCR-single-stranded conformation polymorphism profiling

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P < 0.0001] and least-significant-difference analysis [alpha = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.     

Mechanical soil disturbance as a determinant of arbuscular mycorrhizal fungal communities in semi-natural grassland

While the effect of disturbance on overall abundance and community composition of arbuscular mycorrhizal (AM) fungi has been researched in agricultural fields, less is known about the impact in semi-natural grasslands. We sampled two AM plant species, Festuca brevipila and Plantago lanceolata, from an ongoing grassland restoration experiment that contained replicated plowed and control plots. The AM fungal community in roots was determined using nested PCR and LSU rDNA primers. We identified 38 phylotypes within the Glomeromycota, of which 29 belonged to Glomus A, six to Glomus B, and three to Diversisporaceae. Only three phylotypes were closely related to known morphospecies. Soil disturbance significantly reduced phylotype richness and changed the AM fungal community composition. Most phylotypes, even closely related ones, showed little or no overlap in their distribution and occurred in either the control or disturbed plots. We found no evidence of host preference in this system, except for one phylotype that preferentially seemed to colonize Festuca. Our results show that disturbance imposed a stronger structuring force for AM fungal communities than did host plants in this semi-natural grassland.     

Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P < 0.0001] and least-significant-difference analysis [α = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.     

Mariana Forearc Serpentinite Mud Volcanoes Harbor Novel Communities of Extremophilic Archaea

Extremophilic archaeal communities living in serpentinized muds influenced by pH 12.5 deep-slab derived fluids were detected and their richness and relatedness assessed from across seven serpentinite mud volcanoes located along the Mariana forearc. In addition, samples from two near surface core sections (Holes D and E) at ODP Site 1200 from South Chamorro were subjected to SSU rDNA clone library and phylogenetic analysis resulting in the discovery of several novel operational taxonomic units (OTUs). Five dominant OTUs of Archaea from Hole 1200D and six dominant OTUs of Archaea from Hole 1200E were determined by groups having three or more clones. Terminal-restriction fragment length polymorphism (T-RFLP) analysis revealed all of the dominant OTUs were detected within both clone libraries. Cluster analysis of the T-RFLP data revealed archaeal community structures from sites on Big Blue and Blue Moon to be analogous to the South Chamorro Hole 1200E site. These unique archaeal community fingerprints resulted from an abundance of potential methane-oxidizing and sulfate-reducing phylotypes. This study used deep-sea sediment coring techniques across seven different mud volcanoes along the entire Mariana forearc system. The discovery and detection of both novel Euryarchaeota and Marine Benthic Group B Crenarcheaota phylotypes could be efficacious archaeal indicator populations involved with anaerobic methane oxidation (AMO) and sulfate reduction fueled by deep subsurface serpentinization reactions.     

Colonization and community structure of root-associated microorganisms of Sabina vulgaris with soil depth in a semiarid desert ecosystem with shallow groundwater

Arbuscular mycorrhizal fungi (AMF) have been observed in deep soil layers in arid lands. However, change in AMF community structure with soil depth and vertical distributions of the other root-associated microorganisms are unclear. Here, we examined colonization by AMF and dark septate fungi (DSF), as well as the community structure of AMF and endophytic fungi (EF) and endophytic bacteria (EB) in association with soil depth in a semiarid desert with shallow groundwater. Roots of Sabina vulgaris and soils were collected from surface to groundwater level at 20-cm intervals. Soil chemistry (water content, total N, and available P) and colonization of AMF and DSF were measured. Community structures of AMF, EF, and EB were examined by terminal restriction fragment length polymorphism analysis. AMF colonization decreased with soil depth, although it was mostly higher than 50%. Number of AMF phylotypes decreased with soil depth, but more than five phylotypes were observed at depths up to 100 cm. Number of AMF phylotypes had a significant and positive relationship with soil moisture level within 0-15% of soil water content. DSF colonization was high but limited to soil surface. Number of phylotypes of EF and EB were diverse even in deep soil layers, and the community composition was associated with the colonization and community composition of AMF. This study indicates that AMF species richness in roots decreases but is maintained in deep soil layers in semiarid regions, and change in AMF colonization and community structure associates with community structure of the other root-associated microorganisms.     

Fungi in the Hyporheic Zone of a Springbrook

Eight bimonthly sediment core samples (n = 6) were collected, to a depth of 64 cm, from the hyporheic zone of a springbrook in southern Ontario, Canada. Sediment cores were divided into three to four sections, and organic matter was subdivided into six different categories. Twigs were the most common substrate, followed by roots, cedar leaves, wood, grass, and deciduous leaves. The contributions of deciduous and cedar leaves declined with depth, whereas that of wood increased. On each sampling date and from each section, three randomly chosen substrates >3 cm were examined for conidia of aquatic hyphomycetes. The number of identified species significantly decreased with depth, and was highest on deciduous leaves and lowest on wood. Season had no significant effect on species numbers. DNA from substrates was extracted, amplified with fungal primers, and differentiated into phylotypes with denaturing gradient gel electrophoresis (DGGE). Absence/presence patterns of phylotype were significantly affected by season but not by section level. Both season and section level significantly affected relative densities of the bands of the 10 most common phylotypes. Our data suggest that aquatic hyphomycetes and other fungi readily disperse within the hyporheic zone, and that their relative scarcity in this habitat is due to a lack of suitable substrates.     

Comparative analysis of yellow microbial communities growing on the walls of geographically distinct caves indicates a common core of microorganisms involved in their formation

Morphologically similar microbial communities that often form on the walls of geographically distinct limestone caves have not yet been comparatively studied. Here, we analysed phylotype distribution in yellow microbial community samples obtained from the walls of distinct caves located in Spain, Czech Republic and Slovenia. To infer the level of similarity in microbial community membership, we analysed inserts of 474 16S rRNA gene clones and compared those using statistical tools. The results show that the microbial communities under investigation are composed solely of Bacteria. The obtained phylotypes formed three distinct groups of operational taxonomic units (OTUs). About 60% of obtained sequences formed three core OTUs common to all three sampling sites. These were affiliated with actinobacterial Pseudonocardinae (30-50% of sequences in individual sampling site libraries), but also with gammaproteobacterial Chromatiales (6-25%) and Xanthomonadales (0.5-2.0%). Another 7% of sequences were common to two sampling sites and formed eight OTUs, while the remaining 35% were site specific and corresponded mostly to OTUs containing single sequences. The same pattern was observed when these data were compared with sequence data available from similar studies. This comparison showed that distinct limestone caves support microbial communities composed mostly of phylotypes common to all sampling sites.     

Fungal community composition in the gut of rove beetles (Coleoptera: Staphylinidae) from the Canadian boreal forest reveals possible endosymbiotic interactions for dietary needs

The goal of this study was to investigate the fungal community composition in the gut of Staphylinidae from boreal forest in order to better understand the diversity and the complexity of fungus-insect relationships. DNA gut content analyses of nine abundant rove beetle species (Coleoptera, Staphylinidae) living in the boreal balsam fir forest ecosystem (Montmorency Forest, Quebec, Canada) were performed to identify the fungal taxa present either as endosymbiotic taxa or as a source of nutrition. A total of 42 fungal operational taxonomic units (OTUs) were recorded from the analysis of 441 fungal ITS rDNA sequences recovered from gut extracts. The OTU richness per species ranged between four in Tachinus quebecensis and 16 in Atheta ventricosa. The fungal mycobiota in posterior gut extracts was dominated by Saccharomycetales (12 OTUs), followed by Sordariomycetes (nine OTUs). No significant difference was observed between the OTU richness recorded within each of the three subfamilies of rove beetles investigated. The core mycobiome of the posterior gut extracts was dominated by three OTUs related to yeasts, with ITS sequences having pairwise similarities equal to or greater than 99% with Candida mesenterica, Debaryomyces spp. and Ophiostoma pluriannulatum. These results provide some evidence of the consumer-resource relationships of these beetles. Predominance of yeast and fungal spores in the posterior gut of rove beetles suggests that they may play an important role in their dietary requirements and as endosymbionts.     

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil

In spite of the techniques based on the amplification of 16S rRNA genes (16S rDNA) to compare bacterial communities that are now widely in use in microbial ecology, little is known about the composition of the soybean continuous cropping (CC) and rotational cropping (RC) soil microbial community. To address this, we compared the levels of bacterial community diversity in RC and 5-year CC rhizosphere soil samples. We selected 407 clones in RC and 490 clones in CC for restriction fragment length polymorphism analysis. A total of 123 phylotypes were identified among the 16S rDNA clones, while 78 unique and 21 common phylotypes were identified among the CC soil isolates. Analysis of sequences from a subset of the phylotypes showed that at least 11 bacterial divisions were represented in the clone libraries. The phylotype richness, frequency distribution (evenness), and composition of the two clone libraries were investigated using a variety of diversity indices. Although the analysis of diversity indices and LIBSHUFF comparisons revealed that the compared libraries were not significantly different ( P =0.05) between the RC vs. CC soils, some differences could be observed in terms of specific phyla and groups. We concluded that the group variance was not determined immediately by the cropping system's induction, but was a long-term and slow process.     

Microbial diversity at 83°C in Calcite Springs, Yellowstone National Park: another environment where the Aquificales and "Korarchaeota" coexist

The use of molecular phylogenetic approaches in microbial ecology has revolutionized our view of microbial diversity at high temperatures and led to the proposal of a new kingdom within the Archaea, namely, the "Korarchaeota." We report here the occurrence of another member of this archaeal group and a deeply rooted bacterial sequence from a thermal spring in Yellowstone National Park (USA). The DNA of a mixed community growing at 83°C, pH 7.6, was extracted and the small subunit ribosomal RNA gene (16S rDNA) sequences were obtained using the polymerase chain reaction. The products were cloned and five different phylogenetic types ("phylotypes") were identified: four archaeal phylotypes, designated pBA1, pBA2, pBA3, and pBA5, and only one bacterial phylotype, designated pBB. pBA5 is very closely related to the korarchaeotal phylotype, pJP27, from Obsidian Pool in Yellowstone National Park. The pBB phylotype is a lineage within the Aquificales and, based on 16S rRNA sequence, is different enough from the members of the Aquificales to constitute a different genus. In situ hybridization with bacterial-specific and Aquificales-specific fluorescent oligonucleotide probes indicated the bacterial population dominated the community and most likely contributed significantly to biogeochemical cycling within the community. Received: August 29, 1999 / Accepted: November 16, 1999