Cosmopolitan metapopulations of free-living microbial eukaryotes

Metapopulations of macroscopic organisms tend to be geographically restricted, but free-living protists and other microbial eukaryotes present a different picture. Here we show that most organisms smaller than 1 mm occur worldwide wherever their required habitats are realised. This is a consequence of ubiquitous dispersal driven by huge population sizes, and the consequently low probability of local extinction. Organisms larger than 10 mm are much less abundant, and rarely cosmopolitan. The supporting data, together with the discovery that the 1-10 mm size range accommodates a transition from cosmopolitan to regionally-restricted distribution, were derived from extensive inventories of eukaryotic species in a freshwater pond (1278 species), and a shallow marine bay (785 species). All accessible records were examined to establish the extent of global coverage by these species. Some groups of microbial eukaryotes are severely undersampled (e.g. naked amoebae; marine meiofauna in the southern hemisphere) but this fails to weaken evidence that metapopulations of microbial eukaryotes are cosmopolitan.     

Genome diversity in microbial eukaryotes

The genomic peculiarities among microbial eukaryotes challenge the conventional wisdom of genome evolution. Currently, many studies and textbooks explore principles of genome evolution from a limited number of eukaryotic lineages, focusing often on only a few representative species of plants, animals and fungi. Increasing emphasis on studies of genomes in microbial eukaryotes has and will continue to uncover features that are either not present in the representative species (e.g. hypervariable karyotypes or highly fragmented mitochondrial genomes) or are exaggerated in microbial groups (e.g. chromosomal processing between germline and somatic nuclei). Data for microbial eukaryotes have emerged from recent genome sequencing projects, enabling comparisons of the genomes from diverse lineages across the eukaryotic phylogenetic tree. Some of these features, including amplified rDNAs, subtelomeric rDNAs and reduced genomes, appear to have evolved multiple times within eukaryotes, whereas other features, such as absolute strand polarity, are found only within single lineages.     

Molecular diversity of microbial eukaryotes in sea water from Fildes Peninsula,King George Island,Antarctica

Data on Antarctic coastal sites remain scarce and are generally limited to microscopy; the diversity of coastal Antarctic ecosystems has long been underestimated. The diatom-dominant community in the coastal sea waters of the Fildes Peninsula has been described according to traditional protocols. Molecular diversity of microbial eukaryotes (≤20 µm) from Great Wall Cove and Ardley Cove, Fildes Peninsula, has been determined by Illumina MiSeq2000 sequencing. Inferred metabolisms of summer phytoplankton in the two coves are characterised by autotrophy and heterotrophy. The frequent occurrence of such nanoflagellates as dinoflagellates, Cryptophyta, Stramenopiles, Pyramimonas, Telonema, and Cryothecomonas is predicted to be important in these Antarctic coastal communities. Sea water exchange exists between the two coves when high tide occurs, indicating that there appears to be mixing between the microbial communities in the two coves. Cluster analysis of the microbial eukaryote composition at the phylum and genus levels reveals a conservation of the community composition between the two coves. The inner stations of Great Wall Cove represented by three shoal samples (G1, G2, and G3) are clustered closely together, surrounded by islands and formed into a semi-closed body of water. The samples from the outer stations G4 and G5 of Great Wall Cove, which are separated from the other three Great Wall stations, are more similar to the Ardley Cove samples. Sea water exchange between the outer basins might be the effect of their community compositions. The nanoplankton diversity in Great Wall Cove is richer than in Ardley Cove, according to the α-diversity index.     

Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

Dispersal and species diversity: a meta-analysis

Species diversity in communities of interacting organisms is thought to be enhanced by dispersal, yet mechanisms predicting this have little to say about what effects differing rates of dispersal have on diversity and how dispersal affects diversity at larger spatial scales. I performed meta‐analyses on 23 studies comprising 50 experiments that manipulated species migration and measured community richness or diversity to test three hypotheses: that dispersal increases local diversity; that this effect depends on the rate of dispersal, specifically, that local diversity should be maximized at intermediate dispersal rates or else linearly related to dispersal rate; and that regional diversity may be either unaffected or negatively impacted by dispersal because dispersal tends to homogenize local communities. I found that immigration increased local diversity. Further, in animal studies, diversity appears maximized at intermediate dispersal rates but not with plant studies; however, more standardized studies are needed. Finally, results are ambiguous as to what happens at larger scales, with studies finding either declines or no change in regional diversity with dispersal. Taken together, these results reveal that dispersal has a complex, spatially contingent relationship with patterns of species diversity.     

The microbial diversity in picoplankton enrichment cultures: a molecular screening of marine isolates

Picoplankton bacteria from a North Sea water sample were cultured under a variety of different conditions (nutrients, temperature, light, agitation, adhesion). Fluorescent in situ hybridization (FISH) analysis of the enrichments showed complex communities which were dominated by gamma-Proteobacteria or beta-Proteobacteria, followed by alpha-Proteobacteria and bacteria from the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster. Among 410 isolates, a high degree of diversity was found, both with respect to colony color and morphology and with respect to genetic diversity. Isolated bacteria were classified into the main taxa by a special PCR approach, termed signature PCR (SIG-PCR). It was based on an oligo primer mixture targeting 16S rDNA which yielded PCR products of taxon-specific lengths. Again, gamma-Proteobacteria dominated (48%), followed by alpha-Proteobacteria (20%). beta-Proteobacteria were rarely isolated (eight strains of 410). The CFB cluster comprised the second largest phylum (14%), and 7.5% of all isolates belonged to the high-GC Gram-positives. Thus, isolated bacteria were representative of enrichment communities with the exception of the beta-Proteobacteria, which were detected in high abundance in certain enrichments by FISH but not isolated, and the high-GC Gram-positives, which were cultivated but not detected by FISH. A genomic fingerprinting technique, randomly amplified polymorphic DNA, showed that among 58 CFB isolates only 18 identical genotypes were found, and among the 84 alpha-Proteobacteria only eight identical genotypes were present. The data show the enormous diversity of cultivated bacteria from picoplankton enrichment cultures of one North Sea water sample, which is only a small fraction of the total picoplankton community.     

A meta-analysis of the microbial diversity observed in anaerobic digesters

In this study, the collective microbial diversity in anaerobic digesters was examined using a meta-analysis approach. All 16S rRNA gene sequences recovered from anaerobic digesters available in public databases were retrieved and subjected to phylogenetic and statistical analyses. As of May 2010, 16,519 bacterial and 2869 archaeal sequences were found in GenBank. The bacterial sequences were assigned to 5926 operational taxonomic units (OTUs, based on ?97% sequence identity) representing 28 known bacterial phyla, with Proteobacteria (1590 OTUs), Firmicutes (1352 OTUs), Bacteroidetes (705 OTUs), and Chloroflexi (693 OTUs) being predominant. Archaeal sequences were assigned to 296 OTUs, primarily Methanosaeta and the uncharacterized WSA2 group. Nearly 60% of all sequences could not be classified to any established genus. Rarefaction analysis indicates that approximately 60% of bacterial and 90% of archaeal diversity in anaerobic digesters has been sampled. This analysis of the global bacterial and archaeal diversity in AD systems can guide future studies to further examine the microbial diversity involved in AD and development of comprehensive analytical tools.     

Diversity and distribution of marine microbial eukaryotes in the Arctic Ocean and adjacent seas

We analyzed microbial eukaryote diversity in perennially cold arctic marine waters by using 18S rRNA gene clone libraries. Samples were collected during concurrent oceanographic missions to opposite sides of the Arctic Ocean Basin and encompassed five distinct water masses. Two deep water Arctic Ocean sites and the convergence of the Greenland, Norwegian, and Barents Seas were sampled from 28 August to 2 September 2002. An additional sample was obtained from the Beaufort Sea (Canada) in early October 2002. The ribotypes were diverse, with different communities among sites and between the upper mixed layer and just below the halocline. Eukaryotes from the remote Canada Basin contained new phylotypes belonging to the radiolarian orders Acantharea, Polycystinea, and Taxopodida. A novel group within the photosynthetic stramenopiles was also identified. One sample closest to the interior of the Canada Basin yielded only four major taxa, and all but two of the sequences recovered belonged to the polar diatom Fragilariopsis and a radiolarian. Overall, 42% of the sequences were <98% similar to any sequences in GenBank. Moreover, 15% of these were <95% similar to previously recovered sequences, which is indicative of endemic or undersampled taxa in the North Polar environment. The cold, stable Arctic Ocean is a threatened environment, and climate change could result in significant loss of global microbial biodiversity.     

Electrochemical mutagen screening using microbial chip

Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-d-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities.     

Molecular phylogeny of eukaryotes

Comparisons of ribosomal RNAs and various protein coding genes have contributed to a new view of eukaryote phylogeny. Analyses of paralogous protein coding genes suggest that archaebacteria and eukaryotes are sistergroups. Sequence diversity of small subunit rRNAs in protists by far exceeds that of any multicellular or prokaryote taxon. Remarkably, a group of taxa that lack mitochondria first branches off in the small subunit rRNA tree. The later radiations are formed by a series of clades that were once thought to be more ancestral. Furthermore, tracing of the evolutionary origin of secondary endobiontic events is now possible with sequence comparisons.     

Influence of depth and time on diversity of free-living microbial community in the variably saturated zone of a granitic aquifer

We investigated the temporal and vertical changes in the microbial communities related to hydrological variations an aquifer (Brittany, France). Five water samplings were carried out, spanning three hydrological cycles in the variably and the permanently saturated zones. Seasonal variations in the major anion concentrations (NO3 -, SO4 2- and Cl(-) ) indicated that different physical processes occurred during the recharge process in the two zones. The variably saturated zone is mainly dominated by diffusion and advection processes from the soil, whereas the permanently saturated zone is controlled by moderate advective transfer from the variably saturated zone. Bacterial diversity was investigated by flow cytometry, 16S rRNA and narG genes analyses. Part of this diversity was new in that 6 of the 27 16S rRNA gene sequence phylotypes were unknown even at the class or phylum level. The narG gene analysis did not reveal any clear variation in time or depth within the nitrate reducers' community. In contrast, 16S rRNA gene analyses showed modifications of community composition that could be related to the hydrologic and chemical contrast between the two zones. It was concluded that the physical processes of water transfer could influence bacterial diversity at the soil-aquifer interface.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

Molecular and microscopic diversity of planktonic eukaryotes in the oligotrophic Lake Stechlin (Germany)

The aim of this study was to compare a molecular and a microscopic approach to study the planktonic eukaryotic diversity of an oligotrophic lake. Plankton samples from the temperate Lake Stechlin were assessed in winter and summer 2008 by comparison of 18S rRNA gene clone libraries to light microscopic evaluations. For both approaches identical samples were used. There were remarkable differences between the main groups recovered by the contrasting methods. The microscopic analyses showed predominance of autotrophic planktonic organisms, whereas most of them could not be discovered by the molecular method which resulted in a higher diversity of heterotrophic flagellates. The microscopic survey revealed high diversity of Chlorophyta and Cryptophyta as well as the Stramenopiles groups of Bacillariophyceae and Chrysophyceae. The clone libraries, based on full-length 18S rRNA gene sequences, displayed highest diversity of Alveolata belonging to seven different subclades. Notably, Antarctic Dinophyta-related clones were detected. The occurrence of the marine phagotrophic flagellate Telonema was also documented. Comparing the two sampling seasons, rich diversity suggests that flagellates played an important role in late winter (February), however, there is relatively low diversity in summer (August). The newly discovered molecular diversity of planktonic eukaryotes in Stechlin will help to understand the biodiversity patterns in freshwater lakes.     

Molecular microbial diversity of an agricultural soil in Wisconsin.

A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.     

Reactive oxygen species and development in microbial eukaryotes

Reactive oxygen species (ROS) have been regarded as inevitable harmful by-products of aerobic metabolism. Growing evidence, however, suggests that ROS play important physiological roles. This raises questions about the pathways that different groups of organisms use to produce and sense ROS. In microbial eukaryotes, recent data show (i) increased ROS levels during cell differentiation, (ii) the existence of ROS-producing enzymes, such as NADPH oxidases (NOX), (iii) the involvement of NOX in developmental processes, and (iv) a conservation in the signal-transduction mechanisms used to detect ROS. This shows that manipulation of reactive species, as strategy to regulate cell differentiation, is ubiquitous in eukaryotes and suggests that such strategy was selected early in evolution.     

Molecular diversity and distribution of aromatic hydrocarbon-degrading anaerobes across a landfill leachate plume

Natural attenuation of the mono‐aromates benzene, toluene, ethylbenzene and xylene occurs under iron‐reducing conditions in a leachate‐contaminated aquifer near the Banisveld landfill, the Netherlands. The diversity of mono‐aromate‐degrading microorganisms was studied by targeting functional genes encoding benzylsuccinate synthase α‐subunit (bssA) and 6‐oxocyclohex‐1‐ene‐1‐carbonyl‐CoA hydrolase (bamA). Sixty‐four bssA and 188 bamA variants were sequenced from groundwater sampled along the pollution plume in 1999 and 2004. Species containing bssA sequences closest affiliated (> 91%) with the betaprotebacterium Georgfuchsia toluolica were the dominant alkylbenzene degraders (89% of bssA sequences). bssA genes were found at more than 10‐fold lower copy numbers than bamA genes, of which only a small fraction (< 2%) was closely related to the genes of Georgfuchsia. bamA gene diversity was high and bamA‐based community composition was primarily affected by dissolved organic carbon (DOC) and ferrous iron concentrations. bamA sequences closest related to Geobacteraceae were dominantly (43.2%) observed and the presence of Geobacteraceae‐related bamA sequences was associated with DOC. Our results indicate a key role for specialized Georgfuchsia spp. in the degradation of alkylbenzenes, whereas Geobacteraceae are involved in degradation of aromatics other than toluene and xylene.