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Plant functional group diversity promotes soil protist diversity.

We tested whether effects of plant diversity can propagate through food webs, down to heterotrophic protists not linked directly to plants. To this end we synthesised grassland ecosystems with varying numbers of plant functional groups (FGN) and assessed corresponding changes in testate amoebae communities. The number of plant species was kept constant. When FGN was increased from 1 to 3, species number and total community density of live testate amoebae were enhanced according to a linear and a saturating function, respectively. From FGN 1 to 2, the appearance of new testate amoebae species did not affect the presence of the resident species, whereas, from FGN 2 to 3 about one quarter of the resident testate amoebae species was replaced, without altering the total species number. Overall, density by species increased, while evenness of the testate amoebae community was not affected by FGN; although Trinema lineare, one of the most common species, became more abundant. The observed relationship between plant functional group diversity and testate amoebae diversity could shed new light on the biogeographical distribution patterns of protists.

Amoeba↗

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes.

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.

Animals↗

Protist genetic diversity in the acidic hydrothermal environments of Lassen Volcanic National Park, USA.

We examined eukaryote genetic diversity in the hydrothermal environments of Lassen Volcanic National Park (LVNP), Northern California. We sampled hydrothermal areas of the Bumpass Hell, Sulfur Works, Devil's Kitchen, and Boiling Springs Lake sites, all of which included diverse acidic pools, mud pots, and streams with visible algal mats and biofilms. Temperatures varied from 15 to 85 degrees C and pH from 1.7 to 5.8. DNA extraction methods compared by denaturing gradient gel electrophoresis fingerprinting exhibited similar patterns, and showed limited diversity of eukaryotic small subunit (SSU) rRNA genes compared with prokaryotes. We successfully amplified eukaryotic SSU rRNA genes from most environments up to 68 degrees C. Cloned rDNA sequences reveal acidophilic protists dominate eukaryotes in LVNP hydrothermal environments. Most sites showed phototrophic assemblages dominated by chlorophytes and stramenopiles (diatoms and chrysophytes). Heterotrophic taxa, though less abundant, included diverse alveolates (ciliates), amoebae, and flagellates. Fungi were also found at most sites, and metazoans (hexapods, nematodes, platyhelminths) were sometimes detected in less acidic environments, especially in algal mats. While many cloned rDNA sequences showed 95%-99% identity to known acidophilic isolates or environmental clones from other acidic sites (Rio Tinto), sequence diversity generally declined both with decreasing pH and increasing temperature, and both were controlling physical variables on the abundance and distribution of organisms at our sites. However, a pool at 68 degrees C with pH 1.7 yielded the greatest number of distinct sequences. While some were likely contaminants from nearby cooler sites, we suggest that Lassen's acidic hydrothermal features may harbor novel protists.

Acids↗

From genes to genomes: beyond biodiversity in Spain's Rio Tinto.

Spain's Rio Tinto, or Red River, an example of an extremely acidic (pH 1.7-2.5) environment with a high metal content, teems with prokaryotic and eukaryotic microbial life. Our recent studies based on small-subunit rRNA genes reveal an unexpectedly high eukaryotic phylogenetic diversity in the river when compared to the relatively low prokaryotic diversity. Protists can therefore thrive in and dominate extremely acidic, heavy-metal-laden environments. Further, because we have discovered protistan acidophiles closely related to neutrophiles, we can hypothesize that the transition from neutral to acidic environments occurs rapidly over geological time scales. How have these organisms adapted to such environments? We are currently exploring the alterations in physiological mechanisms that might allow for growth of eukaryotic microbes at acid extremes. To this end, we are isolating phylogenetically diverse protists in order to characterize and compare ion-transporting ATPases from cultured acidophiles with those from neutrophilic counterparts. We predict that special properties of these ion transporters allow protists to survive in the Rio Tinto.

Adenosine Triphosphatases↗

The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases.

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.

Animals↗

The closest unicellular relatives of animals.

Molecular phylogenies support a common ancestry between animals (Metazoa) and Fungi, but the evolutionary descent of the Metazoa from single-celled eukaryotes (protists) and the nature and taxonomic affiliation of these ancestral protists remain elusive. We addressed this question by sequencing complete mitochondrial genomes from taxonomically diverse protists to generate a large body of molecular data for phylogenetic analyses. Trees inferred from multiple concatenated mitochondrial protein sequences demonstrate that animals are specifically affiliated with two morphologically dissimilar unicellular protist taxa: Monosiga brevicollis (Choanoflagellata), a flagellate, and Amoebidium parasiticum (Ichthyosporea), a fungus-like organism. Statistical evaluation of competing evolutionary hypotheses confirms beyond a doubt that Choanoflagellata and multicellular animals share a close sister group relationship, originally proposed more than a century ago on morphological grounds. For the first time, our trees convincingly resolve the currently controversial phylogenetic position of the Ichthyosporea, which the trees place basal to Choanoflagellata and Metazoa but after the divergence of Fungi. Considering these results, we propose the new taxonomic group Holozoa, comprising Ichthyosporea, Choanoflagellata, and Metazoa. Our findings provide insight into the nature of the animal ancestor and have broad implications for our understanding of the evolutionary transition from unicellular protists to multicellular animals.

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↗

Nuclear and nucleomorph SSU rDNA phylogeny in the Cryptophyta and the evolution of cryptophyte diversity.

The plastid-bearing members of the Cryptophyta contain two functional eukaryotic genomes of different phylogenetic origin, residing in the nucleus and in the nucleomorph, respectively. These widespread and diverse protists thus offer a unique opportunity to study the coevolution of two different eukaryotic genomes within one group of organisms. In this study, the SSU rRNA genes of both genomes were PCR-amplified with specific primers and phylogenetic analyses were performed on different data sets using different evolutionary models. The results show that the composition of the principal clades obtained from the phylogenetic analyses of both genes was largely congruent, but striking differences in evolutionary rates were observed. These affected the topologies of the nuclear and nucleomorph phylogenies differently, resulting in long-branch attraction artifacts when simple evolutionary models were applied. Deletion of long-branch taxa stabilized the internal branching order in both phylogenies and resulted in a completely resolved topology in the nucleomorph phylogeny. A comparison of the tree topologies derived from SSU rDNA sequences with characters previously used in cryptophyte systematics revealed that the biliprotein type was congruent, but the type of inner periplast component incongruent, with the molecular trees. The latter is indicative of a hidden cellular dimorphism (cells with two periplast types present in a single clonal strain) of presumably widespread occurrence throughout cryptophyte diversity, which, in consequence, has far-reaching implications for cryptophyte systematics as it is practiced today.

Cell Nucleus↗

Analyses of RNA Polymerase II genes from free-living protists: phylogeny, long branch attraction, and the eukaryotic big bang.

The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant obstacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Polymerase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Ochromonas danica (a heterokont); we have also analyzed the RPB1 gene from the nucleomorph (nm) genome of Guillardia theta (a cryptomonad). Using a variety of phylogenetic methods our analysis shows that RPB1s from Giardia intestinalis and Trichomonas vaginalis are probably subject to intense LBA effects. Thus, the deep branching of these taxa on RPB1 trees is questionable and should not be interpreted as evidence favoring their early divergence. Similar effects are discernable, to a lesser extent, with the Mastigamoeba invertens RPB1 sequence. Upon removal of the outgroup and these problematic sequences, analyses of the remaining RPB1s indicate some resolution among major eukaryotic groups. The most robustly supported higher-level clades are the opisthokonts (animals plus fungi) and the red algae plus the cryptomonad nm-the latter result gives added support to the red algal origin of cryptomonad chloroplasts. Clades comprising Dictyostelium discoideum plus Acanthamoeba castellanii (Amoebozoa) and Ochromonas plus Plasmodium falciparum (chromalveolates) are consistently observed and moderately supported. The clades supported by our RPB1 analyses are congruent with other data, suggesting that bona fide phylogenetic relationships are being resolved. Thus, the RPB1 gene has apparently retained some phylogenetically meaningful signal, making it worthwhile to obtain sequences from more diverse protist taxa. Additional RPB1 data, especially in combination with other genes, should provide further resolution of branching orders among protist groups within the apparently rapid early divergence of eukaryotes.

Evolution, Molecular↗

A multistep process gave rise to RNA polymerase IV of land plants.

Since their discovery in Metazoa, the three nuclear RNA polymerases (RNAPs) have been found in fungi, plants, and diverse protists. In all eukaryotes studied to date, RNAPs I, II, and III collectively transcribe all major RNAs made in the nucleus. We have found genes for the largest subunit (RPD1/RPE1) of a new DNA-dependent RNAP, RNAP IV, in all major land plant taxa and in closely related green algae. Genes for the second-largest subunit (RPD2) of this enzyme were found in all land plants. Phylogenetic study indicates that RNAP IV genes are sister to the corresponding RNAP II genes. Our results show the genesis of RNAP IV to be a multistep process in which the largest and second-largest subunit genes evolved by independent duplication events in the ancestors of Charales and land plants. These findings provide insights into evolutionary mechanisms that can explain the origin of multiple RNAPs in the eukaryotic nucleus.

Amino Acid Sequence↗

Apicoplast genome of the coccidian Eimeria tenella.

Unicellular apicomplexans possess an algal-originated plastid referred to as an apicoplast. Although apicomplexan parasites are comprised of highly diverse protists, the complete apicoplast genome sequences have only been determined from the hematozoan Plasmodium falciparum and cyst-forming coccidian Toxoplasma gondii. Here, we report the third complete sequence of apicoplast genome from the intestinal coccidian Eimeria tenella that may serve as a new drug target against coccidiosis in the livestock. The AT-rich E. tenella plastid genome is a 35-kb circular element. Its gene organization resembles more closely that of T. gondii than P. falciparum. Although the E. tenella plastid genome contains an almost identical set of genes to that found in P. falciparum and T. gondii, its encoded genes share low or moderate homologies with their counterparts in the other two apicomplexans. With the addition of this coccidian plastid genome sequence, we attempted to reexamine the apicoplast genome evolution and performed phylogenetic reconstructions using maximum likelihood and Bayesian inference (BI) methods based on a concatenated dataset of plastid-encoded rpoB, rpoC1 and rpoC2 proteins. All resulting rpo protein trees placed apicoplast as a sister to Euglena within the green lineage. On the other hand, many recent studies based on the organization of plastid genes and some nuclear-encoded plastid proteins have supported a common red algal ancestry of apicomplexan and dinoflagellate plastids. If the apicoplast indeed originated from a red ancestor, the green relationship of apicomplexan genes would probably imply that the ancestral host that gave rise to the (red) apicoplast might have already contained some primary green plastid genes.

Animals↗

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ.

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

Amino Acid Sequence↗

The SSU rDNA coding region of a filose amoeba contains a group I intron lacking the universally conserved G at the 3'-terminus.

We sequenced small subunit ribosomal DNA (rDNA) PCR-fragments of sizes 2.3 kb and 2.9 kb isolated from a culture of the red alga, Porphyra spiralis var. spiralis. Phylogenetic analysis of the 2.3-kb fragment showed that it encoded the sequence of a contaminant filose amoeba. The Nuclearia-like amoeba (named strain N-Por) was identified with scanning electron microscopy. Its rDNA sequence was positioned with strong bootstrap support within a diverse protist assemblage that includes filose amoebae, chlorarachniophytes, cercomonads, and Plasmodiophora brassicae. The rDNA of N-Por contained a group I intron at the conserved 943 position that remarkably, had a U at the 3'-terminus rather than the universally conserved G.

Animals↗

Telonemia, a new protist phylum with affinity to chromist lineages.

Recent molecular investigations of marine samples taken from different environments, including tropical, temperate and polar areas, as well as deep thermal vents, have revealed an unexpectedly high diversity of protists, some of them forming deep-branching clades within important lineages, such as the alveolates and heterokonts. Using the same approach on coastal samples, we have identified a novel group of protist small subunit (SSU) rDNA sequences that do not correspond to any phylogenetic group previously identified. Comparison with other sequences obtained from cultures of heterotrophic protists showed that the environmental sequences grouped together with Telonema, a genus known since 1913 but of uncertain taxonomic affinity. Phylogenetic analyses using four genes (SSU, Hsp90, alpha-tubulin and beta-tubulin), and accounting for gamma- and covarion-distributed substitution rates, revealed Telonema as a distinct group of species branching off close to chromist lineages. Consistent with these gene trees, Telonema possesses ultrastructures revealing both the distinctness of the group and the evolutionary affinity to chromist groups. Altogether, the data suggest that Telonema constitutes a new eukaryotic phylum, here defined as Telonemia, possibly representing a key clade for the understanding of the early evolution of bikont protist groups, such as the proposed chromalveolate supergroup.

Cryptophyta↗

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↗

The nucleariid amoebae: more protists at the animal-fungal boundary.

Nucleariid amoebae are naked amoebae, generally characterized by a spherical or sometimes flattened body with radiating filopodia. Most species preferentially consume algal prey or cyanobacteria. Phylogenetic analyses of the small-subunit rRNA coding regions from four nucleariid amoebae place these species near the origin of the animal-fungal divergence, together with the choanoflagellate-Corallochytrium and the ichthyosporean clades. The species Nuclearia delicatula, N. moebiusi, and N. simplex form a monophyletic group, while ATCC 30864, tentatively but possibly incorrectly assigned to Nuclearia sp., represents a separate line of descent. These nucleariids are unrelated to the lineage containing the testate filose amoebae (Testaceafilosia). Our findings expand the morphological and phylogenetic diversity of protists at the animal-fungal divergence.

Amoeba↗