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Molecular screening of free-living microbial eukaryotes: diversity and distribution using a meta-analysis.

Numerous environmental gene library studies have shown that eukaryote microbial diversity is much greater than expected. Molecular surveys of several 'extreme' and some more anthropomorphically commonplace environments have revealed many previously unsampled micro-eukaryotic lineages. However, it cannot be assumed that all of the sequences recovered from these studies are derived from real organisms, and for those that are, many questions remain about their distribution and ecology. Integrating all available sequence data from these studies reveals patterns of distribution, diversity and evolutionary relationships that are not accessible from independent analyses of the individual surveys and enables us to review the wider implications of such studies.

Animals↗

Multiple cosmopolitan ecotypes within a microbial eukaryote morphospecies.

Microbial eukaryotes that are morphologically indistinguishable (i.e. 'morphospecies') tend to be genetically diverse. While most protist morphospecies have cosmopolitan distribution, it has been suggested that ribotypes (unique rRNA gene sequences) or rRNA sequence clusters do have biogeography and such clusters may correlate with particular (non-morphological) adaptations. We have studied this in the ciliated protozoan morphospecies Cyclidium glaucoma. Fifty-four isolates collected worldwide represented 31 distinct ribotypes. There was no evidence of biogeographic distribution patterns. For example, identical ribotypes occurred in samples from Argentina, Peru, Morocco, Russia and Ukraine; in samples from Denmark and Australia; and in samples from Great Salt Lake and hyperhaline ponds in Spain. The morphospecies Cyclidium glaucoma is euryhaline and occurs in freshwater, brackish water, seawater, and hyperhaline waters. Evidence suggests that one ribotype cluster occurs only in marine or brackish habitats, and another one has so far been found only in hyperhaline habitats. Two clades seem to occur only in freshwater, but one clade includes ribotypes that were found in freshwater as well as in brackish water.

Animals↗

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.

Journal Article↗

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.

Animals↗

Synthetic statistical approach reveals a high degree of richness of microbial eukaryotes in an anoxic water column.

Molecular surveys suggest that communities of microbial eukaryotes are remarkably rich, because even large clone libraries seem to capture only a minority of species. This provides a qualitative picture of protistan richness but does not measure its real extent either locally or globally. Statistical analysis can estimate a community's richness, but the specific methods used to date are not always well grounded in statistical theory. Here we study a large protistan molecular survey from an anoxic water column in the Cariaco Basin (Caribbean Sea). We group individual 18S rRNA gene sequences into operational taxonomic units (OTUs) using different cutoff values for sequence similarity (99 to 50%) and systematically apply parametric models and nonparametric estimators to the OTU frequency data to estimate the total protistan diversity. The parametric models provided statistically sound estimates of protistan richness, with biologically meaningful standard errors, maximal data usage, and extensive model diagnostics and were preferable to the available nonparametric tools. Our clone library exceeded 700 clones but still covered only a minority of species and less than half of the larger protistan clades. Our estimates of total protistan richness portray the target community as very rich at all OTU levels, with hundreds of different populations apparently co-occurring in the small (3-liter) volume of our sample, as well as dozens of clades of the highest taxonomic order. These estimates are among the first for microbial eukaryotes that are obtained using state-of-the-art statistical methods and can serve as benchmark numbers for the local diversity of protists.

Anaerobiosis↗

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.

Animals↗

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).

Anaerobiosis↗

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.

Animals↗

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep.

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.

Animals↗

RACE and RAGE cloning in parasitic microbial eukaryotes.

Many gene-cloning strategies and gene survey often provide partial sequence data. To exploit the information from these partial sequences numerous PCR-based approaches have been developed to clone full-length open reading frames. These approaches can be successful using small quantities of cDNA or genomic DNA as starting material and avoid the need to go through the complex and tedious process of constructing and screening gene libraries. Here we present two of these approaches, called RACE and RAGE, we used to successfully clone partial and full-length ORFs from amitochondriate parasitic microbial eukaryotes. The RACE approach uses cDNA as template for PCR cloning whereas RAGE uses genomic DNA. These two approaches were used to complement each other to provide full-length genes. The amitochondriate microbial eukaryotes we are investigating are of interest from both evolutionary and biomedical perspectives. We have investigated genes of mitochondrial origins in the obligate intracellular parasite called microsporidia. In these organisms spores are the only source of material that can be isolated from host cells and typically yield small amount of mRNA and genomic DNA for cloning. A full-length mitochondrial Hsp70 could be cloned and sequenced and specific antibody raised against a fusion protein. The highly specific antibody allowed us to demonstrate for the first time the presence of mitochondrial-like organelles in microsporidia.

Animals↗

The molecular ecology of microbial eukaryotes unveils a hidden world.

In spite of the great success of small-subunit ribosomal RNA (SSU rRNA)-based studies for the analysis of environmental prokaryotic diversity, this molecular approach has seldom been applied to microbial eukaryotes. Recent molecular surveys of the smallest eukaryotic planktonic fractions at different oceanic surface regions and in deep-sea Antarctic samples revealed an astonishing protist diversity. Many of the phylotypes found in the photic region affiliate with photosynthetic groups that are known to contain picoeukaryotic representatives in the range 1-2 microm. Surprisingly, a vast diversity of presumably heterotrophic or mixotrophic lineages is also found. Among these, several novel lineages of heterokonts, and a large diversity of alveolates clustering in two major groups (Groups I and II), are present at all depths in the water column. Many of these new phylotypes appear biogeographically ubiquitous. These initial studies suggest that a wide diversity of small eukaryotes remains to be discovered not only in the ocean but also in other environments. For both ecology and evolutionary studies, it is predicted that environmental molecular identification of eukaryotes will have a profound impact in the immediate future.

Animals↗

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.

Antioxidants↗

Spatial scaling of microbial eukaryote diversity.

Patterns in the spatial distribution of organisms provide important information about mechanisms that regulate the diversity of life and the complexity of ecosystems. Although microorganisms may comprise much of the Earth's biodiversity and have critical roles in biogeochemistry and ecosystem functioning, little is known about their spatial diversification. Here we present quantitative estimates of microbial community turnover at local and regional scales using the largest spatially explicit microbial diversity data set available (> 10(6) sample pairs). Turnover rates were small across large geographical distances, of similar magnitude when measured within distinct habitats, and did not increase going from one vegetation type to another. The taxa-area relationship of these terrestrial microbial eukaryotes was relatively flat (slope z = 0.074) and consistent with those reported in aquatic habitats. This suggests that despite high local diversity, microorganisms may have only moderate regional diversity. We show how turnover patterns can be used to project taxa-area relationships up to whole continents. Taxa dissimilarities across continents and between them would strengthen these projections. Such data do not yet exist, but would be feasible to collect.

Ascomycota↗

Global dispersal of free-living microbial eukaryote species.

The abundance of individuals in microbial species is so large that dispersal is rarely (if ever) restricted by geographical barriers. This "ubiquitous" dispersal requires an alternative view of the scale and dynamics of biodiversity at the microbial level, wherein global species number is relatively low and local species richness is always sufficient to drive ecosystem functions.

Animals↗

Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species.

Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences. Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent. One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA). Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3' cDNA fragments. To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae. SAGE libraries were constructed from an actively toxic fish-fed culture of P. shumwayae and from a recently toxic alga-fed culture. P. shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries. Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis. Expression of each transcript was confirmed in separate control cultures of toxic P. shumwayae. The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species.

Animals↗

A model of biocomplexity and its application to the analysis of some terrestrial and marsh eukaryotic microbial communities with an emphasis on amoeboid protists.

Biocomplexity theory has become increasingly important in understanding ecosystem dynamics as we realize that the interactions among subunits in a multi-component system often produce elaborate states that are not easily explained in terms of the individual parts of the system. A Euclidean geometric model of biocomplexity is presented and illustrated using protistan communities. The model is based on three quantitative biotic dimensions (indices) for small subsamples (0.01 g) taken from each sample core of substratum: (1) richness of morphospecies expressed as mean count per 0.01 g, (2) spatial diversity of protists expressed as then umber of unique morphospecies (i.e. those occurring in only one of the 0.01-g subsamples and not in any of the other subsamples), and (3) patchiness (non-uniform aggregation) of the distribution of protists across the 0.01-g subsamples. These three indices are mapped into a three-dimensional Euclidean space model, and the position of each point and its geometric distance from the origin are used as a general index of biocomplexity. The usefulness of the model is illustrated by applying it to a range of terrestrial and marsh communities. Within the set of 15 samples examined in this study, the marsh rhizosphere samples are among the most complex.

Amoeba↗